Inhibition of endothelin-1-mediated contraction of hepatic stellate cells by FXR ligand

Abstract

Activation of hepatic stellate cells (HSCs) plays an important role in the development of cirrhosis through the increased production of collagen and the enhanced contractile response to vasoactive mediators such as endothelin-1 (ET-1). The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that is highly expressed in liver, kidneys, adrenals, and intestine. FXR is also expressed in HSCs and activation of FXR in HSCs is associated with significant decreases in collagen production. However, little is known about the roles of FXR in the regulation of contraction of HSCs. We report in this study that treatment of quiescent HSCs with GW4064, a synthetic FXR agonist, significantly inhibited the HSC transdifferentiation, which was associated with an inhibition of the upregulation of ET-1 expression. These GW4064-treated cells also showed reduced contractile response to ET-1 in comparison to HSCs without GW4064 treatment. We have further shown that GW4064 treatment inhibited the ET-1-mediated contraction in fully activated HSCs. To elucidate the potential mechanism we showed that GW4064 inhibited ET-1-mediated activation of Rho/ROCK pathway in activated HSCs. Our studies unveiled a new mechanism that might contribute to the anti-cirrhotic effects of FXR ligands.

Figure 1. GW4064 treatment inhibited transdifferentiation of HSCs.

Freshly isolated rat HSCs were cultured for 7 days in 10% FCS alone (control) or 10% FCS medium containing 1 µmol/L GW4064. The expression of α-SMA mRNA (A) and protein (B) was examined by real-time RT-PCR and immunofluorescence staining, respectively. Data are mean ±SE of 6 experiments. *P<.001 vs. control cells.

Figure 2. GW4064 treatment inhibited the upregulation of ET-1 expression during HSC activation.

Freshly isolated rat HSCs were cultured for 7 days in 10% FCS alone (control) or 10% FCS medium containing 1 µmol/L GW4064. The mRNA expression levels of ET-1 (A), ETB (B) and ETA (C) were examined by real-time RT-PCR, respectively. Data are mean ±SE of 6 experiments. *P<.001 vs. control cells.

Figure 3. GW4064 treatment of HSCs led to reduced contractile response to ET-1.

Freshly isolated rat HSCs were cultured for 7 days in 10% FCS medium alone (control) or 10% FCS medium containing 1 µmol/L GW4064. Collagen gel lattices that contained the control or GW4064-treated HSCs were prepared as described in Materials & Methods . Hydrated collagen lattices were photographed 6 hours after addition of ET-1 (10 nM). Data are mean ±SE of 6 experiments. *P<.001 vs. control cells.

Figure 4. GW4064 treatment inhibited ET-1-induced contraction in fully activated HSCs.

Rat HSCs were isolated and then cultured for 7 days to allow full activation. Collagen gel lattices that contained fully activated HSCs were then prepared. They were treated with DMSO or 1 µmol/L GW4064 for 18 hours. Hydrated collagen lattices were photographed 6 hours after addition of ET-1 (10 nM). Data are mean ±SE of 6 experiments. *P<.001 vs. control cells.

Figure 5. GW4064 treatment inhibited ET-1-induced RhoA activation in activated HSCs.

Activated HSCs were treated with DMSO or 1 µmol/L GW4064 for 18 hours. ET-1 (10 nM) was then added to the medium, and the cells were harvested 10 min later. RhoA-GTP pull-down assay was performed using RhoA-GTP assay kit. RhoA-GTP pull-down and total RhoA fractions were probed by Western blot with anti-RhoA antibody. Blots are representative of experiments performed three times with similar results.

Figure 6. GW4064 treatment inhibited ET-1-induced Rho-kinase activity in activated HSCs.

Activated HSCs were treated with DMSO or 1 µmol/L GW4064 for 18 hours. ET-1 (10 nM) was then added to the medium, and the cells were harvested 10 min later. Phosphorylation of meosin was detected by Western using appropriate phospho-specific antibody. An immunoblot of total meosin present in the cell extracts is shown as a loading control (lower panel). A representative immunoblot from three independent experiments is presented.

Figure 7. GW4064 treatment inhibited ET-1-induced phosphorylation of MLC in activated HSCs.

Activated HSCs were treated with DMSO or 1 µmol/L GW4064 for 18 hours. ET-1 (10 nM) was then added to the medium, and the cells were harvested 10 min later. Phosphorylation of MLC was detected by Western using appropriate phospho-specific antibody. An immunoblot of total MLC present in the cell extracts is shown as a loading control (lower panel). A representative immunoblot from three independent experiments is presented.