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Calcium Signaling and Cell-Proliferation
Abstract
The orderly sequence of events that constitutes the cell cycle is carefully regulated. A part of this regulation depends upon the ubiquitous calcium signalling system. Many growth factors utilize the messenger inositol trisphosphate (InsP3) to set up prolonged calcium signals, often organized in an oscillatory pattern. These repetitive calcium spikes require both the entry of external calcium and its release from internal stores. One function of this calcium signal is to activate the immediate early genes responsible for inducing resting cells (G0) to re-enter the cell cycle. It may also promote the initiation of DNA synthesis at the G1/S transition. Finally, calcium contributes to the completion of the cell cycle by stimulating events at mitosis. The role of calcium in cell proliferation is highlighted by the increasing number of anticancer therapies and immunosuppressant drugs directed towards this calcium signalling pathway.
... Recently, a selective TRPC6 antagonist, BI-749327 (with IC 50 of 13-19 nM for human and animal TRPC6), has been demonstrated to improve renal and cardiac fibrosis (55). BI-749327 is much more selective for TRPC6 than other TRPC channels based on IC 50 for different TRPC channels and other types of cation channels (e.g., 85-fold more selective for TRPC6 than for TRPC3 and 42-fold more selective for TRPC6 than for TRPC7) (55). ...
... An increase in [Ca 2 þ ] cyt , which can rapidly increase nuclear [Ca 2 þ ] (81) and stored [Ca 2 þ ] in the sarcoplasmic and endoplasmic reticulum (82)(83)(84), activates cytoplasmic mitogen-activated protein kinases and nuclear Ca 2 þsensitive transcriptional factors, thereby promoting cell proliferation (20,65,85). The PI3K/AKT/mTOR signaling cascade is one of the major pathways involved in cell proliferation and protein expression (86)(87)(88)(89)(90)(91) and is implicated in the development of PAH/PH (69,92). ...
... An increase in [Ca 2 þ ] cyt in PASMCs is a major trigger for PASMC contraction and pulmonary vasoconstriction (18,19,(99)(100)(101) and is an important stimulus for PASMC proliferation/migration and concentric pulmonary vascular wall thickening or medial hypertrophy (28,85). Receptor-operated Ca 2 þ channels and store-operated Ca 2 þ channels formed in part by TRP channels (including TRPC6) and Orai/ STIM channels are important Ca 2 þ -permeable cation channels responsible for regulating [Ca 2 þ ] cyt in PASMCs and thus pulmonary vasoconstriction and vascular remodeling (20,33,34,62,65,70,102,103). ...
Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Pulmonary vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and pulmonary arterial remodeling due to PASMC proliferation are causes for increased pulmonary vascular resistance in patients with PAH. We and others observed upregulation of TRPC6 channels in PASMC from patients with PAH. An increase in cytosolic Ca ²⁺ concentration ([Ca ²⁺ ] cyt ) in PASMC triggers PASMC contraction and vasoconstriction, while Ca ²⁺ -dependent activation of PI3K/AKT/mTOR pathway is pivotal for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 blocker has yet been developed and tested for treatment of PAH. We sought to investigate whether block of receptor-operated Ca ²⁺ channels or TRPC6 can reverse established PH in mice via inhibiting Ca ²⁺ -dependent activation of AKT/mTOR signaling. Here we report that intrapulmonary application of 2-aminoethyl diphenyl borniate (2-APB), a non-selective blocker of cation channels or BI-749237, a selective blocker of TRPC6, significantly and reversibly inhibited acute hypoxic pulmonary vasoconstriction. Intraperitoneal injection of 2-APB significantly attenuated the development of PH and partially reversed established PH. Oral gavage of the selective TRPC6 blocker BI-749237 reversed established PH by 50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749237 both inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. These results indicates that the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH. BI-749237, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH.
... Members of the Bcl-2 family have been proposed to be regulators of intracellular Ca 2+ signaling in cell survival and cell death by regulating Ca 2+ transport systems located in the ER and mitochondria membranes, and at the plasma membrane [13]. It has been widely demonstrated that Ca 2+ is involved in cell cycle progression [14][15][16]. Ca 2+ controls the cell cycle mainly through the Ca 2+ /calmodulin complex that regulates the expression of cyclin-dependent protein kinases (CDKs), which, in turn, control the cell cycle. However, Ca 2+ fluxes undergo alterations related to checkpoints found throughout the cell cycle [17,18]. ...
... The cell cycle arrest may be temporary or permanent, depending on whether the cellular machinery can repair the failure or not. Ca 2+ signaling also participates in the re-entry of arrested cells (G0) into the cell cycle by the activation of transcription factors such as FOS, JUN, cyclic AMP-response element, and serum response element [16]. ...
Pro-apoptotic multi-domain proteins of the BCL2 family such as BAX and BAK are well known for their important role in the induction of mitochondrial outer membrane permeabilization (MOMP), which is the rate-limiting step of the intrinsic pathway of apoptosis. Human or mouse cells lacking both BAX and BAK (due to a double knockout, DKO) are notoriously resistant to MOMP and cell death induction. Here we report the surprising finding that BAX/BAK DKO cells proliferate less than control cells expressing both BAX and BAK (or either BAX or BAK) when they are driven into tetraploidy by transient exposure to the microtubule inhibitor nocodazole. Mechanistically, in contrast to their BAX/BAK-sufficient controls, tetraploid DKO cells activate a senescent program, as indicated by the overexpression of several cyclin-dependent kinase inhibitors and the activation of β-galactosidase. Moreover, DKO cells manifest alterations in ionomycin-mobilizable endoplasmic reticulum (ER) Ca ²⁺ stores and store-operated Ca ²⁺ entry that are affected by tetraploidization. DKO cells manifested reduced expression of endogenous sarcoplasmic/endoplasmic reticulum Ca ²⁺ ATPase 2a (Serca2a) and transfection-enforced reintroduction of Serca2a, or reintroduction of an ER-targeted variant of BAK into DKO cells reestablished the same pattern of Ca ²⁺ fluxes as observed in BAX/BAK-sufficient control cells. Serca2a reexpression and ER-targeted BAK also abolished the tetraploidy-induced senescence of DKO cells, placing ER Ca ²⁺ fluxes downstream of the regulation of senescence by BAX/BAK. In conclusion, it appears that BAX/BAK prevent the induction of a tetraploidization-associated senescence program. Speculatively, this may contribute to the low incidence of cancers in BAX/BAK DKO mice and explain why human cancers rarely lose the expression of both BAX and BAK.
... (Berridge et al., 2000;Dolmetsch, Lewis, Goodnow, & Healy, 1997;Kuklina, 2013;Srinivasan & Frauwirth, 2009;Zheng, Zha, & Gajewski, 2008). Hierunter fallen unter anderem Proliferation (Berridge, 1995), Differenzierung (Kim et al., 2014; Bei Labormäusen wurde bei der gesamten T-Zellpopulation (Proust, Filburn, Harrison, Buchholz, & Nordin, 1987), isolierten CD4 + T-Zellen (Grossmann et al., 1991;Philosophe & Miller, 1990;Tamura et al., 2000) und CD8 + T-Zellen (Grossmann et al., 1991;Philosophe & Miller, 1990) (1/τ) ± Standardfehler des arithmetischen Mittels für den jeweiligen Ca 2+ -Plateaubereich vor Ca 2+ -Ausstrom für alle Zellen jeder verwendeten jungen (schwarz, n = 4 -5) und alten (rot, n = 4 -7) Maus. Die Stichprobenmenge pro Plateaubereich ist rechtsseitig neben dem Punkt angegeben. ...
... Hierzu wurden Ende der 1980er bis Anfang der 2000er Jahre Publikationen zu einer Verminderung der Ca 2+ -Signale bei der gesamten T-Zellpopulation (Proust et al., 1987), isolierten CD4 + T-Zellen (Grossmann et al., 1991;Philosophe & Miller, 1990;Tamura et al., 2000) und CD8 + T-Zellen (Grossmann et al., 1991;Philosophe & Miller, 1990) Schwarz et al., 2013;Trebak & Kinet, 2019). Somit war zur Zeit der Feststellung der altersbedingt verminderten Ca 2+ -Signale in T-Zellen zwar das Prinzip des kapazitativen Ca 2+ -Einstroms bekannt (Putney, 1986), die Funktionen der Komponenten STIM (Liou et al., 2005;Roos et al., 2005) und Orai Parekh & Putney, 2005;Vig et al., 2006) (Grynkiewicz, Poenie, & Tsien, 1985 Bedeutung (Berridge, 1995;Hoth, 2016;Lu & Means, 1993;Shah et al., 1994). In T-Zellen wurde gezeigt, dass die TZR-vermittelte Expansion vom SOCE und von Calcineurin kontrolliert wird (Vaeth, Maus, et al., 2017). ...
... The activation of potassium channels also has a role to play in maintaining the elevated levels of the cytoplasmic calcium ions because potassium channels sustain the hyperpolarization at the plasma membrane that is necessary for the calcium ions influx [35][36][37]. Calcium ions have several crucial functions inside the cells. Calcium ions act as a second messenger that activates numerous physiological processes including oocytes fertilization, embryonic formation, cell growth, differentiation, proliferation, contraction, secretion, metabolism, gene expression, cell survival, and cell death [38,39]. ...
Potassium voltage-gated channel subfamily a member 2 (Kv1.2, encoded by KCNA2) is highly expressed in the central and peripheral nervous systems. Based on the patch clamp studies, gain-of function (GOF), loss-of-function (LOF), and a mixed type (GOF/LOF) variants can cause different conditions/disorders. KCNA2-related neurological diseases include epilepsy, intellectual disability (ID), attention deficit/hyperactive disorder (ADHD), autism spectrum disorder (ASD), pain as well as autoimmune and movement disorders. Currently, the molecular mechanisms for the reported variants in causing diverse disorders are unknown. Consequently, this review brings up to date the related information regarding the structure and function of Kv1.2 channel, expression patterns, neuronal localizations, and tetramerization as well as important cell and animal models. In addition, it provides updates on human genetic variants, genotype–phenotype correlations especially highlighting the deep insight into clinical prognosis of KCNA2-related developmental and epileptic encephalopathy, mechanisms, and the potential treatment targets for all KCNA2-related neurological disorders.
... This mechanism is usually triggered by the activation of a G-protein coupled receptor, primarily the Gq/11 subtype which leads to the activation of phospholipase C (PLC) cleaving the phosphatidylinositol 4,5 bisphosphate (PIP2) into 1,4,5-inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 then binds to the IP3 receptor (IP3R) in the ER allowing the Ca 2+ to diffuse from the ER to the cytosol [4]. Conversely, Ca 2+ is expelled through transporters and pumps found in the endoplasmic reticulum (ER) or cytoplasmic membrane, which aids in restoring the basal level of free Ca 2+ in the cytoplasm. ...
Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte–astrocyte and astrocyte–neuron interactions occur through cytoplasmic Ca²⁺ level changes that are assessed to determine cell function and response (i.e., drug testing). The evaluation of alterations in intracellular Ca²⁺ levels primarily relies on fluorescence imaging techniques, performed through video recording of cells incubated with Ca²⁺-sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) encompassing a single cell, it is possible to draw conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA—signaling automated analysis, for astrocyte ROI-based fluorescent imaging. This tool is specifically tailored for two wavelengths excited dyes by using two inputs of Ca²⁺ signaling recorded frames/videos and outputting a set of features relevant to the experiment’s conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. Subsequently, SIGAA extracts intracellular Ca²⁺ evolution functions for all identified ROIs detects function transients, and estimates a set of features for each signal. These features closely resemble those obtained through traditional methods and software used thus far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.
... Control over signal transducers such as second messengers can thus be used to pose new questions regarding information embedded in signaling features and to test a diverse range of temporal dynamics. In particular, Ca 2+ is a versatile and ubiquitously present second messenger which profoundly controls cellular processes including proliferation and death [91,92]. Recently, Lai et al [93] used R-GECO [94] and LAR-GECO1.2-mt ...
Cells communicate with each other to jointly regulate cellular processes during cellular differentiation and tissue morphogenesis. This multiscale coordination arises through spatiotemporal activity of morphogens to pattern cell signaling and transcriptional factor activity. This coded information controls cell mechanics, proliferation, and differentiation to shape the growth and morphogenesis of organs. While many of the molecular components and physical interactions have been identified in key model developmental systems, there are still many unresolved questions related to the dynamics involved due to challenges in precisely perturbing and quantitatively measuring signaling dynamics. Recently, a broad range of synthetic optogenetic tools have been developed and employed to quantitatively define relationships between signal transduction and downstream cellular responses. These optogenetic tools can control intracellular activities at the single cell or whole tissue scale to direct subsequent biological processes. In this brief review, we highlight a selected set of studies that develop and implement optogenetic tools to unravel quantitative biophysical mechanisms for tissue growth and morphogenesis across a broad range of biological systems through the manipulation of morphogens, signal transduction cascades, and cell mechanics. More generally, we discuss how optogenetic tools have emerged as a powerful platform for probing and controlling multicellular development.
... The role of cytosolic Ca2+ in regulating VSMC proliferation has received less attention [11,12]. Indeed, increased cytosolic Ca2+ levels ([Ca2+]cyto) and Ca2+ transients are mechanisms by which growth factors stimulate cell proliferation [13][14][15]. ...
Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process.
... High mortality rate of oral cancer is due to its aggressiveness and high metastatic ability, and EMT in OSCC is marked by loss of epithelial markers, high expression of mesenchymal markers and onset of cadherin switching (shift in expression from E-cadherin to N-cadherin) and EGFsignalling pathway was found to the inducer of EMT in OSCC and as EGFR is overexpressed, it leads to continuously activated EMT signalling [87,88]. Calcium signalling is an important regulator for the induction of early response genes of cell cycle, in synthesis and phosphorylation of cyclins [89][90][91] and induce MMPs and focal adhesions to modulate the cytoskeleton during cancer cell migration [11,15]. But in many cancers, this signalling is mis-regulated, leading to enhanced proliferative capabilities and enhanced invasiveness as EMT mediated increase in cytosolic calcium levels were already reported and chelating the intracellular calcium levels reduced the expression of mesenchymal marker VIM and also revered EMT [92]. ...
Understanding the molecular mechanism(s) of small compounds in cellular growth control are essential for using those against the disease(s). Oral cancers exhibit a very high mortality rate due to higher metastatic potential. Aberrant EGFR, RAR, HH signalling, enhanced [Ca2+] and oxidative stress are some of the important characteristics of oral cancer. So, we target these for our study. Herein, we tested the effect of fendiline hydrochloride (FH) as an LTCC Ca2+-channel inhibitor, erismodegib (a SMO inhibitor of HH-signalling) and all-trans retinoic acid (RA) inducer of RAR signalling that causes cellular differentiation. OCT4 activating compound (OAC1) counters differentiation and induces stemness properties. Cytosine β-D arabinofuranoside (Cyto-BDA), a DNA replication inhibitor was used to reduce high proliferative capacity. Treatment of FaDu cells with OAC1, Cyto-BDA and FH increase G0/G1 population by 3%, 20% and 7% respectively, and lead to reduction of cyclin D1, CDK4/6 levels. Erismodegib arrests the cells in S-phase with reduced cyclin-E1&A1 levels, whereas RA-treatment causes G2/M phase arrest with reduced cyclin-B1. There was a decrease in the expression of EGFR and mesenchymal markers, Snail/Slug/Vim/Zeb/Twist, and increased E-cadherin expression in all the drug treatments, indicating a reduction in proliferative signal and EMT. Enhanced MLL2 (Mll4) and reduced EZH2 expression associated overexpression of p53 and p21 were traced out. We conclude that these drugs impact expression of epigenetic modifiers by modulating signalling pathways and the epigenetic modifiers then controls the expression of cell cycle control genes, including p53 and p21.
... A key step in the cell cycle has been reported to be Ca 2+ signaling-dependent [44]. Moreover, the exit from quiescence in the early G1 phase is induced by an increased Ca 2+ concentration [45]. However, cell proliferation cannot be promoted by increasing the Ca 2+ concentration alone, but also requires the participation of several factors, such as MYC [46]. ...
The regulatory role of non-CpG methylation in mammals has been important in whole-genome bisulfite sequencing. It has also been suggested that non-CpG methylation regulates gene expression to affect the development and health of mammals. However, the dynamic regulatory mechanisms of genome-wide, non-CpG methylation during testicular development still require intensive study. In this study, we analyzed the dataset from the whole-genome bisulfite sequencing (WGBS) and the RNA-seq of precocious porcine testicular tissues across two developmental stages (1 and 75 days old) in order to explore the regulatory roles of non-CpG methylation. Our results showed that genes regulated by non-CpG methylation affect the development of testes in multiple pathways. Furthermore, several hub genes that are regulated by non-CpG methylation during testicular development—such as VEGFA, PECAM1, and FZD7—were also identified. We also found that the relative expression of FZD7 was downregulated by the zebularine-induced demethylation of the first exon of FZD7. This regulatory relationship was consistent with the results of the WGBS and RNA-seq analysis. The immature porcine Sertoli cells were transfected with RNAi to mimic the expression patterns of FZD7 during testicular development. The results of the simulation test showed that cell proliferation was significantly impeded and that cell cycle arrest at the G2 phase was caused by the siRNA-induced FZD7 inhibition. We also found that the percentage of early apoptotic Sertoli cells was decreased by transfecting them with the RNAi for FZD7. This indicates that FZD7 is an important factor in linking the proliferation and apoptosis of Sertoli cells. We further demonstrated that Sertoli cells that were treated with the medium collected from apoptotic cells could stimulate proliferation. These findings will contribute to the exploration of the regulatory mechanisms of non-CpG methylation in testicular development and of the relationship between the proliferation and apoptosis of normal somatic cells.
... where the authors showed that non-ER-based CALR can affect calcium signaling (Biwer et al., 2018). Decreased extracellular calcium level hampers cellular communication and eventually results in slower cell growth as imbalance of calcium ions is able to induce the G0 phase of the cell cycle (Berridge, 1995). This is in line with our data on slower cell proliferation of exon-9 KO 293 T cells (data not shown). ...
Mutations in the form of insertions and deletions (INDEL) in the calreticulin gene lead to essential thrombocythemia (ET) which is characterized by the formation of thrombosis. However, the connection between calreticulin INDEL and ET remains largely elusive. Through combined molecular dynamics simulation, clustered regularly interspaced short palindromic repeats (CRISPR) and calcium imaging studies on the wild type and mutated isoforms of calreticulin, the mechanism underlying the calreticulin INDEL induced ET was investigated at the molecular level. Our results demonstrate that mutations in exon-9 could lead to significant conformational variations of calreticulin structure and thereby reducing its interaction with calcium ions due to decreased electrostatic contributions. The consequence of mutations on calreticulin's structural integrity was revealed by identifying the key residues and their roles in calcium binding. Furthermore, mutations implemented by CRISPR-Cas9 in exon-9 showed diminished calcium signaling in HEK-293T cells, which agree well with our in-silico findings. The current study might help in understanding the variations of molecular interactions between calreticulin's exon-9 and calcium ions during physiological and pathological conditions. The results might also provide useful information for designing novel therapeutic approaches targeting ET.
... Ebner et al. (2015) proposed the use of AOA with calcimycin (GM508 Cult-Active) to treat 57 couples with fertilization rate >30% and EDA (no transfer), complete developmental delay (no morula/ blastocyst on D5), or reduced blastocyst formation ( 15%) in at least one ICSI cycle. The authors' hypothesis was based on different reports showing a correlation between cell division and Ca 2þ availability (Berridge, 1995), and reporting that the Ca 2þ fluctuations normally preceding mitotic division, disappear in arrested embryos (Sousa et al., 1996). Moreover, some studies performed in animal models also suggest that altering the Ca 2þ released during oocyte activation burdens both fertilization but also embryo development (Ozil and Huneau, 2001;Ozil et al., 2005Ozil et al., , 2006. ...
Study question:
Can recurrent embryo developmental problems after ICSI be overcome by assisted oocyte activation (AOA)?
Summary answer:
AOA did not improve blastocyst formation in our patient cohort with recurrent embryo developmental problems after ICSI.
What is known already:
The use of AOA to artificially induce calcium (Ca2+) rises by using Ca2+ ionophores (mainly calcimycin and ionomycin) has been reported as very effective in overcoming fertilization failure after ICSI, especially in patients whose Ca2+ dynamics during fertilization are deficient. However, there is only scarce and contradictory literature on the use of AOA to overcome embryo developmental problems after ICSI, and it is not clear whether abnormal Ca2+ patterns during fertilization disturb human preimplantation embryo development. Moreover, poor embryo development after ICSI has also been linked to genetic defects in the subcortical maternal complex (SCMC) genes.
Study design, size, duration:
This prospective cohort single-center study compared ICSI-AOA cycles and previous ICSI cycles in couples with normal fertilization rates (≥60%) but impaired embryonic development (≤15% blastocyst formation) in at least two previous ICSI cycles. In total, 42 couples with embryo developmental problems were included in this study from January 2018 to January 2021.
Participants/materials, setting, methods:
Of the 42 couples included, 17 underwent an ICSI-AOA cycle consisting of CaCl2 injection and double ionomycin exposure. Fertilization, blastocyst development, pregnancy, and live birth rates after ICSI-AOA were compared to previous ICSI cycles. In addition, the calcium pattern induced by the male patient's sperm was investigated by mouse oocyte calcium analysis. Furthermore, all 42 couples underwent genetic screening. Female patients were screened for SCMC genes (TLE6, PADI6, NLRP2, NLRP5, NLRP7, and KHDC3L) and male patients were screened for the sperm-oocyte-activating factor PLCZ1.
Main results and the role of chance:
We compared 17 AOA cycles to 44 previous ICSI cycles from the same patient cohort. After AOA, a total fertilization rate of 68.95% (131/190), a blastocyst development rate of 13.74% (18/131), a pregnancy rate of 29.41% (5/17), and a live birth rate of 23.53% (4/17) were achieved, which was not different from the previous ICSI cycles (76.25% (321/421, P-value = 0.06); 9.35% (30/321, P-value = 0.18), 25.00% (11/44, P-value = 0.75), and 15.91% (7/44, P-value = 0.48), respectively). Calcium analysis showed that patient's sperm induced calcium patterns similar to control sperm samples displaying normal embryo developmental potential. Genetic screening revealed 10 unique heterozygous variants (in NLRP2, NLRP5, NLRP7, TLE6, and PADI6) of uncertain significance (VUS) in 14 females. Variant NLRP5 c.623-12_623-11insTTC (p.?) was identified in two unrelated individuals and variant NLRP2 c.1572T>C (p.Asp524=) was identified in four females. Interestingly, we identified a previously reported homozygous mutation PLCZ1, c.1499C>T (p.Ser500Leu), in a male patient displaying impaired embryonic development, but not showing typical fertilization failure.
Limitations, reasons for caution:
Our strict inclusion criteria, requiring at least two ICSI cycles with impaired embryo development, reduced cycle-to-cycle variability, while the requirement of a lower blastocyst development not influenced by a poor fertilization excluded couples who otherwise would be selective cases for AOA; however, these criteria limited the sample size of this study. Targeted genetic screening might be too restricted to identify a genetic cause underlying the phenotype of poor embryo development for all patients. Moreover, causality of the identified VUS should be further determined.
Wider implications of the findings:
Strong evidence for AOA overcoming impaired embryonic development is still lacking in the literature. Thus far, only one article has reported a beneficial effect of AOA (using calcimycin) compared to previous ICSI cycles in this patient population, whilst two more recent sibling-oocyte control studies (one using calcimycin and the other ionomycin) and our research (using ionomycin) could not corroborate these findings. Although no major abnormalities have been found in children born after AOA, this technique should be reserved for couples with a clear Ca2+-release deficiency. Finally, genetic screening by whole-exome sequencing may reveal novel genes and variants linked to embryo developmental problems and allow the design of more personalized treatment options, such as wild-type complementary RNA or recombinant protein injection.
Study funding/competing interest(s):
This study was supported by the Flemish Fund for Scientific Research (grant FWO.OPR.2015.0032.01 to B.H. and grant no. 1298722N to A.B.). A.C.B., D.B., A.B., V.T., R.P., F.M., I.D.C., L.L., D.S., P.D.S., P.C., and F.V.M. have nothing to disclose. B.H. reports a research grant from the Flemish Fund for Scientific Research and reports being a board member of the Belgian Society for Reproductive Medicine and the Belgian Ethical Committee on embryo research.
Trial registration number:
NCT03354013.
... 7,8 The calcium ions present in the dressing are exchanged for sodium ions present in the serum and wound exudate, triggering the calcium signal transduction pathway through ion exchange and even inducing genes that prompt resting cells to re-enter the cell cycle. 9 Bacterial contamination of skin wounds is responsible for high morbidity and mortality rates globally. The World Health Organization estimates that 11 million burns and scalds of various types occur every year and that 180 000 people die every year from these injuries worldwide. ...
We have developed a multifunctional hydrogel that can carry three synergistic antibiotics commonly used in clinical practice. This hydrogel was discovered to have drug encapsulation efficiencies of 94% for neomycin, 97% for bacitracin, and 88% for polymyxin B. Drug release data indicated that the release profiles of these three antibiotics were different. A swelling test demonstrated that the hydrogel absorbed liquid after the release of its antibiotics until it became saturated, which occurred within 48 h. Moreover, this hydrogel exhibited excellent antibacterial effects against Escherichia coli and Pseudomonas aeruginosa and biocompatibility; it can thus protect a wound from microbial invasion. When the alginate hydrogel is used to cover a wound, the wound can be checked for colonization at any time using ultrasound imaging; this can thus enable the prevention of wound biofilm formation in the early stages of infection. We evaluated the hydrogel against commercially available wound dressings and discovered that these wound dressings did not have the aforementioned desirable features. In conclusion, our multifunctional hydrogel can carry three types of antibiotics simultaneously and is a suitable medium through which an ultrasound can be performed to detect the growth of colonies in wounds. The hydrogel is expected to make a valuable contribution to the prevention of wound infections in the future.
... This mechanism is usually triggered by activation of a G-protein coupled receptor, primarily the Gq/11 subtype which led to the activation of phospholipase C (PLC) cleaving the phosphatidylinositol 4,5 bisphosphate (PIP2) into 1,4,5-inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 then binds to the IP3 receptor (IP3R) in the ER allowing the Ca 2+ to diffuse from the ER to the cytosol [4]. Ca 2+ entering through plasma membrane located channels, also contributes to rises in intracellular Ca 2+ , whereas Ca 2+ exit through transporters and pumps present at the ER or cytoplasm membrane allows restoring of basal levels of free Ca 2+ at the cytoplasm, which in a healthy cell are at the low nanomolar range. ...
Astrocytes are non-neural cells, restricted to the brain and spinal cord, whose functions and morphology depend on their location. Astrocyte-astrocyte and astrocyte-neuron interactions occur through cytoplasmic Ca ²⁺ levels changes, that are assessed to determine cell function and response (i.e. drug testing). Evaluation of changes in intracellular Ca ²⁺ levels is mostly centered on fluorescence imaging approaches, performed through video recording of cells incubated with Ca ²⁺ -sensitive dyes. By observing ion concentration shifts over time in a delimited region of interest (ROI) comprising a single cell, it is possible to attain conclusions on cell responses to specific stimuli. Our work describes a tool named SIGAA – SIGnaling Automated Analysis , for astrocyte ROI-based fluorescent imaging, which is tailored for two wavelengths dyes by using two inputs of Ca ²⁺ signaling recorded frames/videos and outputs a set of features relevant to the experiment’s conclusions and cell characterization. SIGAA performs automatic drift correction for the two recorded videos with a template matching algorithm, followed by astrocyte identification (ROI) using morphological reconstruction techniques. SIGAA then extracts intracellular Ca ²⁺ evolution functions for all identified ROIs, detects function transients, and estimates a set of features for each signal, which are very similar to the ones obtained by the traditional methods and software used so far. SIGAA is a new fully automated tool, which can speed up hour-long studies and analysis to a few minutes, showing reliable results as the validity tests indicate.
... Ca +2 signalling following oocyte activation plays a pivotal role in pronuclear union, in initiating first and subsequent embryonic cleavages (34,35). In the present study the cleavage rates (96.33% ± 11.33 vs. ...
Objective: Achieving pregnancy in poor ovarian response patients is a challenge. Failed fertilization after ICSI, despite normal semen parameters is due to defects in oocyte activation. In-vitro activation of oocytes using Ca+2 agents can be useful in increasing the fertilization rates in these patients. This study aimed to evaluate the efficacy of artificial oocyte activation by calcium ionophores in poor responders in improving fertilization, cleavage, implantation and clinical pregnancy rates.
Materials and methods: This is a prospective, cohort study conducted on 120 patients having poor ovarian response, (POSEIDON criteria) undergoing in-vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment at Southend Fertility and IVF, New Delhi from 1st August 2019 to 31st March 2020. Exclusion criterion was patients with partners with abnormal semen parameters. After OPU patients were randomized into two groups, study group (n=50) underwent ICSI-AOA (ICSI followed by artificial oocyte activation) using calcium ionophore- GM508 Cult-Active Solution) while the controls (n=57) were subjected to ICSI only.
Results: Comparison of ICSI-AOA and ICSI groups showed: (i) number of fertilized oocytes - 2.42 vs. 2.16, p = 0.049 (ii) No. of cleavage stage embryos 2.32 vs. 1.96, p = 0.008 (iii) No. of grade A embryos 1.52 vs. 1.04, p = 0.009 (iv) fertilization rate - 89.00% vs. 83.04%, p = 0.093 (v) cleavage rate - 96.33% vs. 92.55%, p = 0.165 (vi) implantation rate - 27.14% vs. 11.74%, p = 0.098 (vii) clinical pregnancy rate - 34.3% vs. 20.5%, p = 0.167.
Conclusion: The number of fertilized oocytes, grade A embryos and cleavage stage embryos formed after ICSI-AOA were statistically significantly more than ICSI. ICSI-AOA has not shown improvement in fertilization, cleavage, implantation and clinical pregnancy rate. From the present study the conclusive evidence cannot be drawn due to small sample size hence further studies are needed on a larger population.
... The targets of these miRNAs are the genes FOXO3a and FOXO3b, repressing the FoxO signaling pathway and resulting in an anti-apoptotic response [108], in contrast to the one showed by miR-146a. Moreover, miR-21 targets also the phospholipase C (PLC) and phosphatidylinositol 3-kinase (PIK3c) involved in the PIK3-AKT signaling pathway, that modulate positively cell proliferation and growth [109,110]. ...
Extreme environments are characterized by peculiar conditions, such as hypoxia/anoxia, freezing/heat temperatures, and desiccation. With climate change, more and more habitats are facing extreme conditions and living communities are finding ways to adapt in order to survive. In this study, we show several species which have been shown to adapt to marine extreme conditions also via miRNA-mediated responses. miRNAs are a class of small non-coding RNAs that mediate gene regulation via interactions with transcripts. Their action can directly or indirectly regulate pathways that can result in a response to a specific condition. Furthermore, the study of these miRNA-mediated responses could help in the biotechnological field for their application in the development of environmental biomarkers of stress conditions, or in the genetic engineering of algal species for the production of high-value compounds.
... This is mainly because the RE ions (RE 3+ ) have similar chemical structures and properties that are expected to have similar biological properties and activity mechanisms, and can occupy Ca positions and replace bound Ca ions due to their large ionic charge than Ca 2+ , thereby increasing intracellular Ca 2+ [100]. Ca 2+ plays a key role in regulating cell proliferation and differentiation, and an increase in cytoplasmic Ca 2+ concentration is essential and required for cell proliferation [101]. So RE elements can promote extracellular Ca 2+ in cells, thereby increasing cell proliferation. ...
The unique combination of biodegradability, biocompatibility, and functionality of zinc (Zn)-based alloys makes them highly desirable for a wide range of medical applications. However, a long-standing problem associated with this family of biodegradable alloys in the as-cast state is their limited mechanical strength and slow degradation rate. Here we report the development of Zn-xDy (x = 1, 3, and 5 wt.%) alloys with high strength, ductility, cytocompatibility, antibacterial ability, and appropriate degradation rate for biodegradable bone-implant applications. Our results indicate that the mechanical properties of Zn–xDy alloys were effectively improved with increasing Dy addition and hot-rolling due to the second-phase strengthening. The hot-rolled (HR) Zn–3Dy alloy showed the best combined mechanical performance with an ultimate tensile strength of 270.5 MPa, a yield strength of 214.8 MPa, an elongation of 55.1%, and Brinell hardness of 75.9 HB. The corrosion and degradation rates of HR Zn–xDy alloys in Hanks’ solution gradually increased with increasing Dy addition due to the intensification of galvanic corrosion. The HR Zn–3Dy alloy showed high antibacterial ability against S. aureus and cytocompatibility toward MC3T3-E1 cells among all the HR alloys. Overall, the HR Zn–3Dy alloy can be considered a promising biodegradable material for bone implants.
Statement of Significance
This work reports on Zn–xDy (x = 1, 3, and 5%) alloys fabricated by Dy alloying followed by hot-rolling for biodegradable bone-implant applications. Our findings demonstrate that the hot-rolled (HR) Zn–3Dy alloy showed the best combined mechanical performance with an ultimate tensile strength of 270.5 MPa, a yield strength of 214.8 MPa, an elongation of 55.1%, and Brinell hardness of 75.9 HB. The corrosion and degradation rates of HR Zn–xDy alloys in Hanks’ solution gradually increased with increasing Dy addition due to the intensification of galvanic corrosion. Furthermore, the HR Zn–3Dy alloy showed greater antibacterial ability against S. aureus and the best cytocompatibility toward MC3T3-E1 cells among all the HR alloys.
... The serine residue (S303) of the cytoplasmic tail of Trop2 is phosphorylated by protein kinase C (PKC) (Basu, Goldenberg, & Stein, 1995), enabling phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) bound to Trop2-ICD, resulting in inositol triphosphate (IP3) and diacylglycerol (DAG) (Berridge, 1995). The released IP3 interacts with IP3 receptors on the endoplasmic reticulum, causing Ca 2+ stored there to be released (Lupu, Kaznacheyeva, Krishna, Falck, & Bezprozvanny, 1998). ...
Trop2 is a transmembrane glycoprotein and calcium signal transducer with limited expression in normal human tissues. It is consistently overexpressed in a variety of malignant tumors and participates in several oncogenic signaling pathways that lead to tumor development, invasion, and metastasis. As a result, Trop2 has become an attractive therapeutic target in cancer treatment. The anti-Trop2 antibody-drug conjugate (Trodelvy™, sacituzumab govitecan) has been approved to treat metastatic triple-negative breast cancer. However, it is still unclear whether the success observed in Trop2-positive breast cancer could be replicated in other tumor types, owing to the differences in the expression levels and functions of Trop2 across cancer types. In this review, we summarize the recent progress on the structures and functions of Trop2 and highlight the potential diagnostic and therapeutic value of Trop2 beyond breast cancer. In addition, the promising novel Trop2-targeted agents in the clinic were discussed, which will likely alter the therapeutic landscape of Trop2-positive tumors in the future.
... Signaling cascades initiated by intracellular Ca 2+ are ubiquitously employed for the regulation of cell proliferation. This is achieved at several levels including by the promotion of resting G0 cell entry to the cell cycle, activation of the initiation of DNA synthesis at the G1 to S phase transition, and by stimulation of mitosis (90). Thus, in addition to a role in aldosterone production, genes which function in calcium signaling might also mediate cell proliferation, such as those with a calcium binding function such as VSNL1 (visinin-like), CALN1 (calneuron 1) and CLGN (calmegin), which are all significantly upregulated in APAs (70,91,92). ...
Primary aldosteronism is the most common surgically curable form of hypertension. The sporadic forms of the disorder are usually caused by aldosterone overproduction from a unilateral adrenocortical aldosterone-producing adenoma or from bilateral adrenocortical hyperplasia. The main knowledge-advances in disease pathophysiology focus on pathogenic germline and somatic variants that drive the excess aldosterone production. Less clear are the molecular and cellular mechanisms that lead to an increased mass of the adrenal cortex. However, the combined application of transcriptomics, metabolomics, and epigenetics has achieved substantial insight into these processes and uncovered the evolving complexity of disrupted cell growth mechanisms in primary aldosteronism. In this review, we summarize and discuss recent progress in our understanding of mechanisms of cell death, and proliferation in the pathophysiology of primary aldosteronism.
... The relationship between Ca 2+ and gliomas began to draw attention 33 when T-type channels were found to be expressed in the proliferative stage of the cell cycle 34 and that the overexpression of T-type channels could induce the proliferation of glioma cells. 35 Blocking T-type channels Cav3.2, a target in gliomas, could reduce the survival rate of GBM cells and their resistance to temozolomide (TMZ). ...
Gliomas refer to a group of complicated human brain tumors with a low 5‐year survival rate and limited therapeutic options. Extremely low‐frequency pulsed electromagnetic field (ELF‐PEMF) is a specific magnetic field featuring almost no side effects. However, the application of ELF‐PEMF in the treatment of gliomas is rare. This review summarizes five significant underlying mechanisms including calcium ions, autophagy, apoptosis, angiogenesis, and reactive oxygen species, and applications of ELF‐PEMF in glioma treatment from a clinical practice perspective. In addition, the prospects of ELF‐PEMF in combination with conventional therapy for the treatment of gliomas are reviewed. This review benefits any specialists, especially oncologists, interested in this new therapy because it can help treat patients with gliomas properly. Extremely low‐frequency pulsed electromagnetic field (ELF‐PEMF), a specific magnetic field, is regarded as a potential adjuvant therapy for gliomas. This review summarizes significant underlying mechanisms and prospects of EMF‐PEMF on gliomas.
... In all eukaryotic cells, Ca 2+ is required in both the extracellular environment and intracellular stores for cell growth and division [38,39]. The transient increase in the concentration of free Ca 2+ in the cytosol and its spread to the nucleus lead to cell activation, which is involved in the binding of a broad range of stimuli including mitogenic factors and other transcription factors, and further initiates many signal transduction processes [40,41]. ...
Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.
... There is existing literature pertaining to the importance of specific endogenous metals in skin biology and wound healing. Calcium flashes occur within seconds to trigger a healing response (Razzell et al., 2013), while calcium is also widely reported to regulate epidermal stratification and barrier integrity (Elias et al., 2002), platelet activation (Elaïb et al., 2016), cellular migration (Mandeville and Maxfield, 1997;Wei et al., 2009), macrophage phagocytosis (Vashi et al., 2017) and cellular proliferation (Berridge, 1995;Schwarz et al., 2006). Each of these processes is crucial for effective skin wound healing. ...
Endogenous metals are required for all life, orchestrating the action of diverse cellular processes that are crucial for tissue function. The dynamic wound healing response is underpinned by a plethora of such cellular behaviours, occurring in a time-dependent manner. However, the importance of endogenous metals for cutaneous repair remains largely unexplored. Here we combine ICP-MS with tissue-level RNA-sequencing to reveal profound changes in a number of metals, and corresponding metal-regulated genes, across temporal healing in mice. Wound calcium, magnesium, iron, copper and manganese are elevated at 7 days post-wounding, while magnesium, iron, aluminium, manganese and cobalt increase at 14 days post-wounding. At the level of transcription, wound-induced pathways are independently highly enriched for metal-regulated genes, and vice versa. Moreover, specific metals are linked to distinct wound-induced biological processes and converge on key transcriptional regulators in mice and humans. Finally, we reveal a potential role for one newly identified transcriptional regulator, TNF, in calcium-induced epidermal differentiation. Together, these data highlight potential new and diverse roles for metals in cutaneous wound repair, paving the way for further studies to elucidate the contribution of metals to cellular processes in the repair of skin and other tissues.
... It is noted that Ca 2+ oscillations will cease after pronuclear formation [12] and show further changes as the zygote prepares for the first cell division. A close correlation between cell division and calcium availability has been reported [12,13] that alteration in Ca 2+ signaling may be the underlying reason for defects in cell growth and cleavage. In 2005, Wong et al. showed that the application of an ionophore did overcome negative effects caused by Ca 2+ deficiency, e.g., cleavage furrow regression [14]. ...
PurposeTo explore whether artificial oocyte activation (AOA) can improve embryo developmental potentiality and pregnancy outcomes for patients with a history of embryo developmental problem.Methods
This was a retrospective study and candidate patients with embryo development problems were collected. A total of 1422 MII eggs from the enrolled 140 patients were randomized divided equally into 2 groups, half for the AOA group (AOA), and the rest of sibling mature eggs for the control group (non-AOA). The patients were further divided into two subgroups: (1) the rate of good-quality day 3 embryos was 0% (group 1, n = 66); (2) the rate of good-quality day 3 embryos ranged from 1 to 30% (group 2, n = 74).ResultsIn the early embryonic growth, there were no significant differences in the outcomes of AOA and non-AOA groups in terms of normal fertilization rates, cleavage rates, day 3 good-quality embryo rates and available blastocyst rates (72.7% vs. 79.3%, 97.4% vs. 98.0%, 20.1% vs. 19.7%, 6.6% vs. 8.4% in group 1, respectively; 77.7% vs. 81.9%, 98.1% vs. 97.0%, 25.8% vs. 22.1%, 9.6% vs. 9.3% in group 2, respectively). In the late embryonic growth, no significant differences were found in biochemical and clinical pregnancy rates, implantation rates, miscarriage rates, and live-birth rates (50.0% vs. 45.2%, 45.2% vs. 40.5%, 37.3% vs. 31.3%, 10.5% vs. 11.8%, 40.5% vs. 35.7%, respectively) between two groups. In addition, neonatal outcomes were similar in both the groups as well.Conclusion
Our study demonstrated that the AOA using ionomycin 1 h after ICSI did not bring benefits to the early or late development of embryos derived from patients with a history of embryo developmental problems.
... It is known that fertilizing sperm induce oscillations in Ca 2+ levels to initiate the activation of oocytes, and intracellular calcium signaling also plays an essential role in the sequential embryonic development [26]. In addition, a close correlation between cell division and calcium availability has been reported [27,28], that alteration in Ca 2+ signaling may be the underlying reason for defects in cell growth and cleavage. Furthermore Wong et al. showed that the application of ionophore could overcome the negative effects caused by Ca 2+ deficiency, e.g. ...
Background
The application of artificial oocyte activation (AOA) after intracytoplasmic sperm injection (ICSI) is successful in mitigating fertilization failure problems in assisted reproductive technology (ART). Nevertheless, there is no relevant study to investigate whether AOA procedures increase developmental risk by disturbing subsequent gene expression at different embryonic development stages.
Methods
We used a mouse model to explore the influence of AOA treatment on pre- and post-implantation events. Firstly, the developmental potential of embryos with or without AOA treatment were assessed by the rates of fertilization and blastocyst formation. Secondly, transcriptome high-throughput sequencing was performed among the three groups (ICSI, ICSI-AOA and dICSI-AOA groups). The hierarchical clustering and Principal Component Analysis (PCA) analysis were used. Subsequently, Igf2r/Airn methylation analysis were detected using methylation-specific PCR sequencing following bisulfite treatment. Finally, birth rate and birth weight were examined following mouse embryo transfer.
Results
The rates of fertilization and blastocyst formation were significantly lower in oocyte activation-deficient sperm injection group (dICSI group) when compared with the ICSI group (30.8 % vs. 84.4 %, 10.0 % vs. 41.5 %). There were 133 differentially expressed genes (DEGs) between the ICSI-AOA group and ICSI group, and 266 DEGs between the dICSI-AOA group and ICSI group. In addition, the imprinted gene, Igf2r is up regulated in AOA treatment group compared to control group. The Igf2r/Airn imprinted expression model demonstrates that AOA treatment stimulates maternal allele-specific mehtylation spreads at differentially methylated region 2, followed by the initiation of paternal imprinted Airn long non-coding (lnc) RNA, resulting in the up regulated expression of Igf2r . Furthermore, the birth weight of newborn mice originating from AOA group was significantly lower compared to that of ICSI group. The pups born following AOA treatment did not show any other abnormalities during early development. All offspring mated successfully with fertile controls.
Conclusions
AOA treatment affects imprinted gene Igf2r expression and mehtylation states in mouse pre- and post-implantation embryo, which is regulated by the imprinted Airn . Nevertheless, no significant differences were found in post-natal growth of the pups in the present study. It is hoped that this study could provide valuable insights of AOA technology in assisted reproduction biology.
... Numerous studies showed that an increased level of Ca 2+ in cells is a major factor in apoptosis. Therefore, calcium regulation mechanisms, such as this mediated by RyR1, can be a crucial player in the regulation of apoptosis [41][42][43][44]. In contrast, no Ca 2+ sparks were observed in cardiac myocytes after statin treatment, the reason being that RyR2, not RyR1, is present in cardiomyocytes [36,40,45]. ...
Drug-induced myopathies are classified as acquired myopathies caused by exogenous factors. These pathological conditions develop in patients without muscle disease and are triggered by a variety of medicaments, including lipid-lowering drugs (LLDs) such as statins, fibrates, and ezetimibe. Here we summarise the current knowledge gained via studies conducted using various models, such as cell lines and mammalian models, and compare them with the results obtained in zebrafish (Danio rerio) studies. Zebrafish have proven to be an excellent research tool for studying dyslipidaemias as a model of these pathological conditions. This system enables in-vivo characterization of drug and gene candidates to further the understanding of disease aetiology and develop new therapeutic strategies. Our review also considers important environmental issues arising from the indiscriminate use of LLDs worldwide. The widespread use and importance of drugs such as statins and fibrates justify the need for the meticulous study of their mechanism of action and the side effects they cause.
... There are four steps in the cell cycle that are Ca 2+ /CaM dependent: transition from G 0 phase to G 1 phase, transition from G 1 to S (DNA synthesis) phase, transition from G 2 to M (mitosis) phase and the entire mitosis phase (Kahl & Means, 2003). In addition, a rise in cytosolic and nuclear Ca 2+ is also an initial stimulus for many signalling proteins and transcription factors, such as CaMK, MAPK, NFAT, CREB, AP-1 and NF-κB that are associated with cell proliferation (Berridge, 1995;Graef et al., 2001;Sheng et al., 1990). Therefore, halofuginone -mediated inhibition of Ca 2+ influx through various Ca 2+ -permeable cation channels would result efficiently in inhibition of the cell cycle or the Ca 2+ /CaM/ CaMK-dependent PASMC proliferation and, ultimately, regression of pulmonary vascular remodelling. ...
Background and Purpose
Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti‐hypertrophic, anti‐fibrotic and anti‐proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia‐induced pulmonary hypertension (HPH).
Experimental Approach
Patch‐clamp experiments were conducted to examine the activity of voltage‐dependent Ca²⁺ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca²⁺ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2) for 4 weeks were used as model of HPH for in vivo experiments.
Key Results
Halofuginone increased voltage‐gated K⁺ (Kv) currents in PASMCs and K⁺ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03–1 μM) inhibited receptor‐operated Ca²⁺ entry in HEK cells transfected with calcium‐sensing receptor gene and attenuated store‐operated Ca²⁺ entry in PASMCs. Acute (3–5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia‐induced pulmonary vasoconstriction dose‐dependently (0.1–10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg⁻¹, for 2 weeks) partly reversed established PH in mice.
Conclusion and Implications
Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor‐operated and store‐operated Ca²⁺ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation–contraction coupling and anti‐proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.
... El Ca 2+ no sólo participa en los procesos fisiológicos anteriormente discutidos, que son efectos a corto plazo. También produce efectos a largo plazo que implican regulación de la expresión génica (61,62). Se conoce que el aumento de Ca 2+ intracelular lleva a la activación de factores de transcripción. ...
Los canales de calcio son proteínas de membrana que constituyen la vía más importante para el ingreso del ion calcio (Ca2+) a la célula. Al abrirse, permiten el ingreso selectivo del ion, iniciando una variedad de procesos como contracción muscular, secreción endocrina y liberación de neurotransmisores, entre otros. Estas proteínas se agrupan en tres categorías de acuerdo con sus propiedades estructurales y funcionales: (i) Canales de Ca2+ operados por interacción receptor-ligando (ROCC), (ii) Canales activados por parámetros físicos (Transient Receptor Potencial, TRP) y (iii) Canales de Calcio dependientes de voltaje (VDCCs), siendo estos últimos los más estudiados debido a su presencia en células excitables. Dada la importancia de Ca2+ en la fisiología celular, los canales de Ca2+ constituyen un punto de acción farmacológica importante para múltiples tratamientos y, por tanto, son objeto de estudio para el desarrollo de nuevos fármacos. El objetivo de esta revisión es explicar la importancia de los canales de Ca2+ desde una proyección farmacológica, a partir de la exploración documental de artículos publicados hasta la fecha teniendo en cuenta temas relacionados con la estructura de los canales Ca2+, sus propiedades biofísicas, localización celular, funcionamiento y su interacción farmacológica.
... Application of chemotherapy to control these conditions comes with severe side effects affecting the cancer and normal cells simultaneously [12,13]. So, the localized place is the promising way to treat cancer cells and prevent cell proliferation by targeting Ca 2+ signalling [14,15]. The mechanisms behind Ca 2+ channels/transporters or Ca 2+ ATPase pumps are still unclear [16,17]. ...
Background
Identifying ways to reduce the burden of prostate cancer (Pca) or benign prostatic hyperplasia (BPH) is a top research priority. It is a typical entanglement seen in men which is portrayed by trouble in micturition. It stands as a significant problem in our society. Different molecular biomarker has high potential to treat Pca or BPH but also causes serious side effects during treatment.
Main text
The role of calcium signalling in the alteration of different biomarkers of Pca or BPH is important. Therefore, the photoswitch drugs may hold the potential to rebalance the altered calcium signaling cascade and the biomarker levels. Thereby play a significant role in the management of Pca and BPH. Online literature searches such as PubMed, Web of Science, Scopus, and Google Scholar were carried out. The search terms used for this review were photo-pharmacology, photo-switch drug, photodynamic therapy, calcium signalling, etc. Present treatment of Pca or BPH shows absence of selectivity and explicitness which may additionally result in side effects. The new condition of the calcium flagging may offer promising outcomes in restoring the present issues related with prostate malignancy and BPH treatment.
Conclusion
The light-switching calcium channel blockers aim to solve this issue by incorporating photo-switchable calcium channel blockers that may control the signalling pathway related to proliferation and metastasis in prostate cancer without any side effects.
Graphical abstract
Schematic diagram explaining the proposed role of photo-switch therapy in curbing the side effects of active drugs in Pca (prostate cancer) and BPH (benign prostatic hyperplasia). a) Delivery of medication by ordinary strategies and irreversible phototherapy causes side effects during treatment. Utilization of photo-switch drug to control the dynamic and inert condition of the medication can cause the medication impacts as we required in prostate cancer and BPH. b) Support of harmony between the calcium signaling is essential to guarantee ordinary physiology. Increment or abatement in the dimensions of calcium signaling can result in changed physiology. c) Major factors involved in the pathogenesis of BPH; downregulation of vitamin D receptor (VDR) and histone deacetylase (HDAC) can prevent BPH. Similarly, downregulation of α-1 adrenoceptor can reduce muscle contraction, while overexpression of β-3 adrenoceptor in BPH can promote further muscle relaxation in BPH treatment therapy. Inhibition of overexpressed biomarkers in BPH TRPM2-1: transient receptor potential cation channel subfamily M member 1; TRPM2-2: transient receptor potential cation channel subfamily M member 2; Androgens; CXCL5: C-X-C motif chemokine ligand 5; TGFβ-1: transforming growth factor β-1; TXA2; thromboxane-2; NMDA: N -methyl- d -aspartate can be the potential target in BPH therapy.
... The IP3R is a tightly regulated rheostat and a signaling nexus. Ca 2+ released from the ER is required for normal metabolism and proliferation, and its level determines whether cells divide or undergo autophagy or apoptosis [8][9][10][11]. Not surprisingly, many regulators of these processes bind and modulate the IP3R. ...
The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy.
... The use of calcium-alginate as a hydrogel wound dressing dates back to the early 20th century due to its non-mucoadhesive and non-cytotoxic properties [ 11 , 12 ]; more recently, calcium-alginate wound dressings have become available commercially based on their capacity to promote haemostasis after surgical procedures [12] , remove up to 20 times their own weight in wound exudate [13] , and promote the development of healthy granulation tissue [13] . The latter effect is likely the result of ion exchange between calcium in the dressing and sodium in blood serum and wound exudate that activates the calcium signaling pathway and genes responsible for inducing resting cells to re-enter the cell cycle [14] and/or stimulate events throughout mitosis/interphase [15] . ...
While the benefits of both hydrogels and drug delivery to enhance wound healing have been demonstrated, the highly hydrophilic nature of hydrogels creates challenges with respect to the effective loading and delivery of hydrophobic drugs beneficial to wound healing. Herein, we utilize pressurized gas expanded liquid (PGX) technology to produce very high surface area (~200 m²/g) alginate scaffolds and describe a method for loading the scaffolds with ibuprofen (via adsorptive precipitation) and crosslinking them (via calcium chelation) to create a hydrogel suitable for wound treatment and hydrophobic drug delivery. The high surface area of the PGX-processed alginate scaffold facilitates >8 wt% loading of ibuprofen into the scaffold and controlled in vitro ibuprofen release over 12–24 h. In vivo burn wound healing assays demonstrate significantly accelerated healing with ibuprofen-loaded PGX-alginate/calcium scaffolds relative to both hydrogel-only and untreated controls, demonstrating the combined benefits of ibuprofen delivery to suppress inflammation as well as the capacity of the PGX-alginate/calcium hydrogel to maintain wound hydration and facilitate continuous calcium release to the wound. The use of PGX technology to produce highly porous scaffolds with increased surface areas, followed by adsorptive precipitation of a hydrophobic drug onto the scaffolds, offers a highly scalable method of creating medicated wound dressings with high drug loadings.
Statement of Significance
While medicated hydrogel-based wound dressings offer clear advantages in accelerating wound healing, the inherent incompatibility between conventional hydrogels and many poorly water-soluble drugs of relevance in wound healing remains a challenge. Herein, we leveraged supercritical fluids-based strategies to both process and subsequently impregnate alginate, followed by post-crosslinking to form a hydrogel, to create a very high surface area alginate hydrogel scaffold loaded with high hydrophobic drug contents (here, >8 wt% ibuprofen) without the need for any pore-forming additives. The impregnated scaffolds significantly accelerated burn wound healing while also promoting regeneration of the native skin morphology. We anticipate this approach can be leveraged to load clinically-relevant and highly bioavailable dosages of hydrophobic drugs in hydrogels for a broad range of potential applications.
... Calcium is involved in various developmental processes, such as cell differentiation, proliferation, and apoptosis [35][36][37] as well as neurotransmitter release and neuronal membrane excitability [38][39][40]. Essential factors for calcium homeostasis include CaBP-9k [41], which buffer increases in calcium levels [32], such as increases from 10 -7 M in the resting cell to 10 -5 M in the activated cell [42][43][44], and the ER, which regulates the uptake, storage, and mobilization of intracellular calcium [45]. ...
Background/aims:
Calcium homeostasis plays a crucial role in neuronal development and disease. Calbindin-D9k (CaBP-9k) acts as calcium modulators and sensors in various tissues. However, the neurobiological functions of CaBP-9k are unknown.
Methods:
We used CaBP-9k knockout (KO) mice to investigate the roles of these gene in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. We used anatomical and biochemical approaches to characterize functional abnormalities of the brain in the CaBP-9k KO mice.
Results:
We found that the brains of CaBP-9k KO mice have increased APP/β-amyloid, Tau, and α-synuclein accumulation and endoplasmic reticulum (ER) stress-induced apoptosis. Neurons deficient for these CaBP-9k had abnormal intracellular calcium levels and responses. ER stress inhibitor TUDCA reduced ER stress-induced apoptosis and restored ER stress- and apoptosis-related proteins expression to wild-type levels in CaBP-9k KO mice. Furthermore, treatment with TUDCA rescued the abnormal memory and motor behaviors exhibited by older CaBP-9k KO mice.
Conclusion:
Our results suggest that a loss of CaBP-9k may contribute to the onset and progression of neurodegenerative diseases.
... The exact role of calcium in cancer is a conundrum because, on the one hand, releasing calcium is accompanied by activating cytochrome C and leads to a downstream cascade of apoptosis, including apoptosome and other apoptotic intermediates involved in the process of the programmed cell death [176,177]. On the other hand, calcium supports the cell cycle [178][179][180][181] and cellular migration, too [182][183][184]. Therefore, some calcium channels, e.g., Voltage-gated calcium (Ca 2+ ) channels (VGCC), increased in tumors [185][186][187][188][189] while other types of channels were decreased in other tumors (e.g., Ca 2+ -ATPase (SERCA3, PMCA1, PMCA4)) [190][191][192]. ...
Cancer cells and tissues have an aberrant regulation of hydrogen ion dynamics driven by a combination of poor vascular perfusion, regional hypoxia, and increased the flux of carbons through fermentative glycolysis. This leads to extracellular acidosis and intracellular alkalinization. Dysregulated pH dynamics influence cancer cell biology, from cell transformation and tumorigenesis to proliferation, local growth, invasion, and metastasis. Moreover, this dysregulated intracellular pH (pHi) drives a metabolic shift to increased aerobic glycolysis and reduced mitochondrial oxidative phosphorylation, referred to as the Warburg effect, or Warburg metabolism, which is a selective feature of cancer. This metabolic reprogramming confers a thermodynamic advantage on cancer cells and tissues by protecting them against oxidative stress, enhancing their resistance to hypoxia, and allowing a rapid conversion of nutrients into biomass to enable cell proliferation. Indeed, most cancers have increased glucose uptake and lactic acid production. Furthermore, cancer cells have very dysregulated electrolyte balances, and in the interaction of the pH dynamics with electrolyte, dynamics is less well known. In this review, we highlight the interconnected roles of dysregulated pH dynamics and electrolytes imbalance in cancer initiation, progression, adaptation, and in determining the programming and reprogramming of tumor cell metabolism
... Newton suggested that p53 functions as a transcription factor and is responsible for the transactivation and repression of key genes involved in cell growth, apoptosis and the cell cycle [33]. Li et al. pointed out that mTOR is regarded as a central controller of cell growth in response to growth factors, cellular energy and nutrient levels [34].Some researchers gured out that one function of calcium signal is to activate the immediate early genes responsible for inducing resting cells (G0) to re-enter the cell cycle and may also promote the initiation of DNA synthesis at the G1/S transition [35]. According to multiple studies, the TGF-beta pathway promotes granulosa cell proliferation and follicular growth in the antral follicle stage [36][37]. ...
Background Broodiness is a phenomenon that occurs in most avian species and significantly reduces productivity. Several genes are known to play an important role in regulating the progress of reproduction, but the molecular regulation mechanism of broodiness remains unclear. In the current study, via high throughput sequencing, we identified and explored the differentially expressed miRNAs and mRNAs involved in ovarian atrophy. Results We identified a total of 901 mRNAs and 50 miRNAs that were differentially expressed in egg-laying and atrophic ovaries. Among them, numerous DEGs transcripts and target genes for miRNAs were significantly enriched in reproductive processes, cell proliferation, and apoptosis pathways. In addition, a miRNA- gene-pathway network was constructed by considering target relationships and correlation of the expression levels between ovary development-related genes and miRNAs. Conclusions We discovered mRNA and miRNAs transcripts that are candidate regulators of ovary development in broody geese. Our findings expanded our understanding of the functional of miRNAs in ovarian atrophy and demonstrated that RNA-Seq is a powerful tool for examining the molecular mechanism in regulating broodiness.
... Additionally, there is no doubt about the importance of Ca 2+ for cell proliferation not only for immune cells but also in the context of cancer cells [57][58][59][60]. Increased Ca 2+ levels lead to an increased proliferation rate and SOCE plays a central role in its regulation [61]. ...
Ca2+ is a crucial second messenger for proper T cell function. Considering the relevance of Ca2+ signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca2+ signals from elderly individuals are reported. The main Ca2+ entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca2+ signals in untouched and stimulated, but also in central and effector memory CD8+ T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms contribute to the overall reduction in Ca2+ signals of CD8+ T cells of elderly mice: 1) Reduced Ca2+ currents through Orai channels due to decreased expressions of STIMs and Orais. 2) A faster extrusion of Ca2+ owing to an increased expression of PMCA4. The reduced Ca2+ signals correlated with a resistance of the cytotoxic efficiency of CD8+ T cells to varying free [Ca2+]ext with age. In summary, reduced STIM/Orai expression and increased Ca2+ clearing rates following enhanced PMCA4 expression contribute to reduced Ca2+ signals in CD8+ T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca2+ dependency of CTL cytotoxicity.
... Additionally, there is no doubt about the importance of Ca 2+ for cell proliferation not only for immune cells but also in the context of cancer cells [57][58][59][60]. Increased Ca 2+ levels lead to an increased proliferation rate and SOCE plays a central role in its regulation [61]. ...
Ca 2+ is a crucial second messenger for proper T cell function. Considering the relevance of Ca 2+ signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca 2+ signals from elderly individuals are reported. The main Ca 2+ entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca 2+ signals in untouched and stimulated, but also in central and effector memory CD8 + T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms account for the overall reduction in CD8 + T cells of elderly mice: 1) Ca 2+ currents through Orai channels were reduced and decreased mRNA and protein levels of STIMs and Orais were detected. 2) A faster extrusion of Ca 2+ combined with an increased expression of PMCA4. The reduced Ca 2+ signals correlated with a resistance of the cytotoxic efficiency of CD8 + T cells to varying free [Ca 2+ ] ext with age. In summary, reduced STIM/Orai expression and increased pump rates following enhanced PMCA4 expression contribute to reduced Ca 2+ signals in CD8 + T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca 2+ dependency of CTL cytotoxicity.
... These values are subject to 48 change, in particular upon cell stimulation when calcium-selective ion channels 49 open and allow for Ca 2+ influx; thus, setting the basis for calcium signaling as 50 an intricate signal transduction network. Furthermore, calcium signaling is involved 51 in muscle contraction, cell growth, cellular motility, synaptic plasticity, but can also 52 impact apoptosis, the permeability of ion channels and the cytoskeleton [10][11][12][13][14][15][16][17][18]. 53 The concept of Ca 2+ signaling and the importance of Ca 2+ as a ubiquitous 54 second messenger became apparent more than 100 years ago (circa 1883) when 55 studies on heart cells demonstrated that the presence of Ca 2+ was necessary for 56 the contraction of cardiomyocytes [18]. ...
The pioneering work of Richard Altman on the presence of mitochondria in cells set in motion a field of research dedicated to uncovering the secrets of the mitochondria. Despite limitations in studying the structure and function of the mitochondria, advances in our understanding of this organelle prompted the development of potential treatments for various diseases, from neurodegenerative conditions to muscular dystrophy and cancer. As the powerhouses of the cell, the mitochondria represent the essence of cellular life and as such, a selective advantage for cancer cells. Much of the function of the mitochondria relies on Ca²⁺ homeostasis and the presence of effective Ca²⁺ signaling to maintain the balance between mitochondrial function and dysfunction and subsequently, cell survival. Ca²⁺ regulates the mitochondrial respiration rate which in turn increases ATP synthesis, but too much Ca²⁺ can also trigger the mitochondrial apoptosis pathway; however, cancer cells have evolved mechanisms to modulate mitochondrial Ca²⁺ influx and efflux in order to sustain their metabolic demand and ensure their survival. Therefore, targeting the mitochondrial Ca²⁺ signaling involved in the bioenergetic and apoptotic pathways could serve as potential approaches to treat cancer patients. This chapter will review the role of Ca²⁺ signaling in mediating the function of the mitochondria and its involvement in health and disease with special focus on the pathophysiology of cancer.
Zika virus (ZIKV) infection during the first trimester of the pregnancy may lead to Congenital zika syndrome in the neonates. The viral infection hampers foetal brain development and causes microcephaly. Human neural progenitor cells (hNPCs) play an important role in brain development, however they are highly susceptible to ZIKV infection. In this study, we elucidated the molecular mechanisms that lead to cellular alterations in hNPCs due to ZIKV E-protein. We investigated proliferation, differentiation, migration and inflammation in hNPCs, which may lead to microcephaly. In our study, we found that ZIKV E-protein causes cell cycle arrest, decrease in proliferation and increase in mitotic length of the dividing hNPCs. We observed CyclinD1 and upstream molecules (p21 and p53) of the pathway are dysregulated, and intracellular calcium at basal level as well as upon ATP stimulation were reduced following over expression of ZIKV E-protein. ZIKV E-protein transfected hNPCs exhibited pre-mature differentiation with pro-neural genes upregulated. Furthermore, ZIKV E-protein disrupted migrational properties of hNPCs and caused elevated levels of inflammatory chemokines and cytokines. To gain insights into molecular mechanisms of these effects on hNPCs, we explored the possible involvement of long non coding RNAs in ZIKV neuropathogenesis. We have shortlisted lncRNAs associated with differentially expressed genes from publicly available transcriptomic data and found some of those lncRNAs are differentially expressed upon E-protein transfection of hNPCs. Gene ontology analysis suggest these lncRNAs play an important role in regulation of viral life cycle, host's defence response and cell proliferation.
Breast cancer metastasis to bone is a leading killer in breast cancer patients. A type I collagen-modified thread (Col-I@thread) was prepared for 3-dimensional cell culture and breast cancer bone metastasis co-culture device assembly. First, the coating of Col-I on nylon threads for promoting cell adhesion and growth was studied. Through SEM, XPS, and protein concentration measurements, it was found that the lyophilization method remarkably preserved the Col-I activity and the internal structure of the thread, thereby promoting cell attachment and proliferation. RNA-sequencing (RNA-Seq) and quantitative PCR analysis showed that osteoblast cells (MC3T3-E1) grown on Col-I@thread had elevated RUNX2, ALP, OPN, and Col-I gene expression to promote osteoblast differentiation. Single-cell analysis found that osteoblast MC3T3-E1 cells growing on Col-I@thread had higher Ca2+ secretion activity and mineralized nodules, suggesting robust cell activity and bone matrix formation than cells growing on 2D culture plates. Col-I@threads were knotted in an interdigital cross-finger frame to assemble the breast cancer-bone co-culture model. Confocal microscopy and flow cytometry tests quantified the invasive breast cancer cells. Moreover, the thread-based co-culture devices allowed us to isolate the invasive and non-invasive breast cancer cells to compare their molecular characteristics. qPCR results showed that expression of CX43, CXCR5, and CSPG4 genes was significantly increased in breast cancer cells with bone metastasis. Meanwhile, the expression of RUNX2 and OPG genes in osteoblasts was inhibited. The co-culture model based on the Col-I@thread mimics the bone tissue microenvironment to reveal the cross-talk between cancer cells and bone tissue. Moreover, the thread-based co-culture device is easy to fabricate and operate, providing a platform for exploring the cellular and molecular mechanisms of breast cancer bone metastasis, and holds potential for high-throughput screening of anti-breast cancer bone metastasis drugs.
Tebufenpyrad is classified as a pyrazole acaricide and insecticide. It is widely used for several crops, especially in greenhouses, in several countries. While its unfavorable effects on non-target organisms have already been established, relatively little is known about its reproductive toxicity. Therefore, we demonstrated the biochemical effects of tebufenpyrad using porcine trophectoderm and porcine luminal epithelial cells, which are involved in implantation. We found that tebufenpyrad had antiproliferative effects and reduced cell viability. Tebufenpyrad also triggered apoptosis and excessive reactive oxygen species production. Furthermore, it induced cell cycle arrest in the G1 phase and disrupted calcium homeostasis in the cytosol and mitochondria. MAPK signaling pathways and the crosstalk among them were altered following tebufenpyrad treatment. In addition, the migration ability of cells was reduced after treatment with tebufenpyrad. Lastly, tebufenpyrad influenced the expression of genes related to pregnancy. Collectively, these results reveal the mechanism of the biochemical and physiological effects of tebufenpyrad to both trophectoderm and uterine cells and suggest that tebufenpyrad reduces the potential of successful implantation.
The pulmonary circulation is a low-resistance, low-pressure and high-compliance system that allows the lungs to receive entire cardiac output. Pulmonary arterial pressure is a function of cardiac output and pulmonary vascular resistance, while pulmonary vascular resistance is inversely proportional to the fourth power of the intraluminal radius of the pulmonary artery. Therefore, a very small decrease of the pulmonary vascular lumen diameter results in a significant increase in pulmonary vascular resistance and pulmonary arterial pressure. Pulmonary arterial hypertension is a fatal and progressive disease with poor prognosis. Regardless of the initial pathogenic triggers, sustained pulmonary vasoconstriction, concentric vascular remodeling, occlusive intimal lesions, in situ thrombosis and vascular wall stiffening are the major and direct causes for elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension and other forms of pre-capillary pulmonary hypertension. In this review, we aim to discuss the basic principles and physiological mechanisms involved in the regulation of lung vascular hemodynamics and pulmonary vascular function, the changes in the pulmonary vasculature that contribute to the increased vascular resistance and arterial pressure, and the pathogenic mechanisms involved in the development and progression of pulmonary hypertension. We focus on reviewing the pathogenic roles of membrane receptors, ion channels and intracellular Ca ²⁺ signaling in pulmonary vascular smooth muscle cells in the development and progression of pulmonary hypertension.
Background
The transient receptor potential canonical 6 (TRPC6) channel has been studied in pathologies of the hepatobiliary system. Information on the localization of TRPC6-protein in anatomical and histological structures of the human hepatobiliary system in elderly with and without liver disease is lacking.
Methods
Samples were obtained from twelve nitrite pickling salt-ethanol-polyethylene glycol-fixed corpses of the four anatomical liver lobes, collum and the remaining gallbladder, the common bile duct (gender: 5 females, 41.67%; age [years]: median 84, range 20; postmortem interval before fixation [h]: median 60, range 74.15). Orienting histomorphologic assessment was done in a hematoxylin-eosin stain. Immunohistochemical labelling of TRPC6 was carried out following a cross-evaluation scheme. TRPC6-immunoreactivity was assessed regarding intensity (semi-quantification, three independent observers) and area (optical threshold method). Explorative statistical analyses were performed using R (inter-rater-reliability: Fleiss’ kappa; comparisons: Wilcoxon-Rank-sum-test).
Results
Extensive autolysis was found in the liver of one and the biliary samples of three corpses. Extensive liver disease was found in 4 corpses, while 8 corpses only showed age-appropriate degeneration. Only the intrahepatic connective tissue showed no TRPC6-immunoreactivity. Individuals with extensively diseased livers exhibited statistically significantly less TRPC6-immunoreactive area in the bile duct and liver tissue while statistically significant more TRPC6-immunoreactive area in the gallbladder compared to individuals with age-appropriate degeneration of the liver only in the respective organ.
Conclusion
Age-associated degeneration of the hepatobiliary system is likely to be associated with widespread, homogenous TRPC6-expression. Liver disease potentially influences the distribution of TRPC6-protein within the liver and within the biliary tract in elderly.
In cell biology, fluorescent dyes are routinely used for biochemical measurements. The traditional global dye treatment method suffers from low signal-to-noise ratios (SNR), especially when used for detecting a low concentration of ions, and increasing the concentration of fluorescent dyes causes more severe cytotoxicity. Here, we report a robotic technique that controls how a low amount of fluorescent-dye-coated magnetic nanoparticles accurately forms a swarm and increases the fluorescent dye concentration in a local region inside a cell for intracellular measurement. Different from existing magnetic micromanipulation systems that generate large swarms (several microns and above) or that cannot move the generated swarm to an arbitrary position, our system is capable of generating a small swarm (e.g., 1 μm) and accurately positioning the swarm inside a single cell (position control accuracy: 0.76 μm). In experiments, the generated swarm inside the cell showed an SNR 10 times higher than the traditional global dye treatment method. The high-SNR robotic swarm enabled intracellular measurements that had not been possible to achieve with traditional global dye treatment. The robotic swarm technique revealed an apparent pH gradient in a migrating cell and was used to measure the intracellular apparent pH in a single oocyte of living C. elegans. With the position control capability, the swarm was also applied to measure calcium changes at the perinuclear region of a cell before and after mechanical stimulation. The results showed a significant calcium increase after mechanical stimulation, and the calcium increase was regulated by the mechanically sensitive ion channel, PIEZO1.
TRPP2 (PC2, PKD2 or Polycytin-2), encoded by PKD2 gene, belongs to the nonselective cation channel TRP family. Recently, the three-dimensional structure of TRPP2 was constructed. TRPP2 mainly functions in three subcellular compartments: endoplasmic reticulum, plasma membrane and primary cilia. TRPP2 can act as a calcium-activated intracellular calcium release channel on the endoplasmic reticulum. TRPP2 also interacts with other Ca²⁺ release channels to regulate calcium release, like IP3R and RyR2. TRPP2 acts as an ion channel regulated by epidermal growth factor through activation of downstream factors in the plasma membrane. TRPP2 binding to TRPC1 in the plasma membrane or endoplasmic reticulum is associated with mechanosensitivity. In cilium, TRPP2 was found to combine with PKD1 and TRPV4 to form a complex related to mechanosensitivity. Because TRPP2 is involved in regulating intracellular ion concentration, TRPP2 mutations often lead to autosomal dominant polycystic kidney disease, which may also be associated with cardiovascular disease. In this paper, we review the molecular structure of TRPP2, the subcellular localization of TRPP2, the related functions and mechanisms of TRPP2 at different sites, and the diseases related to TRPP2.
Jasmonic acid (JA) is a phytohormone that plays a central role in plant defense against necrotrophic pathogens. JA signaling stimulates the increase of cytosolic calcium ion (Ca2+) and implicates the activity of mitogen-activated protein kinases (MPKs). We previously characterized that Ca2+/calmodulin (CaM) activates MPKs by inhibiting a CaM-regulated dual-specificity protein phosphatase1 (DsPTP1) at the biochemical level. In this study, we reported that Ca2+/CaM-mediated DsPTP1 negatively regulates the resistance to necrotrophic pathogens through the inhibition of JA-responsive MPK6. To elucidate the physiological function of inhibiting DsPTP1 activity by Ca2+/CaM, we constructed transgenic plants overexpressing DsPTP1 wild type (DsPTP1WT OX) and CaM deregulated mutant (DsPTP1K166E OX). Interestingly, the MPK6 activity was significantly reduced in DsPTP1K166E OX plants in response to JA compared to DsPTP1WT OX plants. Moreover, transcript levels of JA-responsive gene PDF1.2 and VSP1 were also highly decreased in DsPTP1K166E OX plants compared to DsPTP1WT OX plants. Furthermore, DsPTP1K166E OX plants showed more susceptibility to necrotrophic pathogens than DsPTP1WT OX plants. Conclusively, these results suggest that Ca2+/CaM activates the JA-responsive MPKs by inhibiting DsPTP1 for the resistance to the necrotrophic pathogen.
Instinctive hierarchically biomineralized structures of various organisms, such as eggs, algae, and magnetotactic bacteria, afford extra protection and distinct performance, which endow fragile organisms with a tenacious ability to adapt and survive. However, spontaneous formation of hybrid materials is difficult for most organisms in nature. Rapid development of chemistry and materials science successfully obtained the combinations of organisms with nanomaterials by biomimetic mineralization thus demonstrating the reproduction of the structures and functions and generation of novel functions that organisms do not possess. The rational design of biomaterial–organism hybridization can control biological recognition, interactions, and metabolism of the organisms. Thus, nanomaterial–organism hybrids represent a next generation of organism engineering with great potential biomedical applications. This review summarizes recent advances in material‐directed organism engineering and is mainly focused on biomimetic mineralization technologies and their outstanding biomedical applications. Three representative types of biomimetic mineralization are systematically introduced, including external mineralization, internal mineralization, and genetic engineering mineralization. The methods involving hybridization of nanomaterials and organisms based on biomimetic mineralization strategies are described. These strategies resulted in applications of various nanomaterial–organism hybrids with multiplex functions in cell engineering, cancer treatment, and vaccine improvement. Unlike classical biological approaches, this material‐based bioregulation is universal, effective, and inexpensive. In particular, instead of traditional medical solutions, the integration of nanomaterials and organisms may exploit novel strategies to solve current biomedical problems.
This article is categorized under:
• Implantable Materials and Surgical Technologies > Nanomaterials and Implants
• Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease
• Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease
Abstract
Rapid development of chemistry and materials science successfully obtained the combinations of organisms with nanomaterials by biomimetic mineralization. This review summarizes recent advances in materials‐directed organism engineering and emphasizes their outstanding biomedical applications.
Nav1.5, encoded by SCN5A, has been associated with metastasis in colorectal cancer (CRC). Here, we investigated the mechanism by which Nav1.5 regulates tumor progression and whether Nav1.5 influences chemosensitivity to 5-fluorouracil (5-FU) in CRCs. CRC cases were evaluated for Nav1.5 expression. Elevated Nav1.5 expression was associated with poor prognosis in CRCs, whereas stage II/III patients with upregulated SCN5A expression could have better survival after receiving 5-FU-based adjuvant chemotherapy. In CRC cells, SCN5A knockdown reduced the proliferation, migration and invasion. According to RNA sequencing, SCN5A knockdown inhibited both the cell cycle and epithelial-mesenchymal transition. In addition, Nav1.5 stabilized the KRas-calmodulin complex to modulate Ras signaling, promoting Ca²⁺ influx through the Na⁺-Ca²⁺ exchanger and Ca²⁺ release-activated calcium channel. Meanwhile, SCN5A knockdown increased the 50% inhibitory concentration to 5-FU by upregulating 5-FU-stimulated apoptosis in CRCs. In conclusion, Nav1.5 could progress to proliferation and metastasis through Ca²⁺/calmodulin-dependent Ras signaling in CRC, and it could also enhance 5-FU-stimulated apoptosis. Clinically, patients with stage II/III CRCs with elevated SCN5A expression demonstrated poor prognosis, yet those patients could benefit more from 5-FU-based chemotherapy than patients with lower SCN5A expression.
Release of Ca²⁺ from intracellular Ca²⁺ stores (Ca²⁺ mobilization) and capacitative Ca²⁺ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca²⁺ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5‐di‐tert‐butylhydroquinone (DBHQ), an inhibitor of the Ca²⁺ pump of intracellular Ca²⁺ stores, and of SK&F 96365, an inhibitor of capacitative Ca²⁺ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [³H]‐thymidine incorporation in a dose‐dependent manner without affecting cell viability or morphology. The inhibition of [³H]‐thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: ∼4 μM) as the blockade of capacitative Ca²⁺ entry in the E3 retinal organ culture. At a concentration of 5 μM SK&F 96365, DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca²⁺ stores also inhibited [³H]‐thymidine incorporation with IC50 values of 20–30 μM in the different cultures. Our results suggest the involvement of Ca²⁺ mobilization and capacitative Ca²⁺ entry in the regulation of DNA synthesis in the developing neural retina.
The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell’s functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCβ pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.
Roles of bioelectrical signals are increasingly recognized in excitable and nonexcitable non-neural tissues. Diverse ion-selective channels, pumps, and gap junctions participate in bioelectrical signaling, including those transporting calcium ions (Ca 2+). Ca 2+ is the most versatile transported ion, because it serves as an electrical charge carrier and a biochemical regulator for multiple molecular binding, enzyme, and transcription activities. We aspire to learn how bioelectrical signals crosstalk to biochemical/biomechanical signals. In this study, we review four recent studies showing how bioelectrical currents and Ca 2+ signaling affect collective dermal cell migration during feather bud elongation, affect chondrogenic differentiation in limb development, couple with mechanical tension in aligning gut smooth muscle, and affect mitochondrial function and skeletal muscle atrophy. We observe bioelectrical signals involved in several developmental and pathological conditions in chickens and mice at multiple spatial scales: cellular, cellular collective, and subcellular. These examples inspire novel concept and approaches for future basic and translational studies.
Certain skin bacteria are able to convert aromatic amino acids (AAA) into trace amines (TA) that act as neuromodulators. Since the human skin and sweat contain a comparatively high content of AAA one can expect that such bacteria are able to produce TA on our skin. Here we show that TA-producing Staphylococcus epidermidis strains expressing SadA are predominant on human skin and that TA accelerate wound healing. In wounded skin, keratinocytes produce epinephrine (EPI) that leads to cell motility inhibition by β2-adrenergic receptor (β2-AR) activation thus delay wound healing. As β2-AR antagonists, TA and dopamine (DOP) abrogate the effect of EPI thus accelerating wound healing both in vitro and in a mouse model. In the mouse model, the S. epidermidis wild type strain accelerates wound healing compared to its ΔsadA mutant. Our study demonstrates that TA-producing S. epidermidis strains present on our skin might be beneficial for wound healing.
Charybdotoxin (ChTX), a K+ channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltage-activated, and three Ca(2+)-activated. To discern the mechanism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca(2+)-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.
Treatment of Swiss 3T3 cells with a subsaturating concentration of recombinant Pasteurella multocida toxin (rPMT) markedly potentiated the production of inositol phosphates induced by bombesin, vasopressin, and endothelin but not by platelet-derived growth factor (PDGF) (AA and BB homodimers). Similarly, the neuropeptides but not PDGF caused a shift in the dose-dependent increase in inositol phosphates induced by rPMT. The rate of accumulation of inositol phosphates induced by bombesin was increased 2-fold by rPMT treatment while that of PDGF was unaffected. rPMT treatment also enhanced bombesin-induced inositol(1,4,5)trisphosphate, the direct product of phosphatidylinositol 4,5-bisphosphate hydrolysis. In contrast, treatment of cells with rPMT had no effect on the tyrosine phosphorylation of phospholipase C gamma. Depletion of protein kinase C increased rPMT-induced inositol phosphates in a manner similar to that observed for bombesin but not PDGF. Thus, rPMT selectively potentiates neuropeptide-mediated inositol phosphate production. The action of rPMT on phosphatidylinositol 4,5-bisphosphate hydrolysis persisted in streptolysin O-permeabilized cells. Addition of guanosine 5'-O-(beta-thiodiphosphate) to permeabilized cells markedly reduced rPMT-induced inositol phosphates in a time- and dose-dependent manner. rPMT also increased the sensitivity of phospholipase C for free calcium. Our results strongly suggest that the action of rPMT facilitates the coupling of G protein to phospholipase C.
L-651,582, 5-amino-[4-(4-chlorobenzoyl)-3,5-dichlorobenzyl]-1,2,3-triazole-4- carboxamide, an antiproliferative and antiparasitic agent previously shown to affect 45Ca2+ uptake into mammalian cells, inhibits both receptor-mediated and voltage-dependent calcium entry in well characterized in vitro systems. Indo 1 fluorescence measurements of cytosolic calcium levels indicate that the drug has no effect on the initial transient release of internal stores of calcium stimulated by fMet-Leu-Phe in rat polymorphonuclear leukocytes. It does decrease the levels maintained subsequently, however, indicating blockage of calcium influx through receptor-operated channels. L-651,582 also blocks the stimulation of leukotriene B4 (LTB4) production by fMet-Leu-Phe with an IC50 = 0.5 micrograms/ml equal to that for calcium entry inhibition. The LTB4 inhibition is likely due to calcium entry inhibition since L-651,582 does not inhibit calmodulin or enzymes producing arachidonate metabolites. L-651,582 also inhibits potassium-stimulated 45Ca2+ influx into GH3 cells with an IC50 of 0.5 microgram/ml, indicating a block of voltage-gated L-type calcium channels. Patch voltage clamp measurements of current through L- and T-type calcium in guinea pig atrial cells also indicate that L-651,582 is a calcium antagonist. Block of L-type calcium channels is voltage-dependent, and the apparent dissociation constant for the high affinity state is 0.2 micrograms/ml. The IC50 for block of T-type calcium channels is 1.4 micrograms/ml. The inhibition of cellular proliferation and the production of arachidonate metabolites by L-651,582 may be the result of the nearly equipotent block of receptor-operated and voltage-gated calcium channels.
Charybdotoxin, a blocker of K+ channels, and the imidazole drug SC38249, a blocker of both voltage- and second messenger-operated Ca2+ channels, were employed in mouse NIH-3T3 fibroblasts overexpressing the epidermal growth factor (EGF) receptor 1) to characterize the ionic events activated by EGF; and 2) to establish the role of those events in cell growth. The [Ca2+]i response by EGF was little changed by charybdotoxin while the parallel hyperpolarization was inhibited in a dose-dependent manner. At high toxin concentrations (greater than 3 x 10(-8) M), the effect of EGF on membrane potential was turned into a persistent depolarization sustained by both Na+ and Ca2+. Pretreatment with 10 microM SC38249 induced only minor changes of the intracellular Ca2+ release by EGF (the process responsible for the initial phase of the [Ca2+]i and membrane potential responses) and blocked the persistent, second phase [Ca2+]i and the hyperpolarization responses, both dependent on Ca2+ influx, as well as the depolarization in the charybdotoxin-pretreated cells. Long term (up to 2-day) treatment with either charybdotoxin or SC38249 failed to affect the viability and growth of unstimulated EGFR-T17 cells. Moreover, in these cells, the ionic responses to EGF were restored after a 30-min incubation in fresh medium. In contrast, growth stimulated by EGF was inhibited, moderately (-20%) by charybdotoxin and markedly (-60%) by SC38249. These results indicate for the first time that both hyperpolarization and, especially, the persistent increase of [Ca2+]i sustained by Ca2+ influx play a role in the activity of EGF, ultimately cooperating with other intracellular events in mitogenesis.
Recent studies indicate multiple mechanisms are involved in Ca2+ stimulation of gene expression. We have used cell-permeable, specific inhibitors of calmodulin-dependent protein kinases (CaM kinases) and phosphatase (calcineurin) to investigate the involvement of these enzymes in transcriptional regulation of three immediate early genes in PC12 cells stimulated with A23187 or KCl. Preincubation of PC12 cells with the CaM kinase inhibitor KN-62 blocked autophosphorylation of CaM kinase II in response to stimulation by the Ca2+ ionophore A23187. KN-62 treatment also resulted in a 60-70% inhibition of Ca(2+)-dependent transcription of c-fos, NGFI-A (zif 268), and NGFI-B (nur 77) as assessed by either Northern or nuclear run-on analyses. Preincubation with the calcineurin inhibitors FK-506 or cyclosporin A strongly enhanced expression of NGFI-A and blocked transcription of NGFI-B, but it had no significant effect on Ca(2+)-stimulated transcription of c-fos. Both FK-506 and KN-62 were specific for Ca(2+)-stimulated transcription as neither effected transcription in response to forskolin or phorbol ester (12-O-tetradecanoylphorbol-13-acetate) treatment. This is the first report of CaM kinase and calcineurin involvement in transcriptional regulation of NGFI-A and NGFI-B. Activation of CaM kinases and calcineurin, in response to elevated intracellular Ca2+, would exert antagonistic effects on transcription of NGFI-A. Since inhibition of either the kinase or phosphatase decreased transcription of NGFI-B by 60-90%, this suggests that each enzyme is necessary but not sufficient for Ca2+ stimulation. These results indicate that CaM kinases and calcineurin can mediate broad and complex regulation of Ca(2+)-stimulated gene expression.
Upon ligand-induced tyrosine phosphorylation, the platelet-derived growth factor (PDGF) receptor (PDGFR) beta subunit associates with PLC-gamma 1, RasGAP, P13K, and a 64 kd protein. To determine the relative role of each of these associated proteins in PDGFR signaling, we constructed a PDGFR mutant (F5) unable to bind any of them and a panel of "add-back" mutants that could bind only one of the receptor-associated proteins. F5 PDGFR failed to activate PLC-gamma 1, P13K, or Ras and was unable to trigger DNA synthesis. Permitting association of F5 PDGFR with either PLC-gamma 1 or P13K restored Ras activation and a mitogenic response. Surprisingly, even though binding of the 64 kd protein almost fully restored Ras activation, it did not rescue the receptor's ability to trigger DNA synthesis. Thus Ras activation is insufficient to trigger PDGF-dependent DNA synthesis, and PLC-gamma 1 and P13K are independent downstream mediators of PDGF's mitogenic signal.
The specific inhibitors of the endoplasmic reticulum Ca2+ pump, thapsigargin and 2,5-di-tert-butylhydroquinone (DBHQ), stimulated reinitiation of DNA synthesis in synergy with either phorbol 12,13-dibutyrate or bombesin in Swiss 3T3 cells. Maximum stimulation was achieved at 0.5 nM thapsigargin and 7.5 microM DBHQ. Kinetics of [3H]thymidine incorporation were consistent with exit from G0 and entry into S phase. Autoradiography of labeled nuclei showed that the increase in [3H]thymidine incorporation was due to an increase in the proportion of cells entering into DNA synthesis. Down-regulation or selective inhibition of protein kinase C abolished this synergistic stimulation of DNA synthesis. Thapsigargin and DBHQ did not potentiate protein kinase C-mediated signals such as direct phosphorylation of myristoylated alanine-rich C-kinase substrate, activation of mitogen-activated protein kinase, and tyrosine phosphorylation of bands 110,000-130,000 and 70,000-80,000. Thapsigargin and DBHQ caused a marked reduction in the ability of bombesin to induce a rapid and transient increase in intracellular Ca2+ via depletion of total cellular Ca2+, measured by 45Ca2+ content. The synergistic stimulation of DNA synthesis by DBHQ and phorbol 12,13-dibutyrate was dependent on a high concentration of extracellular Ca2+ (ED50 = 410 microM) and was preferentially inhibited by the inhibitor of Ca2+ influx econozole. This suggests a role for Ca2+ entry in growth control. This is the first time that either thapsigargin or DBHQ has been shown to stimulate the reinitiation of DNA synthesis in any target cell.
Stimulation of antigen receptors of lymphocytes triggers a transitory release of Ca2+ from internal stores and the opening of a transmembrane Ca2+ conductive pathway. The latter underlies the sustained increase of intracellular free calcium concentration, and it seems to be a key event in the Ca(2+)-dependent biochemical cascade leading to T cell proliferation. Alternatively, pharmacological depletion of internal stores by itself activates Ca2+ influx. This has led to the hypothesis that antigen-triggered Ca2+ influx is secondary to Ca2+ release from internal stores. However, the precise relationship between antigen and Ca2+ release-activated Ca2+ currents remains unclear, particularly since neither of them has been electrophysiologically recorded in normal lymphocytes. Using the whole-cell and the perforated configurations of the patch clamp technique on peripheral blood lymphocytes, we found that a low amplitude Ca(2+)-selective current was triggered when intracellular stores were depleted by stimuli such as the intracellular perfusion of inositol triphosphate or thapsigargin and the extracellular perfusion of ionomycin. A similar current was elicited by the cross-linking of the T cell receptor-CD3 complex. This current displayed an inward rectification below 0 mV and was completely blocked by the divalent cation Cd2+. It was very selective for Ca2+ over Na+ and insensitive to changes in chloride concentration. The physiological relevance of this conductance was investigated with the analysis of abnormal Ca2+ signaling in lymphocytes from a patient suffering from a primary immunodeficiency associated with a defective T cell proliferation. Using fura-2 video imaging, an absence of Ca2+ influx was established in the patient's lymphocytes, whereas the Ca2+ release from internal stores was normal. This was the case whether cells were stimulated physiologically through their antigen receptors or with store depleting pharmacological agents. Most importantly, no Ca(2+)-selective current was elicited in these cells. Our data strongly suggest that the Ca2+ release-activated current underlies the sustained Ca2+ influx during antigenic stimulation and that it plays a key role in the immune function.
Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.
Confocal laser scanning microscopy was used to analyze alterations in nuclear free calcium (Ca2+n) levels induced by platelet-derived growth factor (PDGF) isoforms in BALB/c3T3 fibroblasts loaded with the calcium-sensitive fluorescent indicator Fluo-3. Both AA-PDGF and BB-PDGF caused a transient increase in Ca2+n. Analysis of PDGF-induced Ca2+n alterations as a function of time revealed that BB-PDGF stimulation resulted in the generation of Ca2+n oscillations that diminished over time. The frequency of BB-PDGF-stimulated oscillations was modulated by extracellular Ca2+ and could not be mimicked by increasing intracellular inositol 1,4,5-trisphosphate levels in the absence of growth factor stimulation. Caffeine alone had no effect on Ca2+n levels, but exposure of cells to caffeine after BB-PDGF stimulation augmented Ca2+n oscillations, either by increasing the frequency or reinitiating preexisting oscillations. The genesis of these oscillations in Ca2+n appears to be in the region just outside of the nucleus, as perinuclear cytoplasmic free calcium (Ca2+i) increased just prior to Ca2+n. In contrast, AA-PDGF stimulation resulted in the generation of one or two irregular, transient Ca2+n spikes. Caffeine pretreatment followed by AA-PDGF stimulation resulted in Ca2+n oscillations very similar to those produced by BB-PDGF alone. Additionally, the AA-PDGF and BB-PDGF isoforms appeared to modulate distinct pools of cellular Ca2+, as BB-PDGF was still capable of inducing Ca2+n oscillations subsequent to prior induction of oscillations by AA-PDGF/caffeine. These PDGF isoform-specific changes in nuclear free Ca2+ could serve as a mechanism by which isoform-specific cellular signaling pathways may be manifested by the growth factors.
The gene encoding the homologue of the catalytic subunit of the Ca2+/calmodulin‐regulated protein phosphatase 2B (calcineurin A) has been isolated from Aspergillus nidulans. This gene, cnaA+, is essential in this fungal system. Analysis of growth‐arrested cells following gene disruption by homologous recombination reveals that they are blocked early in the cell cycle. The cnaA+ gene encodes a 2.5 kb mRNA and the deduced protein sequence is highly homologous to the calcineurin A subunit of other species. The mRNA varies in a cell cycle‐dependent manner with maximal levels found early in G1 and considerably before the G1/S boundary. As calmodulin is also essential for A.nidulans cell cycle progression and levels rise before the G1/S boundary, our data suggest that calcineurin may represent a primary target for calmodulin at this cell cycle transition point.
Oocyte activation is a series of events triggered by the fertilizing spermatozoon and necessary for the beginning of the embryonic development. Calcium plays a pivotal role in this process. Here we used confocal laser scanning microscopy to examine the changes in the concentration of intra-cellular free calcium ([Ca²⁺]) in human oocytes after intracytoplasmic sperm injection (ICSI). The first considerable but short (<2 min) increase in [Ca²⁺]1 was detected immediately after the penetration of the micro-injection needle into the ooplasm. This rise by itself did not provoke oocyte activation and was also obtained after the injection of medium without spermatozoa. After a lag period of 4–12 h, oocytes that were subsequently activated initiated a second period of [Ca2⁺]1 changes. These changes were sperm-dependent and followed one of two alternative patterns, a non-oscillatory one and an oscillatory one. The non-oscillatory pattern resembled the changes described previously during parthenogenetic activation of mammalian oocytes. The oscillatory pattern was similar to the changes accompanying normal fertilization in different mammalian species. It is concluded that the initial [Ca²⁺]1 rise provoked by the ICSI procedure is not responsible for oocyte activation, and that a release of a sperm factor(s) is required to initiate this process.
The expression of K+ channels modulates cell proliferation in a variety of tumor cells. Experimental evidence and three putative mechanisms of K+ channel action on cell Proliferation are discussed.
A newly identified subclass of the heterotrimeric GTP binding regulatory protein family, Gq, has been found to be expressed in a diverse range of cell types. We investigated the potential role of this protein in growth factor signal transduction pathways and its potential relationship to the function of other G alpha subclasses. Recent biochemical studies have suggested that Gq regulates the beta 1 isozyme of phospholipase C (PLC beta 1), an effector for some growth factors. By microinjection of inhibitory antibodies specific to distinct G alpha subunits into living cells, we have determined that G alpha q transduces bradykinin- and thrombin-stimulated intracellular calcium transients which are likely to be mediated by PLC beta 1. Moreover, we found that G alpha q function is required for the mitogenic action of both of these growth factors. These results indicate that both thrombin and bradykinin utilize Gq to couple to increases in intracellular calcium, and that Gq is a necessary component of the mitogenic action of these factors. While microinjection of antibodies against G alpha i2 did not abolish calcium transients stimulated by either of these factors, such microinjection prevented DNA synthesis in response to thrombin but not to bradykinin. These data suggest that thrombin-induced mitogenesis requires both Gq and Gi2, whereas bradykinin needs only the former. Thus, different growth factors operating upon the same cell type use overlapping yet distinct sets of G alpha subtypes in mitogenic signal transduction pathways. The direct identification of the coupling of both a pertussis toxin sensitive and insensitive G protein subtype in the mitogenic pathways utilized by thrombin offers an in vivo biochemical clarification of previous results obtained by pharmacologic studies.
We here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca2+-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but lacks its hormone binding domain, HBD, is constitutively localized in the nucleus; the longer one ( ), which contains both NLSs (NL1 + NL2) and the HBS of GR, is normally localized in the cytosol, but is translocated to the nucleus upon treatment with the hormone. When localized to the nucleus, both chimeras give the same estimates of [Ca2+]n, both at rest and upon stimulation with the InsP3 generating agonist histamine. The [Ca2+]n values appear very close, both at rest and upon stimulation, to those of the cytoplasm, measured with cytosolic recombinant aequorin, suggesting that, at least in this cell model, the nuclear membrane does not represent a major barrier to the diffusion of Ca2+ ions, and that the nucleus does not regulate its [Ca2+] independently from the cytosol.
Laser-scanning confocal microscopy was used in conjunction with a highly fluorescent Ca2+ indicator fluo-3 to visualize real-time alterations in the intracellular Ca2+ concentration ([Ca2+]i) in individual living Jurkat T-cells during the first minutes of activation by phytohaemagglutinin (PHA) at the physiological temperature (37°C). With a delay of 30–120 s, PHA induced a strong [Ca2+]i peak in the micromolar range (1–3 μM). The rise in [Ca2+]i lasted for 1–2 minutes, and was followed by a sustained plateau of elevated [Ca2+]i in the 0.2–0.5 μM range. Some cells (10–20%) responded with additional asynchronous 0.5–1.5 μM peaks during the plateau phase. These oscillations continued for 10–20 minutes. The spans of the peaks ranged from 30 to 100 s, intervals between peaks varied from 60 to 300 s. It was shown that the initial [Ca2+]i peak was associated with Ca2+ mobilisation from internal sources, whereas the plateau was maintained by an influx of Ca2+ from external medium. In K+-rich medium or in the presence of quinine, a K+ channel blocker, no secondary response to PHA-activation characterised by an elevated plateau was observed. The data suggest that the Ca2+ influx was dependent on the membrane potential and/or the extracellular K+-concentration. Optical sectioning showed that the intracellular Ca2+ distributed almost homogeneously throughout the cell volume both in control and in PHA-stimulated cells including those exhibiting Ca2+ oscillations. This suggests that Ca2+ signals are localized not only in cytoplasm at the cell plasma membrane but can be also transferred directly into the nucleus.
Patch-clamp recording techniques have made possible the detailed study of ion channels in cells of the immune system. These studies have revealed a number of interesting parallels between channels in the immune and nervous systems, perhaps the most intriguing of which is that channel expression and function is modifiable. Channel plasticity in immune cells takes several forms. The ensemble of K+ channels expressed by T lymphocytes changes in a stereotyped way during development and after, as mature cells interact with mitogens. Adhesion to a solid substrate alters the electrical properties of macrophages. During cell activation, Ca2+- permeable channels are opened by inositol 1,4,5-trisphosphate (IP3) in T cells and by intracellular Ca2+ in neutrophils. These studies indicate that, both during the primary phase of differentiation and during subsequent responses to environmental stimuli, the ‘electrical phenotype’ of cells in the immune system undergoes changes that may be related to cellular behavior.
New advances in the identification of ligands and signal transduction events that induce cell proliferation through tyrosine kinase and G-protein-coupled receptors have extended our understanding of the pathways that control cell proliferation by growth factors.
Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels.
This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular
events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids,
particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products
of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation
of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.
Following receptor activation in Xenopus oocytes, spiral waves of intracellular Ca2+ release were observed. We have identified key molecular elements in the pathway that give rise to Ca2+ excitability. The patterns of Ca2+ release produced by GTP-gamma-S and by inositol 1,4,5-trisphosphate (IP3) are indistinguishable from receptor-induced Ca2+ patterns. The regenerative Ca2+ activity is critically dependent on the presence of IP3 and on the concentration of intracellular Ca2+, but is independent of extracellular Ca2+. Broad regions of the intracellular milieu can be synchronously excited to initiate Ca2+ waves and produce pulsating foci of Ca2+ release. By testing the temperature dependence of wavefront propagation, we provide evidence for an underlying process limited by diffusion, consistent with the elementary theory of excitable media. We propose a model for intracellular Ca2+ signaling in which wave propagation is controlled by IP3-mediated Ca2+ release from internal stores, but is modulated by the cytoplasmic concentration and diffusion of Ca2+.
The results of experiments recently reported from this and other laboratories provide firm support for Heilbrunn's thesis that mitotic events are initiated by transient elevation of intracellular Ca2+, derived from intracellular stores. The ATP-dependent MA Ca2(+)-pump working in concert with an endomembrane Ca2+ channel appears to share the responsibility for regulating these Ca2+ signals. Further results demonstrated a limited time window during which the cell is sensitive to agents that impose mitotic arrest by interfering with transient elevations in intracellular "free" Ca2+ concentration. From this it appears that a discrete, timed increase in cytosolic Ca2+ derived from endomembrane stores is a necessary signal for regulating the onset of NEB, AO, and mitosis. Results from the arrest and release experiments provide support for a model in which Ca2+ is used to coordinate the action of parallel independent and interdependent biochemical pathways whose interaction results in the cytologic events of mitosis. These pathways apparently are operating under the influence of a metabolic "clock" that continues to cycle, at least once, in the absence of a Ca2+ transient sufficient to initiate NEB or AO. The discrete and temporal regulation of this Ca2+ transient through the interaction of the endomembrane Ca2+ pump, an endomembrane Ca2+ channel, and intracellular Ca2(+)-dependent reaction pathways suggest a mechanism incorporating a negative feedback loop to limit the size and duration of the Ca2+ transient and prevent the release of excessive amounts of Ca2+. Deeper understanding of the regulatory mechanism that governs the onset of mitosis requires: (1) quantitative imaging of intracellular Ca2+, especially the Ca2+ signal throughout the cell cycle, with high spatial and temporal resolution; and (2) identifying the molecules responsible for regulating the expression and reception of the Ca2+ signal itself. It is clear that Ca2(+)-dependent pathways are necessary elements of the mitotic process. Molecular candidates for the regulators and regulatees have yet to be identified. The upstream controlling molecules of these transmembrane Ca2+ regulatory elements, as well as the initial mitotic "start" signal, await future identification. Downstream regulation is also clearly indicated, perhaps through regulation of cyclin expression, degradation, or both.
The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT.
Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin.
We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.
It is well known that inositol 1,4,5-trisphosphate binding and release of calcium are mediated by the same protein. Several reports have indicated the location of the inositol 1,4,5-trisphosphate receptor in organelles other than endoplasmic reticulum. Immunocytochemical studies on the subcellular localization of 1,4,5-trisphosphate receptor in the Purkinje cells from two laboratories have given contradictory results regarding the nuclear location of this receptor. In this paper, a high-affinity inositol 1,4,5-[32P]trisphosphate binding site (Kd = 0.11 nM) on nuclei isolated from rat liver and devoid of any microsomal, mitochondrial, or plasma membrane constituents is documented. Furthermore, we present data demonstrating that inositol 1,4,5-trisphosphate is capable of releasing 45Ca2+ from the intact isolated liver nuclei. A rapid and transient release of calcium that was taken up by nuclei in the presence of ATP is observed. The role of inositol 1,4,5-trisphosphate in the coupling between cytoplasmic second messengers and nuclear events activated during signal transduction is postulated.
The release of Ca2+ from intracellular stores by sub-optimal doses of inositol trisphosphate has been shown to be dose-related ('quantal'), and a simple model is proposed here to account for this phenomenon. It is suggested that there is a regulatory Ca2(+)-binding site on, or associated with, the luminal domain of the InsP3 receptor, which allosterically controls Ca2+ efflux, and the affinity for Ca2+ of that site is modulated by InsP3 binding to the cytoplasmic domain of the receptor; a similar mechanism applied to the ryanodine receptor might also explain some aspects of Ca2(+)-induced Ca2+ release. The stimulated entry of Ca2+ into a cell which occurs upon activation of inositide-linked receptors has been variously and confusingly proposed to be regulated by InsP3, InsP4, and/or a 'capacitative' Ca2+ pool; the mechanism of InsP3 receptor action suggested here is shown to lead to a potential reconciliation of all these conflicting proposals.
Recent studies in our laboratory have revealed the existence of an ATP- and calmodulin-dependent Ca2+ uptake system in rat liver nuclei that can promote increases in the free Ca2+ concentration in the nuclear matrix. In the present investigation we show that liver nuclei possess a marked ability to sequester and buffer Ca2+, suggesting a potential role for the nucleus in the regulation of the cytosolic free Ca2+ concentration. In addition, we demonstrate that the intracellular messenger, inositol 1,4,5-trisphosphate [Ins-(1,4,5)P3], stimulates the release of a fraction of the nuclear Ca2+ and transiently lowers the intranuclear free Ca2+ concentration. The Ins(1,4,5)P3-stimulated Ca2+ release is followed by Ca2+ reuptake into an inositol phosphate-insensitive nuclear compartment. Together, these results demonstrate that liver nuclei contain, at least, two Ca2+ pools, one of which is releasable by Ins(1,4,5)P3. These findings are consistent with a role for the nucleus in the modulation of the cytosolic free Ca2+ level by agonists and suggest that the control of the nuclear Ca2+ load by second messengers may participate in the regulation of intranuclear Ca2(+)-dependent processes by hormones and other agents.
Purified resting human T cells can be induced to express the alpha subunit of the interleukin 2 receptor and to proliferate by treatment with 12-O-tetradecanoylphorbol-13-acetate plus ionomycin but not with 1,2-dioctanoylglycerol plus ionomycin. Determination of the translocation of protein kinase C showed that 12-O-tetradecanoylphorbol-13-acetate plus ionomycin caused a prolonged membrane association of the enzyme for more than 4 hr, whereas 1,2-dioctanoylglycerol plus ionomycin induced a transient membrane association, which was maximal at 20 min. Delivery of multiple additions of 1,2-dioctanoylglycerol plus ionomycin to the T cells resulted in progressively increased expression of the alpha subunit of the interleukin 2 receptor and proliferation commensurate with the number of multiple additions delivered, suggesting that prolonged protein kinase C activity is required for T-cell activation.
Two inositol phospholipid-specific phospholipase C (PLC) isozymes (PLC-I and -II) have been purified from bovine brain. When PLC-I or PLC-II was microinjected (100-700 micrograms/ml) into quiescent NIH 3T3 cells, a time- and dose-dependent induction of DNA synthesis occurred, as demonstrated by [3H]thymidine incorporation into nuclear DNA. In addition, approximately to 8 hr after PLC injection, NIH 3T3 fibroblasts appeared spindle-shaped, refractile, and highly vacuolated, displaying a morphology similar to transformed cells. The morphologic transformation was apparent for 26-30 hr after which the injected cells reverted back to a normal phenotype. Microinjected PLC at a high concentration (1 mg/ml) was cytotoxic, dissolving the cytoplasmic membrane and leaving behind cellular ghosts. PLC is a key regulatory enzyme involved in cellular membrane signal transduction. Introduction of exogenous PLC into NIH 3T3 cells by microinjection induced a growth and oncogenic potential, as demonstrated by the ability of microinjected PLC (approximately 10,000 molecules per cell) to override the cellular G0 block, inducing DNA synthesis and morphologic transformation of growth-arrested fibroblast cells.
The exact intracellular mechanisms by which gastrin enhances the proliferation of AR4-2J cells, a tumor pancreatic acinar cell line, are not precisely known. Calcium has long been considered as an intracellular signal involved in growth-regulatory control of many cell types. Moreover, Ca++ channel blockers show growth-suppressing effects in most proliferating cells. In the present study, we analyzed the role of nifedipine, a voltage-dependent Ca++ channel antagonist, on AR4-2J cells which possess well-defined voltage-dependent Ca++ channels. The results showed that 10 nM gastrin induced a transient rise in intracellular calcium (Ca++)i followed by a sustained phase which was dependent upon a Ca++ influx operating through nifedipine-dependent and -independent Ca++ channels. Both influxes are necessary for reloading the agonist-sensitive Cai++ pools. In parallel, we demonstrated that nifedipine at doses of 1 microM and 3 microM preferentially blocked the increase in cell number elicited by 10 nM gastrin and 0.1 microM Bay K 8644, a Ca++ channel agonist, suggesting that voltage-sensitive Ca++ channel activity was required for gastrin-stimulated mitogenesis. Moreover, nifedipine had no effect on the proliferation of AR4-2J cells growing in serum-free medium, indicating that this drug did not simply exert a toxic effect. Therefore, Ca++ influx through voltage-dependent Ca++ channels might be an important initial step representing a component of a synergistic cooperation between different signal transduction pathways involved in gastrin-regulated growth.
Recent research has shown the importance of the spatial and temporal aspects of calcium signals, which depend upon regenerative properties of the inositol trisphosphate and ryanodine receptors that regulate the release of calcium from internal stores. Initiation sites have been found to spontaneously release calcium, recognized as 'hot spots' or 'sparks', and can trigger a wave that spreads through a process of calcium-induced calcium release.
The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate is mediated by activated phosphatidylinositol-specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS-4 and KMS-8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5-trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC-gamma 1 and PLC-delta 1 in KMS-4 and KMS-8 cells. Moreover, we detected the enhancement of protein-tyrosine kinase activity and tyrosine phosphorylation of PLC-gamma 1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis.
A rise in intracellular Ca2+ concentration induces the transcription of a number of eukaryotic genes through transcription factors interacting with calcium response elements. Immediate-early response genes encode proteins that couple extracellular signals to phenotypic alterations by modulating the transcription rates of target genes. Since the activation of early response genes occurs within minutes, this class of genes has served as a paradigm for the understanding of the molecular mechanisms by which external signals are conveyed to the nucleus to induce changes in genetic programs. In this review, we outline the recent information which has been gained specifically on how the Ca2+ messenger system modulates early response gene expression. We also discuss some lines of research with the intent of linking closer Ca2+ homeostasis and gene expression studies which in the past have followed their own separate routes.
Calcium ions (Ca2+) appear to participate in the regulation of several aspects of cell division. Evidence is accumulating that transients or local gradients in the [Ca2+] contribute to different events including nuclear envelope breakdown and reformation, cleavage furrow formation and growth, and cell plate formation. At present there is little direct evidence that Ca2+ transients trigger the onset of anaphase. However, studies with exogenously applied Ca2+ indicate that spindle fibers and the movement of chromosomes at anaphase are exquisitely sensitive to the ion at physiological levels. Although Ca2+ is involved with many processes there are many gaps in our understanding, particularly pertaining to exactly when and where the ion concentration changes are expressed, which events and macromolecules are targeted, and what the processes are that control Ca2+.
The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions.
calmodulin (CaM) is the major calcium-binding protein in all the eukaryotic cells with the exception of skeletal and cardiac muscle. CaM has a Mr around 16 kDa and in higher eukaryotic cells it is encoded by three different genes (CaMI, CaMII and CaMIII) which in spite of displaying different structure and nucleotide sequence, specially in the 5′ and 3′ terminal sequences, all of them encode for exactly the same protein. In yeast and other fungi, only one gene encoding for CaM has been identified. The amino acid sequence of CaM from all the mammalian cells is exactly the same and the homology with CaM from the other eukaryotic species is also very high.
A transient rise in cytoplasmic Ca2+ activity in the sea urchin egg occurs during fertilization due to calcium release from an intracellular store. Using a combination of the Ca2+ sensitive dye Calcium Green dextran and the Ca2+ insensitive dye tetramethylrhodamine dextran we have obtained confocal ratio images of free cytoplasmic calcium distribution during the fertilization calcium wave. We can also trigger calcium release using calcium-releasing agonists such as InsP3, ryanodine and cADP-ribose. Calcium levels are in all cases similar within nucleus and in the cytoplasm. A striking result from confocal calcium imaging is that the fertilization calcium wave is not the only spatio-temporal calcium signal observed after fertilization. In fact, a second calcium wave propagates through the egg as pronuclear migration begins; this wave also originates at the point of sperm entry. A global calcium increase is also recorded during the fusion of the male and female pronuclei. We conclude that calcium concentrations in the nucleus are similar to those in the cytoplasm during these calcium transients, that a remnant at the point of sperm entry can originate a second propagating calcium wave and that a global calcium transient occurs at the time of pronuclear fusion.
We here describe the measurement of nuclear Ca2+ concentration ([Ca2+]n) with targeted recombinant aequorin. Two aequorin chimeras have been constructed, composed of the Ca(2+)-sensitive photoprotein and two different portions of the glucocorticoid hormone receptor (GR). The shorter chimera (nuAEQ), which contains the nuclear localization signal (NLS) NL1 of GR, but lacks its hormone binding domain, HBD, is constitutively localized in the nucleus; the longer one (nu/cytAEQ), which contains both NLSs (NL1 + NL2) and the HBS of GR, is normally localized in the cytosol, but is translocated to the nucleus upon treatment with the hormone. When localized to the nucleus, both chimeras give the same estimates of [Ca2+]n, both at rest and upon stimulation with the InsP3 generating agonist histamine. The [Ca2+]n values appear very close, both at rest and upon stimulation, to those of the cytoplasm, measured with cytosolic recombinant aequorin, suggesting that, at least in this cell model, the nuclear membrane does not represent a major barrier to the diffusion of Ca2+ ions, and that the nucleus does not regulate its [Ca2+] independently from the cytosol.
The temporal and spatial organization of [Ca2+] changes within the nucleus of Fura-2 loaded hepatocytes maintained in primary culture has been investigated. Vasopressin stimulation induced oscillatory waves of cytosolic free [Ca2+] increase, which propagated freely through the nuclear region. Based on the amplitude of the Fura-2 signals from this region, the morphology of the hepatocyte nucleus and the rapid penetration of the nucleus by injected Fura-2, it can be concluded that the nuclear Ca2+ responses reflect changes occurring within the nucleoplasm. Intranuclear Ca2+ increases occurred as waves that appear to be directed by the Ca2+ waves passing through the surrounding cytoplasm. The apparent velocity of Ca2+ waves was higher in the nucleoplasm than in the cytoplasm (19.5 +/- 2.9 versus 11.0 +/- 1.1 microns/s). The nucleoplasm does not contain vesicular Ca2+ stores that might be released by Ins(1,4,5)P3. However, the nuclear envelope functions as a Ca2+ store that is sensitive to mobilization by Ins(1,4,5)P3. We conclude that the [Ca2+] in the nucleoplasm of the hepatocyte is close to equilibrium with the cytosolic [Ca2+] and that oscillatory waves of cytosolic [Ca2+] are closely paralleled by similar [Ca2+] changes in the nucleoplasm. The nuclear envelope is a component of the intracellular Ins(1,4,5)P3-sensitive Ca2+ storage pool and may serve as a reservoir for [Ca2+] elevations within the nucleus.
The free calcium concentration in nucleus ([Ca2+]n) and in cytoplasm ([Ca2+]c) of single cells were estimated by confocal laser microscopy using the Ca(2+)-indicator Indo-1. It is shown that in various cell types a nucleo-cytosolic Ca(2+)-gradient is present at rest and during stimulation. The direction and the extent of the nucleo-cytosolic Ca(2+)-gradient may vary with the cell type, differentiation status, phosphorylation conditions and also with the type of agonist. Evidence is given for the role of extra- and intranuclear storage sites as well as for Ca(2+)-influx. Finally potential artefactual interference with the measurements is discussed.
The activity of PIP kinase (1-phosphatidylinositol 4-phosphate 5-kinase; EC 2.7.1.68), the second ATP-utilizing enzyme of 1,4,5-trisphosphate and diacylglycerol biosynthesis, was determined in the rat in a spectrum of transplantable solid hepatomas of different growth rates and in normal tissues of high and low cell renewal rates. In a standard isotopic method developed for the assay, the enzyme activity was linear with time for 4 min and proportional with protein concentration over a range of 0.05 to 1 mg per 0.135-ml reaction mixture. The apparent Km for the substrate PIP (phosphatidylinositol 4-phosphate) and for ATP and Mg2+ in normal liver were 0.06, 0.5, and 4.2 mM, respectively, and in rapidly growing hepatoma 3924A, 0.08, 0.7, and 7.1 mM. The kinase activity in adult Wistar rat liver was 0.046 +/- 0.003 nmol/h/mg protein. In hepatomas of slow and intermediate growth rates, PIP kinase activity increased 3.3-9.7-fold, and in hepatoma 3924A, it was elevated 45-fold over that of normal liver. When hepatoma 3924A cells were plated and expressed their proliferative program, enzyme activity increased 4.3-fold in mid-log phase. To further clarify the linkage between PIP kinase activity and proliferation, enzyme activity was determined in rat organs of high and low cell renewal capacity. The PIP kinase activity in rat thymus, bone marrow, spleen, and testes was 5.4-, 6.3-, 4.8- and 4.3-fold higher, respectively, than in normal rat liver; in lung, brain, skeletal muscle, renal cortex, and heart, the activities were low. In all tissues examined, the activity of PIP kinase was 4.6 to 18% of that of phosphatidylinositol kinase. Since enzymes of crucial significance frequently have short half-lives, the decay rates of PIP kinase were examined in liver, bone marrow, and hepatoma 3924A in rats injected with cycloheximide, which inhibits protein biosynthesis. In cycloheximide-treated animals, PIP kinase had the shortest decay rate (t1/2 = 0.12 h) in comparison with eight enzymes of purine and pyrimidine biosynthesis of rat bone marrow (t1/2 = 0.6 to 4.3 h). In liver and solid hepatoma 3924A, the activity of PIP kinase was degraded less rapidly (t1/2 = 5 h). The relationship of PIP kinase activity with proliferation and transformation is apparent in the high activity in thymus, bone marrow, spleen, and testes and in the increased activities in the rat hepatomas of different growth rates. The coordinate increases in phosphatidylinositol and PIP kinase activities suggest that the capacity for signal transduction is heightened in cancer cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) are calcium-regulating second messenger molecules generated following the binding of a wide range of hormones and growth factors to their receptors. The actions of these messengers, which play important roles in the regulation of cell proliferation as well as in other signaling pathways, are terminated by the action of a 5-phosphomonoesterase (5-PME) enzyme. We have assayed this enzyme in normal and malignant hemopoietic cells. Extracts from normal bone marrow cells and peripheral blood mononuclear cells (PBMNC) degraded [3H]IP3 at rates of 74.5 (+/- 3.4) and 84.5 (+/- 7.9) pmol/min/micrograms protein, respectively. PME activity in 10/13 (77%) acute lymphoblastic leukemia samples were significantly below the normal range and the enzyme was completely undetectable in three (23%) of these. Enzyme activity in 8/9 (89%) chronic lymphocytic leukemia samples were below the normal range, being undetectable in three of these (33%). Nine of 24 (38%) acute myeloid leukemia samples contained low 5-PME levels, which was undetectable in one sample. Reduced 5-PME activity was detected in 2/7 (28%) of chronic granulocytic leukemia samples. The data here are consistent with the hypothesis that a reduced rate of degradation of IP3 and IP4 in some leukemia cells may result in the aberrant operation of signaling pathways, possibly including those involved in the control of cell proliferation.
The role of increases in intracellular calcium levels on tyrosine phosphorylation in human B lymphocytes was studied. Stimulation of normal, resting B lymphocytes or B lymphoblastoid cells with the calcium ionophores ionomycin or A23187 induced the tyrosine phosphorylation and the enzymatic activation of microtubule-associated protein-2 kinase (MAP2-K). Treatment of these cells with PMA induced tyrosine phosphorylation of a protein with the identical mobility, as well as the enzymatic activation of MAP2-K. Stimulation of these cells with ionomycin also resulted in increased ribosomal S6 kinase activity. Activation of MAP2-K in B lymphocytes by calcium ionophore was rapid (detectable within 1 min), transient (returning to background levels by 45 min), and dependent on extracellular calcium. These results demonstrate that transmembrane calcium flux induced by calcium ionophore results in the tyrosine phosphorylation and enzymatic activation of MAP2-K in human B cells.
A calcineurin (type 2B)-like protein phosphatase gene designated ppb1+ was isolated from the fission yeast Schizosaccharomyces pombe. The predicted amino acid sequence was 57% identical to rat PP2B alpha. ppb1 null mutant could form colonies at 33 degrees C but the size of the colonies was small at 22 degrees C. Cytokinesis was greatly delayed at 22 degrees C, and a large number of multi-septate cells were produced. The cell polarity control was impaired, causing branched cells. ppb1 null was virtually sterile. These phenotypes were rescued by a plasmid carrying the ppb1+ gene. Multi-septate cells were also produced in wild type at 22 degrees C by cyclosporin A, an inhibitor of calcineurin. This drug effect was enhanced in stst1 null mutant, which was hypersensitive to various drugs and cations. ppb1 null was not affected by cyclosporin A, consistent with the hypothesis that ppb1 is its target. Double-mutant analysis indicated that ppb1 had a function related to that of two other phosphatases, type 1-like dis2 and 2A-like ppa2.ppb1 null-sts1 null showed the severe multi-septate phenotype in the absence of cyclosporin A. ppb1+ and sts1+ gene functions are related. The double mutant ppb1-sts5 was lethal, indicating that the ppb1+ gene shared an essential function with the sts5+ gene. Overexpression of ppb1+ caused anomalies in cell and nuclear shape, microtubule arrays and spindle pole body positioning in interphase cells. Thus the ppb1+ gene appears to be involved in cytokinesis, mating, transport, nuclear and spindle pole body positioning, and cell shape.