Cross-Species RNA-Seq Study Comparing Transcriptomes of Enriched Osteocyte Populations in the Tibia and Skull.

Local site-specific differences between bones in different regions of the skeleton account for their different properties and functions. To identify mechanisms behind these differences, we have performed a cross-species study comparing RNA transcriptomes of cranial and tibial osteocytes, from bones with very different primary functions and physiological responses, collected from the same individual mouse, rat, and rhesus macaque. Bioinformatic analysis was performed to identify 32 genes changed in the same direction between sites and shared across all three species. Several well-established key genes in bone growth and remodeling were upregulated in the tibias of all three species (BMP7, DKK1, FGF1, FRZB, SOST). Many of them associate or crosstalk with the Wnt signaling pathway. These results suggest Wnt signaling-related candidates for different control of regulatory mechanisms in bone homeostasis in the skull and tibia and indicate a different balance between genetically determined structure and feedback mechanisms to strains induced by mechanical loading at the different sites.

Connexin43 Mediated Delivery of ADAMTS5 Targeting siRNAs from Mesenchymal Stem Cells to Synovial Fibroblasts.

Osteoarthritis is a joint-destructive disease that has no effective cure. Human mesenchymal stem cells (hMSCs) could offer therapeutic benefit in the treatment of arthritic diseases by suppressing inflammation and permitting tissue regeneration, but first these cells must overcome the catabolic environment of the diseased joint. Likewise, gene therapy also offers therapeutic promise given its ability to directly modulate key catabolic factors that mediate joint deterioration, although it too has limitations. In the current study, we explore an approach that combines hMSCs and gene therapy. Specifically, we test the use of hMSC as a vehicle to deliver ADAMTS5 (an aggrecanase with a key role in osteoarthritis)-targeting siRNAs to SW982 synovial fibroblast-like cells via connexin43 containing gap junctions. Accordingly, we transduced hMSCs with ADAMTS5-targeting shRNA or non-targeted shRNA, and co-cultured them with synovial fibroblasts to allow delivery of siRNAs from hMSC to synovial fibroblasts. We found that co-culture of hMSCs-shRNA-ADAMTS5 and synovial fibroblasts reduced ADAMTS5 expression relative to co-culture of hMSCs-shRNA-control and synovial fibroblasts. Furthermore, ADAMTS5 was specifically reduced in the synovial fibroblasts populations as determined by fluorescence-activated cell sorting, suggesting transfer of the siRNA between cells. To test if Cx43-containing gap junctions are involved in the transfer of siRNA, we co-cultured hMSCs-shRNA-ADAMTS5 cells with synovial fibroblasts in which connexin43 was knocked down. Under these conditions, ADAMTS5 levels were not inhibited by co-culture, indicating that connexin43 mediates the delivery of siRNA from hMSCs to synovial fibroblasts. In total, our findings demonstrate that hMSCs can function as donor cells to host and deliver siRNAs to synovial fibroblasts via connexin43 gap junction in vitro. These data may have implications in the combination of hMSCs and gene therapy to treat diseases like osteoarthritis, in vivo.

Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture.

BACKGROUND: Recent work has shown that the gap junction protein connexin43 (Cx43) is upregulated in cells of the joint during osteoarthritis (OA). Here we examined if the OA-associated increase in Cx43 expression impacts the function of synovial fibroblasts by contributing to the production of catabolic and inflammatory factors that exacerbate joint destruction in arthritic disease. METHODS: Using rabbit and human synovial fibroblast cell lines, we examined the effects of Cx43 overexpression and Cx43 siRNA-mediated knockdown on the gene expression of OA-associated matrix metalloproteinases (MMP1 and MMP13), aggrecanases (ADAMTS4 and ADAMTS5), and inflammatory factors (IL1, IL6 and PTGS2) by quantitative real time RT-PCR. We examined collagenase activity in conditioned media of cultured synovial cells following Cx43 overexpression. Lastly, we assessed the interplay between Cx43 and the NFκB cascade by western blotting and gene expression studies. RESULTS: Increasing Cx43 expression enhanced the gene expression of MMP1, MMP13, ADAMTS4, ADAMTS5, IL1, IL6 and PTGS2 and increased the secretion of collagenases into conditioned media of cultured synovial fibroblasts. Conversely, knockdown of Cx43 decreased expression of many of these catabolic and inflammatory genes. Modulation of Cx43 expression altered the phosphorylation of the NFκB subunit, p65, and inhibition of NFκB with chemical inhibitors blocked the effects of increased Cx43 expression on the mRNA levels of a subset of these catabolic and inflammatory genes. CONCLUSIONS: Increasing or decreasing Cx43 expression alone was sufficient to alter the levels of catabolic and inflammatory genes expressed by synovial cells. The NFκB cascade mediated the effect of Cx43 on the expression of a subset of these OA-associated genes. As such, Cx43 may be involved in joint pathology during OA, and targeting Cx43 expression or function may be a viable therapeutic strategy to attenuate the catabolic and inflammatory environment of the joint during OA.

Induction of chondrogenic differentiation in mesenchymal stem cells by TGF-beta cross-linked to collagen-PLLA [poly(L-lactic acid)] scaffold by transglutaminase 2.

Transglutaminase-mediated cross-linking has been employed to optimize the mechanical properties and stability of tissue scaffolds. We have characterized tissue transglutaminase (TG2)-mediated cross-linking as a useful tool to deliver biologically-active TGF to mesenchymal stem cells (MSCs) and direct their differentiation towards a chondrogenic lineage. TGF-ß3 is irreversibly cross-linked by TG2 to collagen type II-coated poly(L-lactic acid) nanofibrous scaffolds and activates Smad phosphorylation and Smad-dependent expression of a luciferase reporter. Human bone marrow-derived MSCs cultured on these scaffolds deposit cartilaginous matrix after 14 days of culture at 50 % efficiency compared to chondrogenesis in the presence of soluble TGF-ß3. These findings are significant because they suggest a novel approach for the programming of MSCs in a spatially controlled manner by immobilizing biologically active TGF-ß3 via cross-linking to a collagen-coated polymeric scaffold.

The regulation of runt-related transcription factor 2 by fibroblast growth factor-2 and connexin43 requires the inositol polyphosphate/protein kinase Cδ cascade.

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 runt-related transcription factor 2 (Runx2). In this study, we examined the contribution of the phospholipase Cγ1/inositol polyphosphate/protein kinase C delta (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 inositol 1,3,4,5-tetrakisphosphate (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 inositol hexakisphosphate kinase (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.

Implication for transglutaminase 2-mediated activation of ß-catenin signaling in neointimal vascular smooth muscle cells in chronic cardiac allograft rejection.

BACKGROUND: Cardiac allograft vasculopathy (CAV) remains the main cause of long-term transplant rejection. CAV is characterized by hyperproliferation of vascular smooth muscle cells (VSMCs). Canonical ß-catenin signaling is a critical regulator of VSMC proliferation in development; however, the role of this pathway and its regulation in CAV progression are obscure. We investigated the activity of ß-catenin signaling and the role for a putative activating ligand, transglutaminase 2 (TG2), in chronic cardiac rejection. METHODS: Hearts from Bm12 mice were transplanted into C57BL/6 mice (class II mismatch), and allografts were harvested 8 weeks after transplantation. Accumulation and sub-cellular distribution of ß-catenin protein and expression of several components of ß-catenin signaling were analyzed as hallmarks of pathway activation. In vitro, platelet-derived growth factor treatment was used to mimic the inflammatory milieu in VSMC and organotypic heart slice cultures. RESULTS: Activation of ß-catenin in allografts compared with isografts or naïve hearts was evidenced by the augmented expression of ß-catenin target genes, as well as the accumulation and nuclear localization of the ß-catenin protein in VSMCs of the occluded allograft vessels. Expression of TG2, an activator of ß-catenin signaling in VSMCs, was dramatically increased in allografts. Further, our ex vivo data demonstrate that TG2 is required for VSMC proliferation and for ß-catenin activation by platelet-derived growth factor in cardiac tissue. CONCLUSIONS: ß-Catenin signaling is activated in occluded vessels in murine cardiac allografts. TG2 is implicated as an endogenous activator of this signaling pathway and may therefore have a role in the pathogenesis of CAV during chronic allograft rejection.

ERK acts in parallel to PKCδ to mediate the connexin43-dependent potentiation of Runx2 activity by FGF2 in MC3T3 osteoblasts.

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-δ (PKCδ) activation to affect Runx2 activity. In the present study, we show by luciferase reporter assays that the ERK signaling cascade acts in parallel to PKCδ to modulate Runx2 activity downstream of the Cx43-dependent amplification of FGF2 signaling. The PKCδ-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 PKCδ, 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 18ß-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 PKCδ pathways in the coordination of the osteoblast response to FGF2 among populations of osteoblasts.

The transcriptional activity of osterix requires the recruitment of Sp1 to the osteocalcin proximal promoter.

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.

Induction of an osteocyte-like phenotype by fibroblast growth factor-2.

The purpose of this study was to characterize the molecular phenotype that occurs during the profound morphological shift of cultured osteogenic cells upon treatment with fibroblast growth factor-2 (FGF2). A time course of treatment with FGF2 was performed on an osteoblast cell line, primary bone marrow stromal cells and an osteocyte-like cell line. Morphologic changes were recorded, and gene profiling was carried out by real time PCR. By 8h of FGF2 treatment, there is a striking morphological shift of osteoblast and stromal cells to an elongated dendritic-like morphology that is remindful of osteocytes. In osteoblasts treated with FGF2, this morphologic shift is preceded by an induction of several osteocyte markers, including dentin matrix protein 1 (>20-fold) and E11 (>5-fold). There is a transient increase in the gene expression of sclerostin (3.5-fold) and PHEX (2.5-fold). Sclerostin regulation by FGF2 is complex, as gene expression becomes markedly inhibited by FGF2 at times points after 8h of treatment before rebounding at day 12. Analogous modulation of osteocyte markers is seen in bone marrow stromal cells and MLO-Y4 osteocyte-like cells. In conclusion, this study shows that FGF2 can regulate the transition of osteogenic cells towards the osteocyte lineage, as well as, regulate the expression of critical genes in osteocytes.

Effect of endothelin-1 on osteoblastic differentiation is modified by the level of connexin43: comparative study on calvarial osteoblastic cells isolated from Cx43+/- and Cx43+/+ mice.

Bone is a dynamic tissue that undergoes a precise remodeling process involving resorptive osteoclastic cells and bone-forming osteoblastic (OB) cells. The functional imbalance of either of these cell types can lead to severe skeletal diseases. The proliferation and differentiation of OB cells play a major role in bone development and turnover. These cellular processes are coordinated by connexin43 (Cx43)-based gap-junctional intercellular communication (GJIC) and by soluble factors such as endothelin-1 (ET-1). We have used the Cx43 heterozygous (Cx43(+/-)) murine model to study the possible cross-talk between Cx43 and ET-1 in cultured calvarial OB cells. On microcomputed tomographic analysis of 3-day-old pups, Cx43(+/-) mice showed hypomineralized calvaria in comparison with their Cx43(+/+) littermates. Characterization of cultured OB cells clearly demonstrated the effect of the partial deletion of the Cx43 gene on its expression, on GJIC, and subsequently on OB differentiation. In this model, ET-1 (10(-8) M) lost its mitogenic action in Cx43(+/-) OB cells compared with Cx43(+/+) cells. Moreover, a correlation between the inhibition of cell differentiation by ET-1 and the decreased amount and function of Cx43 was found in Cx43(+/+) OB cells but not in their Cx43(+/-) counterparts. Thus, as Cx43 is linked to OB differentiation, our data indicate that this mitogenic ET-1 peptide has pronounced effects on fully differentiated OB cells. With respect to roles in mechanotransduction and OB differentiation, Cx43 might modulate osteoblastic sensitivity to soluble factors.

Interaction of connexin43 and protein kinase C-delta during FGF2 signaling.

BACKGROUND: We have recently demonstrated that modulation of the gap junction protein, connexin43, can affect the response of osteoblasts to fibroblast growth factor 2 in a protein kinase C-delta-dependent manner. Others have shown that the C-terminal tail of connexin43 serves as a docking platform for signaling complexes. It is unknown whether protein kinase C-delta can physically interact with connexin43. RESULTS: In the present study, we investigate by immunofluorescent co-detection and biochemical examination the interaction between Cx43 and protein kinase C-delta. We establish that protein kinase C-delta physically interacts with connexin43 during fibroblast growth factor 2 signaling, and that protein kinase C delta preferentially co-precipitates phosphorylated connexin43. Further, we show by pull down assay that protein kinase C-delta associates with the C-terminal tail of connexin43. CONCLUSIONS: Connexin43 can serve as a direct docking platform for the recruitment of protein kinase C-delta in order to affect fibroblast growth factor 2 signaling in osteoblasts. These data expand the list of signal molecules that assemble on the connexin43 C-terminal tail and provide a critical context to understand how gap junctions modify signal transduction cascades in order to impact cell function.

Interleukin-1beta increases gap junctional communication among synovial fibroblasts via the extracellular-signal-regulated kinase pathway.

BACKGROUND INFORMATION: The gap junction protein, Cx43 (connexin 43), has been implicated in the aetiology of osteoarthritis. Studies have revealed that the size and number of gap junctions increase in synovial biopsies from patients with osteoarthritis. Furthermore, pharmacological inhibition of Cx43 function has been shown to reduce IL-1beta (interleukin-1beta)-induced metalloproteinase production by synovial fibroblasts in vitro. RESULTS: In the present study, we examined the link between IL-1beta and Cx43 function. We demonstrated that treatment of a rabbit synovial fibroblast cell line with IL-1beta markedly increased the level of the Cx43 protein in a concentration- and time-dependent manner. The impact on Cx43 protein levels appeared to occur post-transcriptionally, as mRNA levels are unaffected by IL-1beta administration. Additionally, we showed by fluorescence microscopy that IL-1beta alters the cellular distribution of Cx43 to cell-cell junctions and is concomitant with a striking increase in gap junction communication. Furthermore, we demonstrated that the increase in Cx43 protein, and the associated change in protein localization and gap junction communication following IL-1beta treatment, are dependent upon activation of the ERK (extracellular-signal-regulated kinase) signalling cascade. CONCLUSION: These data show that IL-1beta acts through the ERK signalling cascade to alter the expression and function of Cx43 in synovial fibroblasts.

Connexin43 potentiates osteoblast responsiveness to fibroblast growth factor 2 via a protein kinase C-delta/Runx2-dependent mechanism.

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 (PKCdelta) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCdelta translocates to the nucleus, PKCdelta 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 PKCdelta and Runx2 function.

Endothelin-1 inhibits human osteoblastic cell differentiation: influence of connexin-43 expression level.

Gap junctional intercellular communication (GJIC) permits coordinated cellular activities during developmental and differentiation processes. In bone, the involvement of the gap junctional protein, connexin-43 (Cx43), and of GJIC in osteoblastic differentiation and mineralization of the extracellular matrix has been previously demonstrated. Former studies have shown that endothelin-1 (ET-1) was also implicated in the control of osteoblastic proliferation and differentiation. However, depending on the cellular models, ET-1 has been shown to decrease or increase osteoblastic differentiation markers. As no data were available on the ET-1 effect on GJIC and Cx43 expression in osteoblastic cells, we analyzed here the possible crosstalk between Cx43 and ET-1 in a human cell line (hFOB 1.19) which displays different Cx43 expression levels and phenotypes when cultured at 33.5 or 39 degrees C. The presence of ET-1 (10(-8) M) for 2-12 days of culture did not significantly alter the proliferation rate of hFOB cells whatever their phenotype. In contrast, ET-1 induced a differential inhibitory effect on the biochemical differentiation markers (alkaline phosphatase activity and osteocalcin expression) with a significant reduction in the differentiated phenotype at 39 degrees C, whereas no effects were measured at 33.5 degrees C. The inhibitory effect was linked to a decrease of GJIC and of Cx43 both at transcriptional and protein levels. Altogether, our results suggest that Cx43 expression level could influence the action of ET-1 on human osteoblastic cell differentiation. Our data also indicate that the gap junctional protein could play a pivotal role in the response of osteoblasts to mitogenic factors implicated in bone pathologies.