Gap Junctional Regulation of Signal Transduction in Bone Cells

Abstract

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.

INTRODUCTION

The normal development and maintenance of skeletal tissue is dependent on the tightly coordinated activity of osteoblasts, osteoclasts, and osteocytes. Osteoblasts form new bone, osteoclasts resorb bone and osteocytes seem to coordinate the activation of these two cell types. In order for the bone embedded osteocytes (Fig. 1A,C) to direct bone formation and resorption on bone surfaces, there is an obvious need for these cells to signal over a substantial distance, impeded by the presence of a mineralized matrix (Fig 1A,B). This is accomplished both by the release of soluble signals (e.g., RANKL, osteoprotegerin and sclerostin) and by direct cell-to-cell communication through gap junctions. Indeed, osteocytes have an extensive network of long, dendritic-like cell processes that extend through the canaliculi of bone, where they physically interconnect with adjacent osteocytes and with osteogenic cells on the bone surface via connexin43 (Cx43)-containing gap junctions (Fig.1C). Likewise, surface osteoblasts, osteoprogenitors and bone lining cells express Cx43 and form functional gap junctions among each other as with osteocytes. The result is a functional syncytium of interconnected cells throughout bone that acts in concert to orchestrate the formation and turnover of bone.

Figure 1.

Interconnected network of osteoblasts and osteocytes. A) Osteocytes (arrows) are embedded in bone and yet they direct the activity of osteoprogenitor (asterisks) cells on the bone surface. B) Immunofluorescence of Cx43 (green) shows expression at the osteocyte cell body (arrows), as well as throughout the canalicular network extending through the mineralized cortical bone. Cx43 is also abundantly expressed by osteoblasts on the bone surface (asterisks). C) Illustration of osteocytes embedded in bone. Long dendritic-like processes, enable contact between osteocytes and surface osteoblasts. Cx43 containing gap junctions form between the osteocytes and osteoblasts, (a.) which allows the exchange of molecules between the cells. Osteocytes are also known to express gap junction hemichannels (b.), that allow for the release of factors into the extracellular space. The regulation of bone resorption by osteoclasts is mediated by osteoblast/osteocyte produced RankL and OPG. The balance of these factors in the control of osteoclast formation is a target of Cx43.

A number of compelling studies over the past decade have demonstrated that gap junctions play an integral role in coordinating the activities of these different cell types in bone. Specifically, Cx43 can regulate osteoblast differentiation, bone formation and bone resorption. Mechanistic details are emerging as to how gap junctional intercellular communication (GJIC) impacts the cellular processes that affect skeletal homeostasis. Understanding how Cx43 effects these processes is critically important as there are diverse mechanisms by which Cx43 can carry out its function in these cells, each with distinct consequences, some that enhance bone quality (osteo-anabolic) and others that are detrimental (osteo-catabolic). This review revisits some of the key findings highlighting the important role of gap junctions in bone and will emphasize the emerging details and recent contributions towards elucidating the molecular mechanisms by which Cx43 gap junctions regulate signal transduction pathways, gene expression, mechanotransduction and cell survival in bone cells.

GAP JUNCTIONS AND SKELETAL FUNCTION

Gap junctions, which are made up of connexin protein monomers, are specialized intercellular membrane channels that function as aqueous conduits between the cytoplasm of contiguous cells. Gap junctions permit the direct exchange of ions, nucleotides, small molecules and second messengers between neighboring cells. Furthermore, gap junction channels do not function in isolation, but rather, aggregate into gap junction plaques that can contain thousands of channels and range from <100 nm to several micrometers in diameter [1]. Accordingly, GJIC can provide the means for a dynamic, interconnected network of cells. In addition to classic GJIC, unopposed gap junction hemichannels exist at the membrane, where they function as direct conduits between the cytosol and extracellular milieu [2, 3].

The most abundantly expressed gap junction protein in bone is Cx43, encoded by the Gja1 gene. Gap junction channels comprised of Cx43 typically permit the passage of molecules less than ~1.2 kDa in size, whereas larger molecules, proteins and nucleic acids are generally restricted from passage. Cells of the osteoblast lineage also express other connexins, including Cx45, Cx40, Cx46 and Cx37, however the contributions of these gap junction proteins to skeletal homeostasis are still unclear.

Cx43, osteoblast differentiation, and skeletal development

Several lines of evidence underscore the importance of Cx43 in bone. First, mutation of GJA1 results in oculodentodigital dysplasia (ODDD), a pleiotropic hereditary disease characterized by craniofacial, neurologic, limb and ocular abnormalities [4, 5]. Mouse models of ODDD have shown that these mutations frequently lead to dominant negative action of Cx43 and results in changes to bone geometry, bone microarchitecture and osteopenia [6, 7]. In addition, mutation of GJA1 can lead to craniometaphyseal dysplasia with skeletal phenotypes distinct from ODDD [8]. Second, genetic ablation of Gja1 either globally or in conditional knockout models within the osteoblast lineage consistently results in a skeletal phenotype, including delayed osteoblast differentiation, osteopenia, and changes in skeletal geometry that are remindful of those observed in patients with ODDD, including broadening of the circumference of long bones with an expansion of the marrow cavity and often with thinned cortex [9-14]. The expansion of the cross sectional area and change in geometry of the long bones in ODDD and/or Gja1 genetic ablation in bone are strikingly similar to the skeletal phenotype of aging and disuse [12]. Third, Cx43 has been shown to be an important modulator of the ability of bone and bone cells to respond to mechanical cues, hormonal and growth factor stimulation and to facilitate bone healing following fracture [10, 11, 15, 16]. These topics of the in vivo effects of Cx43 are reviewed in greater detail elsewhere [17, 18]. We will focus on the molecular underpinnings for the remainder of this review.

The involvement of Cx43 in the processes that control bone cell function and ultimately bone quality is conspicuously complex, with differential responses based on the context of the effect. For example, loss of Cx43 differentially modulates the response of bone cells on the periosteal and endosteal surface of bone in response to mechanical loading [11]. Somewhat paradoxically, loss of Cx43 reduces the anabolic effect of mechanical load and yet also blunts the effects of mechanical unloading or perhaps even aging induced bone loss [19, 20]. This implies that Cx43 transmits signals that can be either osteo-anabolic or osteo-catabolic, depending on the context such as aging, mechanical loading or unloading, or even location (i.e., differential effects on the periosteal and endosteal surfaces of bone) [21]. This complexity underscores the need to understand the specific details of how Cx43 affects bone cells and bone remodeling and raises several important questions. What are the second messengers and effectors of the osteo-anabolic effects of Cx43 on bone? How do these differ from the effectors of the osteo-catabolic actions? Can we selectively regulate the ability to communicate and/or respond to some signals passed through gap junctions but not others? Understanding the molecular mechanisms by which Cx43 can modulate bone cell function in a context dependent manner is critical to the development of treatments that modulate these connexin-regulated pathways to enhance or maintain bone quality.

MOLECULAR MECHANISMS OF CX43 SIGNAL TRANSDUCTION IN BONE

One of the most common models for how Cx43 function affects cell function is related to classic GJIC. In this model, the passage of second messengers among cells by gap junctions converges on signaling pathways downstream of the gap junction [22]. Specifically, a target cell can respond to stimuli by producing second messengers that further participate in downstream signaling pathways to elicit effects on gene transcription and cell function. In addition to evoking a response in the targeted cell, these second messengers can also pass through gap junctions and elicit the same response in neighboring recipient cells. The ability to communicate or share these second messengers results in a concerted and amplified activation of signaling cascades among a population of cells. Indeed, work from our laboratory has used this model to explain how enhancing GJIC can enhance signal transduction cascades and gene expression in bone cells [17]. According to this model, if cells share second messenger signals via gap junctions, then a greater number of cells can respond to the cue that initiated the signaling event. Conversely, loss of Cx43 would limit signal exchange via gap junctional communication resulting in delays in osteoblast differentiation and function and leading to the skeletal phenotypes observed in mouse models (reviewed in [21]). Similarly, in the context of a bone catabolic environment such as hind limb unloading, disuse or aging [11, 20, 23, 24], Cx43 deletion may halt the communication and prevent the amplification of catabolic cues, reducing bone destruction and preserving bone quality.

Admittedly, such a model lacks critical mechanistic details related to how Cx43 impacts bone cell function. For example, what are the biologically relevant second messengers that are passing through Cx43 gap junctions? Do connexins function simply in the formation of a passive channel for signal diffusion, or do they actively participate in signaling cascades that control osteoblast differentiation and function? Further, to what extent do connexins function in non-junctional roles such as hemichannels to modulate cellular function? And what are the effectors mediating the effects of Cx43 on bone homeostasis? Which of these effectors and second messenger signals are osteo-anabolic? Which are osteo-catabolic? To date, the most progress has been with regard to potential osteo-anabolic signaling cascades that are enhanced by Cx43 expression.

Cx43 and Osteo-anabolic Signaling Pathways

One way that Cx43 regulates bone is through the control of osteoblast differentiation and bone cell activity, as well as through the expression of factors (e.g., RANKL and osteoprotegerin) controlling bone resorption by osteoclasts (Fig 1C). Cell autonomous defects in cells of the osteoblast lineage underlie at least a subset of the reduced bone quality caused by Cx43 gene deletion. It seems there is an intrinsic inability of osteoblasts to optimally express the genes necessary for matrix production, mineralization, the suppression of bone resorption (osteoprotegerin) and progression through the osteogenic lineage in the absence of Cx43 [9, 10]. This highlights that in certain contexts Cx43 is decidedly osteo-anabolic. In vitro, alterations in Cx43 expression or function modulate the expression of several osteoblast genes [25-30]. With respect to how Cx43 regulates gene expression, osteoblast differentiation and function, the effectors and signaling pathways are complex. It is clear that there is no single effector of Cx43’s action on bone cells.

One set of mechanisms responsible for Cx43-mediated regulation of osteoblast differentiation and gene expression involves the transcription factors Runx2 and Osterix (Sp7), both of which are master regulators of osteogenesis. Cx43 overexpression or knockdown modulates the transcriptional activity of both Runx2 and Osterix in MC3T3-E1 cells, thereby providing a potential means by which Cx43 can impact the wide spectrum of osteoblast gene transcription and osteoblast differentiation [31-34]. Two of the downstream effectors of Cx43-activated signaling responsible for modulating Runx2 and Osterix activity have been identified (Fig 2). In the case of Runx2, Cx43 affects the activation of extracellular signal-regulated kinase (ERK) and protein kinase C delta (PKCδ), both of which work in parallel to enhance Runx2 activity [28, 29, 33]. ERK and PKCδ phosphorylate Runx2, increasing its transcriptional activity [35-38]. Interestingly, ERK and PKCδ physically complex with Cx43, suggesting a point of spatial signaling control [34, 39, 40]. Consistent with the action of Cx43 in this context as a classic gap junction rather than via hemichannel activity, disruption of GJIC by pharmacological inhibition or culturing of cells in low density abrogates the Cx43-dependent effects on Runx2 activity [33].

Figure 2.

Overview of Cx43-associated signaling pathways. This schematic illustrates some of the identified signaling factors that are regulated downstream of Cx43 in osteoblasts and osteocytes. A) Cx43 impacts osteogenic gene expression via the activation of either ERK or PKCδ, which have been shown to interact with Cx43. The transmission of a signal via Cx43 gap junctions (Cx43 GJ) activates ERK or PKCδ, which leads to the activation of osteogenic transcription factors Runx2 or Osterix in the nucleus. In zebrafish, Cx43 GJIC can also induce the expression of Semaphorin3d, which gets secreted and interacts with surface receptors Neuropilin 2a (Nrp2a) and Plexin a3 (PlxnA3) on other cells. This releases the inhibitory effect of Nrp2a and PlxnA3 on the expression of genes that regulate cell proliferation and joint formation respectively, thus allowing Semaphorin3a to mediate the inductive effect of Cx43 on bony fin formation in zebrafish. B) Mechanical force can trigger the opening of Cx43 hemichannels (Cx43 HC) by stimulating PI3K signaling. PI3K acts on integrin α5β1 and induces a conformational change that drives the opening of Cx43 HCs and the release of osteo-anabolic factors such as prostaglandin E2 (PGE₂) into the extracellular space. PGE₂ can then bind to its cognate EP2 receptor, which stimulates β-catenin accumulation through cAMP/PKA and PI3K/AKT signaling. β-catenin can then induce the expression of osteogenic genes. C) Cx43 HCs can also mediate the anti-apoptotic effects of bisphosphonates and parathyroid hormone (PTH) on osteocytes. In response to bisphosphonates, Cx43 HCs can trigger the activation of Src/ERK signaling, resulting in the activation of anti-apoptotic proteins p90^RSK and C/EBPβ. Conversely, during PTH signaling, Cx43 HCs can associate with βarrestin, thus preventing it from inhibiting the action of the parathyroid hormone 1 receptor and allows for the continued propagation of cell survival signals. Crosstalk also exists where the integrin α5β1 conformational change in response to mechanical force can also stimulate focal adhesion kinase (FAK) and Src signaling and promote cell survival, and β-catenin can also regulate genes responsible for cell survival.

Similarly, Cx43-dependent modulation of ERK regulates the recruitment of Osterix (as part of an Sp1-containing complex) to osteoblast promoters [31, 32]. Cx43 regulation of Sp1 has similarly been reported in other cell types as well [41]. Taken together, these findings demonstrate that Cx43 regulates the ERK and PKCδ signaling pathways, converging on the activity of osteogenic effector molecules Runx2 and Osterix with clear effects on osteoblast gene expression, which explains, at least in part, the osteo-anabolic effects of Cx43 on bone.

An important question is, what is the identity of the second messenger signals being shared by bone cells via Cx43 to carry out these effects on transcription? Recent work has identified inositol polyphosphates as biologically relevant second messengers that may be a key signal communicated by Cx43 among bone cells that impacts Runx2 activation by activating PKCδ [42]. In Cx43-overexpressing MC3T3-E1 cells treated with FGF2, inhibition of phospholipase C gamma1 (PLCγ1), inositol phosphate multikinase and inositol hexakisphosphate kinase 1 (IP6K1), which produce inositol polyphosphate second messengers such as IP₃, IP₅, IP₆ and IP₇, results in less PKCδ phosphorylation and consequently abrogates the Cx43-dependent enhancement in Runx2 activity. Considering that IP₃ passes through Cx43 gap junctions [43], this provides evidence into a potential second messenger upstream of Cx43 that is communicated through gap junctions and activates PKCδ in adjacent bone cells. Inositol polyphosphates are probably one of many second messengers of biologic importance that are communicated through Cx43 channels that positively or negatively effect bone cell function.

There is evidence that control of gene expression and bone cell function by Cx43 is evolutionarily conserved. In zebrafish, loss-of-function and gain-of-function mutations in the gene encoding Cx43 affects the length of skeletally-derived fin segments resulting in distinct short fin and long fin phenotypes, respectively [44, 45]. Cx43 expression in the regenerating fin rays of zebrafish affects the expression of a key secreted factor, semaphorin3d [46] (Fig 2). Semaphorin3d can then signal through two other factors, neuropilin2a and plexin a3, which in turn regulates proliferation and joint formation by skeletal precursor cells that ultimately control bone tissue morphogenesis and fin growth [46]. Accompanying these findings is the intriguing observation that the upregulation of semaphorin3d does not occur in the proliferating Cx43-positive blastema cells responsible for bony fin growth, but rather in a non-proximal tissue compartment of the fin ray containing lateral skeletal precursor cells, emphasizing the importance of the cell-to-cell signaling and coordinated multicellular function in this model [47]. As such, semaphorin3d is a critical downstream effector of Cx43-controlled bony fin growth.

Cumulatively, these identified molecular mechanisms provide compelling evidence as to how Cx43 affects signal transduction cascades, such as ERK and PKCδ, in order to regulate downstream osteogenic processes such as gene transcription. Further, these studies provide insights as to why this “information sharing” of second messengers carries great consequence towards osteoblast function and bone physiology. In brief, it seems that Cx43 not only participates in the gap junctional transmission of second messengers between cells, but Cx43 also reinforces the signaling pathways activated by these second messengers in adjacent cells, thereby increasing the activation of these signaling pathways downstream of the gap junction channel. Many other mediators (both second messengers and signal pathways) that affect gene expression may exist downstream of Cx43 in bone cells, and identification of these mediators is important in developing a complete picture of the signaling factors that contribute to the role of Cx43 in bone.

Cx43 Signaling during Mechanotransduction

A second means by which Cx43 affects bone is via its role in transducing mechanical load signals among bone cells. An important cue that regulates the anabolic and catabolic action of bone cells on the skeleton is mechanical stress. Bone is amazingly adept at modulating the skeletal microarchitecture to adapt to changing demands in mechanical load. A series of seminal works on the effect of Cx43 gene deletion on the mechano-responsiveness of bone have revealed the importance of Cx43 in translating both osteo-anabolic and osteo-catabolic mechanical responses from osteocytes into the remodeling of bone [11, 19, 20, 23, 48]. In vitro, osteoblasts and osteocytes can respond to mechanical perturbation and/or fluid flow-induced mechanical stress by producing calcium oscillations that can propagate among cells in a Cx43-dependent manner [49-51]. These Cx43-dependent calcium oscillations between osteocytes and osteoblasts can be reproduced ex vivo in intact bone in response to mechanical stress on the cells [52, 53]. Thus, calcium or effectors of calcium signaling, such as IP₃, may be second messengers used by bone cells to transduce mechanical load signals though gap junctions. It is not yet known if these pathways then converge upon the ERK or PKCδ pathways to regulate osteoblast gene expression via Runx2 or Osterix or if they take their own unique path to modulate bone cell function.

In addition to classic cell-to-cell communication through gap junction channels, several studies suggest that Cx43 hemichannels contribute to mechanical signaling in osteocytes. In response to fluid flow-induced mechanical stress, MLO-Y4 osteocyte-like cells release prostaglandin E2 (PGE₂) via opening of Cx43 hemichannels [54, 55] (Fig 2). The opening of hemichannels occurs as a result of a physical interaction between Cx43 hemichannels and α5β1 integrins [56]. In this context, fluid flow-induced mechanical stress results in the PI3K-dependent phosphorylation of α5β1, inducing a conformation change in the integrin, opening the Cx43 hemichannel. Subsequently, PGE₂ can be released through the Cx43 hemichannel. With regard to mechano-signaling in bone, PGE₂ signals through its cognate EP2 receptors, which induces the activation of PI3K/AKT pathway and accumulation of cAMP [57, 58]. Both cAMP/PKA- and PI3K/AKT-dependent pathways converge on β-catenin signaling, a key effector of mechanical load responses by bone cells [59-62]. PGE₂ also induces Cx43 expression and GJIC in osteocytes and osteoblasts [58, 63]. Thus, PGE₂ 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. It should be noted that there are two levels of controversy with respect to Cx43 hemichannel function in bone. First, the in vivo relevance of Cx43 hemichannels is unclear, as characterization of mice expressing an ODDD-causing Cx43 mutant, which has defective GJIC but hyperactive hemichannel function, largely phenocopies the Cx43 conditional knockout models with respect to bone [7, 12]. Second, bone cells isolated from Cx43-deficient mice are as effective as wild type bone cells at releasing PGE₂ in response to fluid flow [64]. In fact, in this study the authors convincingly demonstrate pannexin1 activity rather than Cx43 hemichannel activity was required for PGE₂ release. Additional work, particularly using in vivo models, is necessary to clarify the role of GJIC, Cx43 hemichannels and pannexins in mechanotransduction in bone. Regardless, it is clear that Cx43 plays a large role in how bone homeostasis is controlled not only during osteo-anabolic activities, such as mechanical stress induced by exercise, but also during osteo-catabolic activities, such as the removal of mechanical signals as a result of exposure to microgravity or disuse. Little is known about the nature of these osteo-catabolic signals that are propagated through Cx43 to impact bone quality.

Cx43 Mechanisms of Osteocyte Apoptosis and Survival

A third means by which Cx43 impacts bone quality is through regulation of osteocyte apoptosis. Osteocyte death is exacerbated in various catabolic bone syndromes [65, 66]. Increased apoptosis of osteocytes may compromise the mechanosensory and communicative function of bone cells, leading to increased bone resorption and low bone mass [67, 68]. Hence, there is great interest in determining what regulates osteocyte death and the downstream consequences on bone remodeling.

In mouse models, conditional deletion of Cx43 from late stage osteoblast and osteocytes results in increased osteocyte apoptosis [14, 20]. Perplexingly, no increase in osteocyte death is observed in Cx43 conditional knockout mice with deletion of Cx43 in the entire osteoblast lineage (rather than just late stage osteoblasts and osteocytes) [12]. The reasons for this discrepancy are not clear, as all of these models lack Cx43 in the osteocyte compartment, but perhaps it implies that GJIC between osteoprogenitor cells and osteocytes produces survival signals that are in direct opposition to those exchange by osteocytes and late osteoblasts. Undoubtedly, clarification of this point is critical to understanding all of the ways that Cx43 can regulate bone homeostasis.

Additionally, Cx43 can influence cell survival downstream of specific cues. Cx43 is required for the anti-apoptotic action of bisphosphonates and parathyroid hormone (PTH) on osteocytes and osteoblasts [69, 70]. In the case of bisphosphonates, the opening of Cx43 hemichannels by the indirect action of bisphosphonates leads to the activation of ERK-dependent cell survival signals that inactivate the pro-apoptotic effector BAD and drive the anti-apoptotic actions of C/EBPβ [69, 71]. With respect to PTH, Cx43 can mediate cell survival signals by forming a complex with β-arrestin, effectively sequestering β-arrestin and permitting sustained action of the parathyroid hormone 1 receptor (PTHR1) and the accumulation of intracellular cAMP accumulation, leading to enhanced cell survival [72]. The implication that cAMP is sufficient to induce osteocyte survival, even in the absence of Cx43, establishes that cAMP may be another biologically relevant second messenger transmitted by Cx43 gap junctions.

The Cx43 C-terminus and an Active Role for Cx43 in Signaling

A particularly exciting role for Cx43 as an active participant in signal transduction and not just a passive channel for second messenger diffusion is emerging. The C-terminal (CT) domain of Cx43, which is the least conserved of the connexin protein domains, has been shown to bind to and/or be the target of numerous signaling complexes [39, 73]. While much of the focus of the gap junction field has been on how modulation of the connexin by these signal complexes can regulate the open/closed state of gap junction channels (signaling to the gap junction), recent evidence demonstrates a role for signaling from the Cx43 gap junction, as well.

Several in vitro structure-function studies show that the Cx43 C-terminus plays an important role in signaling and function of bone cells. Overexpression of a full-length Cx43 construct, but not a Cx43 mutant construct that lacks the C-terminus, enhances the expression of osteoblast genes and supports the Cx43-dependent potentiation of FGF2 signaling on Runx2 activity, despite both constructs being able to support similar levels of GJIC [34]. Importantly, not only is the C-terminus required for the signaling and transcriptional effects of Cx43 overexpression on bone cells, the C-terminus is insufficient to mimic the effects of Cx43 and is antagonistic to signaling via the ERK and PKCδ pathways and to osteoblast gene expression. This requirement for the C-terminus is consistent with the fact that PKCδ complexes with Cx43 via its C-terminus domain and upon activation translocates from Cx43 to the nucleus where it interacts with Runx2 [40, 42]. This demonstrates that both the communication function (Cx43 channel) and the C-terminus of Cx43 are required for Cx43 to affect bone cells. Likewise, α5 integrin-dependent opening of Cx43 hemichannels following fluid flow requires the intact Cx43, while the Cx43 C-terminus is antagonistic to hemichannel opening [56]. Also, a similar Cx43δCT construct abolishes the bisphosphonate- and PTH-induced anti-apoptotic effect mediated by Cx43, although in this context GJIC may not be required [70, 71].

These experiments suggest that Cx43 serves, not only to exchange second messengers between cells, but also as a “docking platform” for signaling complexes. In the three dimensional context of the cell, location is important, and thus the ability to bring signaling effector molecules (e.g., PKCδ) to the unique cellular microdomain (i.e., the membrane bound gap junction plaque) where second messengers (e.g., IP₇, cAMP) are exchanged is logical. Therefore, intercellular communication and signal complex association with the gap junction channel may be intertwined functions, and the biologic activity of Cx43 may be determined, not only by the charge and size selective permeability of gap junctions, but also by the locally recruited signaling complexes associated with the Cx43 C-terminus.

This model may explain, at least in part, the need for 20+ connexin proteins, which often have overlapping permeability and yet often serve unique biologic roles. Among connexins, the highly variant C-terminus is primarily the site of signaling complexes interactions. Extrapolating this model, it is possible that each connexin may recruit its own unique subset of signaling complexes, providing specific function to each connexin. While intriguing, these hypotheses still require rigorous testing, but they highlight a novel role of Cx43 as an active regulator of signaling and not just a passive channel for the intercellular diffusion of molecules that are small enough to pass through.

CONCLUSION AND FUTURE PERSPECTIVES

In summary, Cx43 is crucial for skeletal homeostasis, regulating bone cell function by actively participating in the regulation of signaling, gene expression, cell survival and the ability to respond to diverse cues, such as mechanical stress. The means by which Cx43 carries out these functions are numerous and are context specific, communicating both osteo-anabolic and osteo-catabolic signals within the skeletal network of cells. Indeed, even within the largely anabolic signaling network, second messengers like inositol polyphosphates (e.g., IP₃ and IP₇), calcium and cAMP have all been implicated (Fig 2). These messengers converge on effectors including PKCδ, ERK, β-catenin, PI3K and PKA.

Despite significant advances, much work remains to be done. The links between the second messengers that are communicated by Cx43 and cell function need to be defined more clearly. The nature of osteo-catabolic signals communicated by Cx43 needs to be established. The identity of biologically relevant second messengers must be determined. These studies will be critical for understanding how Cx43 ultimately affects bone quality. This knowledge may permit us to modulate Cx43-dependent processes to increase bone cell function and bone mass accumulation in order to prevent or treat diseases of skeletal fragility. Understanding the coordination of osteoblast, osteocyte and osteoclasts networks is vital to the understanding diseases of skeletal metabolism.

Interventions that can impact bone biology based on protein-protein interactions and structure function analysis of Cx43 are certainly feasible and have been successful in other tissues. For example, the association of the Cx43 C-terminus to the tight junction protein zonula occludens-1 (ZO-1) has been extensively studied [39, 74-77]. Recently, a mimetic peptide (alphaCT1) that affects Cx43 C-terminus interactions with ZO-1 has been demonstrated to be effective at improving corneal wound healing, reducing scar tissue and improving healing time, and attenuating injury-induced cardiac arrhythmia [77-79]. Accordingly, such molecular dissections of Cx43 structure and function can have important therapeutic potential.