Cloning and expression of SLC10A4, a putative organic anion transport protein

INTRODUCTION Bile acids, the major solute in bile, are physiologically important for promoting bile flow and facilitating the absorption of dietary lipids[1,2]. Bile acids are also involved in cholesterol homeostasis, xenobiotic excretion, as well as apop…     

Functional characterization of genetic variants in the apical sodium-dependent bile acid transporter (ASBT; SLC10A2)

Background and Aim: The major transporter responsible for bile acid uptake from the intestinal lumen is the apical sodium‐dependent bile acid transporter (ASBT, SLC10A2). Analysis of the SLC10A2 gene has identified a variety of sequence variants including coding region single nucleotide polymorphisms (SNPs) that may influence bile acid homeostasis/intestinal function. In this study, we systematically characterized the effect of coding SNPs on SLC10A2 protein expression and bile acid transport activity. Methods: Single nucleotide polymorphisms in SLC10A2 from genomic DNA of ethnically‐defined healthy individuals were identified using a polymerase chain reaction (PCR)‐based temperature gradient capillary electrophoresis (TGCE) system. A heterologous gene expression system was used to assess transport activity of SLC10A2 nonsynonymous variants and missense mutations. Total and cell surface protein expression of wild‐type and variant ASBT was assessed by Western blot analysis and immunofluorescence confocal microscopy. Expression of ASBT mRNA and protein was also measured in human intestinal samples. Results: The studies revealed two nonsynonymous SNPs, 292G>A and 431G>A, with partially impaired in vitro taurocholate transport. A novel variant, 790A>G, was also shown to exhibit near complete loss of taurocholate transport, similar to the previously identified ASBT missense mutations. Examination of ASBT protein expression revealed no significant differences in expression or trafficking to the cell surface among variants versus wild‐type ASBT. Analysis of ASBT mRNA and protein expression in human intestinal samples revealed modest intersubject variability. Conclusions: Genome sequencing and in vitro studies reveal the presence of multiple functionally relevant variants in SLC10A2 that may influence bile acid homeostasis and physiology.     

Cholangiocyte bile salt transporters in cholesterol gallstone-susceptible and resistant inbred mouse strains

Background and Aim: We investigated the dietary and gender influences on the expression and functionality of cholangiocyte bile salt transporters and development of biliary hyperplasia in cholesterol gallstone‐susceptible C57L/J and resistant AKR/J mice. Methods: C57L and AKR mice were fed chow, a lithogenic diet, or a cholic acid‐containing diet for 14 days. Expression of cholangiocyte bile salt transporter proteins ASBT (SLC10A2), ILBP (FABP6), and MRP3 (ABCC3) were studied by Western blot analysis. Taurocholate uptake studies were performed using microperfusion of isolated bile duct units. The pre‐ and post‐perfusion taurocholate concentrations were analyzed by high performance liquid chromatography. Biliary proliferation in liver sections was scored. Results: The lithogenic diet induced ductular proliferation in C57L mice. On chow, SLC10A2 and ABCC3 were overexpressed in male and female C57L compared to AKR mice. A lithogenic diet reduced the expressions of FABP6 in both male and female C57L mice, SLC10A2 in female C57L mice, and ABCC3 in male C57L mice. These alterations in transporter expressions were not associated with changes in taurocholate uptake. The lithogenic diet induced biliary hyperplasia and reduced bile salt transporter expressions in C57L mice. Conclusions: Although bile salt uptake was not increased in the bile duct unit, we speculate that the biliary hyperplasia on the lithogenic diet may lead to an increase in intrahepatic bile salt recycling during cholesterol cholelithogenesis.     

Taurocholate transport by hepatic and intestinal bile acid transporters is independent of FIC1 overexpression in Madin-Darby canine kidney cells

BACKGROUND: Mutations in the human familial intrahepatic cholestasis gene, FIC1, result in progressive familial intrahepatic cholestasis type 1 in children and benign recurrent intrahepatic cholestasis. The present study was performed to determine whether FIC1 transports bile acids and/or influences the activity of apical bile acid transporters. METHODS: The apical secretion assay utilized transfected Madin-Darby canine kidney (MDCK) cells, which stably express the bile acid uptake protein, Na+/taurocholate co-transporting polypeptide (NTCP). These cells were then transiently transfected with FIC1 and/or the bile salt export pump (BSEP) and were grown on Transwell filters to form a polarized monolayer. [(3)H]Taurocholate was added to the basal medium and the taurocholate secretion was measured in the apical medium. A second assay, apical uptake assays, utilized polarized MDCK-II cells, which were transiently transfected with FIC1, FIC1 mutants and/or the apical sodium-dependent bile acid transporter (ASBT). [(3)H]Taurocholate was added to the apical media and intracellular uptake of taurocholate was measured. RESULTS: Apical secretion assays: FIC1 expression in MDCK/NTCP cells had no effect on taurocholate secretion compared to controls. In contrast, apical secretion of taurocholate in BSEP-transfected cells was approximately twofold higher than in non-transfected MDCK/NTCP cells (P < 0.01). The BSEP-mediated secretion was unaffected by co-transfection with FIC1. Apical uptake assays: taurocholate uptake in ASBT expressing cells was 6.5-fold higher than in controls and was unaffected by co-transfection of cells with FIC1 or FIC1 mutants. CONCLUSIONS: These results indicate that FIC1 does not transport taurocholate and, when overexpressed in MDCK cells, had no effect on the function of BSEP or ABST.     

Expression of the liver Na-independent organic anion transporting polypeptide (oatp-1) in rats with bile duct ligation

Background/Aims: In rats with cholestasis due to bile duct ligation, the expression of the Na⁺-dependent taurocholate co-transporting polypeptide, the major uptake system for conjugated bile acids in hepatocytes, is down-regulated. Our purpose was to examine the expression of the organic anion transporting polypeptide, a Na⁺-independent uptake system for bile acids and organic anions, in rats with bile duct ligation, and to compare the expression of organic anion transporting polypeptide to that of Na⁺-dependent taurocholate co-transporting polypeptide.Methods: Rats with bile duct ligation were studied after 1, 3 or 7 days. The expression of organic anion transporting polypeptide and Na⁺-dependent taurocholate co-transporting polypeptide proteins was examined by Western blot analysis and steady-state mRNA levels were determined by Northern blot analysis using cDNAs encoding organic anion transporting polypeptide and Na⁺-dependent taurocholate co-transporting polypeptide. Sham-operated animals were used as controls.Results: The expression of organic anion transporting polypeptide protein was slightly, but not significantly, decreased 1 day after ligation (10.3%); it was markedly decreased after 3 days (56.9%; p<0.03) and 7 days (45.8%; p<0.05) compared to sham-operated animals. Steady-state mRNA levels of organic anion transporting polypeptide was decreased by 79.7% (p<0.04), 48.8% (p<0.02) and 57.4% (p<0.02) after 1, 3 and 7 days respectively. For comparison, Na⁺-dependent taurocholate co-transporting polypeptide protein and mRNA levels were decreased by 73.8% (p<0.03) and 70.0% (p<0.05) at 1 day and remained low after 3 and 7 days.Conclusions: In rats with bile duct ligation, the expression of organic anion transporting polypeptide protein and mRNA is down-regulated. Down-regulation of organic anion transporting polypeptide seems less pronounced than that of Na⁺-dependent taurocholate co-transporting polypeptide. Nevertheless, it could contribute to a decreased uptake of potentially toxic bile acids or organic anions in this situation.     

SLC10A1 S267F variant influences susceptibility to HBV infection and reduces cholesterol level by impairing bile acid uptake

The SLC10A1 Ser267Phe (S267F) variant has been reported to severely inhibit hepatitis B virus (HBV) infection and taurocholate transport activity. This study aimed to clarify the effects of this variant on HBV infection and bile acid metabolism. SLC10A1 S267F was genotyped in 2907 HBV‐exposed subjects (including HBV persistent carriers and spontaneously recovered subjects) and 1364 unexposed subjects (HBV marker‐negative subjects), followed by replication I, comprising 914 exposed subjects and 1123 unexposed subjects, and replication II, comprising 355 children born to HBsAg‐positive mothers (226 HBV‐infected children and 129 controls). Intriguingly, SLC10A1 AA was observed only in the unexposed group, but not in the exposed group. The SLC10A1 A allele consistently decreased HBV infection risk compared with the G allele (OR = 0.76, 95% CI: 0.64‐0.90 in combined samples). In addition, children with the SLC10A1 GA genotype had a reduced risk of perinatal transmission (OR = 0.31, 95% CI: 0.14‐0.71). Moreover, unexposed subjects with the SLC10A1 AA genotype exhibited decreased serum total cholesterol and low‐density lipoprotein cholesterol compared to those with the GG or GA genotypes (P = 2.975 × 10^?4 and 0.004, respectively). The study highlighted the role of the SLC10A1 S267F variant in the loss of the ability to support HBV infection and taurocholate transport activity. Subjects with the AA genotype may escape from HBV infection and present decreased cholesterol levels as a consequence of impaired bile acid uptake.     

Pancreatic regenerating protein (reg I) and reg I receptor mRNA are upregulated in rat pancreas after induction of acute pancreatitis

AIM: Pancreatic regenerating protein (reg Ⅰ) stimulates pancreatic regeneration after pancreatectomy and is mitogenic to ductal and β-cells. This suggests that reg Ⅰ and its receptor may play a role in recovery after pancreatic injury. We hypothesized that reg Ⅰ and its receptor are induced in acute pancreatitis.METHODS: Acute pancreatitis was induced in male Wistar rats by retrograde injection of 3% sodium taurocholate into the pancreatic duct. Pancreata and serum were collected 12, 24, and 36 hours after injection and from normal controls (4 rats/group). Reg Ⅰ receptor mRNA, serum reg Ⅰ protein, and tissue reg Ⅰ protein levels were determined by Northern analysis, enzymelinked immunosorbent assay (ELISA), and Western analysis, respectively. Immunohistochemistry was used to localize changes in reg Ⅰ and its receptor.RESULTS: Serum amylase levels and histology confirmed necrotizing pancreatitis in taurocholate treated rats. There was no statistically significant change in serum reg Ⅰ concentrations from controls. However,Western blot demonstrated increased tissue levels of reg Ⅰ at 24 and 36 h. This increase was localized primarily to the acinar cells and the ductal cells by immunohistochemistry. Northern blot demonstrated a significant increase in reg Ⅰ receptor mRNA expression with pancreatitis. Immunohistochemistry localized this increase to the ductal cells, islets, and acinar cells.CONCLUSION: Acute pancreatitis results in increased tissue reg Ⅰ protein levels localized to the acinar and ductal cells, and a parallel threefold induction of reg Ⅰ receptor in the ductal cells, islets, and acinar cells. These changes suggest that induction of reg Ⅰ and its receptor may be important for recovery from acute pancreatitis.     

Pancreatic regenerating protein (reg Ⅰ) and reg Ⅰ receptor mRNA are upregulated in rat pancreas after induction of acute pancreatitis

AIM: Pancreatic regenerating protein (reg Ⅰ) stimulates pancreatic regeneration after pancreatectomy and is mitogenic to ductal and β-cells. This suggests that reg Ⅰ and its receptor may play a role in recovery after pancreatic injury. We hypothesized that reg Ⅰ and its receptor are induced in acute pancreatitis.METHODS: Acute pancreatitis was induced in male Wistar rats by retrograde injection of 3% sodium taurocholate into the pancreatic duct. Pancreata and serum were collected 12, 24, and 36 hours after injection and from normal controls (4 rats/group). Reg Ⅰ receptor mRNA, serum reg Ⅰ protein, and tissue reg Ⅰ protein levels were determined by Northern analysis, enzymelinked immunosorbent assay (ELISA), and Western analysis, respectively. Immunohistochemistry was used to localize changes in reg Ⅰ and its receptor.RESULTS: Serum amylase levels and histology confirmed necrotizing pancreatitis in taurocholate treated rats. There was no statistically significant change in serum reg Ⅰ concentrations from controls. However,Western blot demonstrated increased tissue levels of reg Ⅰ at 24 and 36 h. This increase was localized primarily to the acinar cells and the ductal cells by immunohistochemistry. Northern blot demonstrated a significant increase in reg Ⅰ receptor mRNA expression with pancreatitis. Immunohistochemistry localized this increase to the ductal cells, islets, and acinar cells.CONCLUSION: Acute pancreatitis results in increased tissue reg Ⅰ protein levels localized to the acinar and ductal cells, and a parallel threefold induction of reg Ⅰ receptor in the ductal cells, islets, and acinar cells. These changes suggest that induction of reg Ⅰ and its receptor may be important for recovery from acute pancreatitis.     

Tissue-specific and ubiquitous expression of fibrinogen gamma-chain mRNA

Fibrinogen gamma-chains are derived from differential mRNA splicing resulting in polypeptide that differ in their carboxyterminal sequences as well as tissue distribution. We examined the expression of the rat fibrinogen gamma-chain mRNAs to determine the nature of the differential tissue expression of the gamma-fibrinogens. Here we demonstrate that the expression of the rat gamma B mRNA is tissue-specific. Northern blot analysis indicated that gamma A mRNA was expressed in liver, lung, brain and marrow, while the alternatively spliced gamma B mRNA was expressed only in liver. Three distinct full-length species of gamma B mRNA were identified in liver, indicative of multiple sites for polyadenylation that is suggestive of regulation at the level of 3' RNA processing. The restricted expression of gamma B mRNA in liver was contrasted by the ubiquitous expression of the gamma-chain promoter in brain and lung tissues that are not known for production of plasma coagulation proteins. The results of in situ hybridization showed that only gamma A mRNA was found in lung, localized to bronchiolar epithelial cells and chondrocytes but not smooth muscle or endothelial cells. Tissue-specific regulation of the gamma-chain gene results in compartmentalization of gamma B-fibrinogen to the circulation.     

Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system.

Liver parenchymal cells continuously extract high amounts of bile acids from portal blood plasma. This uptake process is mediated by a Na+/bile acid cotransport system. A cDNA encoding the rat liver bile acid uptake system has been isolated by expression cloning in Xenopus laevis oocytes. The cloned transporter is strictly sodium-dependent and can be inhibited by various non-bile-acid organic compounds. Sequence analysis of the cDNA revealed an open reading frame of 1086 nucleotides coding for a protein of 362 amino acids (calculated molecular mass 39 kDa) with five possible N-linked glycosylation sites and seven putative transmembrane domains. Translation experiments in vitro and in oocytes indicate that the transporter is indeed glycosylated and that its polypeptide backbone has an apparent molecular mass of 33-35 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with mRNA species from rat kidney and intestine as well as from liver tissues of mouse, guinea pig, rabbit, and man.     

Normal or increased bile acid uptake in isolated mucosa from patients with bile acid malabsorption

INTRODUCTION: Bile acid malabsorption as reflected by an abnormal Se-labelled homocholic acid-taurine (SeHCAT) test is associated with diarrhoea, but the mechanisms and cause-and-effect relations are unclear. OBJECTIVES: Primarily, to determine whether there is a reduced active bile acid uptake in the terminal ileum in patients with bile acid malabsorption. Secondarily, to study the linkage between bile acid malabsorption and hepatic bile acid synthesis. METHODS: Ileal biopsies were taken from patients with diarrhoea and from controls with normal bowel habits. Maximal active bile acid uptake was assessed in ileal biopsies using a previously validated technique based on uptake of C-labelled taurocholate. To monitor the hepatic synthesis, 7alpha-hydroxy-4-cholesten-3-one, a bile acid precursor, was assayed in blood. The SeHCAT-retention test was used to diagnose bile acid malabsorption. RESULTS: The taurocholate uptake in specimens from diarrhoea patients was higher compared with the controls [median, 7.7 (n=53) vs 6.1 micromol/g per min (n=17)] (P<0.01) but no difference was seen between those with bile acid malabsorption (n=18) versus diarrhoea with a normal SeHCAT test (n=23). The SeHCAT values and 7alpha-hydroxy-4-cholesten-3-one were inversely correlated. CONCLUSIONS: The data do not support bile acid malabsorption being due to a reduced active bile acid uptake capacity in the terminal ileum.