In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKC␦) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKC␦ translocates to the nucleus, PKC␦ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKC␦ and Runx2 function. INTRODUCTIONBone formation and remodeling is a tightly organized and dynamic process that requires the coordinated action of osteoblasts, osteocytes, and osteoclasts to maintain bone homeostasis. It is hypothesized that osteoblasts and osteocytes coordinate their activities, at least in part, through direct cell-to-cell communication through gap junctions. Gap junctions are composed of connexins, a family of transmembrane proteins that constitute the intercellular gap junction channels (Beyer et al., 1990). Six connexins assemble to make up the gap junction hemichannel on the plasma membrane of one cell, which docks with a hemichannel on an adjacent cell to form an aqueous gap junction channel. The resultant gap junctions provide direct conduits for the passage of ions and other low molecular weight molecules, including second messengers, among cells.The gap junction protein connexin43 (Cx43) is abundantly expressed in both osteoblasts and osteocytes, where it has been hypothesized to transmit hormonal signals, mechanical load, and growth factor cues among cells in order to coordinate the synthesis of new bone (reviewed in Stains and Civitelli, 2005a;Jiang et al., 2007). Genetic ablation of gja1, the gene encoding Cx43, in mice leads to a severe delay in the ossification of both intramembranous and endochondral derived skeletal elements during embryonic development (Lecanda et al., 2000). The bones of these animals are remarkably brittle, and the osteoblasts isolated from the Cx43 null animals are dysfunctional, with reduced osteogenic and mineralizing capacity (Lecanda et al., 2000). These Cx43-deficient mice die at birth because of a defect in cardiac f...

Background: HMGA2 expression has been shown to be associated with enhanced selective chemosensitivity towards the topoisomerase (topo) II inhibitor, doxorubicin, in cancer cells. Although the roles of signaling cascades and proteins as regulatory factors in development, neoplasia and adaptation to the environment are becoming well established, evidence for the involvement of regulatory small RNA molecules, such as microRNAs (miRNAs) as important regulators of both transcriptional and posttranscriptional gene silencing is presently mounting.

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...

In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1 +/− controls, male Gja1 −/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition.

Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.

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.

Hsp47, Grp78, and Grp94 have been implicated with procollagen maturation events. In particular, Hsp47 has been shown to nascent procollagen alpha 1(I) chains in the course of synthesis and/or translocation into the endoplasmic reticulum (ER). Although, Hsp47 binding to gelatin and collagen has previously been suggested to be independent of ATP. Grp78 and Grp94 are known to dissociate from its substrates by an ATP-dependent release mechanism. The early association of Hsp47 with procollagen and its relatively late release suggested that other chaperones, Grp78 and Grp94, interact successively or concurrently with Hsp47. Herein, we examined how these events occur in cells metabolically stressed by depletion of ATP. In cells depleted of ATP, the release of Hsp47, Grp78, and Grp94 from maturing procollagen is delayed. Thus, in cells experiencing metabolic stress, newly synthesized procollagen unable to properly fold became stably bound to a complex of molecular chaperones. In that Hsp47, Grp78, and Grp94 could be recovered with nascent procollagen and as oligomers in ATP depleted cells suggests that these chaperones function in a series of coupled or successive reactions.