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Title: The intersecting function of Connexin 43 and Runx2 in bone
Buo_umaryland_0373D_10887.pdf  (4.63 MB)
Authors: Buo, Atum Michael Lionel
Advisors: Stains, Joseph P.
Date: 2017
Abstract: Bone is formed and maintained through the tightly coordinated activities of osteoblasts, osteoclasts, and osteocytes. Connexin 43 (Cx43), the most predominantly expressed gap junction protein in bone, facilitates the coordination of bone cell function via the gap junction-mediated sharing of second messengers throughout the osteoblast-osteocyte network. However, the underlying mechanistic details explaining how Cx43 converts shared second messengers into signals that induce essential osteogenic processes are largely unclear. Therefore, this work investigates whether Cx43 impacts osteoblast differentiation and bone quality by regulating Runx2, the master transcriptional regulator of osteogenesis. To assess this in vitro, we devised a reproducible Cre/LoxP-based system to delete the Cx43 gene (Gja1) from murine primary osteoblasts, and hypothesized that adenoviral overexpression of Runx2 in these cells would be able to rescue the defective osteogenesis caused by loss of Cx43. To assess this in vivo, we used a compound hemizygous breeding strategy to generate mice that are doubly hemizygous for the Cx43 gene (Gja1) and the Runx2 gene (Cx43+/- Runx2+/-), and then utilized micro-CT scanning on femurs and skulls to analyze the skeletal phenotype at 8 weeks of age. We hypothesized that if Cx43 and Runx2 indeed functionally intersect to regulate osteogenesis in vivo, then the dual hemizygosity of both Cx43 and Runx2 should manifest a skeletal phenotype not visible in wild-type or singly hemizygous animals. Our findings reveal that overexpressing Runx2 in Cx43-depleted cells rescues osteoblast differentiation by restoring osteocalcin gene expression and reducing proliferation. Additionally, cortical bones of compound Gja1+/- Runx2+/- mice, in comparison to wildtype and singly Gja1+/- and Runx2+/- littermate controls, have a marked increase in tissue area, a widened marrow cavity due to increased bone resorption, and a striking increase in porosity. Furthermore, the compound mice display cranial defects not observable in the other genotypes, and the calvarial osteoblasts from the compound mice also possess a hyperproliferative defect. Together, these findings strongly indicate that Cx43 and Runx2 functionally intersect to regulate osteogenesis in vitro and in vivo and provide greater insight towards understanding how Cx43 impacts bone quality.
Subject Keywords: connexin
Bone and Bones
Gap Junctions
Description: University of Maryland, Baltimore. Molecular Medicine. Ph.D. 2017
Type: dissertation
Appears in Collections:Theses and Dissertations Graduate School
Theses and Dissertations School of Medicine

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