Dysregulation of nectin-2 in the testicular cells: An explanation of cadmium-induced male infertility

Nectin-2, a junction molecule, is found at the basal and apical ectoplasmic specializations (ES) for the formation of the blood-testis barrier (BTB) (constituted by tight junctions and basal ES) and Sertoli-spermatid adhesion. Loss of nectin-2 causes male infertility, suggesting nectin-2-based ES is crucial for spermatogenesis. Cadmium (Cd) has been known to induce severe testicular injury. Recent evidence has shown that the basal ES at the BTB and apical ES are the targets of Cd, suggesting that unique junction protein at the ES may explain why testis is more susceptible than other tissues. Since nectin-2 is expressed exclusively at the ES, it is highly possible that nectin-2 is the direct target of Cd. In this study, we investigate if nectin-2 is the target protein of Cd toxicity and the mechanism on how Cd down-regulates nectin-2 to achieve ES disruption. Our results revealed that Cd suppresses nectin-2 at transcriptional and post-translational levels. Inhibitor and shRNA knockdown have shown that Cd induces nectin-2 protein degradation via clathrin-dependent endocytosis. Immunofluorescence staining and endocytosis assays further confirmed that nectin-2 internalization is promoted upon Cd treatment. Besides, Cd directly represses nectin-2 transcription. EMSA and ChIP assays showed that Cd inhibits the binding of positive regulators to nectin-2 promoter. siRNA and overexpression analyses have demonstrated that Cd reduces the expression and binding affinity of positive regulators for transcription. Taken together, nectin-2 is the direct molecular target of Cd and its disruptive effects are mediated via direct repressing nectin-2 transcription and endocytosis of nectin-2 for degradation.