The orphan nuclear hormone receptor SHP has been proposed to have a key role in the negative feedback regulation of bile acid production. Consistent with this, mice lacking the SHP gene exhibit mild defects in bile acid homeostasis and fail to repress cholesterol 7-alpha-hydroxylase expression in response to a specific agonist for the bile acid receptor FXR. However, this repression is retained in SHP null mice fed bile acids, demonstrating the existence of compensatory repression pathways of bile acid signaling. We provide evidence for two such pathways, based on activation of the xenobiotic receptor PXR or the c-Jun N-terminal kinase JNK. We conclude that redundant mechanisms regulate this critical aspect of cholesterol homeostasis.

Brief periods of in vitro hypoxia/ischemia induce apoptosis of cultured renal epithelial cells, but the underlying mechanisms remain unknown. We show that partial ATP depletion (≈10–65% of control) results in a duration-dependent induction of apoptosis in Madin-Darby canine kidney (MDCK) cells, as evidenced by internucleosomal DNA cleavage (DNA laddering and in situ nick end labeling), morphological changes (cell shrinkage), and plasma membrane alterations (externalization of phosphatidylserine). The ATP-depleted cells display a significant upregulation of Fas, Fas ligand, and the Fas-associating protein with death domain (FADD). Exogenous application of stimulatory Fas monoclonal antibodies also induces apoptosis in nonischemic MDCK cells, indicating that they retain Fas-dependent pathways of programmed cell death. Furthermore, cleavage of poly(ADP)ribose polymerase (PARP) is evident after ATP depletion, indicating activation of caspases. Indeed, the apoptotic cells display a significant increase in caspase-8 (FLICE) activity. Finally, apoptosis induced by ATP depletion is ameliorated by pretreatment with inhibitors of caspase-8 (IETD), caspase-1 (YVAD), or caspase-3 (DEVD) but is not affected by inhibitors of serine proteases (TPCK). Our results indicate that partial ATP depletion of MDCK cells results in apoptosis and that Fas- and caspase-mediated pathways may play a critical role.

Background & aims Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study. Methods Hepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic) acid, PPADS), and liver regeneration after hepatectomy was analyzed. Results A robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction. Conclusion Extracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

Bile flow is rapidly and markedly reduced in hepatic inflammation, correlating with suppression of critical hepatic bile acid transporter gene expression, including the principal hepatic bile acid importer, the Na ؉ /taurocholate co-transporting polypeptide (Ntcp, Slc10a1). Endotoxin treatment of rats and interleukin-1␤ (IL-1␤) treatment of liver-derived HepG2 cells leads to a marked decline in the nuclear binding activity of a main Ntcp gene regulator, the nuclear receptor heterodimer retinoid X receptor:retinoic acid receptor (RXR:RAR). How IL-1␤ signaling leads to reduced RXR:RAR nuclear binding activity is unknown, and we sought to determine whether mitogen-activated protein kinase (

Partial hepatectomy leads to an orchestrated regenerative response, activating a cascade of cell signaling events necessary for cell cycle progression and proliferation of hepatocytes. However, the identity of the humoral factors that trigger the activation of these pathways in the concerted regenerative response in hepatocytes remains elusive. In recent years, extracellular ATP has emerged as a rapidly acting signaling molecule that influences a variety of liver functions, but its role in hepatocyte growth and regeneration is unknown. In this study, we sought to determine if purinergic signaling can lead to the activation of c-jun N-terminal kinase (

As the obligate heterodimer partner to class II nuclear receptors, the retinoid X receptor ␣ (RXR␣) plays a vital physiological role in the regulation of multiple hepatic functions, including bile formation, intermediary metabolism, and endobiotic/xenobiotic detoxification. Many RXR␣-regulated genes are themselves suppressed in inflamed liver via unknown mechanisms, which constitute a substantial component of the negative hepatic acute phase response. In this study we show that RXR␣, generally considered a stable nuclear resident protein, undergoes rapid nuclear export in response to signals initiated by the pro-inflammatory cytokine interleukin-1␤ (IL-1␤), a central activator of the acute phase response. Within 30 min of exposure to IL-1␤, nuclear levels of RXR␣ are markedly suppressed in human liver-derived HepG2 cells, temporally coinciding with its appearance in the cytoplasm. The nuclear residence of RXR␣ is maintained by inhibiting c-jun N-terminal kinase (JNK, curcumin or SP600125) or CRM-1-mediated nuclear export (Leptomycin B). Pretreatment with the proteasome inhibitor MG132 blocks IL-1␤-mediated reductions in nuclear RXR␣ levels while increasing accumulation in the cytoplasm. Mutational studies identify one residue, serine 260, a JNK phosphoacceptor site whose phosphorylation status had an unknown role in RXR␣ function, as critical for IL-1␤-mediated nuclear export of transfected human RXR␣-green fluorescent fusion constructs. These findings indicate that inflammation-mediated cell signaling leads to rapid and profound reductions in nuclear RXR␣ levels, via a multistep, JNK-dependent mechanism involving Ser 260 , nuclear export, and proteasomal degradation. Thus, inflammation-meditated cell signaling targets RXR␣ for nuclear export and degradation; a potential mechanism that explains the broad suppression of RXR␣-dependent gene expression in the inflamed liver.After injury or infection, the liver participates in a program of modified gene expression known collectively as the acute phase response (APR) 2 (1). A wide variety of hepatic functions are altered during the APR, much of which occurs by cytokine-mediated activation or suppression of target gene transcription. Among the principal hepatic physiologic processes inhibited during the negative hepatic APR are genes involved in endobiotic/xenobiotic metabolism, glucose homeostasis, and bile formation, which then leads to cholestasis. Activation of the negative hepatic APR leads to cholestasis by decreasing bile salt synthesis (2), reducing canalicular bile salt export (3), and suppressing bile salt import, the latter of which occurs primarily by transcriptional down-regulation of the sodium-dependent taurocholate co-transporting polypeptide (Slc10A1) (4, 5). Recent evidence suggests that cytokine-mediated activation of cell signaling pathways during the APR leads to this coordinated response (6). One possibility is targeted repression of the essential heterodimer partner for type II nuclear receptor, RXR␣ (NR2B1), which is an attractive mechanism to ...

Cardiac dysfunction is a major cause of morbidity and mortality in patients with end-stage liver disease, yet the mechanisms remain largely unknown. We hypothesized that the complex interrelated impairments in cardiac structure and function secondary to progression of liver diseases involve alterations in signaling pathways engaged in cardiac energy metabolism and hypertrophy, augmented by direct effects of high circulating levels of bile acids. Biliary fibrosis was induced in male C57BL/6J mice by feeding a 0.1% 3, 5-diethoxycarbonyl-1, 4-dihydroxychollidine (DDC) supplemented diet. After 3 weeks, mice underwent live imaging (DEXA scanning, 2DEcho, EKG, cardiac MRI), exercise treadmill testing and histological and biochemical analyses of livers and hearts. Compared to chow-fed mice, DDC-fed mice fatigued earlier on the treadmill, with reduced VO2. Marked changes were identified electrophysiologically (bradycardia and prolonged QTc) and functionally (hyperdynamic left ventricular (LV) contractility along with increased LV thickness). Hearts of DDC-fed mice showed hypertrophic signaling (activation of AKT, inhibition of GSK3β and a 20-fold upregulation of β myosin heavy chain RNA) and elevated Gsα/Giα ratio. Genes regulating cardiac fatty acid oxidation pathways were suppressed, along with a 3-fold increase in myocardial glycogen content. Treatment of mouse cardiomyocytes (which express the membrane bile acid receptor TGR5) with potent natural TGR5 agonists, taurochenodeoxycholic acid and lithocholic acid, activated AKT and inhibited GSK3β, similar to the changes seen in DDC-fed mouse hearts. This provides support for a novel mechanism whereby circulating natural bile acids can induce signaling pathways in heart associated with hypertrophy. In conclusion, 3 weeks of DDC feeding-induced biliary fibrosis leads to multiple functional, metabolic, electrophysiological and hypertrophic adaptations in the mouse heart recapitulating some of the features of human cirrhotic cardiomyopathy.

The rat ileal apical Na+-dependent bile acid transporter (ASBT) and the liver Na+-taurocholate cotransporting polypeptide (Ntcp) are members of a new family of anion transporters. These transport proteins share limited sequence homology and almost identical predicted secondary structures but are localized to the apical surface of ileal enterocytes and the sinusoidal surface of hepatocytes, respectively. Stably transfected Madin-Darby canine kidney (MDCK) cells appropriately localized wild-type ASBT and Ntcp apically and basolaterally as assessed by functional activity and immunocytochemical localization studies. Truncated and chimeric transporters were used to determine the functional importance of the cytoplasmic tail in bile acid transport activity and membrane localization. Two cDNAs were created encoding a truncated transporter in which the 56-amino-acid COOH-terminal tail of Ntcp was removed or substituted with an eight-amino-acid epitope FLAG. For both mutants there was some loss of fidelity in basolateral sorting in that ∼75% of each protein was delivered to the basolateral surface compared with ∼90% of the wild-type Ntcp protein. In contrast, deletion of the cytoplasmic tail of ASBT led to complete loss of transport activity and sorting to the apical membrane. An Ntcp chimera in which the 56-amino-acid COOH-terminal tail of Ntcp was replaced with the 40-amino-acid cytoplasmic tail of ASBT was largely redirected (82.4 ± 3.9%) to the apical domain of stably transfected MDCK cells, based on polarity of bile acid transport activity and localization by confocal immunofluorescence microscopy. These results indicate that a predominant signal for sorting of the Ntcp protein to the basolateral domain is located in a region outside of the cytoplasmic tail. These studies have further shown that a novel apical sorting signal is localized to the cytoplasmic tail of ASBT and that it is transferable and capable of redirecting a protein normally sorted to the basolateral surface to the apical domain of MDCK cells.