Role of cytokines in the regulation of cell junction dynamics in the testis

Abstract

During spermatogenesis, developing germ cells must migrate across the blood-testis barrier (BTB) and enter the adluminal compartment for further development. Throughout this process, extensive junction restructuring occurs at Sertoli-Sertoli and Sertoli-germ cell interfaces. Cytokines are known to play crucial roles in regulating testicular cell junction dynamics at different regulatory levels. However, the mechanism of cytokine-mediated regulation on newly identified junction molecules remains unclear. In this dissertation, the molecular mechanisms on how cytokines regulate the junction proteins of immunoglobulin superfamily (IgSF) including coxsackievirus and adenovirus receptor (CAR), nectin-like molecule-2 (Necl-2) and Necl-4 in testicular cells were studied.

CAR is expressed on Sertoli and germ cells. It mediates both homophilic and heterophilic interaction for Sertoli-germ cell adhesion. It was found that combined treatment of interferon-γ (IFN-γ) and tumor necrosis factor α (TNFα) reduced CAR mRNA and protein levels, and caused the disappearance of CAR from germ cell interface. IFN-γ+TNFα promoted CAR protein degradation via ubiquitin-proteasome pathway. In addition, IFN-γ+TNFα reduced CAR mRNA through regulating the binding of NF-κB subunits and SP/KLF proteins to CAR promoter. Collectively, these results demonstrated for the first time the potential mechanism utilized by IFN-γ+TNFα to exert their effects during testicular inflammation.

Necl-2 is exclusively expressed by spermatogenic cells in the testis. In this study, it was demonstrated that transforming growth factor-β1 (TGF-β1) down-regulated Necl-2 mRNA and protein levels, and caused the disappearance of Necl-2 from cell surface. Using inhibitors and shRNAs, it was found that TGF-β1 induced Necl-2 protein degradation through clathrin-dependent endocytosis. Endocytosis assay further confirmed that TGF-β1 accelerated the internalization of Necl-2 to cytosol. Moreover, TGF-β1 repressed Necl-2 gene transcription in Smad-dependent manner. Taken together, these results unraveled the mechanism of how TGF-β1 regulates Necl-2 expression to achieve timely junction restructuring during spermatogenesis. Necl-4 has been detected in Sertoli cells, but little is known about its regulation in the testis. It was found that TNFα down-regulated Necl-4 mRNA and protein levels. Inhibitor studies suggested that both caveolin-dependent endocytosis and ubiquitin-proteasome pathway were involved in TNFα-induced Necl-4 protein degradation. Co-immunoprecipitation indicated that Necl-4 was physically associated with Par3, Par6, aPKC and Cdc42 which are the major components of polarity complex in mouse testis. Further study was shown that TNFα reduced the expression of Par3, and altered the binding between Necl-4 and Par complex in Sertoli cells. These results suggested that Necl-4-mediated cell adhesion could be disrupted by TNFα via reducing its expression and altering its interaction with Par complex.

Studies reported herein suggest that junction proteins of the IgSF are precisely regulated by cytokines at transcriptional and post-translational levels. These results further enrich current understanding on how junction dynamics are regulated during spermatogenesis.