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Mitochondrial connexin 43 (mtCx43) response to oxidative stress in primary osteocytes. (A) Osteocytes from Csf1 ± have an increased reactive oxygen species (ROS) level by DCF staining. Data collected from three animals in each group, each point indicated the ROS level from each individual well. Scale bar: 50 µm. Two-tailed t-test was conducted, ****p<0.0001. (B) Osteocytes from Csf1 ± mice have an increased co-localization of succinate dehydrogenase (SDHA, green) and Cx43 (red). Primary osteocytes were stained with SDHA and Cx43 antibodies. Scale bar: 20 µm. Coefficient coFigure 8 continued on next page
Source publication
Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stres...
Contexts in source publication
Context 1
... data 1. Raw oxygen consumption rate (OCR) data of Seahorse assay for Figure 5. primary osteocytes with decreased CSF1 ( Figure 8A). Furthermore, the significant increase of co-localization of Cx43 and SDHA suggested the increased migration of Cx43 to the mitochondrial in Csf1 ± compared with the wild-type (WT) (Csf1 +/+ ) cells ( Figure 8B). ...
Context 2
... oxygen consumption rate (OCR) data of Seahorse assay for Figure 5. primary osteocytes with decreased CSF1 ( Figure 8A). Furthermore, the significant increase of co-localization of Cx43 and SDHA suggested the increased migration of Cx43 to the mitochondrial in Csf1 ± compared with the wild-type (WT) (Csf1 +/+ ) cells ( Figure 8B). The result recapitulated the mitochondria migration and accumulation of Cx43 in oxidized osteocytes in vivo. ...
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... data 1. Raw data of DCF staining and succinate dehydrogenase (SDHA), connexin 43 (Cx43) co-localization analysis in primary osteocytes isolated from Csf1 +/+ and Csf1 ± for Figure 8A and B. ...
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... the translocation of Cx43 to mitochondria in osteocytes after oxidative stress acts as a cell 'rescue mission' by promoting ATP production through increased mitochondrial membrane potential and proton gradient through activation of mitochondrial complexes. The localization and interaction of complex V ATPase may aid in the transfer of ATP into the cytosol in protecting cells against oxidative stress ( Figure 8C). ...
Citations
... Connexins (Cxs), or gap junction proteins, are transmembrane proteins that form gap junctions (GJs) and hemichannels between two neighboring cells to transfer and exchange small molecules up to 1200 Daltons [1][2][3]. Twenty-one members of the connexin family have already been recognized in humans [4], each playing a vital role in maintaining organizational balance, regulating cell growth and facilitating cell differentiation [5][6][7][8]. Connexin 43 (Cx43), encoded by the GJA1 gene, is a highly studied and abundant member using R v3.6.3, and the visualization of the data was performed using the ggplot2 package. We used the Wilcoxon signed rank test for the analysis of data between two groups, which was considered to have statistical significance if p < 0.05. ...
... Connexins, as a family of large pore channel-forming proteins, have critical functions in facilitating the transfer of small signaling molecules between adjacent cells [6,7,11,44]. Recent advances in connexin research have revealed their crucial contribution to cancer development and progression through modulation of cell proliferation [45], apoptosis [46], and cellular localization [11,14]. ...
Background and Objectives: Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods: Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results: The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein–protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions: GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.
... Suggested that YME1L helped buffer oxidative stress in HG conditions. Furthermore, uncontrolled oxidative stress is known to interrupt ATP production in the mitochondria [33]. In the present study, we demonstrated that YME1L overexpression was able to reverse HG-induced suppression of ATP production in HK2 cells (Fig. 5J), while YME1L silencing led to an even greater decrease in ATP production (Fig. 5K). ...
Background
The senescence of renal tubular epithelial cells (RTECs) is crucial in the progression of diabetic kidney disease (DKD). Accumulating evidence suggests a close association between insufficient mitophagy and RTEC senescence. Yeast mitochondrial escape 1-like 1 (YME1L), an inner mitochondrial membrane metalloprotease, maintains mitochondrial integrity. Its functions in DKD remain unclear. Here, we investigated whether YME1L can prevent the progression of DKD by regulating mitophagy and cellular senescence.
Methods
We analyzed YME1L expression in renal tubules of DKD patients and mice, explored transcriptomic changes associated with YME1L overexpression in RTECs, and assessed its impact on RTEC senescence and renal dysfunction using an HFD/STZ-induced DKD mouse model. Tubule-specific overexpression of YME1L was achieved through the use of recombinant adeno-associated virus 2/9 (rAAV 2/9). We conducted both in vivo and in vitro experiments to evaluate the effects of YME1L overexpression on mitophagy and mitochondrial function. Furthermore, we performed LC–MS/MS analysis to identify potential protein interactions involving YME1L and elucidate the underlying mechanisms.
Results
Our findings revealed a significant decrease in YME1L expression in the renal tubules of DKD patients and mice. However, tubule-specific overexpression of YME1L significantly alleviated RTEC senescence and renal dysfunction in the HFD/STZ-induced DKD mouse model. Moreover, YME1L overexpression exhibited positive effects on enhancing mitophagy and improving mitochondrial function both in vivo and in vitro. Mechanistically, our LC–MS/MS analysis uncovered a crucial mitophagy receptor, BCL2-like 13 (BCL2L13), as an interacting partner of YME1L. Furthermore, YME1L was found to promote the phosphorylation of BCL2L13, highlighting its role in regulating mitophagy.
Conclusions
This study provides compelling evidence that YME1L plays a critical role in protecting RTECs from cellular senescence and impeding the progression of DKD. Overexpression of YME1L demonstrated significant therapeutic potential by ameliorating both RTEC senescence and renal dysfunction in the DKD mice. Moreover, our findings indicate that YME1L enhances mitophagy and improves mitochondrial function, potentially through its interaction with BCL2L13 and subsequent phosphorylation. These novel insights into the protective mechanisms of YME1L offer a promising strategy for developing therapies targeting DKD.
... Moreover, Cx43 and Panx1 functioning is also modulated by several inflammatory/toxic agents, including TNF-α, interferon gamma (IFN-γ) or amyloid-beta protein [129]. Similarly, Cx43 participates to ROS spreading among cells [131]. In addition, increased surface levels of Panx1 and Cx43 correlates with channel opening and microglial activation [132]. ...
Background
Redox imbalance and inflammation have been proposed as the principal mechanisms of damage in the auditory system, resulting in functional alterations and hearing loss. Microglia and astrocytes play a crucial role in mediating oxidative/inflammatory injury in the central nervous system; however, the role of glial cells in the auditory damage is still elusive.
Objectives
Here we investigated glial-mediated responses to toxic injury in peripheral and central structures of the auditory pathway, i.e., the cochlea and the auditory cortex (ACx), in rats exposed to styrene, a volatile compound with well-known oto/neurotoxic properties.
Methods
Male adult Wistar rats were treated with styrene (400 mg/kg daily for 3 weeks, 5/days a week). Electrophysiological, morphological, immunofluorescence and molecular analyses were performed in both the cochlea and the ACx to evaluate the mechanisms underlying styrene-induced oto/neurotoxicity in the auditory system.
Results
We showed that the oto/neurotoxic insult induced by styrene increases oxidative stress in both cochlea and ACx. This was associated with macrophages and glial cell activation, increased expression of inflammatory markers (i.e., pro-inflammatory cytokines and chemokine receptors) and alterations in connexin (Cxs) and pannexin (Panx) expression, likely responsible for dysregulation of the microglia/astrocyte network. Specifically, we found downregulation of Cx26 and Cx30 in the cochlea, and high level of Cx43 and Panx1 in the ACx.
Conclusions
Collectively, our results provide novel evidence on the role of immune and glial cell activation in the oxidative/inflammatory damage induced by styrene in the auditory system at both peripheral and central levels, also involving alterations of gap junction networks. Our data suggest that targeting glial cells and connexin/pannexin expression might be useful to attenuate oxidative/inflammatory damage in the auditory system.
... In summary, there is growing evidence that Cx43 is a key component of intracellular mechanisms responsible for signaling in bone in response to pharmacological, hormonal, and mechanical stimuli. Under HO-induced oxidative stress, mitochondrial Cx43 (mtCx43) enhances mitochondrial ATP production to protect cells from oxidative stress by participating in the maintenance of the proton gradient between the membrane space and the matrix (88). This mechanism may be the result of small molecule release and/or the activation of other signaling pathways (6). ...
Background and Objective
Connexin 43 (Cx43) is the main gap junction (GJ) protein and hemichannel protein in bone tissue. It is involved in the formation of hemichannels and GJs and establishes channels that can communicate directly to exchange substances and signals, affecting the structure and function of osteocytes. CX43 is very important for the normal development of bone tissue and the establishment and balance of bone reconstruction. However, the molecular mechanisms by which CX43 regulates osteoblast function and homeostasis have been less well studied, and this article provides a review of research in this area.
Methods
We searched the PubMed, EMBASE, Cochrane Library, and Web of Science databases for studies published up to June 2023 using the keywords Connexin 43/Cx43 and Osteocytes. Screening of literatures according to inclusion and exclusion guidelines and summarized the results.
Key Content and Findings
Osteocytes, osteoblasts, and osteoclasts all express Cx43 and form an overall network through the interaction between GJs. Cx43 is not only involved in the mechanical response of bone tissue but also in the regulation of signal transduction, which could provide new molecular markers and novel targets for the treatment of certain bone diseases.
Conclusions
Cx43 is expressed in osteoblasts, osteoclasts, and osteoclasts and plays an important role in regulating the function, signal transduction, and mechanotransduction of osteocytes. This review offers a new contribution to the literature by summarizing the relationship between Cx43, a key protein of bone tissue, and osteoblasts.
... As of now, there are still no patch-clamp data available on Cx43-formed channels in mitoplasts [139]. Instead, putative Cx43-formed channel-like structures within mitochondria have been investigated using indirect methods [140,141]. The role of mtCx43 is not fully understood; however, some studies suggest that mtCx43 may influence cellular processes related to tissue injury, inflammation, and fibrosis. ...
... Studies largely support the role of mtCx43 as a critical modulator of apoptosis. Recent work from our group demonstrates the translocation of Cx43 to mitochondria in bone osteocytes after oxidative damage by promoting ATP production through increased mitochondrial membrane potential and enhancement of the proton gradient through its interaction with mitochondrial complex V [141]. Additionally, the increased ATP level in the cytosol protects the cells against oxidative stress, which depletes cytosolic ATP, leading to cell apoptosis [141]. ...
... Recent work from our group demonstrates the translocation of Cx43 to mitochondria in bone osteocytes after oxidative damage by promoting ATP production through increased mitochondrial membrane potential and enhancement of the proton gradient through its interaction with mitochondrial complex V [141]. Additionally, the increased ATP level in the cytosol protects the cells against oxidative stress, which depletes cytosolic ATP, leading to cell apoptosis [141]. ATP production and ROS generation are known to be involved in tissue injury and inflammation [145,146]. ...
Fibrosis initially appears as a normal response to damage, where activated fibroblasts produce large amounts of the extracellular matrix (ECM) during the wound healing process to assist in the repair of injured tissue. However, the excessive accumulation of the ECM, unresolved by remodeling mechanisms, leads to organ dysfunction. Connexins, a family of transmembrane channel proteins, are widely recognized for their major roles in fibrosis, the epithelial–mesenchymal transition (EMT), and wound healing. Efforts have been made in recent years to identify novel mediators and targets for this regulation. Connexins form gap junctions and hemichannels, mediating communications between neighboring cells and inside and outside of cells, respectively. Recent evidence suggests that connexins, beyond forming channels, possess channel-independent functions in fibrosis, the EMT, and wound healing. One crucial channel-independent function is their role as the primary functional component for cell adhesion. Other channel-independent functions of connexins involve their roles in mitochondria and exosomes. This review summarizes the latest advances in the channel-dependent and independent roles of connexins in fibrosis, the EMT, and wound healing, with a particular focus on eye diseases, emphasizing their potential as novel, promising therapeutic targets.
... ACSL1 is mainly responsible for regulating lipid metabolism and fatty acid oxidation balance (Quan et al. 2021), while ALDH4A1 and GCSH play important roles in amino acid metabolism, and are responsible for regulating the production and transport of proline and glycine, respectively (Lorenzo et al. 2021). ATP5MF is an important component in the regulation of oxidative phosphorylation: Complex 5 (Zhang et al. 2022). BIK is directly related to mitochondrial apoptosis (Chinnadurai et al. 2008). ...
Background
Renal clear cell carcinoma (RCC) is a common cancer in urinary system with increasing incidence. At present, targeted therapy and immunotherapy are the main therapeutic programs in clinical therapy. To develop novel drugs and provide new ideas for clinical therapy, the identification of potential ccRCC subtypes and potential target genes or pathways has become a current research focus.
Aim
The aim of this study was to explore the underlying mechanisms of mitochondrial function in ccRCC. This regulatory pathway is closely related to tumor development and metastasis in ccRCC patients, and their abnormal changes may affect the prognosis of cancer patients. Therefore, we decided to construct a prognostic model of ccRCC patients based on mitochondrial regulatory genes, aiming to provide new methods and ideas for clinical therapy.
Result
The 5-year survival prediction model based on iterative LASSO reached 0.746, and the cox model based on coxph reached C-index = 0.77, integrated c/D AUC = 0.61, and integrated brier score = 0.14. The rsf model based on randomForestSRC was built with C-index = 0.82, integrated c/D AUC = 0.69, and integrated brier score = 0.11. The results show that mitochondrial regulatory pathway is a potential target pathway for clinical therapy of ccRCC, which can provide guidelines for clinical targeted therapy, immunotherapy and other first-line therapy.
... Wen et al. [47] found a significant increase in dephosphorylated Cx43 at Serine 368 at the intercalated discs in the myocardial tissue of a cocaine abuse rat model, compared to control. Moreover, Cx43 has been suggested to be essential for the regulation of cardiomyocytes homeostasis, oxygen consumption, the regulation of potassium fluxes, and oxidative stress response, therefore concurring to cardio-protection [51,52]. ...
Cocaine abuse is a serious public health problem as this drug exerts a plethora of functional and histopathological changes that potentially lead to death. Cocaine causes complex multiorgan toxicity, including in the heart where the blockade of the sodium channels causes increased catecholamine levels and alteration in calcium homeostasis, thus inducing an increased oxygen demand. Moreover, there is evidence to suggest that mitochondria alterations play a crucial role in the development of cocaine cardiotoxicity. We performed a systematic review according to the Preferred Reporting Items for Systemic Reviews and Meta-Analysis (PRISMA) scheme to evaluate the mitochondrial mechanisms determining cocaine cardiotoxicity. Among the initial 106 articles from the Pubmed database and the 17 articles identified through citation searching, 14 final relevant studies were extensively reviewed. Thirteen articles included animal models and reported the alteration of specific mitochondria-dependent mechanisms such as reduced energy production, imbalance of membrane potential, increased oxidative stress, and promotion of apoptosis. However, only one study evaluated human cocaine overdose samples and observed the role of cocaine in oxidative stress and the induction of apoptosis though mitochondria. Understanding the complex processes mediated by mitochondria through forensic analysis and experimental models is crucial for identifying potential therapeutic targets to mitigate or reverse cocaine cardiotoxicity in humans.
... Functional mitochondrial connexin-based channels have been reported that are capable of dye uptake potentially allowing them to serve as regulatable conduits for potassium and calcium influx (141,144). Dye uptake experiments further suggest that Cx43 forms functional mitochondrial hemichannels with enhanced dye uptake occurring in response to increased delivery of Cx43 to the mitochondria due to oxidative stress (145). Furthermore, Cx43 has been suggested to regulate mitochondrial calcium uptake (via interactions with the mitochondrial calcium uniporter) (146), mitochondrial respiration and ATP production (145,147), reactive oxygen species production (145,148), and the mitochondrial membrane potential (145). ...
... Dye uptake experiments further suggest that Cx43 forms functional mitochondrial hemichannels with enhanced dye uptake occurring in response to increased delivery of Cx43 to the mitochondria due to oxidative stress (145). Furthermore, Cx43 has been suggested to regulate mitochondrial calcium uptake (via interactions with the mitochondrial calcium uniporter) (146), mitochondrial respiration and ATP production (145,147), reactive oxygen species production (145,148), and the mitochondrial membrane potential (145). In keeping with these findings, Cx40 ablation and overexpression experiments in endothelial cells indicate that Cx40 regulates mitochondrial calcium homeostasis and reactive oxygen species formation (138). ...
... Dye uptake experiments further suggest that Cx43 forms functional mitochondrial hemichannels with enhanced dye uptake occurring in response to increased delivery of Cx43 to the mitochondria due to oxidative stress (145). Furthermore, Cx43 has been suggested to regulate mitochondrial calcium uptake (via interactions with the mitochondrial calcium uniporter) (146), mitochondrial respiration and ATP production (145,147), reactive oxygen species production (145,148), and the mitochondrial membrane potential (145). In keeping with these findings, Cx40 ablation and overexpression experiments in endothelial cells indicate that Cx40 regulates mitochondrial calcium homeostasis and reactive oxygen species formation (138). ...
Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions.
... While contact sites have been demonstrated to occur between most organelles [1], including mitochondria, contact sites between mitochondria and organelles composed of gap junction proteins has received little consideration. However, the finding of members of the gap junction family of proteins, connexins, on mitochondrial membranes [2][3][4][5] and the reports of mitochondrial contacts with gap junction plaques at the plasma membrane and vesicles composed of connexins in the cytoplasm [6][7][8][9] has opened a new realm of possible functions for connexins, outside of their primary cell-cell communication functions. We will discuss connexins in general and then go on to present what is known about mitochondrial contacts with connexincontaining membranes and mitochondrial connexins. ...
... In this model, Cx43 derived from the gap junction plaques, annular gap junction membranes or other sources would be translocated into the outer mitochondria membrane via the HSP90 and TOM 20 translocation complex [70,74]. Interaction with translocase of the inner membrane (TIM) would then result in the translocation of Cx43 to the inner mitochondrial membrane where it remains in monomeric form or is assembled into functional hemichannels by an unknown mechanism [5,74]. The hemichannels, once in the inner membrane, could then participate in different mitochondrial functions. ...
... In addition, the interaction of the hemichannels with the K + channel could increase potassium flux [87,88]. Furthermore, hemichannels may directly block reactive oxygen species (ROS) production [5]. The possible combined effect of inhibiting ROS and apoptosis could lead to ischemic preconditioning. ...
Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell–cell communication. Cell–cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell–cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins’ functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.
... There is some evidence to suggest that CX43 gene expression is associated with cellular oxidative stress. For example, studies have shown that increased expression of CX43 can lead to improved antioxidant defense mechanisms and reduced oxidative stress in cells (17). Additionally, CX43 gene expression has been linked with the regulation of cellular signaling pathways that play a role in antioxidant defense(18). ...
Background
Brilliant cresyl blue (BCB) staining can stain oocytes and differentiated oocytes will lead to different developmental outcomes. This technique has been studied in multiple species, but it is still unclear whether buffalo oocytes can be used for developmental potential prediction through BCB staining methods. This study used the BCB staining method to group buffalo oocytes (BCB + and BCB-) and perform in vitro maturation, in vitro fertilization, and embryo culture. By statistical analysis, the effect of BCB staining on predicting the developmental potential of buffalo oocytes will be explored. At the same time, molecular biology techniques will be used to detect gap junction protein and oxidative stress-related indicators to explore the molecular mechanism of BCB staining predicting oocyte cell developmental potential.
Methods
The oocytes were divided into BCB + and BCB- groups using BCB staining technique. And then mainly uses in vitro maturation, in vitro fertilization and embryo culture techniques of buffalo oocytes to analyze their developmental potential, and uses immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels, and qPCR to detect the expression levels of the antioxidant related genes SOD2 and GPX1.
Results
Our results showed that the in vitro maturation rate, embryo cleavage rate, and blastocyst rate of buffalo oocytes in the BCB + group were significantly higher than those in the BCB- group and the control group (P < 0.05). The expression level of CX43 protein in the BCB + group was higher than that in the BCB- group both before and after maturation (P < 0.05). The intensity of ROS in the BCB + group was significantly lower than that in the BCB- group (P < 0.05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB + group were significantly higher than those in the BCB- group (P < 0.05).
Conclusions
BCB staining can effectively predict the developmental potential of buffalo oocytes. The results of BCB staining are positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the ROS level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes may be closely related to antioxidant activity.
