Connexin43 and Runx2 Interact to Affect Cortical Bone Geometry, Skeletal Development, and Osteoblast and Osteoclast Function.

The coupling of osteoblasts and osteocytes by connexin43 (Cx43) gap junctions permits the sharing of second messengers that coordinate bone cell function and cortical bone acquisition. However, details of how Cx43 converts shared second messengers into signals that converge onto essential osteogenic processes are incomplete. Here, we use in vitro and in vivo methods to show that Cx43 and Runx2 functionally interact to regulate osteoblast gene expression and proliferation, ultimately affecting cortical bone properties. Using compound hemizygous mice for the Gja1 (Cx43) and Runx2 genes, we observed a skeletal phenotype not visible in wild-type or singly hemizygous animals. Cortical bone analysis by micro-computed tomography (µCT) revealed that 8-week-old male, compound Gja1+/- Runx2+/- mice have a marked increase in cross-sectional area, endosteal and periosteal bone perimeter, and an increase in porosity compared to controls. These compound Gja1+/- Runx2+/- mice closely approximate the cortical bone phenotypes seen in osteoblast-specific Gja1-conditional knockout models. Furthermore, µCT analysis of skulls revealed an altered interparietal bone geometry in compound hemizygotes. Consistent with this finding, Alizarin red/Alcian blue staining of 2-day-old Gja1+/- Runx2+/- neonates showed a hypomorphic interparietal bone, an exacerbation of the open fontanelles, and a further reduction in the hypoplastic clavicles compared to Runx2+/- neonates. Expression of osteoblast genes, including osteocalcin, osterix, periostin, and Hsp47, was markedly reduced in tibial RNA extracts from compound hemizygous mice, and osteoblasts from compound hemizygous mice exhibited increased proliferative capacity. Further, the reduced osteocalcin expression and hyperproliferative nature of osteoblasts from Cx43 deficient mice was rescued by Runx2 expression. In summary, these findings provide evidence that Cx43 and Runx2 functionally intersect in vivo to regulate cortical bone properties and affect osteoblast differentiation and proliferation, and likely contributes to aspects of the skeletal phenotype of Cx43 conditional knockout mice. © 2017 American Society for Bone and Mineral Research.

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.