4. of MAPK signaling is required for upregulation of Egr-1 by bile acids in hepatocytes and for upregulation of Egr-1 in the liver during cholestasis. These studies suggest that inhibition of MAPK signaling may be a novel therapy to prevent upregulation of Egr-1 in liver during cholestasis. Keywords:Early growth response factor-1, Bile acids, Mitogen-activated protein kinases, Farnesoid X receptor, Inflammation, Cholestasis == Introduction == Cholestasis is a condition that occurs when bile flow from the liver is disrupted (Li and Crawford, 2004). This results in increased concentrations of toxic bile acids in the liver and blood (Lindblad et al., 1977;Setchell et al., 1997). If cholestasis is left untreated, this disease results in hepatocellular injury, bile duct proliferation, and activation of hepatic stellate cells and portal fibroblasts, leading to fibrosis (Gujral et al., 2003;Ramadori and Saile, 2004). Over time as this disease progresses, the liver becomes cirrhotic and the patient will be in danger of liver failure (Paumgartner, 2006). Inflammation, consisting primarily of neutrophils, has been shown to play an integral role in the development of hepatocellular injury during cholestasis (Gujral et al., 2003;Gujral et al., 2004) Neutrophil accumulation has been shown to occur in animals and humans with obstructive jaundice (Gulubova, 1998;Yamashiki et al., 1998;Neuman et al., 2002). Studies from our laboratory have shown that early growth response factor-1 (Egr-1), a transcription factor, is important for inflammation and injury during cholestasis (Kim et al., 2006). In these studies, Egr-1 was upregulated in hepatocytes of mice subjected to bile duct ligation (BDL), a model of cholestatic liver disease. Furthermore, Egr-1 knockout mice subjected to bile duct ligation had reduced liver injury, fewer neutrophils in the liver, and reduced expression of proinflammatory mediators compared to wild-type mice (Kim et al., 2006). These studies suggested Ferrostatin-1 (Fer-1) that Egr-1 is a key mediator of inflammation in the liver during cholestasis. Our results demonstrated further that the bile acid, deoxycholic acid (DCA), increased Egr-1 protein levels in primary mouse hepatocytes (Kim et al., 2006). This suggested that the stimulus for upregulation of Egr-1 in liver during cholestasis may be bile acids. What remains unknown, however, is the molecular mechanism by which Ferrostatin-1 (Fer-1) bile acids increase Egr-1 levels in hepatocytes. Identification of the signaling pathways that Ferrostatin-1 (Fer-1) stimulate upregulation of Egr-1 could lead to the development of therapeutics that attenuate the inflammatory response in the liver during cholestasis. Bile acids modulate gene expression in hepatocytes by several mechanisms. For example, bile acids activate the nuclear receptor, farnesoid X receptor (FXR). Upon bile acid binding, FXR heterodimerizes with RXR (Makishima et al., 1999) and binds to response elements in the promoters of genes, and modulates gene transcription by suppressing or enhancing gene expression (Claudel et al., 2005). In addition to FXR, bile acids modulate gene expression by activating mitogen-activated protein kinase (MAPK) signaling (Rao et al., 2002). Bile acids stimulate ligand-independent activation of the epidermal growth factor receptor which stimulates phosphorylation and activation of raf kinase, MEK, and Erk1/2 (Rao et al., 2002). Whether activation of FXR and/or MAPK signaling is required for upregulation of Egr-1 by bile acids is not known. Accordingly, the hypothesis was tested SPP1 Ferrostatin-1 (Fer-1) that upregulation of Egr-1 in hepatocytesin vitroby bile acids andin vivoduring cholestasis requires FXR and/or MAPK signaling. == Materials and Methods == == Animals care == C57BL/6 mice (Harlan, Madison, WI) and FXR knockout mice with a congenic C57BL/6 background were used for all studies. Generation of the FXR knockout mice was described previously (Sinal et al., 2000). Mice were maintained on a 12-h light/dark cycle under controlled temperature (18-21C) and humidity. Food (Rodent Chow; Harlan-Teklad, Madison, WI) and tap water were allowed ad libitum. All procedures on animals were carried out in accordance with the Guide for the Care and Use of Laboratory Animals promulgated by the National Institutes of Health and were approved by the institutional IACUC committee at the University Ferrostatin-1 (Fer-1) of Kansas Medical Center. == Hepatocyte Isolation == Hepatocytes were isolated from the livers of mice by collagenase perfusion as described in detail by us previously (Kim et al., 2006). The hepatocytes were cultured in Williams’ medium.