Transactivation of the human apical sodium-dependent bile acid transporter gene by human serum.
Using a luciferase reporter assay we found that human serum transactivated the ileal apical sodium-dependent bile acid transporter (ASBT) promoter three to fourfold. Confirming this effect, addition of human serum to both Caco-2 cells and fresh human ileal biopsies caused an approximate 2.0-fold increase in endogenous ASBT mRNA production. Alteration of non-esterified fatty acid (NEFA) content and cortisol content did not affect the transactivation potential of serum. Site-directed mutagenesis of response elements for corticosteroid, peroxisome proliferation-activated alpha (PPARalpha), hepatocyte nuclear factor 1alpha (HNF1alpha), and retinoic acid (RAR/RXR) did not affect transactivation potential of serum. Three putative serum response elements (SRE) were identified on the promoter, but all were determined inactive using site-directed mutagenesis and electrophoretic mobility shift assay. Promoter deletion analysis demonstrated that >80% of the response to serum was located within the last 273 bp of the 5'-UTR, an area containing one of two activate protein 1 (AP-1) response elements. Site-directed mutagenesis of this downstream AP-1 response element reduced the effect of serum on the promoter by about 50% while full deletion of the response element completely eliminated the effect of serum. These studies demonstrate that one or more constituents of human stimulate ASBT gene expression largely via the down-stream AP-1 response element.
Duane
William C
WC
Department of Medicine, Veterans Affairs Medical Center and University of Minnesota, Minneapolis, MN 55417, USA. duane006@umn.edu
Xiong
Wendy
W
Lofgren
Jennifer
J
D000818:Animals; D018938:Caco-2 Cells; D002352:Carrier Proteins / Q000494:pharmacology; D002645:Chickens; D003345:Corticosterone; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology; D017930:Genes, Reporter / Q000187:drug effects; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D007082:Ileum / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D015735:Mifepristone / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D029363:Organic Anion Transporters, Sodium-Dependent / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D044967:Serum / Q000502:physiology*; D027602:Serum Response Element / Q000187:drug effects / Q000502:physiology; D027981:Symporters / Q000235:genetics* / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005230:Fatty Acids, Nonesterified; D029363:Organic Anion Transporters, Sodium-Dependent; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D027981:Symporters; D018808:Transcription Factor AP-1; C071908:sodium-bile acid cotransporter; D015735:Mifepristone; D008156:Luciferases; D003345:Corticosterone; D006854:Hydrocortisone