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Gap junctions are intercellular channels formed by hexamers of connexins in one cell that dock with a hexameric array of connexins on an adjacent cell, forming an aqueous pore between the two cells. Gap junctions permit the direct intercellular exchange of ions, small molecules, and second messengers. In addition, gap junctions can function as unopposed hemichannels, serving as a direct conduit between the cytosol and extracellular fluid.
In bone, osteoblasts and osteocytes are highly interconnected via gap junctions composed primarily of connexin43 (Cx43). In these cells, Cx43 has been shown to play an important role in transmitting hormonal-induced signals, mechanical load–induced signals, and growth factor–induced signals and, ultimately, in bone mass acquisition via both classic cell-to-cell communication through gap junctions or via hemichannel activity.[1, 2] Mutations in Gja1, the gene encoding Cx43, result in the pleiotropic disorder oculodentodigital dysplasia, which includes numerous skeletal manifestations. Mouse models of oculodentodigital dysplasia and Cx43 genetic ablation (both globally and osteoblast-specific conditional knockout models) have underscored the fundamental importance of Cx43 in skeletal function and bone mass acquisition.[4-11] Loss or disruption of Cx43 in these mouse models profoundly impairs osteoblast function and responsiveness to anabolic hormones and mechanical load, typically resulting in osteopenic bone. Indeed, modulation of Cx43 affects signaling transduction cascades, impacting osteoblast and osteocyte survival and/or gene expression.[12-19]
Despite the critical role of Cx43 as a regulator of bone mass, the complex molecular mechanisms by which Cx43 regulates osteoblast function are only beginning to emerge. Critical questions remain to be answered, such as what is the identity of the second messengers communicated by Cx43 gap junctions, what are the targets of these second messengers, and how do they regulate osteoblast/osteocyte function?
Ultimately, by defining the molecular pathways by which Cx43 regulates osteoblast function, we can identify the biologically relevant second messengers that are being communicated among osteogenic cells and gain insight into how osteoblasts and osteocytes coordinate their activities to form new bone. Toward this end, we have focused our attention on signaling via the well-defined fibroblast growth factor-2 (FGF2) signaling pathway in osteoblasts.
FGF2 is an important regulator of skeletal tissue with complex action, acting at several stages of differentiation to differentially affect osteoblast function.[20, 21] FGF2 signals through its cognate FGF receptors (FGFRs) to activate several signaling cascades, including phospholipase Cγ1 (PLCγ1). In osteoblasts, FGF2 signaling converges on runt-related transcription factor 2 (Runx2), an important regulator of osteoblast differentiation.[23-26]
In our attempts to identify the molecular mechanisms by which Cx43 regulates osteoblasts, we have shown that alterations of Cx43 expression in osteoblasts can impact their responsiveness to FGF2, by modulating the transcriptional activity of Runx2 in a protein kinase C delta (PKCδ) and extracellular signal-regulated kinase (ERK)-dependent manner.[12, 27, 28] Further, we have shown that the ability of Cx43 to potentiate Runx2 activity requires gap junctional communication, because gap junction channel blockers and cell culture at low density, in which cell-to-cell contacts are kept at a minimum, abrogates the effects of Cx43 overexpression on osteoblast responsiveness to FGF2. In addition we have shown that Cx43 overexpression in MC3T3 cells enhances the percentage of cells responding to FGF2, underscoring that Cx43 is permitting the communication of signals between cells.[12, 28] In this study, we examined the hypothesis that the amplification of FGF2-regulated signaling cascades by Cx43 occurs as a result of second messengers generated downstream of PLCγ1 that are communicated by Cx43 gap junction channels to affect the expression of osteoblast genes.
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In this study, we show that the PLCγ1/IPMK/IP6K/PKCδ pathway is required for the modulation of Runx2 activity by FGF2, as well as for the Cx43-mediated potentiation of that response. These data demonstrate for the first time that higher-order InsPs (eg, InsP5 and InsP7), which are products of IPMK and IP6K activities, are mediators of the FGF2 response in osteoblastic cells. Further, this is the first study to show that IP6Ks regulate Runx2 activity and affects the expression of osteoblast genes.
Notably, the impact of disruption of the PLCγ1/IPMK/IP6K/PKCδ pathway consistently has a stronger effect on the Cx43 amplified response (greater-fold inhibition) than it does on the response in the absence of overexpressed Cx43, strongly suggesting that a factor in this pathway mediates the effects of Cx43 on Runx2. Further, since we have previously shown that the effects of Cx43 on FGF2-signaling requires functional gap junctional communication, this hints that inositol polyphosphates produced by the action of IPMK and IP6K1 may be biologically relevant second messengers communicated by Cx43. Alternately, these inositol polyphosphates may simply lie along the pathway in which gap junctional communication participates without necessarily being directly communicated. Future studies will need to be done to directly test the ability of higher order InsPs to transverse gap junctions and stimulate signaling in a coupled cell. That said, InsPs such as InsP7 do meet the necessary criteria for a molecule capable of passing through a Cx43 channel. InsPs are soluble in cytoplasm and could pass through the aqueous pore of a gap junction channel. Indeed, the molecular weight and charge of InsPs are consistent with the known permeability of Cx43-containing gap junctions, which permit the passage of molecules under ∼1000 molecular weight with a preference for negatively charged molecules. The molecular weight of the backbone inositol (C6H12O6) is ∼180, whereas the pyrophosphorylated InsP7 has a molecular weight of ∼740, making them capable of being transmitted through Cx43 gap junctions. Indeed, InsP3 is communicated through Cx43 gap junctions.
Related to the permeability and size of a gap junction communicated second messenger, the synergistic activities of Cx43 on FGF2 signaling cannot be recapitulated by Cx45, which has the opposing effect of Cx43 on signaling, gene transcription, and Runx2 activity in osteoblasts.[12-14, 48, 49] Cx45 forms a gap junction with a smaller pore size, reduced permeability, and greater selectively for positively charged molecules[50, 51]; and is thus unlikely to support communication of the large, negatively charged higher order InsPs. This reinforces the hypothesis that InsPs are a biologically relevant second messenger that are propagated by Cx43 channels.
It is worth mentioning that we do not intend to suggest a lack of importance of other inositol polyphosphates (or other second messengers) in gap junction communication, but our current data support a role for IP6Ks and specifically IP6K1. It is unclear if the minimal effect of IP6K2 knockdown on FGF2 signaling and the Cx43-dependent regulation of Runx2 transcriptional activity is because IP6K2 plays only a minor role in this cascade or if knockdown was insufficient to expose an effect on Runx2. Notably, overexpression of IP6K2 enhanced both the FGF2 response of Runx2 and the potentiation of this response by Cx43 overexpression, indicating that IP6K2 can contribute to Runx2 regulation even if it may not required for this action. Further, it is possible that IP6K2 (and IP6K1) may play important roles in Cx43 signaling independent of Runx2 activity. Indeed, IP6K2 is known to be an inducer of apoptosis,[52-54] an outcome associated with loss of Cx43 function in bone cells.[9, 16, 55] Future studies will require examination of these effects in primary osteoblasts isolated from IP6K1 and IP6K2 null mice, as we were unable to achieve greater than 50% knockdown of IP6K1 or IP6K2 with various siRNAs.
Based on these data and our other published data, we propose a speculative model (Fig. 6) in which FGF2 binds to its cognate FGFR in one cell, activating PLCγ1, leading to DAG and InsP3 generation. The action of IMPK and IP6Ks convert the InsP3 into higher-order InsPs. The action of IP6K1 and the second messenger InsP7 are required for the translocation of PKCδ into the nucleus, where it interacts with Runx2, promoting the expression of Runx2-responsive osteogenic genes. Further, InsP7 can diffuse through Cx43 gap junctions to initiate PKCδ activation and Runx2-dependent transcription in an adjacent cell. In fact, we have shown that PKCδ is transiently recruited to the Cx43 channel prior to nuclear translocation following FGF2 treatment. Thus, Cx43 is a docking platform for the signal complex that responds to the communicated second messenger. The cell-to-cell communication of the second messengers causes the population of osteoblasts to respond more robustly to the extracellular cue (FGF2) than would happen in the absence of gap-junctional communication. In contrast, when Cx43 expression is reduced, such as in Cx43 knockout models, the response of a population of cells is blunted, because information is not shared among the cells. As a result, there is a reduction in osteoblast activation, gene expression, and bone formation by the cell population. Such an effect may underlie the skeletal phenotype of the Cx43 deletion models. These concepts are consistent with our previous data, revealing an increase in the number of osteoblastic cells responding to a cue in the presence of overexpressed Cx43[12, 28] and the need for Cx43 function and direct cell-to-cell contacts for the potentiation of FGF2 responses by these cells. Future studies will be needed to definitively establish that InsP7 is passed through Cx43 gap junction channels, and that it is required to reactivate signaling in the adjacent, gap junction coupled cell.
Figure 6. Model of Cx43 potentiated Runx2 activity via an inositol pyrophosphate second messenger following FGF2 stimulation. Upon binding to its receptor (FGFR) in one cell, FGF2 activates PLCγ1, generating DAG and InsP3 (IP3). Subsequently, the activity of IPMK and IP6K1 leads to the production of inositol polyphosphates and pyrophosphates (InsPs), such as InsP6 (IP6) and InsP7 (IP7). The InsPs activate PKCδ, which translocates to the nucleus where it interacts with Runx2, increasing its transcriptional activity and driving the expression of osteoblast genes. In addition, we speculate that these small, water-soluble second messengers may be communicated to adjacent cells via Cx43 containing gap junctions. In the coupled cell, PKCδ, which is locally recruited to the Cx43-containing gap junction channel, can reinitiate signaling in this cell, independent of direct stimulation by FGF2, resulting in a potentiation of the response among coupled cells.
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It is possible that this mechanism can be extrapolated to other extracellular cues, such as the responsiveness to mechanical strain and growth factor and hormonal signaling. Indeed, the conditional genetic ablation of Cx43 in cells of the osteoblast lineage makes these mice refractory to the anabolic effects of intermittent parathyroid hormone, as well as mechanical loading and unloading. Notably, a similar knockout model has reported an increase in the osteoanabolic response to mechanical loading in human osteocalcin–cyclic recombinase (hOCN-Cre)-driven Cx43 conditional knockout mice, suggesting that there is considerable complexity in the signals communicated by Cx43. As we have mentioned, it is likely that multiple second messengers and signaling pathways lie downstream of Cx43 activity, some anabolic and some catabolic to bone. Further, these second messengers and signaling pathways will undoubtedly be dependent upon the nature of the extracellular cue. InsP signaling likely represents only a subset of these pathways.
In total, these data extend our understanding of FGF2 signaling and the convergence of the PLCγ1/IPMK/IP6K/PKCδ pathway on Runx2 activity in osteoblasts. Also, these data indicate a role for InsPs in osteogenic differentiation. Further, these data show that IPMK and IP6K1 are required for the Cx43-dependent potentiation of Runx2 activity in response to FGF2, suggesting that higher order InsPs, such as InsP7, are likely biologically relevant second messengers that are communicated by Cx43 gap junctions in osteoblasts to regulate the expression of osteoblast genes. This role may extend to osteogenic cues beyond FGF2 and could explain the defective osteogenesis and skeletal phenotypes observed in many of the Cx43 genetic deletion or mutation models.