Interrupting tumor necrosis factor-alpha signaling prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND: We have recently reported a mouse model of PN-associated cholestasis (PNAC) in which combining intestinal inflammation and PN infusion results in cholestasis, hepatic macrophage activation, and transcriptional suppression of canalicular bile acid, bilirubin and sterol transporters Abcb11, Abcc2 and Abcg5/8. The aim of this study was to examine the role of TNFα in promoting PNAC in mice. METHODS: First, recombinant TNFα was administered to mice as well as in hepatocyte cell culture. Second, Tnfr1/2KO or wild-type (WT) mice were exposed to dextran sulfate sodium (DSS) for 4 days followed by soy-oil lipid emulsion-based PN infusion through a central venous catheter for 14 days (DSS-PN). Finally, WT/DSS-PN mice were also infused with infliximab at 10 mg/kg on days 3 and 10 of PN. PNAC was defined by increased serum aspartate aminotransferase, alanine aminotransferase, total bile acids, and bilirubin. RESULTS: Intraperitoneal injection of TNFα into WT mice or TNFα treatment of Huh7 hepatocarcinoma cells and primary mouse hepatocytes suppressed messenger RNA (mRNA) transcription of bile (Abcb11, Abcc2]) and sterol transporters (Abcg5/8) and their regulators Nr1h3 and Nr1h4. DSS-PN mice with PNAC had increased hepatic TNFα mRNA expression and significant reduction of mRNA expression of Abcb11, Abcc2, Abcg5/8, Nr1h3, and Nr1h4. In contrast, PNAC development was prevented and mRNA expression normalized in both Tnfr1/2KO /DSS-PN mice and DSS-PN mice treated with infliximab. CONCLUSIONS: TNFα is a key mediator in the pathogenesis of PNAC through suppression of hepatocyte Abcb11, Abcc2, and Abcg5/8. Pharmacologic targeting of TNFα as a therapeutic strategy for PNAC thus deserves further investigation.

Pharmacologic activation of hepatic farnesoid X receptor prevents parenteral nutrition-associated cholestasis in mice.

BACKGROUND AND AIMS: Parenteral nutrition (PN)-associated cholestasis (PNAC) complicates the care of patients with intestinal failure. In PNAC, phytosterol containing PN synergizes with intestinal injury and IL-1ß derived from activated hepatic macrophages to suppress hepatocyte farnesoid X receptor (FXR) signaling and promote PNAC. We hypothesized that pharmacological activation of FXR would prevent PNAC in a mouse model. APPROACH AND RESULTS: To induce PNAC, male C57BL/6 mice were subjected to intestinal injury (2% dextran sulfate sodium [DSS] for 4 days) followed by central venous catheterization and 14-day infusion of PN with or without the FXR agonist GW4064. Following sacrifice, hepatocellular injury, inflammation, and biliary and sterol transporter expression were determined. GW4064 (30 mg/kg/day) added to PN on days 4-14 prevented hepatic injury and cholestasis; reversed the suppressed mRNA expression of nuclear receptor subfamily 1, group H, member 4 (Nr1h4)/FXR, ATP-binding cassette subfamily B member 11 (Abcb11)/bile salt export pump, ATP-binding cassette subfamily C member 2 (Abcc2), ATP binding cassette subfamily B member 4(Abcb4), and ATP-binding cassette subfamily G members 5/8(Abcg5/8); and normalized serum bile acids. Chromatin immunoprecipitation of liver showed that GW4064 increased FXR binding to the Abcb11 promoter. Furthermore, GW4064 prevented DSS-PN-induced hepatic macrophage accumulation, hepatic expression of genes associated with macrophage recruitment and activation (ll-1b, C-C motif chemokine receptor 2, integrin subunit alpha M, lymphocyte antigen 6 complex locus C), and hepatic macrophage cytokine transcription in response to lipopolysaccharide in vitro. In primary mouse hepatocytes, GW4064 activated transcription of FXR canonical targets, irrespective of IL-1ß exposure. Intestinal inflammation and ileal mRNAs (Nr1h4, Fgf15, and organic solute transporter alpha) were not different among groups, supporting a liver-specific effect of GW4064 in this model. CONCLUSIONS: GW4064 prevents PNAC in mice through restoration of hepatic FXR signaling, resulting in increased expression of canalicular bile and of sterol and phospholipid transporters and suppression of macrophage recruitment and activation. These data support augmenting FXR activity as a therapeutic strategy to alleviate or prevent PNAC.

miR-199a-5p inhibits the expression of ABCB11 in obstructive cholestasis.

ATP-binding cassette, subfamily B member 11 (ABCB11) is an efflux transporter for bile acids on the liver canalicular membrane. The expression of this transporter is reduced in cholestasis; however, the mechanisms contributing to this reduction are unclear. In this study, we sought to determine whether miR-199a-5p contributes to the depletion of ABCB11/Abcb11 in cholestasis in mice. In a microRNA (miRNA) screen of mouse liver after common bile duct ligation (CBDL), we found that miR-199a-5p was significantly upregulated by approximately fourfold. In silico analysis predicted that miR-199a-5p would target the 3'-untranslated region (3'-UTR) of ABCB11/Abcb11 mRNA. The expression of ABCB11-3'-UTR luciferase construct in Huh-7 cells was markedly inhibited by cotransfection of a miRNA-199a-5p mimic, which was reversed by an miRNA-199a-5p mimic inhibitor. We also show treatment of mice after CBDL with the potent nuclear receptor FXR agonist obeticholic acid (OCA) significantly increased Abcb11 mRNA and protein and decreased miR-199a-5p expression. Computational mapping revealed a well-conserved FXR-binding site (FXRE) in the promoter of the gene encoding miR-199a-5, termed miR199a-2. Electromobility shift, chromatin immunoprecipitation, and miR199a-2 promoter-luciferase assays confirmed that this binding site was functional. Finally, CBDL in mice led to depletion of nuclear repressor NcoR1 binding at the miR199a-2 promoter, which facilitates transcription of miR199a-2. In CBDL mice treated with OCA, NcoR1 recruitment to the miR199a-2 FXRE was maintained at levels found in sham-operated mice. In conclusion, we demonstrate that miR-199a-5p is involved in regulating ABCB11/Abcb11 expression, is aberrantly upregulated in obstructive cholestasis, and is downregulated by the FXR agonist OCA.

Mutation Analysis and Disease Features at Presentation in a Multi-Center Cohort of Children With Monogenic Cholestasis.

OBJECTIVES: To advance our understanding of monogenic forms of intrahepatic cholestasis. METHODS: Analyses included participants with pathogenic biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 11 (ABCB11) (bile salt export pump; BSEP) or adenosine triphosphatase (ATPase) phospholipid transporting 8B1 (ATP8B1) (familial intrahepatic cholestasis; FIC1), or those with monoallelic or biallelic mutations in adenosine triphosphate (ATP)-binding cassette subfamily B member 4 (ABCB4) (multidrug resistance; MDR3), prospectively enrolled in the Longitudinal Study of Genetic Causes of Intrahepatic Cholestasis (LOGIC; NCT00571272) between November 2007 and December 2013. Summary statistics were calculated to describe baseline demographics, history, anthropometrics, laboratory values, and mutation data. RESULTS: Ninety-eight participants with FIC1 (n = 26), BSEP (n = 53, including 8 with biallelic truncating mutations [severe] and 10 with p.E297G or p.D482G [mild]), or MDR3 (n = 19, including four monoallelic) deficiency were analyzed. Thirty-five had a surgical interruption of the enterohepatic circulation (sEHC), including 10 who underwent liver transplant (LT) after sEHC. Onset of symptoms occurred by age 2 years in most with FIC1 and BSEP deficiency, but was later and more variable for MDR3. Pruritus was nearly universal in FIC1 and BSEP deficiency. In participants with native liver, failure to thrive was common in FIC1 deficiency, high ALT was common in BSEP deficiency, and thrombocytopenia was common in MDR3 deficiency. sEHC was successful after more than 1 year in 7 of 19 participants with FIC1 and BSEP deficiency. History of LT was most common in BSEP deficiency. Of 102 mutations identified, 43 were not previously reported. CONCLUSIONS: In this cohort, BSEP deficiency appears to be correlated with a more severe disease course. Genotype-phenotype correlations in these diseases are not straightforward and will require the study of larger cohorts.

Up-regulation of miR-let7a-5p Leads to Decreased Expression of ABCC2 in Obstructive Cholestasis.

Adenosine triphosphate-binding cassette subfamily C member 2 (ABCC2/Abcc2) is critically important to biliary excretion of many endobiotic and xenobiotic compounds, and is a major driving force for bile acid-independent bile flow. Abcc2 expression is reduced at the messenger RNA (mRNA) and protein levels in various forms of experimental cholestasis. In a microRNA (miRNA) screen of mouse liver after biliary obstruction, we found that miRNA let7a-5p was significantly up-regulated approximately 4-fold. Similarly, ABCC2 mRNA was depleted and miRNA let7a-5p was elevated over 4-fold in livers of children with biliary atresia compared with normal livers. In silico analysis predicted that let7a-5p would target the 3' untranslated region (3' UTR) of ABCC2/Abcc2 RNA. The objective of this study was to determine whether let7a-5p contributes to the depletion of ABCC2/Abcc2 in cholestasis. To demonstrate the functional importance of miRNA let7a-5p in regulating the expression of ABCC2, co-transfection of a let7a-5p mimic and an ABCC2-3' UTR luciferase construct into Huh-7 cells led to a marked inhibition of luciferase activity by about 60%-70% compared with controls, which was reversed by a let7a-5p mimic inhibitor. Expression of this mimic led to a significant decrease in endogenous ABCC2 mRNA and protein levels in a Huh-7 liver cell line, which could be blocked by expression of a let7a-5p mimic inhibitor. Injection of a lentivirus let7a-5p inhibitor into normal mouse liver or into mouse liver after common bile duct ligation led to a significant increase in endogenous Abcc2 mRNA and protein levels and a depletion of let7a-5p mRNA levels compared with untreated, saline-injected livers or livers treated with an inactive lentivirus control. Conclusion: These studies demonstrate that miR-let7a-5p is involved in regulating ABCC2/Abcc2 expression, and is aberrantly up-regulated in obstructive cholestasis.

Impact of Steroid Therapy on Early Growth in Infants with Biliary Atresia: The Multicenter Steroids in Biliary Atresia Randomized Trial.

OBJECTIVE: To investigate the impact of corticosteroid therapy on the growth of participants in the Steroids in Biliary Atresia Randomized Trial (START) conducted through the Childhood Liver Disease Research Network. The primary analysis in START indicated that steroids did not have a beneficial effect on drainage in a cohort of infants with biliary atresia. We hypothesized that steroids would have a detrimental effect on growth in these infants. STUDY DESIGN: A total of 140 infants were enrolled in START, with 70 randomized to each treatment arm: steroid and placebo. Length, weight, and head circumference were obtained at baseline and follow-up visits to 24 months of age. RESULTS: Patients treated with steroids had significantly lower length and head circumference z scores during the first 3 months post-hepatoportoenterostomy (HPE), and significantly lower weight until 12 months. Growth trajectories in the steroid and placebo arms differed significantly for length (P < .0001), weight (P = .009), and head circumference (P < .0001) with the largest impact noted for those with successful HPE. Growth trajectory for head circumference was significantly lower in patients treated with steroids irrespective of HPE status, but recovered during the second 6 months of life. CONCLUSIONS: Steroid therapy following HPE in patients with biliary atresia is associated with impaired length, weight, and head circumference growth trajectories for at least 6 months post-HPE, especially impacting infants with successful bile drainage. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00294684.

Macrophage-derived IL-1ß/NF-κB signaling mediates parenteral nutrition-associated cholestasis.

In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1ß in PNAC. Pharmacological antagonism of  IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1ß) prevents PNAC in mice. IL-1ß increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1ß, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Nuclear factor-κB regulates the expression of multiple genes encoding liver transport proteins.

In this study we identified the mechanisms underlying the inhibitory effects of NF-κB on the expression of genes encoding multiple liver transport proteins. Well-conserved NF-κB binding sites were found in the promoters of farnesoid X receptor (FXR)-target genes. An electromobility shift assay (EMSA) demonstrated the specific interaction between the NF-κB p65 protein and a (32)P-labeled BSEP NF-κB response element (NF-κBE). Chromatin immunoprecipitation (ChIP) analysis confirmed binding of NF-κB p65 to the BSEP locus but not the FXRE in vitro. NF-κB p65 overexpression in Huh-7 cells markedly repressed FXR/RXR transactivation of the BSEP, ABCG5/G8, MRP2, and FXR promoters, which was totally reversed by expression of the IκBα super-repressor. NF-κB interacted directly with FXR on coimmunoprecipitation, suggesting another level for the inhibitory effects of NF-κB on FXR-target genes. In vivo ChIP analysis with liver nuclei obtained from mice after 3 days of common bile duct ligation (BDL) or 6 h post-lipopolysaccharide (LPS) injection showed a markedly increased recruitment of NF-κB p65 to the Bsep promoter compared with controls. There was also increased recruitment of the corepressor silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and histone deacetylase (HDAC)3 and HDAC2 to the NF-κB sites. We also found that NF-κB p65 was recruited to NF-κB binding sites in the promoters of organic solute transporter, OSTα and OSTß, and unexpectedly activated rather than repressed gene expression. In mouse liver after BDL NF-κB recruitment to Ostα and Ostß promoters was associated with increased binding of the potent coactivator cAMP response element binding protein (CREB)-binding protein (CBP)/p300 to the NF-κBE and depletion of CBP/p300 at the FXR element. Overall, these studies demonstrate a novel role for NF-κB in adaptation to obstructive and LPS-induced cholestasis acting through recruitment to specific NF-κB binding sites in the promoters of FXR-target genes and possibly through direct interaction with FXR.

Deposition of 5-Methylcytosine on Enhancer RNAs Enables the Coactivator Function of PGC-1α.

The Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) is a transcriptional co-activator that plays a central role in adapted metabolic responses. PGC-1α is dynamically methylated and unmethylated at the residue K779 by the methyltransferase SET7/9 and the Lysine Specific Demethylase 1A (LSD1), respectively. Interactions of methylated PGC-1α[K779me] with the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, the Mediator members MED1 and MED17, and the NOP2/Sun RNA methytransferase 7 (NSUN7) reinforce transcription, and are concomitant with the m(5)C mark on enhancer RNAs (eRNAs). Consistently, loss of Set7/9 and NSun7 in liver cell model systems resulted in depletion of the PGC-1α target genes Pfkl, Sirt5, Idh3b, and Hmox2, which was accompanied by a decrease in the eRNAs levels associated with these loci. Enrichment of m(5)C within eRNA species coincides with metabolic stress of fasting in vivo. Collectively, these findings illustrate the complex epigenetic circuitry imposed by PGC-1α at the eRNA level to fine-tune energy metabolism.

CHD6 regulates the topological arrangement of the CFTR locus.

The control of transcription is regulated through the well-coordinated spatial and temporal interactions between distal genomic regulatory elements required for specialized cell-type and developmental gene expression programs. With recent findings CFTR has served as a model to understand the principles that govern genome-wide and topological organization of distal intra-chromosomal contacts as it relates to transcriptional control. This is due to the extensive characterization of the DNase hypersensitivity sites, modification of chromatin, transcription factor binding sites and the arrangement of these sites in CFTR consistent with the restrictive expression in epithelial cell types. Here, we identified CHD6 from a screen among several chromatin-remodeling proteins as a putative epigenetic modulator of CFTR expression. Moreover, our findings of CTCF interactions with CHD6 are consistent with the role described previously for CTCF in CFTR regulation. Our results now reveal that the CHD6 protein lies within the infrastructure of multiple transcriptional complexes, such as the FACT, PBAF, PAF1C, Mediator, SMC/Cohesion and MLL complexes. This model underlies the fundamental role CHD6 facilitates by tethering cis-acting regulatory elements of CFTR in proximity to these multi-subunit transcriptional protein complexes. Finally, we indicate that CHD6 structurally coordinates a three-dimensional stricture between intragenic elements of CFTR bound by several cell-type specific transcription factors, such as CDX2, SOX18, HNF4α and HNF1α. Therefore, our results reveal new insights into the epigenetic regulation of CFTR expression, whereas the manipulation of CFTR gene topology could be considered for treating specific indications of cystic fibrosis and/or pancreatitis.

Use of corticosteroids after hepatoportoenterostomy for bile drainage in infants with biliary atresia: the START randomized clinical trial.

IMPORTANCE: Biliary atresia is the most common cause of end-stage liver disease in children. Controversy exists as to whether use of steroids after hepatoportoenterostomy improves clinical outcome. OBJECTIVE: To determine whether the addition of high-dose corticosteroids after hepatoportoenterostomy is superior to surgery alone in improving biliary drainage and survival with the native liver. DESIGN, SETTING, AND PATIENTS: The multicenter, double-blind Steroids in Biliary Atresia Randomized Trial (START) was conducted in 140 infants (mean age, 2.3 months) between September 2005 and February 2011 in the United States; follow-up ended in January 2013. INTERVENTIONS: Participants were randomized to receive intravenous methylprednisolone (4 mg/kg/d for 2 weeks) and oral prednisolone (2 mg/kg/d for 2 weeks) followed by a tapering protocol for 9 weeks (n = 70) or placebo (n = 70) initiated within 72 hours of hepatoportoenterostomy. MAIN OUTCOMES AND MEASURES: The primary end point (powered to detect a 25% absolute treatment difference) was the percentage of participants with a serum total bilirubin level of less than 1.5 mg/dL with his/her native liver at 6 months posthepatoportoenterostomy. Secondary outcomes included survival with native liver at 24 months of age and serious adverse events. RESULTS: The proportion of participants with improved bile drainage was not statistically significantly improved by steroids at 6 months posthepatoportoenterostomy (58.6% [41/70] of steroids group vs 48.6% [34/70] of placebo group; adjusted relative risk, 1.14 [95% CI, 0.83 to 1.57]; P = .43). The adjusted absolute risk difference was 8.7% (95% CI, -10.4% to 27.7%). Transplant-free survival was 58.7% in the steroids group vs 59.4% in the placebo group (adjusted hazard ratio, 1.0 [95% CI, 0.6 to 1.8]; P = .99) at 24 months of age. The percentage of participants with serious adverse events was 81.4% [57/70] of the steroids group and 80.0% [56/70] of the placebo group (P > .99); however, participants receiving steroids had an earlier time of onset of their first serious adverse event by 30 days posthepatoportoenterostomy (37.2% [95% CI, 26.9% to 50.0%] of steroids group vs 19.0% [95% CI, 11.5% to 30.4%] of placebo group; P = .008). CONCLUSIONS AND RELEVANCE: Among infants with biliary atresia who have undergone hepatoportoenterostomy, high-dose steroid therapy following surgery did not result in statistically significant treatment differences in bile drainage at 6 months, although a small clinical benefit could not be excluded. Steroid treatment was associated with earlier onset of serious adverse events in children with biliary atresia. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00294684.

A novel RARα/CAR-mediated mechanism for regulation of human organic solute transporter-ß gene expression.

The organic solute transporter-α/ß (OSTα/ß) is a heteromeric transporter that is essential for bile acid and sterol disposition and for the enterohepatic circulation. To better understand the mechanism underlying OST gene regulation, the effects of retinoic acid (RA) on OSTα/ß gene expression were investigated. The results show a dose-dependent induction of OSTß but not OSTα expression in both Huh7 and HepG2 cells by RA treatment. A novel functional RA receptor response element (RARE; so-called DR5) in the promoter of OSTß gene was identified. The interaction of RARα/RXRα with the RARE was verified by electrophoretic mobility shift and chromatin immunoprecipitation assays and its functional importance by hOSTß promoter activation in luciferase reporter assays. The studies demonstrated that the RARE is also a constitutive androstane receptor (CAR) binding site for OSTß gene regulation. These results suggest that OSTß is a target of both FXR-mediated (by binding to IR-1 element) and RARα- and CAR-mediated (by binding to DR5 element) gene regulation pathways. In summary, this study has uncovered a novel RARE (DR5) element in the promoter of OSTß that binds RARα or CAR heterodimerized with RXRα and appears to function synergistically with the IR-1 element to provide maximal induction of OSTß in response to RA. These findings demonstrate a role for RARα and CAR in controlling OSTß expression levels.

SUMOylation of the farnesoid X receptor (FXR) regulates the expression of FXR target genes.

BACKGROUND: Small ubiquitin-like modifiers (SUMO) are covalently conjugated to other proteins including nuclear receptors leading to modification of various cellular processes. RESULTS: Ligand-dependent SUMOylation of farnesoid X receptor (FXR) negatively regulates the expression of its target genes. CONCLUSION: SUMO modification attenuates the capacity of FXR to function as a transcriptional activator. SIGNIFICANCE: Defining post-translation modification of FXR bySUMOis important to understanding how this nuclear receptor functions in health and disease. The farnesoid X receptor (FXR) belongs to a family of ligand-activated transcription factors that regulate many aspects of metabolism including bile acid homeostasis. Here we show that FXR is covalently modified by the small ubiquitin-like modifier (Sumo1), an important regulator of cell signaling and transcription. Well conserved consensus sites at lysine 122 and 275 in the AF-1 and ligand binding domains, respectively, of FXR were subject to SUMOylation in vitro and in vivo. Chromatin immunoprecipitation (ChIP) analysis showed that Sumo1 was recruited to the bile salt export pump (BSEP), the small heterodimer partner (SHP), and the OSTα-OSTß organic solute transporter loci in a ligand-dependent fashion. Sequential chromatin immunoprecipitation (ChIP-ReChIP) verified the concurrent binding of FXR and Sumo1 to the BSEP and SHP promoters. Overexpression of Sumo1 markedly decreased binding and/or recruitment of FXR to the BSEP and SHP promoters on ChIP-ReChIP. SUMOylation did not have an apparent effect on nuclear localization of FXR. Expression of Sumo1 markedly inhibited the ligand-dependent, transactivation of BSEP and SHP promoters by FXR/retinoid X receptor α (RXRα) in HepG2 cells. In contrast, mutations that abolished SUMOylation of FXR or siRNA knockdown of Sumo1 expression augmented the transactivation of BSEP and SHP promoters by FXR. Pathways for SUMOylation were significantly altered during obstructive cholestasis with differential Sumo1 recruitment to the promoters of FXR target genes. In conclusion, FXR is subject to SUMOylation that regulates its capacity to transactivate its target genes in normal liver and during obstructive cholestasis.

Identification of functionally relevant lysine residues that modulate human farnesoid X receptor activation.

Base amino acid lysine residues play an important role in regulation of nuclear receptors [e.g., farnesyl X receptor (FXR)], leading to enhanced or suppressed biologic activity. To understand the molecular mechanisms and the subsequent effects in modulating FXR functions in diverse biologic processes, we individually replaced eight highly conserved lysine residues of human FXR (hFXR) with arginine. The effects of each mutated FXR on target gene activation, subcellular localization, protein-protein association, and protein-DNA interaction were investigated. Results demonstrated that K122R, K210R, K339R, and K460R mutants of hFXR significantly impaired target gene [organic solute transporter α/ß and bile salt export pump (BSEP)] promoter reporter activity in a ligand-dependent fashion. None of the four mutants affected the nuclear localization of FXR. Protein interaction studies show that K210R slightly but significantly decreased FXR/retinoid X receptor (RXR) binding affinity but enhanced the interaction of FXR with lysine methyltransferase Set7/9 by ∼21%. K460R decreased the FXR interaction with Set7/9 by ∼45% but had no significant effects on interaction with RXR. Electrophoretic mobility shift assays demonstrated that hFXR-K210R and -K339R reduced the protein-DNA (IR1 element at hBSEP promoter) binding affinity by ∼80 and ∼90%, respectively. Computational-based protein modeling studies were consistent with these results and provided further insights into the potential underlying mechanisms responsible for these results. In conclusion, four highly conserved lysine residues of hFXR, K122, K210, K339, and K460, have been identified that play a critical role in FXR target gene regulation and molecular interaction (protein-protein and protein-DNA).

Human Organic Solute Transporter (hOST): protein interaction and membrane sorting process.

The human organic solute transporter (hOST) is a heterodimer composed of alpha and beta subunits. Physical association of hOSTα and ß subunits is essential for their polarized basolateral plasma membrane localization and function in the export of bile acids and steroids. To understand the role of carboxyl- and amino-tails of OSTß and mechanisms underlying membrane localization of hOST, the effects of tail deletion of the hOSTß subunit and biological reagents on membrane distribution and transport function of hOST were investigated in stably transfected MDCK cells. After deletion of 35 amino acids from the amino-tail of hOSTß, the efflux transport activity and polarized membrane distribution of the truncated hOSTß was abolished. A co-immunoprecipitation study verified that the amino-tail of hOSTß is essential for the association with hOSTα subunit. Treatments with acytochalasin D (interrupting ctin-filaments), bafilomycin A1 (inhibiting vacuolar H(+)-ATPase), brefeldin A (disrupting the Golgi complex), and calphostin C (inhibiting protein kinase C), significantly disrupted the polarized membrane distribution of hOST and markedly reduced transport activity in stably transfected MDCK cells. In summary, the 35 amino acid amino-terminal fragment of hOSTß contains critical information for interaction with the hOSTα subunit and subsequent trafficking to the plasma membrane. These studies suggest that the membrane sorting process of hOST is mediated by a bafilomycin A1-sensitive vesicular pathway that is associated with the actin-cytoskeleton network. The membrane localization of hOST is also partially mediated through a brefeldin A sensitive mechanism, which controls its transit from the ER to Golgi and is regulated by PKC.

Efficacy of fat-soluble vitamin supplementation in infants with biliary atresia.

OBJECTIVE: Cholestasis predisposes to fat-soluble vitamin (FSV) deficiencies. A liquid multiple FSV preparation made with tocopheryl polyethylene glycol-1000 succinate (TPGS) is frequently used in infants with biliary atresia (BA) because of ease of administration and presumed efficacy. In this prospective multicenter study, we assessed the prevalence of FSV deficiency in infants with BA who received this FSV/TPGS preparation. METHODS: Infants received FSV/TPGS coadministered with additional vitamin K as routine clinical care in a randomized double-blinded, placebo-controlled trial of corticosteroid therapy after hepatoportoenterostomy (HPE) for BA (identifier NCT 00294684). Levels of FSV, retinol binding protein, total serum lipids, and total bilirubin (TB) were measured 1, 3, and 6 months after HPE. RESULTS: Ninety-two infants with BA were enrolled in this study. Biochemical evidence of FSV insufficiency was common at all time points for vitamin A (29%-36% of patients), vitamin D (21%-37%), vitamin K (10%-22%), and vitamin E (16%-18%). Vitamin levels were inversely correlated with serum TB levels. Biochemical FSV insufficiency was much more common (15%-100% for the different vitamins) in infants whose TB was ≥2 mg/dL. At 3 and 6 months post HPE, only 3 of 24 and 0 of 23 infants, respectively, with TB >2 mg/dL were sufficient in all FSV. CONCLUSIONS: Biochemical FSV insufficiency is commonly observed in infants with BA and persistent cholestasis despite administration of a TPGS containing liquid multiple FSV preparation. Individual vitamin supplementation and careful monitoring are warranted in infants with BA, especially those with TB >2 mg/dL.

miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity.

Bile secretion is essential for whole body sterol homeostasis. Loss-of-function mutations in specific canalicular transporters in the hepatocyte disrupt bile flow and result in cholestasis. We show that two of these transporters, ABCB11 and ATP8B1, are functional targets of miR-33, a micro-RNA that is expressed from within an intron of SREBP-2. Consequently, manipulation of miR-33 levels in vivo with adenovirus or with antisense oligonucleotides results in changes in bile secretion and bile recovery from the gallbladder. Using radiolabelled cholesterol, we show that systemic silencing of miR-33 leads to increased sterols in bile and enhanced reverse cholesterol transport in vivo. Finally, we report that simvastatin causes, in a dose-dependent manner, profound hepatotoxicity and lethality in mice fed a lithogenic diet. These latter results are reminiscent of the recurrent cholestasis found in some patients prescribed statins. Importantly, pretreatment of mice with anti-miR-33 oligonucleotides rescues the hepatotoxic phenotype. Therefore, we conclude that miR-33 mediates some of the undesired, hepatotoxic effects of statins.