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Connexins are essential for the communication of cells among themselves and with their environment. Connexin hexamers assemble at the plasma membrane to form hemichannels that allow the exchange of cellular contents with the extracellular milieu. In addition, hemichannels expressed in neighboring cells align to form gap junction channels that mediate the exchange of contents among cells. Connexin 43 (Cx43) is the most abundant connexin expressed in bone cells and its deletion in all tissues leads to osteoblast dysfunction, as evidenced by reduced expression of osteoblast markers and delayed ossification. Moreover, Cx43 is essential for the survival of osteocytes; and mice lacking Cx43 in these cells exhibit increased prevalence of osteocyte apoptosis and empty lacunae in cortical bone. Work of several groups for the past few years has unveiled the role of Cx43 on the response of bone cells to a variety of stimuli. Thus, the preservation of the viability of osteoblasts and osteocytes by the anti-osteoporotic drugs bisphosphonates depends on Cx43 expression in vitro and in vivo. This survival effect does not require cell-to-cell communication and is mediated by unopposed hemichannels. Cx43 hemichannels are also required for the release of prostaglandins and ATP by osteocytes induced by mechanical stimulation in vitro. More recent evidence showed that the cAMP-mediated survival effect of parathyroid hormone (PTH) also requires Cx43 expression. Moreover, the hormone does not increase bone mineral content in mice haploinsufficient for Cx43 or lacking Cx43 in osteoblastic cells. Since inhibition of osteoblast apoptosis contributes, at least in part, to bone anabolism by PTH, the lack of response to the hormone might be due to the requirement of Cx43 for the effect of PTH on osteoblast survival. In summary, mounting evidence indicate that Cx43 is a key component of the intracellular machinery responsible for the transduction of signals in the skeleton in response to pharmacologic, hormonal and mechanical stimuli.
Bone cells communicate with each other to properly respond to hormonal and mechanical stimuli. One of the mechanism by which cells this communication is achieved is through gap junctions – clusters of channels that open only transiently and allow the exchange of small (<1 kDalton) molecules between adjacent cells 1. A gap junction channel is formed by two hemichannels provided by each adjacent cell; and each hemichannel is composed of six molecules of connexin (Figure 1). Hemichannels are also present in unopposed cell membranes and their opening allows exchange of cytoplasmic contents with the extracellular fluid 2,3. Besides its participation in gap junctions and hemichannels, Cx43 might affect cellular functions by interacting with structural and signaling intracellular molecules (summarized in Table 1). The majority of these interactions are localized in areas of the cytoplasmic C-terminal tail of Cx43, which does not participate in channel formation 4,5. Moreover, it has been shown that the C-terminus domain of Cx43 (in the absence of channel forming domains of the protein) can inhibit cell proliferation when transfected to HeLa or HEK293 cells 6,7, or to human glioma cells, in which it also increases cell migration 8.
Early studies on bone sections and isolated bone cells showed that gap junctions and connexins are expressed in all the cells types in bone including pre-osteoblasts, osteoblasts, osteocytes and osteoclasts 9-16. This manuscript reviews the evidence collected during the past years pointing towards Cx43 as an essential component of signaling pathways activated in osteoblasts and osteocytes.
The connexin family comprises approximately 21 proteins that exhibit high homology 5. Each connexin molecule is formed by 4 transmembrane domains, 2 extracellular and 1 intracellular loop, and N-terminus and C-terminus tails (Figure 1). Among the members of the connexin family, Cx43, is the most highly expressed in bone cells, although Cx45 and Cx37 have also been detected 10,17. Inhibition of connexin channel opening or reduced Cx43 function leads to reduced expression of osteoblast specific genes 18,19 and to decreased osteoclast precursor fusion and osteoclastic bone resorption in vitro 20-22. Mice lacking Cx43 ubiquitously die within hours after birth due to cardiac malformations precluding the study of the adult skeleton 23. Nevertheless, neonatal bones exhibit delayed ossification and osteoblastic cells show low expression of osteoblast markers and deficient mineralization compared to wild type cells 24. Similarly, immortalized calvaria cells derived from Cx43−/− mice show delay differentiation and mineralization in culture 25. In addition, mice expressing ubiquitously Cx43 mutants associated with oculodentodigital dysplasia (ODDD), which is unable to form gap junctions and acts as dominant negative for wild type Cx43, exhibit low bone mass and decrease bone strength 26-28. Moreover, mice in which Cx43 was deleted exclusively from osteochondroprogenitor cells (by using the Dermo1 promoter) have a severe phenotype, with decreased trabecular bone mass and cortical thickness 29. In contrast, deletion of Cx43 from early osteoblastic cells expressing the 2.3 kb fragment of the collagen1a1 promoter (Cx43fl/-;ColCre mice) have a less pronounce phenotype, with mild reduction in bone volume, osteoblast number and bone mass, when compared to control littermates up to 6 months of age 30. Furthermore, we have found that mice in which Cx43 has been deleted specifically in mature osteoblasts and osteocytes by expression of the Cre recombinase under the control of the human osteocalcin promoter (Cx43fl/-;OCNCre mice) exhibit indistinguishable growth, body weight and bone mass (at least between 2 and 4.5 month of age), when compared to littermates expressing Cx43 in osteoblasts and osteocytes 31. Consistent with the requirement of Cx43 expression in early progenitors, but not mature osteoblast, for full skeletal development, a Cx43 ODDD mutant only prevents osteoblast differentiation when present as a germline mutation and not when it is expressed after osteoblast commitment 32. Overall, these findings indicate that Cx43 expression in osteoblast precursors (but not mature osteoblasts or osteocytes) is required for normal development of the skeleton.
In addition to Cx43 participation in cell-to-cell communication, we and others have shown that osteoblasts and osteocytes express functional hemichannels that allow the communication of the cells with the extracellular medium 33-35. This evidence, together with findings in other cell types 3,36-39, indicate that connexin hemichannels have physiological functions, in addition to be an intermediate in the formation of gap junction channels 3. Moreover, Cx43 might also function as a scaffold, necessary for the transduction of intracellular signaling. Indeed, the interaction of Src with Cx43 is required for the anti-apoptotic effect of the anti-osteoporotic drugs bisphosphonates on osteoblasts and osteocytes 33 and Cx43 interacts with βarrestin to modulate parathyroid hormone (PTH) signaling 40. Another protein that interacts with the C-terminal domain of Cx43 is the nephroblastoma overexpressed gene CCN3/NOV, a cell growth suppressor that in turn may be responsible for the tumor suppressor properties of Cx43 over-expression 4,41-43. Whether Cx43 is involved in the reported effects of CCN3/NOV on osteoblastogenesis and BMP and Wnt signaling remains unknown 44.
In addition to inhibiting osteoclast function, the recognized mechanism by which bisphosphonates stop bone loss, these agents prevent apoptosis of osteoblasts and osteocytes 45, via a mechanism different from inhibition of the mevalonate pathway or conversion into toxic metabolites, the mechanisms of action of bisphosphonates on osteoclasts 46 (Figure 2). Thus, anti-apoptosis by bisphosphonates is observed at concentrations about 3 orders of magnitude lower than those required to promote osteoclast apoptosis in vitro 45,47-50. Moreover, the anti-apoptotic effect of bisphosphonates on osteoblasts and osteocytes is exerted not only by traditional bisphosphonates but also by compounds such as IG9402 that do not affect osteoclasts and do not inhibit the mevalonate pathway 45,49-53.
Mechanistic studies demonstrated that bisphosphonates induce the opening of Cx43 hemichannels, but do not affect gap junctions, leading to activation of the survival kinases ERKs in osteoblasts and osteocytes 33,45 (Figure 3). Cx43, but not other connexins, confers the anti-apoptotic response to bisphosphonates. Similar to other ERK activators, bisphosphonates induce activation of the kinase Src. However, unlike most ERKs activating stimuli that induce nuclear accumulation of the kinases, bisphosphonate-activated ERKs are retained outside the nucleus. This leads to the activation of the cytoplasmic ERK substrate p90RSK 33,54.
We found that bisphosphonates increase the association of Cx43 with βarrestin, a scaffolding protein that binds to clathrin, and via this association, induce G protein-coupled receptor internalization and cell desensitization to the corresponding agonist. In addition, βarrestins associate with signaling molecules, and modulate cell motility, chemotaxis and apoptosis 55. Moreover, recent evidence indicates that βarrestins are responsible for cytoplasmic retention of ERKs and inhibition of the activation of ERK nuclear targets induced by some ligands of G protein-coupled receptors 56-58. Consistent with a role of βarrestins in bisphosphonate-induced extranuclear retention of ERKs, cytoplasmic retention of ERKs and anti-apoptosis induced by the drugs are abolished in cells expressing a dominant negative form of βarrestin that prevents the interaction of endogenous βarrestin with clathrin 59. Based on this evidence, we propose that opening of Cx43 hemichannels by bisphosphonates results in the formation of a complex containing Cx43, ERKs, βarrestin and clathrin (Figure 3). This complex is responsible for the retention of ERKs outside the nucleus, leading to activation of the cytoplasmic target of ERKs p90RSK followed by phosphorylation of BAD and C/EBPβ, resulting in osteoblast and osteocyte survival.
Studies of the last decade have shown that Cx43 is a target of, and mediate the effect of mechanical stimulation in osteoblastic cells. Thus, mechanical loading increases Cx43 expression in osteoblasts and osteocytes in vitro and in murine tibiae in vivo 60-62, and increases gap junction communication and leads to opening of Cx43 hemichannels in vitro 35,60,61. It has been also shown that Cx43 expression and opening of hemichannels are required for mechanical stimulation-induced release of PGE2 in osteocytic cells 35,63, which may be involved in the anabolic effect of mechanical forces on the skeleton 64-66. More recent evidence suggests that ATP release through Cx43 hemichannels and the consequent stimulation of purinergic receptor, rather than direct release of PGE2 through the hemichannels, is required for PGE2 release induced by mechanical stimulation in osteocytic cells 67. Taken together, these pieces of evidence suggest that the response to mechanical stimulation depends on Cx43 expression. Consistent with this, Cx43fl/-;ColCre mice deficient in Cx43 in osteoblasts and osteocytes, exhibit an attenuated response to the anabolic action of mechanical stimulation 68. Thus, loading of the tibia resulted in significantly lower increase in endocortical bone formation rate and mineral apposition rate in Cx43fl/-;ColCre mice, as compared to wild type littermates. However, the mechanism by which Cx43 mediates the response to mechanical loading remains unknown.
Daily injections of PTH increase osteoblast number and bone mass 69,70. Studies in mice have shown that the increase in osteoblast number in cancellous bone is due, at least in part, to inhibition of osteoblast apoptosis 71. Similarly, PTH related protein (PTHrP), the other ligand of the PTH1 receptor, as well as constitutive activation of this receptor in transgenic mice, also increases osteoblast number and decreases the prevalence of osteoblast apoptosis 72-74. This anti-apoptotic effect of PTH and PTHrP have been reproduced by us and others in murine and human osteoblastic cell lines 71,75-77. Mechanistic studies showed that the anti-apoptotic effect of PTH on osteoblasts requires the activation of the cAMP/PKA pathway, the phosphorylation of the pro-apoptotic protein BAD and the activity of the transcription factors CREB and Runx2 71,76. Strikingly, PTH does not prevent apoptosis in osteoblastic cells expressing dominant negative forms of Cx43 or in which Cx43 expression was silenced using small hairpin RNA 40. Moreover, the response to PTH on cAMP production is blunted in osteoblastic cells in which Cx43 expression has been reduced using anti-sense cDNA 78. Cx43 expression appears to be required to obtain a full anabolic response to intermittent PTH administration in mice 30,79. Thus, PTH does not increase bone mass, bone formation and osteoblast number when administered to heterozygous Cx43 deficient mice (Cx43+/−) 79. Consistent with this, intermittent PTH administration does not increase bone mineral content in mice lacking Cx43 in osteoblastic cells 30. Taken together, these pieces of evidence suggests that intermittent PTH administration does not result in a full anabolic response in mice lacking Cx43 due to the inability of the hormone to prevent apoptosis of osteoblastic cells in the absence of Cx43. Further studies are required to address this possibility.
Mounting evidence suggests the importance of Cx43 for the development and function of several cell types, including bone cells. In addition to its well-recognized function in cell-to-cell communication, Cx43 hemichannels are also active in unopposed plasma membranes, mediating the exchange of molecules between the cells and the extracellular milieu 3,80. Small molecules released through hemichannels might transmit signals from one cell to others in the vicinity, without requiring direct cell-to-cell contact. Cx43 exhibits also channel independent functions, acting as a scaffolding protein with the ability to foster interactions among molecules of different signaling pathways, thereby regulating intracellular signaling 81. The importance of Cx43 expression in bone has been demonstrated by the phenotype of embryos of Cx43 null mice 24. However, the role of Cx43 and, in particular Cx43 hemichannels, in the response of bone to different stimuli, is far from being completely understood. We propose that Cx43 has a central role in the response of the skeleton to pharmacologic, hormonal and mechanical stimuli (Figure 4). Understanding the mechanism of the contribution of Cx43 on the effect of bone-acting stimuli will provide new opportunities to improve the treatment of conditions with increased bone fragility.
This research was supported by the National Institutes of Health (R01-AR053643) and by the National Osteoporosis Foundation (2007 NOF Research Grants Program).