Nectin-like molecule-2 (Necl-2), a junction molecule, is exclusively expressed 
by spermatogenic cells. It mediates homophilic interaction between germ cells 
and heterophilic interaction between Sertoli and germ cells. Knockout studies 
have shown that loss of Necl-2 causes male infertility, suggesting Necl-2-based 
cell adhesion is crucial for spermatogenesis. Transforming growth factor-βs 
(TGF-βs) are crucial for regulating cell junction restructuring that are 
required for spermatogenesis. In the present study, we aim to investigate the 
mechanism on how TGF-β1 regulates Necl-2 expression to achieve timely junction 
restructuring in the seminiferous epithelium during spermatogenesis. We have 
demonstrated that TGF-β1 reduces Necl-2 mRNA and protein levels at both 
transcriptional and post-translational levels. Using inhibitor and clathrin 
shRNA, we have revealed that TGF-β1 induces Necl-2 protein degradation via 
clathrin-dependent endocytosis. Endocytosis assays further confirmed that TGF-β1 
accelerates the internalization of Necl-2 protein to cytosol. Immunofluorescence 
staining also revealed that TGF-β1 effectively removes Necl-2 from cell-cell 
interface. In addition, TGF-β1 reduces Necl-2 mRNA via down-regulating Necl-2 
promoter activity. Mutational studies coupled with knockdown experiments have 
shown that TGF-β1-induced Necl-2 repression requires activation of Smad 
proteins. EMSA and ChIP assays further confirmed that TGF-β1 promotes the 
binding of Smad proteins onto MyoD and CCAATa motifs in vitro and in vivo. Taken 
together, TGF-β1 is a potent cytokine that provides an effective mechanism in 
controlling Necl-2 expression in the testis via Smad-dependent gene repression 
and clathrin-mediated endocytosis.