TNFα stimulates the proliferation of immature Sertoli cells by attenuating UPS-degradation of cyclin D1 and leads to the delay of BTB maturation in pubertal rats

Backgrounds: The Sertoli cell (SC) which plays a vital role during spermatogenesis is a known target of physiological and pathological factors affecting testicular development. TNFα participates in the blood-testis barrier (BTB) reconstruction, cell Apoptosis and inflammatory response by recognizing receptors on SC. TNFα has also been shown to induce the proliferation of immature SC in vitro, yet the mechanism still remains unclarified.

Objectives: This study was designed to investigate the effect of TNFα on BTB development during puberty and the underlying mechanisms of TNFα-induced immature SC proliferation.

Materials and methods: Immature male SD rats of postnatal day (PND) 12 were intraperitoneally injected with TNFα. Biotin-labeled method was used to detect permeability of the developing BTB after TNFα treatment, and the distribution of occludin and JAM-A were detected by immunofluorescence. SCs isolated from SD rats of PND10 were cultured in vitro and treated with TNFα. Cell proliferation rate was reflected by CCK-8 and EdU assay. Immunoblot and qPCR were used to detect the expression of PCNA, Fbxo4 and cyclin D1. Immunoprecipitation was used to detect the ubiquitination of cyclin D1 and the interaction between Fbxo4 and cyclin D1. PDTC was applied to detect the effect of NFκB activity inhibition on TNFα-induced SC proliferation. The adenoviral recombinant plasmid containing rat Fbxo4 gene was constructed to investigate the effect of Fbxo4 overexpression on SC proliferation promoted by TNFα.

Results: The in vivo experiment revealed a significant delay of BTB maturation in pubertal rats caused by exogenous TNFα. TNFα (10 ng/ml) treatment in vitro was found to promote the proliferation of immature SCs, accompanied with increased NFκB activity and cyclin D1 protein level. The level of Fbxo4 and ubiquitination of cyclin D1 were decreased after TNFα treatment. Inhibitor of NFκB or overexpression of Fbxo4 could both reverse the TNFα-induced proliferation of immature SCs, meanwhile restore the ubiquitin-proteasome system (UPS)-dependent degradation of cyclin D1. Overexpression of Fbxo4 could not affect the activation of NFκB caused by TNFα.

Conclusion: These results indicate that TNFα inhibits the ubiquitination and degradation of cyclin D1 through the NFκB pathway, thereby promoting the proliferation of immature SC in vitro and inducing the delay of BTB maturation in pubertal rats. This article is protected by copyright. All rights reserved.

BTB; Sertoli cell; TGFα; cyclin D1; proliferation.