Transcriptional and post-translational regulations of nectin-like molecule-2 in spermatogenic cells under the control of transforming growth factor-β1

Abstract

Transforming growth factor-β (TGF-β) is a known family of cytokine that regulates cell junction restructuring required for germ cell development and movement in the testis. Previous studies have shown that it is important to regulate the restructuring of the blood-testis barrier constituted by an array of tight and adherens junctions between adjacent sertoli cells. In this study, we extend our investigation to examine if TGF-β1 regulates the restructuring of cell junctions between sertoli and germ cells. Nectin-like molecule-2 (necl-2) is found in spermatogenic cells and is involved in Sertoli-germ cell interaction, we therefore examine if TGF-β1 exert its effect on Sertoli-germ cell junction restructuring by modulating the expression of necl-2 in spermatogenic cells. In this study, we have found that TGF-β1 (5 ng/ml) significantly downregulates both mRNA and protein levels of necl-2 in mouse germ cell cell line, GC-1 spg cells. The reduction of necl-2 mRNA mediated by TGF-β1 is at transcriptional level, but not post-transcriptional level. Luciferase reporter assays have confirmed that TGF-β1 downregulates necl-2 promoter activity and siRNA analyses have shown that knockdown of Smad3 and Smad4 abolish TGF-β1-mediated necl-2 gene repression. Electromobility shift assays have shown that TGF-β1 activates and promotes the binding of Smad3 and Smad4 to the cis-acting motifs including two CCAAT and MyoD motifs that lie within nt-159 and -1 of the promoter region. Apart from transcriptional regulation, TGF-β1 exerts its negative effect on necl-2 expression via post-translational regulation. Cycloheximide treatment coupled with immunoblotting has shown that TGF-β1 speed-ups the turnover of necl-2 protein. By inhibitor and shRNA approaches targeting targeting clathrin, we have confirmed that TGF-β1 promotes necl-2 degradation by activating clathrin-dependent endocytosis.