A number of nuclear receptors have been reported to show inhibitory effects on HSC activation. These include retinoid X receptor, PPARs, pregnane-X-receptor, and FXR. Treatment with the respective ligands has been shown to inhibit the activation of the stellate cells and decrease the fibrotic changes in animal models of liver injury. However little information is available on the roles of nuclear receptors in the regulation of stellate cell contraction. As an initial step to address this question we studied the effect of GW4064, a synthetic FXR ligand, on the expression of ET-1 and its corresponding receptors on stellate cells. As shown in Fig. 2, there was a significant increase in the mRNA expression level of ET-1 during the process of stellate cell activation. This upregulation in ET-1 expression was significantly inhibited by GW4064 treatment. We have previously shown that activation of FXR inhibited both basal and LPS-stimulated ET-1 production in endothelial cells. Functional promoter assays including electrophoretic mobility shift assay and chromatin L-Ornithine immunoprecipitation assay suggest that FXR inhibits ET-1 expression via interference with NF- kB/AP-1 signaling. It is likely that FXR regulates ET-1 expression in stellate cells via a similar mechanism. Despite the inhibitory effect on ET-1 expression, GW4064 showed no effect on the expression of either ETA or ETB receptor in stellate cells. A study by Chi and colleagues showed that retinoic acid exerted an inhibitory effect on ETB expression in stellate cells but showed no effect on either ET-1 or Lomitapide Mesylate ETA expression. Clearly, different nuclear receptors regulate stellate cell activation via different mechanisms. The freshly isolated stellate cells that were continuously treated with GW4064 for 7 days showed reduced contractile response to ET-1. This is unlikely due to changes in the expression levels of ET-1 receptors as shown in our RT-PCR studies. It is likely due to the fact that GW4064-treated cells are less activated and are equipped with less active contractile mechanism. It has been shown that a-SMA is involved in the stellate cell contraction. GW4064-treated cells have significantly reduced levels of a-SMA expression compared to fully activated stellate cells, which could account for, at least partially, the reduced contractile response to ET-1. After demonstrating a reduced contractile response in GW4064-treated, partially activated stellate cells we further observed a similar inhibitory effect of GW4064 in fully activated stellate cells. To understand the underlying mechanism we focused on examining the effect of GW4064 on RhoA/Rho kinase signaling as this pathway plays a key role in stellate cell contraction. Our preliminary study with quantitative RT-PCR showed no effect of GW4064 on the mRNA expression levels of either ETA or ETB. Thus it is unlikely that GW4064 inhibits stellate cell contractile response to ET-1 via modulating the expression of ET receptors at transcriptional level. We then examined if GW6064 affects RhoA activation. Western analysis clearly showed that the ET-1-mediated activation of RhoA, measured as pull down of active GTP-RhoA, was significantly inhibited by GW4064 treatment. So far, the mechanism responsible for this inhibition is unclear. Preliminary studies showed that there were no significant changes in the mRNA expression levels of the receptors and several GTPase-activating proteins and guanine nucleotide exchange factors following treatment with GW4064. It remains to be determined if GW4064 treatment can induce any changes in the expression of these molecules at protein level.