Anti-apoptotic effect of adrenomedullin gene delivery on Leydig cells by suppressing TGF-β1 via the Hippo signaling pathway

Abstract

This study aims to establish whether adrenomedullin (ADM) is capable to restore the steroidogenic functions of Leydig cells by suppressing transforming growth factor-β1 (TGF-β1) through Hippo signaling. Primary Leydig cells were treated with lipopolysaccharide (LPS), an adeno-associated virus vector that expressed ADM (Ad-ADM) or sh-RNA of TGF-β1 (Ad-sh-TGF-β1). The cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were determined for steroidogenic enzymes, TGF-β1, RhoA, YAP, TAZ and TEAD1. The role of Ad-ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Similar to Ad-sh-TGF-β1, Ad-ADM mitigated the decline in the number of Leydig cells and plasma concentrations of testosterone by restoring the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD. Similar to Ad-sh-TGF-β1, Ad-ADM not only inhibited the LPS-induced cytotoxicity and cell apoptosis but also restored the gene and protein levels of SF-1, LRH1, NUR77, StAR, P450scc, 3β-HSD, CYP17 and 17β-HSD, along with the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-β1, Ad-ADM improved LPS-induced TGF-β1 expression. In addition, Ad-ADM suppressed RhoA activation, enhanced the phosphorylation of YAP and TAZ, reduced the expression of TEAD1 which interacted with HDAC5 and then bound to TGF-β1 gene promoter in LPS-exposed Leydig cells. It is thus suspected that ADM can exert anti-apoptotic effect to restore the steroidogenic functions of Leydig cells by suppressing TGF-β1 through Hippo signaling.