Exendin-4, a glucagon-like peptide receptor agonist, facilitates osteoblast differentiation via connexin43

Purpose: To investigate whether exendin-4 (Ex-4), a glucagon-like peptide 1 receptor (GLP-1R) agonist, affects connexin 43 (Cx43) expression in osteoblasts, and determine the specific mechanism underlying Cx43 modulation by Ex-4. Methods: Osteoblast-like MC3T3-E1 cells were treated with Ex-4 with or without GLP-1R antagonist. We assessed Cx43 expression using RT-PCR, western blotting, and confocal microscopy; visualized intercellular communication using Lucifer yellow dye transfer assay; evaluated osteoblast differentiation using alkaline phosphatase and Alizarin red S (ARS) staining. Cx43 silencing or overexpression was investigated via RNA-interference or adenovirus infection. The mechanism underlying Cx43 regulation by Ex-4 was determined via treatment with either Src kinase inhibitor, KX2–391, Akt activator, sc79, or inhibitor, LY294002. Results: Ex-4 treatment enhanced Cx43 expression and gap junctional intercellular communication in MC3T3-E1 cells. GLP-1R antagonist pretreatment abrogated the induction of Cx43 expression. Cx43 silencing significantly decreased ARS staining intensity in Ex-4-treated cells, whereas overexpression enhanced cell differentiation. Treatment with KX2–391 reduced both the Ex-4-induced increase of Cx43 expression and p-Akt protein levels. sc79 upregulated Cx43 expression, while LY294002 attenuated Cx43 upregulation by Ex-4. Conclusions: Induced Cx43 expression in osteoblasts via the Src-Akt signaling pathway illustrates the underlying mechanism for promoting osteoblast differentiation by Ex-4.