833 Bile acid mediated activation of JNK requires the bile acid nuclear receptor FXR

833 Bile acid mediated activation of JNK requires the bile acid nuclear receptor FXR

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003 AASLD ABSTRACTS elucidated. The specific sulfation state of cell surface h e p a r a n sulfate glycosamin...

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HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

elucidated. The specific sulfation state of cell surface h e p a r a n sulfate glycosaminoglycans (HSGAGs) has b e e n shown to mediate signaling by growth factors, such as FGF2 signaling. We hypothesized that a novel differentially-expressed sulfatase, hSulfl, regulates FGF and HGF signaling in hepatocellular carcinomas by desulfating cell surface h e p a r a n sulfate glycosaminoglycans. Methods: The subcellular location of hSulfl and the sulfation state of cell surface h e p a r a n sulfate glycosaminoglycans were assessed by immunocytochemistry, hSulfl sulfatase activity was m e a s u r e d using 4-methylumbelliferyl-sulfate as substrate. FGF2 and HGF signaling were assessed using antibodies against native and phosphorylated forms of FGF receptor1, c-Met and p44/42 mitogen activated protein kinase. Cell growth were m e a s u r e d by the MTF assay and counting viable cells. Apoptosis was quantitated by fluorescence microscopy and flow cytometry. Results: Low hSulfl expression was associated with increased sulfation of cell surface h e p a r a n sulfate glycosaminoglycans, e n h a n c e m e n t of both FGF and HGF-mediated signaling and growth of hepatocellular carcinoma cells. Conversely, forced expression of hSulfl decreased sulfation of cell surface h e p a r a n sulfate glycosaminoglycans and abrogated FGF2 and HGF signaling. High hSulfl expressing hepatocellular carcinoma cells were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulfl into hSulfl-negative cells restored sensitivity to staurosporine or cisplatin. Conclusions: The results suggest that hSulfl inactivation contributes to hepatocarcinogenesis by enhancing growth factor signaling and resistance to apoptosis. Disclosures: Ileana Aderca - No relationships to disclose Jeremy R Chien - No relationships to disclose Eddie L Greene - No relationships to disclose Jin-Ping Lai - No relationships to disclose Tori A Matthews - No relationships to disclose Damian P Montoya - No relationships to disclose Lewis R Roberts No relationships to disclose Viji Shridhar - No relationships to disclose David I Smith No relationships to disclose Julie K Staub - No relationships to disclose -

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BILE ACID MEDIATED ACTIVATION OF JNK REQUIRES THE BILE ACID NUCLEAR RECEPTOR FXR. Amethyst C

Kurbegov, Sundararajah Thevananther, Andrew M Perry, Saul J Karpen, Baylor College of Medicine, Houston, TX Background: Bile acid (BA) homeostasis is a crucial aspect of enteric and hepatic function, contributing to proper nutrient absorption, total body lipid metabolism, and toxin protection. BA accumulation within hepatocytes leads to apoptosis and necrosis. Recent research has shown that hepatocytes, via multiple mechanisms, have clear self-protective molecular responses to elevated levels of bile acids. Bile acids appear to act as signaling molecules within hepatocytes, interacting with both nuclear receptors and signal transduction pathways. The key BA nuclear receptor is the

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recently described farnesoid X receptor (FXR) whose endogenous ligands are the primary bile acids chenodeoxycholic acid (CDCA) and cholic acid (CA). FXR coordinates a variety of responses to bile acids, including suppression of BA import and synthesis via the transcriptional repressor small heterodimer protein (SHP). In addition to activating FXR, bile acids also can stimulate signal transduction pathways, including the c-Jun N-terminal kinase (JNK) subgroup of the mitogen-activated protein (MAP) kinases. Direct treatment of hepatocytes with bile acids results in JNK activation and JNK-dependent suppression of BA synthesis (Gupta et al, JBC, 2001). We have recently shown that JNK activation also leads to suppression of BA import via transcriptional down-regulation of the main BA importer, Ntcp (Li et al, JBC, 2002). This response to BA is maintained in SHP -/- mice, as JNK remains activated and BA import and synthesis remain suppressed in these mice (Wang et al, Dev Cell, 2002). FXR -/- mice fed CA, however, do not show the characteristic protective response to bile acids seen in both wild type and SHP -/- mice and quickly die of BA-induced liver failure. This suggests that, in addition to being unable to activate SHP, FXR -/- mice also may have impaired JNK signaling. We hypothesized that FXR is required for bile acid activation of the JNK signaling pathway. Methods: Traditional CA feeds are a chronic model of cholestasis, not suited for studying a rapid, acute-phase response to bile acids. C o m m o n bile duct ligation, another standard model of cholestasis, combines b o t h BA and inflammatory stressors on the liver, making this model unsuitable for a strictly BA-focused study. Thus, in order to study acute liver responses to hydrophobic bile acid stress, we established an intraperitoneal (IP) m e t h o d of bile acid delivery. Dose and time-response curves were established in FXR+/+ mice. Single dose injections from 25 to 100 mg/kg were given at a variety of time points from 0 to 360 minutes to determine the optimal conditions for maximal JNK activation. Results: Maximal JNK activation was seen with a dose of 50 mg/kg of CDCA (2mg/ml) at a time point of 90 minutes following injection. FXR+/+ and FXR -/- mice were treated according to this regimen with CDCA or saline vehicle. Animals were vigorous and showed no overt toxicity from the bile acid injections. Livers were harvested, whole cell extracts were prepared, and western blots were performed for levels of phospho-JNK (the activated form of JNK) and total JNK. Saline injected mice did not show JNK activation in either group. FXR + / + mice injected with CDCA showed clear upregulation of phospho-JNK at 90 minutes following injection. FXR -/- mice tested at 90 minutes, however, did not show JNK activation in response to CDCA. Summary and Conclusions: Bile acids acutely activate JNK, and this activation requires the presence of the nuclear receptor FXR. This represents a novel interaction between nuclear receptor and signal transduction pathways that directly impacts upon our understanding of the hepatic response to cholestasis. Disclosures: Saul J Karpen - No relationships to disclose Amethyst C Kurbegov - No relationships to disclose Andrew M Perry - No relationships to disclose Sundararajah Thevananther - No relationships to disclose