Role of AMPK in the expression of tight junction proteins in heat-treated porcine Sertoli cells

Abstract

Hyperthermia can cause dysfunction of the tight junctions (TJs) in testes. Adenosine 5'-monophosphate-activated protein kinase (AMPK) participates in the regulation of TJs in testis. However, whether AMPK regulates the expression of TJ proteins in the response of Sertoli cells to heat treatment remains unknown. We subjected Sertoli cells from 3-week-old piglets to heat treatment (43 °C, 30 min), which decreased cell viability, and increased the early apoptosis rate. These effects were reversible and the cells gradually recovered to normal viability at 48 h post-heat treatment. Expression of TJ proteins (claudin 11, JAMA, occludin, and ZO1) was detected in immature porcine Sertoli cells. The mRNA and protein levels of TJ proteins significantly decreased at 1 h after heat exposure, but recovered with increasing recovery time. Additionally, the expression of claudin 11 in the cytoplasm was also markedly decreased by heat treatment. AMPK phosphorylation, the cellular ATP level, and Ca²⁺/calmodulin-dependent protein kinase kinase B (CaMKKB) level, but not the liver kinase B1 (LKB1) level, were downregulated by heat treatment. More importantly, activation or overexpression of AMPK, which is a regulator of the assembly of TJs, partially rescued the heat treatment-induced downregulation of TJ proteins. By contrast, AMPK knockdown using small interfering RNA (siRNA) further decreased the expression levels of TJ proteins. In addition, claudin 11 was almost undetectable post heat treatment. Collectively, this study demonstrated that heat treatment could reversibly perturb the expression of TJ proteins in immature porcine Sertoli cells by inhibiting the AMPK signaling pathway.

Keywords: AMPK; Heat stress; Porcine; Sertoli cell; Tight junction protein.