Background: The intrahepatic biliary tree is formed by large, senescent cholangiocytes and small, more primordial (less senescent) cholangiocytes. PSC is characterized by biliary injury that contribute to liver fibrosis. Cholangiocyte senescence (a condition in which cells are irreversibly arrested at the G1 phase) contributes to liver fibrosis in PSC. EMT has been associated with liver diseases. We have previously shown that GnRH induces biliary damage and liver fibrosis (by interaction with GnRHRl) in normal and cholestatic models. Thus, we tested the hypothesis that GnRH/GnRHR axis activates fibrosis in PSC by changes in EMT and senescence in different‐sized cholangiocytes. Methods: The studies were performed in: (i) serum and cDNA from total liver from normal and early and late stage male PSC patients; and (ii) small and large cholangiocytes isolated by laser capture microscopy (LCM) from normal WT mice treated with saline or GnRH (250 ng/Kg BW) for 1 wk and Mdr2‐/‐ mice. We evaluated: (i) GnRH serum levels in the selected mouse models and PSC (early and late stage male) patients by ELISA kits; (ii) the mRNA expression of (a) GnRH, GnRHRl and the markers for EMT, vimentin, N‐cad‐ herin (Cdh2), S100a4; (b) epithelial phenotypes, E‐cadherin (Cdhl), fibronectin 1 (Fn‐1); (c) senescence, CCL2, pi 6, p21 and pi 8; and (d) fibrosis, a‐SMA, collagen‐la and TIMP‐1 by qPCR. In vitro, we treated small and large murine biliary lines with GnRH (100 nmol/L, 24 hr) before evaluating the expression of EMT, epithelial, senescence and fibrosis markers by qPCR. Results: There was increased: (i) GnRH serum levels in WT mice treated with GnRH, in Mdr2‐/‐ mice and PSC (early/late stage) patients compared to their controls; (ii) mRNA expression of GnRH and GnRHRl, EMT, epithelial, senescence and fibrosis markers in total liver samples from PSC (early/late stage) patients; (iii) expression of EMT and fibrosis (but not senescence) markers in small LCM‐isolated cholangiocytes; and senescence and fibrosis (but not EMT) phenotypes in large LCM‐isolated cholangiocytes from normal WT mice (treated with GnRH) and Mdr2‐/‐ mice compared to their control groups. In vitro, GnRH increased the expression of fibrosis and EMT in small biliary lines and the expression of fibrosis and senescence in large biliary lines. Conclusion: We demonstrated enhanced GnRH serum levels and increased biliary expression of the GnRH/GnRHR/fibrosis axis in PSC models and PSC human samples. While small cholangiocytes contribute to fibrosis through enhanced EMT, large cholangiocytes participate in liver fibrosis by enhanced senescence. Modulation of the GnRH/GnRHR axis may be key for the management of PSC.
Gonadotropin-releasing hormone (GnRH) contributes to liver fibrosis in primary sclerosing cholangitis (PSC) through enhanced epithelial-mesenchymal transition (EMT) in small but increased senescence in large cholangiocytes
FRANCHITTO, Antonio;
2016-01-01
Abstract
Background: The intrahepatic biliary tree is formed by large, senescent cholangiocytes and small, more primordial (less senescent) cholangiocytes. PSC is characterized by biliary injury that contribute to liver fibrosis. Cholangiocyte senescence (a condition in which cells are irreversibly arrested at the G1 phase) contributes to liver fibrosis in PSC. EMT has been associated with liver diseases. We have previously shown that GnRH induces biliary damage and liver fibrosis (by interaction with GnRHRl) in normal and cholestatic models. Thus, we tested the hypothesis that GnRH/GnRHR axis activates fibrosis in PSC by changes in EMT and senescence in different‐sized cholangiocytes. Methods: The studies were performed in: (i) serum and cDNA from total liver from normal and early and late stage male PSC patients; and (ii) small and large cholangiocytes isolated by laser capture microscopy (LCM) from normal WT mice treated with saline or GnRH (250 ng/Kg BW) for 1 wk and Mdr2‐/‐ mice. We evaluated: (i) GnRH serum levels in the selected mouse models and PSC (early and late stage male) patients by ELISA kits; (ii) the mRNA expression of (a) GnRH, GnRHRl and the markers for EMT, vimentin, N‐cad‐ herin (Cdh2), S100a4; (b) epithelial phenotypes, E‐cadherin (Cdhl), fibronectin 1 (Fn‐1); (c) senescence, CCL2, pi 6, p21 and pi 8; and (d) fibrosis, a‐SMA, collagen‐la and TIMP‐1 by qPCR. In vitro, we treated small and large murine biliary lines with GnRH (100 nmol/L, 24 hr) before evaluating the expression of EMT, epithelial, senescence and fibrosis markers by qPCR. Results: There was increased: (i) GnRH serum levels in WT mice treated with GnRH, in Mdr2‐/‐ mice and PSC (early/late stage) patients compared to their controls; (ii) mRNA expression of GnRH and GnRHRl, EMT, epithelial, senescence and fibrosis markers in total liver samples from PSC (early/late stage) patients; (iii) expression of EMT and fibrosis (but not senescence) markers in small LCM‐isolated cholangiocytes; and senescence and fibrosis (but not EMT) phenotypes in large LCM‐isolated cholangiocytes from normal WT mice (treated with GnRH) and Mdr2‐/‐ mice compared to their control groups. In vitro, GnRH increased the expression of fibrosis and EMT in small biliary lines and the expression of fibrosis and senescence in large biliary lines. Conclusion: We demonstrated enhanced GnRH serum levels and increased biliary expression of the GnRH/GnRHR/fibrosis axis in PSC models and PSC human samples. While small cholangiocytes contribute to fibrosis through enhanced EMT, large cholangiocytes participate in liver fibrosis by enhanced senescence. Modulation of the GnRH/GnRHR axis may be key for the management of PSC.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
