Session: Cell-Cell Junctions IIPoster presentationJunctional 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.]published_or_final_versio
