Transforming Growth Factor-β3-Mediated Regulation of Junctional Adhesion Molecule-B (JAM-B) in Testicular Cells

Abstract

Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells as well as between Sertoli and germ cells in the testis. The expression of JAM-B is highly regulated to facilitate the passage of developing germ cells across the blood-testis barrier as well as the release of mature spermatids. Transforming growth factor beta (TGF-β) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the influence of TGF-β3 on the expression of JAM-B as well as the underlying mechanisms. TGF-β3 (5 ng/ml) treatment coupled with RT-PCR and immunoblot analyses have shown that TGF-β3 down-regulates JAM-B expression on mRNA and protein levels in a timedependent manner in mouse Sertoli cell line, MSC-1 cells. Cycloheximide assay further indicates that the reduction of JAM-B protein by TGF-β3 is mediated via post-translational modification. Moreover, the involvement of ubiquitin-proteasome pathway in TGF-β3-mediated JAM-B protein destabilization was demonstrated by proteasome inhibitor, MG-132, treatment and ubiquitin siRNA knockdown assays. Furthermore, co-immunoprecipitation (Co-IP) assay has further confirmed that JAM-B protein is conjugated by a chain of ubiquitin upon TGF-β3 stimulation in the presence of MG-132. TGF-β3 also speeds up the degradation of JAM-B through Smad-dependent pathway. As knockdown of Smad3 and/or Smad4 effectively abolish TGF-β3-mediated JAM-B degradation. Taken together, the involvement of both ubiquitinproteasome pathway and Smad-dependent signalling are essential for TGF-β3-mediated JAM-B regulation in mouse Sertoli cells. [This work was supported by Hong Kong Research Grants Council (HKU772009 and HKU773710) and CRCG Seed Funding for Basic Research.]