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

Abstract

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 microCT 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, microCT 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.

Connexin 43 Modulates Osteogenic Differentiation of Bone Marrow Stromal Cells Through GSK-3beta/Beta-Catenin Signaling Pathways

“…The activity of GSK-3beta can be inhibited by phosphorylation at Ser-9 [61]. Furthermore, the impact of Cx43 on beta-catenin activity and GSK are also well established [56, 57, 62], whereas the mechanism by which the defects in Cx43 cause skeletal development deficiency has not been shown [17]. Our research involves the roles of Cx43 in skeletal development.…”

Parathyroid-Targeted Overexpression of Regulator of G-Protein Signaling 5 (RGS5) Causes Hyperparathyroidism in Transgenic Mice

“…Assessment of mouse bone morphology by μCT was performed and are reported according to guidelines published in Bouxsein and colleagues . Femurs from 12‐month‐old male and female mice were scanned at a 10 μm voxel size, using a Skyscan 1172 system (Bruker MicroCT, Kontich, Belgium), as described previously . The following settings were applied: X‐ray power and tube current were 60 kV and 0.167 mA, respectively.…”

“…Final images were generated from an average of 10 pictures taken at 0.9° angles. Bone microarchitectural patterns were assessed in the cortical and trabecular regions of femurs in all mice using Skyscan software packages (NRecon, CtAn, and Dataviewer), as described previously …”

“…(21) Femurs from 12-month-old male and female mice were scanned at a 10 mm voxel size, using a Skyscan 1172 system (Bruker MicroCT, Kontich, Belgium), as described previously. (22,23) The following settings were applied: X-ray power and tube current were 60 kV and 0.167 mA, respectively. Beam hardening (20%) was reduced with a 0.5 mm aluminum filter, ring artifact reduction was set at 5, and exposure time was 5.9 seconds.…”

Diminished Canonical β-Catenin Signaling During Osteoblast Differentiation Contributes to Osteopenia in Progeria

“…Femurs were dissected from 8‐week‐old male Zmpste24+/+ and Zmpste24‐/‐ mice and fixed in 4% PFA for 2 to 4 days, then transferred to 70% ethanol. Three‐dimensional micro‐CT was performed on the femurs of each genotype using SkyScan 1172 (Bruker, Kontich, Belgium), as described . The skeletal parameters assessed by micro‐CT followed published nomenclature guidelines .…”