• Connexins as Active Regulators of Signal Transduction in Bone

      Moorer, Megan Carmell; Stains, Joseph P.; 0000-0001-6581-3502 (2018)
      Intercellular communication by gap junctions plays an important role in achieving peak bone mass and determining bone quality. Deletion of the gap junction protein connexin43 (Cx43) in mice impairs bone quality (osteopenia), and results in a cortical structure resembling aging and disuse. Although it is clear that Cx43 plays a role in osteoblast function and bone mineralization, the molecular mechanism underlying the specific roles of Cx43 are not well defined. Contrastingly, osteoblasts also express connexin45 (Cx45) and its role in osteoblasts has not been extensively studied and nothing is known about its role in skeletal tissue in vivo. Cx43 and Cx45 form gap junctions with different molecular permeability, or pore sizes (<1kD <0.3kD) and seem to have opposing functions in bone biology. While gap junctions are generally thought of as passive conduits for small molecules to be shared between cells, growing evidence indicates that connexins actively contribute to downstream signaling. Modulation of Cx43 expression impacts PKCδ, ERK1/2 and β-catenin signaling pathways in vitro, which influence osteoblast gene expression and function. The regulation of signal transduction downstream of intercellular communication may involve assembly of a specific subset of effectors to the gap junction. The work presented in this thesis examines the influence of the C-terminus (CT) of connexins on osteoblast signaling and function through physical interactions with effectors like β-catenin, ERK(1/2), CREB, and PKCδ, both in vivo and in vitro. The central hypothesis of this work is that in addition to having distinct molecular permeability, each connexin can assemble a unique "interactome" of locally recruited signaling machinery that can affect downstream signaling from the gap junction and ultimately, bone cell function. We characterized the skeletal phenotype of a Cx43 truncation mouse model. The absence of the Cx43 CT in mice resulted in an osteopenic skeletal phenotype analogous to osteoblast conditional deletion of the entire Cx43 gene including cortical thinning and increased cross-sectional area, defective signaling, reduced gene expression, and altered collagen processing. These data imply that Cx43-containing gap junctions not only exchange signals, but also recruit signaling machinery to the Cx43 CT domain to optimally affect cell signaling, cell function, and bone acquisition. We also determined the requirement for both pore permeability (pore function) and signal effector protein recruitment (tail function) to Cx43 and Cx45 in osteoblast signaling and function using chimeric constructs composed of portions of Cx43 and Cx45. By luciferase reporter assays, western blot analysis, and qPCR, we found that, in general, both the Cx43 pore and Cx43 tail are required for optimal osteoblast signaling and gene expression, as Cx45 overexpression was ineffective or inhibitory in all aspects, while Cx43 overexpression stimulated signaling and gene expression. The chimeras largely mimicked Cx45 or had an intermediate effect. We also further defined the interactomes of Cx43 and Cx45 to determine their requirement for affecting osteoblast function. Through co-immunoprecipitation, our data shows binding of ERK(1/2), CAMKII, and β-catenin to Cx43 but not to Cx45, which is consistent with the idea of differential interactomes. In total, these data imply that each connexin can differentially regulate downstream signaling and gene expression from the gap junction by local recruitment of different signaling effector molecules to each connexin's CT in order to affect bone cell function and bone modeling and remodeling.
    • Cyclic AMP (cAMP) as a Putative Second Messenger Communicated by Gap Junctions in Bone

      Anderson, Hidayah M.; Stains, Joseph P. (2014)
      Skeletal homeostasis is crucial to maintaining bone quality. In the absence of balanced bone formation and bone resorption, skeletal disease develops. Numerous studies show that the gap junction protein connexin43 (Cx43) plays a role in the function of bone forming osteoblasts and osteocytes, as well as the coordination of bone resorption by these cells. Little is known about how Cx43 influence skeletal homeostasis. In particular, the identities of the second messenger signals that are communicated by bone cells to regulate skeletal homeostasis are unclear. Here, we assessed the role of cAMP as a biologically relevant second messenger communicated by Cx43 containing gap junctions among bone cells and its regulation of genes involved in skeletal homeostasis, including sclerostin and RANKL in UMR106- cells. Methods: UMR106-cells were cultured and transfected with different plasmid constructs to manipulate cAMP levels (constitutively active GalphaS), as well as Cx43 expression. Luciferase reporter assays and western blots were used to assess cAMP-dependent signaling. Regulation of the SOST and RANKL were assessed by quantitative RT-PCR. Results: The in vitro studies conducted have shown the synergism between Cx43 and G-alphaS, which generates cAMP, confirming the amplified signal when these two were expressed together. Further, a modified parachute assay revealed that cell-to-cell contact was required for the sharing of cAMP-dependent signals by Cx43 expressing bone cells. Cx43 and cAMP combine to potently increase RANKL (a stimulator of bone resorption) expression and suppress SOST expression (An inhibitor of bone formation). Conclusion: There was up regulated signals when Cx43 and G-alphaS were co-expressed implying the role of the second messenger cAMP in cell-to-cell communication by bone cells. In addition, these signals converged on the expression of RANKL and sclerostin, two genes that play a major role in bone turnover. Accordingly, this pathway may represent one of the ways that gap junctions regulate skeletal homeostasis and could be a target of therapeutic intervention for skeletal disease.
    • The intersecting function of Connexin 43 and Runx2 in bone

      Buo, Atum Michael Lionel; Stains, Joseph P.; 0000-0001-9610-2623 (2017)
      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.
    • The role of the phospholipase C/inositol phosphate pathway in cell-cell communication between osteoblasts

      Luciotti, Maria; Stains, Joseph P. (2010)
      Osteoblasts are mesenchymal-derived cells within bone responsible for bone formation. During the highly regulated process of bone remodeling, osteoblasts are simultaneously activated in local areas undergoing the bone formative phase. Direct cell-to-cell communication between osteoblasts is facilitated by gap junctions, protein-based transcellular channels that allow the diffusion of ions and small signaling molecules. Osteocalcin is the most osteoblast specific gene whose promoter is positively regulated by the transcription factor Runx2, a master regulator of osteoblast differentiation. Fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling activates the phospholipase C (PLC) cascade resulting in inositol phosphate production, and the FGF2-dependent transcription of Runx2 that is mediated by protein kinase C-delta (PKCδ). The goal of this thesis is to investigate the role of the phospholipase C/inositol phosphate pathway in the Cx43-gap junction mediated amplification of cell responsiveness to FGF2 and to gain insight on the identity of the second messenger signaling molecule. We hypothesize that inositol phosphates are key second messengers that diffuse through gap junctions and mediate the synergistic activation of Runx2 transcription among gap junction coupled osteoblasts.