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The nuclear receptor FXR is the sensor of physiological levels of enterohepatic bile acids, the end products of cholesterol catabolism. Here we report crystal structures of the FXR ligand binding domain in complex with coactivator peptide and two different bile acids. An unusual A/B ring juncture, a feature associated with bile acids and no other steroids, provides ligand discrimination and triggers a pi-cation switch that activates FXR. Helix 12, the activation function 2 of the receptor, adopts the agonist conformation and stabilizes coactivator peptide binding. FXR is able to interact simultaneously with two coactivator motifs, providing a mechanism for enhanced binding of coactivators through intermolecular contacts between their LXXLL sequences. These FXR complexes provide direct insights into the design of therapeutic bile acids for treatment of hyperlipidemia and cholestasis.
Previously, we have demonstrated that deoxycholic acid (DCA)-induced signaling of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in primary hepatocytes is a protective response. In the present study, we examined the roles of the ERK and c-Jun NH(2)-terminal kinase (JNK) pathways, and downstream transcription factors, in the survival response of hepatocytes. DCA caused activation of the ERK1/2 and JNK1/2 pathways. Inhibition of either DCA-induced ERK1/2 or DCA-induced JNK1/2 signaling enhanced the apoptotic response of hepatocytes. Further analyses demonstrated that DCA-induced JNK2 signaling was cytoprotective whereas DCA-induced JNK1 signaling was cytotoxic. DCA-induced ERK1/2 activation was responsible for increased DNA binding of C/EBPbeta, CREB, and c-Jun/AP-1. Inhibition of C/EBPbeta, CREB, and c-Jun function promoted apoptosis following DCA treatment, and the level of apoptosis was further increased in the case of CREB and c-Jun, but not C/EBPbeta, by inhibition of MEK1/2. The combined loss of CREB and c-Jun function or of C/EBPbeta and c-Jun function enhanced DCA-induced apoptosis above the levels resulting from the loss of either factor individually; however, these effects were less than additive. Loss of c-Jun or CREB function correlated with increased expression of FAS death receptor and PUMA and decreased expression of c-FLIP-(L) and c-FLIP-(S), proteins previously implicated in the modulation of the cellular apoptotic response. Collectively, these data demonstrate that multiple DCA-induced signaling pathways and transcription factors control hepatocyte survival.
Intracellular signaling mediated by phosphatidylinositol 3-kinase (PI3K) is important for a number of cellular processes and is stimulated by a variety of hormones, including insulin and leptin. A histochemical method for assessment of PI3K signaling would be an important advance in identifying specific cells in histologically complex organs that are regulated by growth factors and peptide hormones. However, current methods for detecting PI3K activity require either homogenization of the tissue or cells or the ability to transfect probes that bind to phosphatidylinositol 3,4,5 trisphosphate (PIP3), the reaction product of PI3K catalysis. Here we report the validation of an immunocytochemical method to detect changes in PI3K activity, using a recently developed monoclonal antibody to PIP3, in paraformaldehyde-fixed bovine aortic endothelial cells (BAECs) in culture and in hepatocytes of intact rat liver. Treatment with either insulin or leptin increased BAEC PIP3 immunoreactivity, and these effects were blocked by pretreatment with PI3K inhibitors. Furthermore, infusion of insulin into the hepatic portal vein of fasted rats caused an increase of PIP3 immunostaining in hepatocytes that was associated with increased serine phosphorylation of the downstream signaling molecule protein kinase B/Akt (PKB/Akt). We conclude that immunocytochemical PIP3 staining can detect changes in PI3K activation induced by insulin and leptin in cell culture and intact liver.
The importance of hepatocyte-derived apolipoprotein (apo) E in the clearance of remnant lipoproteins in the liver is controversial. To address this controversy, we compared remnant clearance in two mouse models in which apoE is primarily derived either from hepatocytes or from an extrahepatic source. Hypomorphic apoE mice universally express reduced levels of apoE in all tissues, with the liver remaining the primary source of apoE. This mouse model of hepatocyte-derived apoE was compared with Apoe(-/-) mice transplanted with mouse bone marrow as a model of primarily non-hepatocyte-derived apoE. Immunohistochemical analysis of liver sections revealed that only the hepatocyte-derived apoE model had detectable levels of apoE on hepatic sinusoidal surfaces. The non-hepatocyte-derived apoE model with plasma apoE levels similar to those in the hepatocyte-derived model had 2-fold more total plasma cholesterol, 4-fold more total plasma triglycerides, and 8-fold higher levels of apoB48, similar to Apoe(-/-) mice. Both the hepatocyte-derived and the non-hepatocyte-derived apoE models had delayed clearance of an infused bolus of (125)I-labeled remnants compared with wild-type mice. However, after 3 h, plasma remnants reached wild-type levels only in the hepatocyte-derived apoE model, which had accumulated 70 +/- 5% of wild-type levels of remnants in the liver while the non-hepatocyte-derived apoE model had accumulated only 38 +/- 4%. These results demonstrate the existence of a role for both hepatically derived and localized apoE in remnant clearance. This role likely represents the enrichment of remnants sequestered on hepatocyte, with hepatocyte-derived apoE, facilitating their receptor-mediated internalization.
Internalization of apoE-containing very low density protein (VLDL) by hepatocytes in vivo and in vitro leads to apoE recycling and resecretion. Because of the role of apoE in VLDL metabolism, apoE recycling may influence lipoprotein assembly or remnant uptake. However, apoE is also a HDL protein, and apoE recycling may be related to reverse cholesterol transport. To investigate apoE recycling, apoE(-/-) mouse hepatocytes were incubated (pulsed) with wild-type mouse lipoproteins, and cells and media were collected at chase periods up to 24 h. When cells were pulsed with VLDL, apoE was resecreted within 30 min. Although the mass of apoE in the media decreased with time, it could be detected up to 24 h after the pulse. Intact intracellular apoE was also detectable 24 h after the pulse. ApoE was also resecreted when cells were pulsed with HDL. When apoA-I was included in the chase media after a pulse with VLDL, apoE resecretion increased 4-fold. Furthermore, human apoE was resecreted from wild-type mouse hepatocytes after a pulse with human VLDL. Finally, apoE was resecreted from mouse peritoneal macrophages after pulsing with VLDL. We conclude that 1) HDL apoE recycles in a quantitatively comparable fashion to VLDL apoE; 2) apoE recycling can be modulated by extracellular apoA-I but is not affected by endogenous apoE; and 3) recycling occurs in macrophages as well as in hepatocytes, suggesting that the process is not cell-specific.
Rifampin, a member of the rifamycin class of antibiotics, is well known for its ability to induce drug-metabolizing enzymes and transporters, through activation of the pregnane X receptor. Available data suggest rifampin entry into hepatocytes may be transporter-mediated. Accordingly, it is therefore plausible that modulation of the achievable intracellular concentration of rifampin by drug uptake transporters would influence the degree of induction. In this study, we expressed an array of known hepatic uptake transporters to show the key hepatic rifampin uptake transporters are liver-specific members of the organic anion transporting polypeptide family (OATP). Indeed, both OATP-C and OATP8 seemed capable of mediating rifampin uptake into HeLa cells. OATP-C, however, seemed to have far greater affinity and capacity for rifampin transport. In addition, several allelic variants of OATP-C known to be present among European and African Americans were found to have markedly decreased rifampin transport activity. In cell-based, transactivation assays, OATP-C expression was associated with increased cellular rifampin retention as well as potentiation of PXR reporter gene activity. This is the first demonstration of an uptake transporter such as OATP-C, in modulating PXR function, and sheds important new insight into our understanding of the molecular determinants of PXR-mediated inductive processes.
The ability of N-methyldithiocarbamate (NMDC) to generate methylisothiocyanate and HS(-) together with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure than its dialkyl analog,N,N-dimethyldithiocarbamate (DMDC). To assess this possibility, cell culture, perfused liver, and in vivo studies were performed to delineate differences in the hepatotoxicity and thiol interactions of NMDC and DMDC in the rat. The role of methylisothiocyanate and HS(-) in NMDC-induced hepatotoxicity was evaluated and glutathione interactions characterized through analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and S-methylthiocarbamoylglutathione (GSMITC) using HPLC and liquid chromatography tandem mass spectrometry (LC/MS/MS). Following oral administration, centrilobular hepatocyte necrosis and enzyme leakage was observed for NMDC but not for DMDC. Dose dependent decreases of intracellular GSH were produced by both dithiocarbamates in primary hepatocytes but DMDC appeared to deplete GSH through the generation of GSSG whereas NMDC produced GSMITC consistent with the generation of a methylisothiocyanate intermediate. In primary hepatocytes, both NMDC and DMDC cytotoxicity was increased by prior depletion of intracellular GSH and diminished by prior supplementation of GSH. The results obtained using perfused livers were similar for NMDC in that elevated levels of GSMITC were detected in the bile; however, DMDC produced only a modest increase of GSSG over controls that was not significantly different to that produced by NMDC. Results obtained from isolated liver mitochondria and primary hepatocytes were not consistent with NMDC producing HS(-)-mediated inhibition of mitochondrial respiration. These data support a greater potential for hepatotoxicity to result following oral exposure to NMDC relative to DMDC and that glutathione may play a role in cytoprotection for NMDC, presumably through detoxification of a methylisothiocyanate metabolite.
The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was investigated in primary human and rat hepatocytes. The genotoxic metabolites 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx) and 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which are formed by cytochrome P4501A2 (CYP1A2), were detected as stable N(2)-glucuronide and N(2)- and N(3)-glucuronide conjugates, respectively. These products accounted for as much as 10% of the amount of MeIQx and 60% of PhIP added to human hepatocytes. Significantly lower amounts of these products were formed in rat hepatocytes. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl-sulfamic acid (MeIQx-N(2)-SO(3)H) and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx-N(2)-Gl), as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx) were also identified. A novel CYP1A2-derived metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH) and was the predominant metabolite formed in human hepatocytes exposed to MeIQx at levels approaching human exposure. Unlike human hepatocytes, rat cell preparations, even following pretreatment with the potent CYP1A1/CYP1A2 inducer 3-methylcholanthrene (3-MC) did not produce IQx-8-COOH but did catalyze the formation of 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline (5-HO-MeIQx) as a major CYP-mediated detoxication product. In the case of PhIP, direct glucuronidation of the N(2) and N(3) positions also occurred in human and rat hepatocytes. Glucuronide and sulfate conjugates of 2-amino-4'-hydroxy-1-methyl-6-phenylimidazo[4,5-b]pyridine (4'-HO-PhIP) were detected as relatively minor metabolites in human hepatocytes but were the major products formed in rat hepatocytes, accounting for up to 50% of the metabolism. Rat CYP1A2, but not the human ortholog, significantly contributes to 4'-hydroxylation of PhIP. Important differences exist between human and rat liver enzymes in catalytic activity and regioselectivity of MeIQx and PhIP metabolism. Some human hepatocyte preparations are more active at transforming MeIQx and PhIP to a genotoxic species than rat hepatocytes pretreated with potent inducer 3-MC. These pronounced interspecies differences in metabolism of MeIQx and PhIP may affect the biological activity of these mutagens and must be considered when assessing human health risk.
Factor XI is the zymogen of a plasma protease produced primarily in liver that is required for normal blood coagulation. We cloned approximately 2600 base pairs of the human factor XI gene upstream of exon one, identified transcription start sites, and conducted a functional analysis. Luciferase reporter assays demonstrate that the 381 base pairs upstream of exon one are sufficient for maximum promoter activity in HepG2 hepatocellular carcinoma cells. The removal of 19 base pairs between -381 and -363 results in a nearly complete loss of promoter activity. This region contains the sequence ACTTTG, a motif required for binding of the transcription factor hepatocyte nuclear factor 4alpha (HNF-4alpha) to the promoters of several genes. Gel mobility shift assays using HepG2 or rat hepatocyte nuclear extract confirm HNF-4alpha binds between bp -375 and -360. Scrambling the ACTTTG motif completely abolishes promoter activity in luciferase assays. The factor XI promoter functions poorly when transfected into HeLa carcinoma cells, and gel mobility shift experiments with HeLa nuclear extracts demonstrate no HNF-4alpha binding to the ACTTTG sequence. When a rat HNF-4alpha expression construct is co-transfected into HeLa cells, factor XI promoter activity is enhanced approximately 10-fold. We conclude that HNF-4alpha is required for hepatocyte-specific expression of factor XI.
Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.
(c)2002 Elsevier Science.