29 Increased CK synthesis and phosphorylation occurred in hepatocytes in acinar zone 1 and in the vicinity of bile infarcts that are areas with probably the highest bile acid concentrations and resulting toxic stress. hepatocellular CK expression and phosphorylation in cholestasis may be caused by retention of toxic bile acids and reflect a hepatocellular stress response with potential beneficial effects. Cholestatic liver disorders were found to be associated with profound alterations of all three components of the hepatocyte cytoskeleton, namely microtubules, microfilaments, and cytokeratin (CK) intermediate filaments (IFs). 1-3 Hydrophobic bile acids accumulate during cholestasis and affect microtubular motors, such as kinesin and dynein, resulting in impaired vesicle movement to the canalicular membrane and reduced bile flow. 1-4 Disruption of microfilaments, which normally form a contractile web around the bile canaliculus and regulate tight junction permeability, results in dilated canaliculi, leaky tight junctions, and loss of microvilli. All these changes may not only be consequences of but also contribute to cholestasis. 1-4 Hepatocytic IFs consisting of CK 8 and CK 18 mechanically support the bile canaliculus (pericanalicular sheath) but their role in bile secretion remains unclear. 5,6 Increased density of IFs in experimental obstructive cholestasis in rats results in the thickening of the pericanalicular sheath and has mainly been attributed to increased canalicular pressure as a result of biliary obstruction. 7-9 Tubacin However, primary biliary cirrhosis and disorders associated with nonmechanical intrahepatic Tubacin cholestasis, such as alcoholic hepatitis, nonalcoholic steatohepatitis, and Wilsons disease, are also associated with profound cytoskeletal alterations including the formation of Mallory bodies (MBs), which are cytoplasmic inclusions in hepatocytes consisting of CKs and non-CK components. 10,11 The CK-IF Tubacin network has long been considered as being a rather static structure responsible for mechanical stability of cells. More recently, however, the importance of CKs for maintenance of functional integrity of hepatocytes has been demonstrated in several gene knockout mouse models. 11-16 Mutations of CKs in mice are associated with higher susceptibility to hepatotoxins, indicating that CKs may also have important nonmechanical functions, such as defense against toxic injury. 12,16 The importance of CK mutations for the pathogenesis of human liver diseases has been emphasized by the recent finding of mutations in the CK 8 gene in patients with cryptogenic cirrhosis. 17 In addition, hyperphosphorylation of CKs seems to be involved in cytoprotection against toxic stress. 6,11,15,18 Marked overexpression and hyperphosphorylation of CK 8 and CK 18 were also demonstrated in human alcoholic hepatitis, which is also frequently accompanied by cholestasis. 18,19 Therefore, the aim of this Tubacin study was to investigate whether bile Tubacin acids play a causal role in overexpression and phosphorylation of CK 8 and CK 18. For this purpose we studied expression and phosphorylation of CKs in different mouse models associated with elevated bile acid levels. Materials and Methods Animals and Materials Male Swiss Albino mice (strain Him OF1 SPF) were obtained from the Institute of Laboratory Animal Research, University of Vienna School of Medicine, Himberg, Austria, housed with a 12-hour light-dark cycle and permitted consumption of water and a standard mouse diet (Marek, Vienna, Austria). Experiments were performed with 2-month-old mice weighing 25 to 30 g. The experiments described in this article were approved by the local ethics committee and followed the criteria outlined in the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences) as published by the National Institutes of Health (NIH publication 86-23, revised 1985). Cholic acid NFKBIA (CA) was obtained from Aldrich (Steinheim, Germany), ursodeoxycholic acid (UDCA) was kindly provided by the Falk Foundation (Freiburg, Germany). Animal Experiments Common Bile Duct Ligation (CBDL) All surgical procedures were performed under sterile conditions. To study the effects of obstructive cholestasis on hepatic CK expression, the common bile duct was ligated under general anesthesia (10 mg Avertin intraperitoneally) close to the liver hilum immediately below the bifurcation and dissected between the ligatures as described previously. 20 Cholecystectomy was performed after ligation of the cystic duct. Controls underwent a sham operation with exposure, but without ligation of the common bile duct and removal of the gallbladder. The livers were excised under.