Ann L. Craddock

The Heteromeric Organic Solute Transporter α-β, Ostα-Ostβ, Is an Ileal Basolateral Bile Acid Transporter

Bile acids are transported across the ileal enterocyte brush border membrane by the well characterized apical sodium-dependent bile acid transporter (Asbt) Slc10a2; however, the carrier(s) responsible for transporting bile acids across the ileocyte basolateral membrane into the portal circulation have not been fully identified. Transcriptional profiling of wild type and Slc10a2 null mice was employed to identify a new candidate basolateral bile acid carrier, the heteromeric organic solute transporter (Ost)α-Ostβ. By Northern blot analysis, Ostα and Ostβ mRNA was detected only in mouse kidney and intestine, mirroring the horizontal gradient of expression of Asbt in the gastrointestinal tract. Analysis of Ostα and Ostβ protein expression by immunohistochemistry localized both subunits to the basolateral surface of the mouse ileal enterocyte. The transport properties of Ostα-Ostβ were analyzed in stably transfected Madin-Darby canine kidney cells. Co-expression of mouse Ostα-Ostβ, but not the individual subunits, stimulated Na+-independent bile acid uptake and the apical-to-basolateral transport of taurocholate. In contrast, basolateral-to-apical transport was not affected by Ostα-Ostβ expression. Co-expression of Ostα and Ostβ was required to convert the Ostα subunit to a mature glycosylated endoglycosidase H-resistant form, suggesting that co-expression facilitates the trafficking of Ostα through the Golgi apparatus. Immunolocalization studies showed that co-expression was necessary for plasma membrane expression of both Ostα and Ostβ. These results demonstrate that the mouse Ostα-Ostβ heteromeric transporter is a basolateral bile acid carrier and may be responsible for bile acid efflux in ileum and other ASBT-expressing tissues.

Expression and transport properties of the human ileal and renal sodium-dependent bile acid transporter

The enterohepatic circulation of bile acids is maintained by Na+-dependent transport mechanisms. To better understand these processes, a full-length human ileal Na+-bile acid cotransporter cDNA was identified using rapid amplification of cDNA ends and genomic cloning techniques. Using Northern blot analysis to determine its tissue expression, we readily detected the ileal Na+-bile acid cotransporter mRNA in terminal ileum and kidney. Direct cloning and mapping of the transcriptional start sites confirmed that the kidney cDNA was identical to the ileal Na+-bile acid cotransporter. In transiently transfected COS cells, ileal Na+-bile acid cotransporter-mediated taurocholate uptake was strictly Na+ dependent and chloride independent. Analysis of the substrate specificity in transfected COS or CHO cells showed that both conjugated and unconjugated bile acids are efficiently transported. When the inhibition constants for other potential substrates such as estrone-3-sulfate were determined, the ileal Na+-bile acid cotransporter exhibited a narrower substrate specificity than the related liver Na+-bile acid cotransporter. Whereas the multispecific liver Na+-bile acid cotransporter may participate in hepatic clearance of organic anion metabolites and xenobiotics, the ileal and renal Na+-bile acid cotransporter retains a narrow specificity for reclamation of bile acids.

Targeted Deletion of the Ileal Bile Acid Transporter Eliminates Enterohepatic Cycling of Bile Acids in Mice

The ileal apical sodium bile acid cotransporter participates in the enterohepatic circulation of bile acids. In patients with primary bile acid malabsorption, mutations in the ileal bile acid transporter gene (Slc10a2) lead to congenital diarrhea, steatorrhea, and reduced plasma cholesterol levels. To elucidate the quantitative role of Slc10a2 in intestinal bile acid absorption, the Slc10a2 gene was disrupted by homologous recombination in mice. Animals heterozygous (Slc10a2+/–) and homozygous (Slc10a2–/–) for this mutation were physically indistinguishable from wild type mice. In the Slc10a2–/– mice, fecal bile acid excretion was elevated 10- to 20-fold and was not further increased by feeding a bile acid binding resin. Despite increased bile acid synthesis, the bile acid pool size was decreased by 80% and selectively enriched in cholic acid in the Slc10a2–/– mice. On a low fat diet, the Slc10a2–/– mice did not have steatorrhea. Fecal neutral sterol excretion was increased only 3-fold, and intestinal cholesterol absorption was reduced only 20%, indicating that the smaller cholic acid-enriched bile acid pool was sufficient to facilitate intestinal lipid absorption. Liver cholesteryl ester content was reduced by 50% in Slc10a2–/– mice, and unexpectedly plasma high density lipoprotein cholesterol levels were slightly elevated. These data indicate that Slc10a2 is essential for efficient intestinal absorption of bile acids and that alternative absorptive mechanisms are unable to compensate for loss of Slc10a2 function.

The organic solute transporter α-β, Ostα-Ostβ, is essential for intestinal bile acid transport and homeostasis

The apical sodium-dependent bile acid transporter (Asbt) is responsible for transport across the intestinal brush border membrane; however, the carrier(s) responsible for basolateral bile acid export into the portal circulation remains to be determined. Although the heteromeric organic solute transporter Ostα-Ostβ exhibits many properties predicted for a candidate intestinal basolateral bile acid transporter, the in vivo functions of Ostα-Ostβ have not been investigated. To determine the role of Ostα-Ostβ in intestinal bile acid absorption, the Ostα gene was disrupted by homologous recombination in mice. Ostα−/− mice were physically indistinguishable from wild-type mice. In everted gut sac experiments, transileal transport of taurocholate was reduced by >80% in Ostα−/− vs. wild-type mice; the residual taurocholate transport was further reduced to near-background levels in gut sacs prepared from Ostα−/−Mrp3−/− mice. The bile acid pool size was significantly reduced (>65%) in Ostα−/− mice, but fecal bile acid excretion was not elevated. The decreased pool size in Ostα−/− mice resulted from reduced hepatic Cyp7a1 expression that was inversely correlated with ileal expression of fibroblast growth factor 15 (FGF15). These data indicate that Ostα-Ostβ is essential for intestinal bile acid transport in mice. Unlike a block in intestinal apical bile acid uptake, genetic ablation of basolateral bile acid export disrupts the classical homeostatic control of hepatic bile acid biosynthesis.

APOL1 Renal-Risk Variants Induce Mitochondrial Dysfunction

APOL1 G1 and G2 variants facilitate kidney disease in blacks. To elucidate the pathways whereby these variants contribute to disease pathogenesis, we established HEK293 cell lines stably expressing doxycycline-inducible (Tet-on) reference APOL1 G0 or the G1 and G2 renal-risk variants, and used Illumina human HT-12 v4 arrays and Affymetrix HTA 2.0 arrays to generate global gene expression data with doxycycline induction. Significantly altered pathways identified through bioinformatics analyses involved mitochondrial function; results from immunoblotting, immunofluorescence, and functional assays validated these findings. Overexpression of APOL1 by doxycycline induction in HEK293 Tet-on G1 and G2 cells led to impaired mitochondrial function, with markedly reduced maximum respiration rate, reserve respiration capacity, and mitochondrial membrane potential. Impaired mitochondrial function occurred before intracellular potassium depletion or reduced cell viability occurred. Analysis of global gene expression profiles in nondiseased primary proximal tubule cells from black patients revealed that the nicotinate phosphoribosyltransferase gene, responsible for NAD biosynthesis, was among the top downregulated transcripts in cells with two APOL1 renal-risk variants compared with those without renal-risk variants; nicotinate phosphoribosyltransferase also displayed gene expression patterns linked to mitochondrial dysfunction in HEK293 Tet-on APOL1 cell pathway analyses. These results suggest a pivotal role for mitochondrial dysfunction in APOL1-associated kidney disease.

Gene Variability between Perineural-Positive and Perineural-Negative Squamous Cell Skin Cancers

AIM To identify differentially expressed genes (DEGs) between perineural invasion-positive (PP) and -negative (PN) cutaneous squamous cell cancers (CSCC). MATERIALS/METHODS Forty CSCC samples with and without perineural invasion were processed for RNA isolation and hybridization to Affymetrix-U219 DNA microarrays. Raw gene expression data were normalized by Robust Multi-array Averaging (RMA) and log2 transformed. Gene expression-based classification models were created and accuracies evaluated using leave-one-out cross-validation. RESULTS At a stringent limma p-value (p<0.001), 24 genes were differentially expressed between PP and PN samples. The cross-validated performance of the eight classification models exhibited a mean accuracy of 85-95%. Diagonal linear discriminant was most accurate at 95%, followed by Bayesian compound covariate at 94%. The poorest accuracy (85%) was observed for 1-Nearest neighbor and Support vector machines. CONCLUSION Gene expression may distinguish between PP and PN CSCC. Understanding these gene patterns may potentiate more timely diagnosis of perineural invasion and guide comprehensive therapies.

Expression Cloning of Receptor Ligand Transporters

Publisher Summary This chapter focuses on the expression cloning of receptor ligand transporters using mammalian cell systems. Expression cloning in mammalian cells, or Xenopus oocytes, is a powerful method to isolate cDNA clones that encode functional proteins for which no amino acid sequences exist. These methods have been especially useful for the membrane transporters that tend to be very hydrophobic proteins and, as such, refractory to conventional protein purification techniques. The ultimate success or failure of expression cloning depends on a number of factors that must be considered before embarking on what may become a quixotic quest. Expression cloning has proven to be a powerful alternative approach. Whereas protein purification relies on differences in physical properties to achieve isolation, expression cloning takes advantage of the high degree of substrate specificity exhibited by different transporters. Expression cloning has been used to isolate a wide variety of transporters including the hepatic and ileal sodium–bile acid cotransporters. The expression-cloning strategy requires an extremely sensitive assay for the target activity. Optimally, the chosen expression system should lack the biological activity in question, but possess the ability to express that activity. With expression cloning in Xenopus oocytes, the target activity can often be expressed and detected after the injection of poly(A) + RNA.