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

To investigate whether adrenomedullin (ADM) rescues the steroidogenic functions of Leydig cells by suppressing transforming growth factor-β1 (TGF-β1) via the 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). Cell viability and medium concentrations of testosterone were detected. Gene expression and protein levels were detected for steroidogenic enzymes, TGF-β1, RhoA, YAP, TAZ and TEAD1. Role of Ad-ADM in the regulation of TGF-β1 promoter was confirmed by ChIP and Co-IP. Like Ad-sh-TGF-β1, Ad-ADM reversed the decreased number of Leydig cells and plasma concentrations of testosterone by rescuing 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 inhibited the LPS-induced cytotoxicity and cell apoptosis and rescued the gene and protein levels of SF-1, LRH1, Nur77, StAR, P450scc, 3β-HSD, CYP17, 17β-HSD and the medium concentrations of testosterone in LPS-induced Leydig cells. Like Ad-sh-TGF-β1, Ad-ADM ameliorated LPS-induced TGF-β1 expression. In addition, Ad-ADM inhibited RhoA activation, enhanced the phosphorylation of YAP and TAZ, decreased TEAD1 expression which interacted with HDAC5 and then bound to TGF-β1 gene promoter in LPS-exposed Leydig cells. ADM may exert anti-apoptotic effect to rescue the steroidogenic functions of Leydig cells by suppressing TGF-β1 via the Hippo signaling.