The role of the Smad2/3/4 signaling pathway in osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells

Abstract

Background: ClC-3 chloride channels promote osteogenic differentiation. Transforming growth factor-β1 (TGF-β1) and its receptors are closely related to ClC-3 chloride channels, and canonical TGF-β1 signaling is largely mediated by Smad proteins. The current study aimed to explore the role of the Smad2/3/4 signaling pathway in the mechanism by which ClC-3 chloride channels regulate osteogenic differentiation in osteoblasts. Methods: First, real-time PCR and western blotting were used to detect the expression of Smad and mitogen-activated protein kinase (MAPK) proteins in response to ClC-3 chloride channels. Second, immunocytochemistry, coimmunoprecipitation (Co-IP) and immunofluorescence analyses were conducted to assess formation of the Smad2/3/4 complex and its translocation to the nucleus. Finally, markers of osteogenic differentiation were determined by real-time PCR, western blotting, ALP assays and Alizarin Red S staining. Results: ClC-3 chloride channels knockdown led to increased expression of Smad2/3 but no significant change in p38 or Erk1/2. Furthermore, ClC-3 chloride channels knockdown resulted in increases in the formation of the Smad2/3/4 complex and its translocation to the nucleus. In contrast, the inhibition of TGF-β1 receptors decreased the expression of Smad2, Smad3, p38, and Erk1/2 and the formation of the Smad2/3/4 complex. Finally, the expression of osteogenesis-related markers were decreased upon ClC-3 and Smad2/3/4 knockdown, but the degree to which these parameters were altered was decreased upon the knockdown of ClC-3 and Smad2/3/4 together compared to independent knockdown of ClC-3 or Smad2/3/4. Conclusions: The Smad2/3 proteins respond to changes in ClC-3 chloride channels. The Smad2/3/4 signaling pathway inhibits osteogenic differentiation regulation by ClC-3 chloride channels in MC3T3-E1 cells.