The gap junction protein, connexin43 (Cx43), plays an important role in skeletal biology. Previously, we have shown that Cx43 can enhance the signaling and transcriptional response to fibroblast growth factor 2 (FGF2) in osteoblasts by increasing protein kinase C-␦ (PKC␦) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKC␦ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKC␦-independent activation of ERK by FGF2 was confirmed by Western blotting, as was the Cx43-dependent enhancement of ERK activation. Consistent with our prior observations for PKC␦, flow cytometry analyses show that Cx43 overexpression enhances the percentage of phospho-ERK-positive cells in response to FGF2, supporting the notion that shared signals among gap junction-coupled cells result in the enhanced response to FGF2. Western blots and luciferase reporter assays performed on osteoblasts cultured under low-density and high-density conditions revealed that cell-cell contacts are required for Cx43 to amplify ERK activation and gene transcription. Similarly, inhibition of gap junctional communication with the channel blocker 18␤-glycyrrhetinic acid attenuates the Cx43-dependent enhancement of Runx2-transcriptional activity. In total, these data underscore the importance of cell-cell communication and activation of the ERK and PKC␦ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts. gap junctions; osteoblast; signal transduction; fibroblast growth factor; protein kinase C THE GAP JUNCTION PROTEIN connexin43 (Cx43) is abundantly expressed in osteoblasts and osteocytes and has been shown to be fundamentally important to skeletal function (5,22,41). Mutations in Gja1, the gene encoding Cx43, cause the pleiotropic disorder oculodentodigital dysplasia (ODDD) (35, 36), which has several skeletal manifestations. While the bone mass phenotype of patients with ODDD has not been reported, two mouse models of ODDD have markedly reduced bone mass (7,9). Similarly, genetic ablation of Cx43 in mouse models leads to delayed ossification, markedly reduced peak bone mass, insensitivity to osteoanabolic interventions, such as intermittent parathyroid hormone administration and mechanical load, and a generalized reduction in osteoblast differentiation and mineralizing capacity (4,15,27,48). Conversely, overexpression of Cx43 in cultured osteoblasts enhances osteogenic capacity and responsiveness to extracellular cues (14,26,28,39), suggesting that the degree of Cx43 expression regulates the full elaboration of the osteoblast phenotype. In addition, Cx43 has been implicated in the mechanosensing and signaling by osteocytes in vitro (3,12,19,37,40,45,49). Despite the relevance of Cx43 to bone, the complex molecular mechanisms by which Cx43 regulates skeletal function and osteoblast/ osteocyte biology are only beginning to emerge.Previously, we have shown that modulation of...

The transcription factor osterix (Osx/Sp7) is required for osteogenic differentiation and bone formation in vivo. While Osx can act at canonical Sp1 DNA-binding sites and/or interact with NFATc1 to cooperatively regulate transcription in some osteoblast promoters, little is known about the molecular details by which Osx regulates osteocalcin (OCN) transcription. We previously identified in the OCN proximal promoter a minimal C/T-rich motif, termed OCN-CxRE (connexin-response element) that binds Sp1 and Sp3 in a gap junction-dependent manner. In the present study, we hypothesized that Osx could act via this non-canonical Sp1/Sp3-binding element to regulate OCN transcription. OCN promoter luciferase reporter assays show that Osx alone is an insufficient activator that requires Sp1, but not Sp3, to synergistically stimulate OCN promoter activity. Moreover, promoter deletion analyses demonstrate that both the Sp1/Sp3-binding OCN-CxRE (−70 to −57) and the −92 to −87 region of the OCN proximal promoter are critical for Osx/Sp1 synergistic activities. Our data show that Sp1 influences Osx activity by enhancing Osx occupancy on the OCN promoter, perhaps via physical interactions between the two transcription factors. Finally, alteration of the expression of the gap junction protein connexin43 modulates the recruitment of both Sp1 and Osx to the OCN promoter. In total, our data are strongly in support of Sp1 as an essential transcription factor required for Osx recruitment and transactivation of the OCN promoter. Further, these data lend insight into a mechanism by which alteration of connexin43 impacts osteogenesis in vitro and in vivo.

Connexin43 (Cx43) plays a critical role in osteoblast function and bone mass accrual, yet the identity of the second messengers communicated by Cx43 gap junctions, the targets of these second messengers and how they regulate osteoblast function remain largely unknown. We have shown that alterations of Cx43 expression in osteoblasts can impact the responsiveness to fibroblast growth factor-2 (FGF2), by modulating the transcriptional activity of Runx2. In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/PKCδ cascade to the Cx43-dependent transcriptional response of MC3T3 osteoblasts to FGF2. Knockdown of expression and/or inhibition of function of phospholipase Cγ1, inositol polyphosphate multikinase, which generates InsP4 and InsP5, and inositol hexakisphosphate kinase 1/2, which generates inositol pyrophosphates, prevented the ability of Cx43 to potentiate FGF2-induced signaling through Runx2. Conversely, overexpression of phospholipase Cγ1 and inositol hexakisphosphate kinase 1/2 enhanced FGF2 activation of Runx2 and the effect of Cx43 overexpression on this response. Disruption of these pathways blocked the nuclear accumulation of PKCδ and the FGF2-dependent interaction of PKCδ and Runx2, reducing Runx2 transcriptional activity. These data reveal that FGF2-signaling involves the inositol polyphosphate cascade, including IP6K, and demonstrate that IP6K regulates Runx2 and osteoblast gene expression. Additionally, these data implicate the water-soluble inositol polyphosphates as mediators of the Cx43-dependent amplification of the osteoblast response to FGF2, and suggest that these low molecular weight second messengers may be biologically relevant mediators of osteoblast function that are communicated by Cx43-gap junctions.

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.