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Effect of PGE 2 and PTH on Cx43 mRNA of UMR 106-01 cells. Confluent cultures were incubated with either 10 6 M PGE 2 or 10 7 M PTH for the indicated times, and total RNA extracted and separated, as detailed in Material and Methods. Membranes were hybridized with a cDNA probe for Cx43, then
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Connexin43 (Cx43) forms gap junctions that mediate intercellular communication between osteoblasts. We have examined the effects of prostaglandin E2 (PGE2) and parathyroid hormone (PTH) on gap junctional communication in the rat osteogenic sarcoma cells UMR 106-01. Incubation with either PGE2 or PTH rapidly (within 30 min) increased transfer of neg...
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... UMR 106-01 cells incubated with either 10 6 M PGE 2 or 10 7 M PTH for different times. Both PGE 2 and PTH induced time-dependent increases of steady-state Cx43 mRNA with similar time- courses. An increase of Cx43 mRNA abundance was detectable after approximately 2 h of incu- bation with either agonist, and the effect lasted for at least 4-6 h (Fig. 3). Importantly, no changes were detected in the level of Cx43 mRNA after 30 min incubation with PGE 2 or PTH, a time at which dye coupling was en- hanced (see above). Significant increases of Cx43 mRNA (2.5-1.5-fold of baseline) were obtained in UMR 106-01 cells with PTH concentrations as low as 10 9 M. Lower doses were ...
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... Previous studies reported sex-related differences in the bone anabolic response to loading. 70,71 To our knowledge, androgen could inhibit the mechanical sensitivity of bone in male mice, 72 while activation of estrogen enhances the osteogenic response to mechanical loading in female mice. 24,27,73 The investigation will be continued with female mice, especially in ovariectomized mice, a relevant model for post-menopausal women with high morbidity of osteopenia and osteoporosis. ...
Physical mechanical stimulation can maintain and even increase bone mass. Here, we report an important role of osteocytic integrin α5 in regulating the anabolic response of bone to mechanical loading using an Itga5 conditional gene knockout (cKO) mouse model. Integrin α5 gene deletion increased apoptotic osteocytes and reduced cortical anabolic responses to tibial compression including decreased endosteal osteoblasts and bone formation, and increased endosteal osteoclasts and bone resorption, contributing to the decreased bone area fraction and biomechanical properties, leading to an enlarged bone marrow area in cKO mice. Similar disruption of anabolic responses to mechanical loading was also detected in cKO trabecular bone. Moreover, integrin α5 deficiency impeded load-induced Cx43 hemichannel opening, and production and release of PGE2, an anabolic factor, resulting in attenuated effects of the loading on catabolic sclerostin (SOST) reduction and anabolic β-catenin increase. Together, this study shows an indispensable role of integrin α5 in osteocytes in the anabolic action of mechanical loading on skeletal tissue through activation of hemichannels and PGE2-evoked gene expression. Integrin α5 could act as a potential new therapeutic target for bone loss, especially in the elderly population with impeded mechanical sensitivity.
... It was found that Cx43 modulates the stimulatory action of PTH on matrix production by MC3T3-E1 cells [32]. Cx43 gene expression between osteoblasts is up regulated by PTH treatment [33], and the interference with Cx43mediated GJIC disrupts both PTH-induced cAMP accumulation and osteoblast differentiation [32,34,35]. Besides, the anabolic response to PTH is attenuated in mice with an osteoblast-specific deletion of Cx43 [36]. ...
Estrogen has garnered considerable attention because of its importance in bone mass maintenance and the efficacy of hormone therapy in combating postmenopausal osteoporosis. Gap junctions are membrane spanning protein channels present on the surfaces of adjacent cells. They enable neighboring cells to physically link, which facilitate intercellular communication by allowing passage of small molecules from cell to cell in a process known as Gap Junctional Intercellular Communication (GJIC), can make the relevant cells function as a whole. An in vitro experiment combined with microarray analyses has identified novel genes in the response of MC3T3-E1 cells to an appropriate concentration of 17-β estradiol and the results of microarray showed that gap junction alpha-1 (Gja1) in the gap junction pathway was significantly elevated. We studied the effect of 17-β estradiol on the proliferation and differentiation of MC3T3-E1 cells disrupting of GJIC among osteoblasts with the chemical inhibitor, 18α-Glycyrrhetinic Acid (AGA). And we found that an appropriate concentration and duration of 17-β estradiol increased Methyl Thiazol Tetrazolium (MTT) values, Alkaline Phosphatase (ALP) activity and Runt-related transcription factor 2 (Runx2) proteins and gene expression and facilitated the mineralization of extracellular matrix. However, the promoting effect of 17-β estradiol on the proliferation and differentiation of MC3T3-E1 cells was weakened under the action of AGA. Therefore, we suggest that 17-β estradiol promotes MC3T3-E1’s proliferation, differentiation and functions associated with Cx43-based GJIC, but gap junction is not the only signaling pathway that mediates the influence of 17-β estradiol on osteoblasts. The specific regulatory mechanism has yet to be researched.
... Cx43 also plays an important role in the effect of antiosteoporotic medications such as parathyroid hormone (PTH) or bisphosphonate. PTH increases gap junctional communication between osteoblasts by modulating Cx43 expression or function [20]. Mice lacking Cx43 in osteoblastic cells exhibit a deficient response to intermittent PTH administration [21]. ...
The inhibition of proliferation or functional alteration of osteoblasts by glucocorticoids (GCs) has been recognized as an important etiology of GC-induced osteoporosis (GIO). Connexin 43 (Cx43) is the most abundant connexin isoform in bone cells and plays important roles in bone remodeling. Despite the important role of Cx43 in bone homeostasis and the prevalence of GIO, the direct action of GCs on Cx43 expression in osteoblasts has been poorly described. The aim of the present study was to evaluate how GCs affect Cx43 expression in osteoblasts. Dexamethasone (Dex) treatment decreased expression of Cx43 RNA and protein in MC3T3-E1 mouse osteoblastic cells. Reduction of Cx43 expression by Dex was dependent on the glucocorticoid receptor (GR), as it was abolished by pretreatment with a GR blocker. Treatment with PTH (1-34), a medication used for GIO management, counteracted the suppression of Cx43 by Dex. Akt or mTOR signaling modulators revealed the involvement of the Akt/mTOR signaling pathway in Dex-induced reduction of Cx43 expression. Moreover, overexpression of Cx43 significantly attenuated Dex-inhibited cell viability and proliferation, as evidenced by MTT and bromodeoxyuridine (BrdU) incorporation assay of MC3T3-E1 cells. To account for possible species or cell type differences, human primary osteoblasts were treated with Dex and similar downregulation of Cx43 by Dex was observed. In addition, immunofluorescent staining for Cx43 further demonstrated an apparent decrease in Dex-treated human osteoblasts, while analysis of lucifer yellow propagation revealed reduced gap junction intercellular communication by Dex. Collectively, these findings indicate that GCs suppress Cx43 expression in osteoblasts via GR and the Akt/mTOR signaling pathway and overexpression of Cx43 may, at least in part, rescue osteoblasts from GC-induced reductions in proliferation.
... However, whether or not Cx43-dependent communication of cAMP occurs at a level sufficient to elicit a biological response in coupled bone cells has not been demonstrated. Similarly, PGE2 has been shown to regulate the abundance of Cx43 in bone cells (14)(15)(16)(17), but whether or not cAMP produced by PGE2 is then communicated through this gap junction-coupled network of cells and is capable of eliciting a biological response has not been explicitly tested. Indeed, if cAMP is a biologically relevant second messenger that permits coordination of osteoblast activity, then it is essential that not only must cAMP pass between gap junction coupled cells but also must impact signaling and cell function. ...
... In fact, Cx43 hemichannels have been shown to be involved in the release of PGE2 from mechanically stimulated osteocytes (55,62,63). Further, PGE2 has been shown to increase Cx43 expression and regulate gap junctional communication among osteoblasts and osteocytes (15,17,64). The subsequent gap junction dependent communication of the signals downstream of PGE2 release, including cAMP, may be an important regulator network for controlling the autocrine and paracrine action of PGE2 in bone formation. ...
Connexin43 (Cx43) containing gap junctions play an important role in bone homeostasis, yet little is known about the second messengers communicated by Cx43 among bone cells. Here, we used MC3T3-E1 pre-osteoblasts and UMR106 rat osteosarcoma cells to test the hypothesis that cAMP is a second messenger communicated by bone cells through Cx43 containing gap junctions in a manner that is sufficient to impact osteoblast function. Overexpression of Cx43 markedly enhanced the activity of a cAMP-response element driven transcriptional luciferase reporter (CRE-luc) and increased phospho-CREB and phospho-ERK1/2 levels following expression of a constitutively active Gsα or by treatment with prostaglandin E2 (PGE2), 3-Isobutyl–1-methyl xanthine (IBMX) or forskolin. The Cx43-dependent potentiation of signaling in PGE2 treated cells was not accompanied by a further increase in cAMP levels, suggesting that the cAMP was shared between cells rather than Cx43 enhancing cAMP production. To support this, we developed a novel assay in which one set of cells expressing constitutively active Gsα (donor cells) were co-cultured with a second set of cells expressing a CRE-luc reporter (acceptor cells). Using this assay, activation of a CRE-luc reporter in the acceptor cells was both Cx43- and cell contact-dependent, indicating communication of cAMP among cells. Finally, we showed that Cx43 increased the cAMP-dependent mRNA expression of receptor activator of nuclear factor kappa B ligand (RANKL) and enhanced the repression of the sclerostin mRNA, implying a potential mechanism for the modulation of tissue remodeling. In total, these data demonstrate that Cx43 can communicate cAMP between cells and, more importantly, that the communicated cAMP is sufficient to impact signal transduction cascades and the expression of key bone effector molecules between interconnected cells.
... While few signaling pathways underlying connexin gene expression have been identified across various cell types, gene transcription has been shown to be regulated by a variety of molecular treatments. For example, Cx43 transcription is elevated by treatment with cAMP elevating agents [76], glucocorticoids [77], estrogen [78], parathyroid hormone, and prostaglandin [79]. Transcriptional regulation of Cx43 may also be transactivated by the Wnt-1 signaling pathways, however further study is required to determine which signaling elements are involved [80]. ...
Multi-cellular systems require complex signaling mechanisms for proper tissue function, to mediate signaling between cells in close proximity and at distances. This holds true for the islets of Langerhans, which are multicellular micro-organs located in the pancreas responsible for glycemic control, through secretion of insulin and other hormones. Coupling of electrical and metabolic signaling between islet β-cells is required for proper insulin secretion and effective glycemic control. β-cell specific coupling is established through gap junctions composed of connexin36, which results in coordinated insulin release across the islet. Islet connexins have been implicated in both Type-1 and Type-2 diabetes; however a clear link remains to be determined. The goal of this review is to discuss recent discoveries regarding the role of connexins in regulating insulin secretion, the regulation of connexins within the islet, and recent studies which support a role for connexins in diabetes. Further studies which investigate the regulation of connexins in the islet and their role in diabetes may lead to novel diabetes therapies which regulate islet function and β-cell survival through modulation of gap junction coupling.
... Both cAMP/PKA-and PI3K/AKT-dependent pathways converge on bcatenin signaling, a key effector of mechanical load responses by bone cells [59][60][61][62]. PGE 2 also induces Cx43 expression and GJIC in osteocytes and osteoblasts [58,63]. Thus, PGE 2 may function both as an osteoanabolic mediator of Cx43 hemichannel function, while also enhancing GJIC, increasing the propagation of intercellular mediators of mechanical load among bone cells. ...
The role of gap junctions, particularly that of connexin43 (Cx43), has become an area of increasing interest in bone physiology. An abundance of studies have shown that Cx43 influences the function of osteoblasts and osteocytes, which ultimately impacts bone mass acquisition and skeletal homeostasis. However, the molecular details underlying how Cx43 regulates bone are only coming into focus and have proven to be more complex than originally thought. In this review, we focus on the diverse molecular mechanisms by which Cx43 gap junctions and hemichannels regulate cell signaling pathways, gene expression, mechanotransduction and cell survival in bone cells. This review will highlight key signaling factors that have been identified as downstream effectors of Cx43 and the impact of these pathways on distinct osteoblast and osteocyte functions.
... The Cx43 indicated more intense staining in OSS groups in the absence of OM compared with the control. Gap junction communication is important in bone cells [21], where the channels are involved in mechanical transmission [22][23][24], induction of cytokines in osteoblasts [25], and coordination of hormonal responses [26,27]. In osteoblast-like cells in vitro, Cx43 is the dominant connexin subtype and likely plays an important role in normal skeletal development [28][29][30]. ...
... Connexins play a major role in response to many mechanical, electrical, chemical, and hormonal stimuli and help regulate cell homeostasis as well as calcium signaling and differentiation [26,48,49]. Therefore, controlling fluid flow like OSS can also potentially induce the opening of Cx43 hemichannels in osteocytes and other bone cells allowing for enhanced cell-cell communication and bone formation [50][51][52]. ...
Cellular behavior is dependent on a variety of physical cues required for normal tissue function. In order to mimic native tissue environments, human alveolar bone-derived mesenchymal stem cells (hABMSCs) were exposed to orbital shear stress (OSS) in a low-speed orbital shaker. The synergistic effects of OSS on proliferation and differentiation of hABMSCs were investigated. In particular, we induced the osteoblastic differentiation of hABMSCs cultured in the absence of OM by exposing hABMSCs to OSS (0.86-1.51 dyne/cm(2)). Activation of Cx43 was associated with exposure of hABMSCs to OSS. The viability of cells stimulated for 10, 30, 60, 120, and 180 min/day increased by approximately 10% compared with that of control. The OSS groups with stimulation of 10, 30, and 60 min/day had more intense mineralized nodules compared with the control group. In quantification of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2) protein, VEGF protein levels under stimulation for 10, 60, and 180 min/day and BMP-2 levels under stimulation for 60, 120, and 180 min/day were significantly different compared with those of the control. In conclusion, the results indicated that exposing hABMSCs to OSS enhanced their differentiation and maturation.
... This was found particularly for gap junctions composed of Cx43 and Cx40 (Cruciani and Mikalsen 2002;Harris 2001;Olk et al. 2009;van Rijen et al. 2000). Many factors, including increasing levels of intracellular cAMP, lead to the regulation of gap junction coupling (Civitelli et al. 1998;Murray and Shah 1998). ...
Previous data showed that dipyridamole enhanced gap junction coupling in vascular endothelial and smooth muscle cell lines by a cAMP-dependent mechanism. The present study investigates the level at which dipyridamole affects gap junction coupling. In the GM-7373 endothelial cell line, scrape loading/dye transfer experiments revealed a rapid increase in gap junction coupling induced during the first 6 h of dipyridamole treatment, followed by a slow increase induced by further incubation. Immunostaining analyses showed that the rapid enhancement of gap junction coupling correlated with an increased amount of Cx43 gap junction plaques and a reduced amount of Cx43 containing vesicles, while the amount of Cx43 mRNA or protein was not changed during this period, as found by semiquantitative RT-PCR and Western blot. Additionally, brefeldin A did not block this short-term-induced enhancement of gap junction coupling. Along with the dipyridamole-induced long-term enhancement of gap junction coupling, the amount of Cx43 mRNA and protein additionally to the amount of Cx43 gap junction plaques were increased. Furthermore, the anti-Cx43 antibody detected only two bands at 42 kDa and 44 kDa in control cells and cells treated with dipyridamole for 6 h, while long-term dipyridamole-treated cells showed a third band at 46 kDa. We propose that a dipyridamole-induced cAMP synthesis increased gap junction coupling in the GM-7373 endothelial cell line at different levels: the short-term effect is related to already oligomerised connexins beyond the Golgi apparatus and the long-term effect involves new expression and synthesis as well as posttranslational modification of Cx43.
... Cx43 modulates the differentiation and secretory activity of bone forming cells [12,[19][20][21]. Cx43 also modulates parathyroid hormone stimulatory action on matrix production by MC3T3 cells [22]; and Cx43 gene expression is stimulated by prostaglandin E2 and parathyroid hormone, critical factors for systemic and local control in bone remodeling [23,24]. Further, the anabolic response to PTH is attenuated in mice with an osteoblastspecific deletion of Cx43 [19]. ...
... Based on the established function of Cx43 in osteoblast differentiation and function [22,30], this result further underlines a positive role of risedronate on bone forming cells. Cx43 expression and function is upregulated by other proosteogenic factors, such as prostaglandin E2, parathyroid hormone and bone morphogenetic protein-2 [24,31]; and is also modulated by PI3K/AKT [32]. Furthermore, Runx2, Osterix and Dlx5, critical transcription factors for osteoblastic differentiation, also stimulate Cx43 transcription. ...
Bisphosphonates are potent antiresorptive drugs which have antifracture efficacy by reducing bone turnover rate and increasing bone mineral density. In addition to inhibiting osteoclast function, bisphosphonates have been reported to also promote survival of osteocyte and osteoblast via an anti-apoptotic effect, mediated by opening of hemi-gap junction channels formed by connexin43 (Cx43). In this study, we investigated the effect of risedronate, one amino-bisphosphonate, on osteoblast differentiation and Cx43 expression using the mesenchymal cell line C2C12. Risedronate dose-dependently increased the activity of osterix (OSE)-luciferase containing Runx2 response element with highest activity at 50 μM. The activity of osteocalcin (OC)- and bone sialoprotein (BSP)-luciferase reporters, markers of osteoblast differentiation, were also increased by risedronate. When risedronate and BMP2 were used in combination, alkaline phosphatase (ALP) activity increased to a larger extent than when BMP2 was used alone. Risedronate as well as the pro-osteogenic transcription factors, Runx2, Osterix or Dlx5, increased transcriptional activity of the Cx43 promoter in a dose-dependent manner. In the presence of Runx2 or Dlx5, risedronate had an additive effect on Cx43 promoter activity. Accordingly, risedronate increased protein expression of Cx43, Runx2, Osterix, and Dlx5. These results suggest that risedronate promotes osteoblastic differentiation and positively regulates Cx43 gene transcription.
... Increasing cAMP positively correlates with increased GJIC, and it is through cAMP signaling that PTH acutely stimulates GJIC [44] . We have previously demonstrated that osteoblastic cells from mature and old rats are less capable of generating cAMP in response to PTH or CTX compared to osteoblastic cells from young rats [27] . ...
Aging demonstrates deleterious effects upon the skeleton which can predispose an individual to osteoporosis and related fractures. Despite the well-documented evidence that aging decreases bone formation, there remains little understanding whereby cellular aging alters skeletal homeostasis. We, and others, have previously demonstrated that gap junctions-membrane-spanning channels that allow direct cell-to-cell conductance of small signaling molecules-are critically involved in osteoblast differentiation and skeletal homeostasis. We examined whether the capacity of rat osteoblastic cells to form gap junctions and respond to known modulators of gap junction intercellular communication (GJIC) was dependent on the age of the animal from which they were isolated. We observed no effect of age upon osteoblastic Cx43 mRNA, protein or GJIC. We also examined age-related changes in PTH-stimulated GJIC. PTH demonstrated age-dependent effects upon GJIC: Osteoblastic cells from young rats increased GJIC in response to PTH, whereas there was no change in GJIC in response to PTH in osteoblastic cells from mature or old rats. PTH-stimulated GJIC occurred independently of changes in Cx43 mRNA or protein expression. Cholera toxin significantly increased GJIC in osteoblastic cells from young rats compared to those from mature and old rats. These data demonstrate an age-related impairment in the capacity of osteoblastic cells to generate functional gap junctions in response to PTH, and suggest that an age-related defect in G protein-coupled adenylate cyclase activity at least partially contributes to decreased PTH-stimulated GJIC. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1979-1984, 2012.
