Role of hypoxia inducible factor-1α in the regulation of the cancer-specific variant of organic anion transporting polypeptide 1B3 (OATP1B3), in colon and pancreatic cancer.
Organic anion transporting polypeptide 1B3 (OATP1B3) was initially considered to be a liver-specific transporter, mediating the uptake of a variety of endogenous and xenobiotic substances. Over the past decade, several investigations reported that OATP1B3 is also expressed across multiple types of cancers. Only recently, our laboratory and others demonstrated the identity of cancer-specific OATP1B3 variants (csOATP1B3) arising from the use of an alternative transcription initiation site, different from the wildtype (WT) OATP1B3 expressed in the normal liver. However, the mechanisms regulating the expression of csOATP1B3 remained unknown. In our current study, we investigated the role of hypoxia and the involvement of hypoxia inducible factor-1α (HIF-1α) in regulating the transcription of csOATP1B3. Our RT-PCR and immunoblotting results indicated that csOATP1B3, but not WT OATP1B3, can be induced in response to ambient or chemical hypoxia (upon exposure to 1% O₂ or cobalt chloride). Reporter assays with deletion and mutated constructs of the csOATP1B3 promoter revealed a functional hypoxia response element (HRE) located in the proximal upstream region. Constructs harboring the HRE displayed the upregulated reporter gene expression in response to hypoxia, but not when mutated. Electrophoretic mobility shift assays using a biotin-labeled csOATP1B3 promoter HRE probe indicated the binding of HIF-1α, which was blocked by an excess of unlabeled csOATP1B3 probe. Furthermore, siRNA-based knockdown of HIF-1α caused a substantial decrease in the expression level of csOATP1B3. Taken together, these findings demonstrate that the transcription of csOATP1B3 is actively engaged during hypoxia, through a commonly utilized pathway involving HIF-1α.
Department of Biological Sciences, Konkuk University, Seoul 143-701, Republic of Korea.
Songhee
S
Han
Kyungbo
K
Kim
Nilay
N
Thakkar
Donghak
D
Kim
Wooin
W
Lee
D017398:Alternative Splicing; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003035:Cobalt / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases; D009928:Organ Specificity; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics* / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D015398:Signal Transduction; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D014158:Transcription, Genetic
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D029362:Organic Anion Transporters, Sodium-Independent; D034741:RNA, Small Interfering; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D003035:Cobalt; D008156:Luciferases; C018021:cobaltous chloride; D010100:Oxygen
Colon cancer; EMSA; HIF-1; HRE; Hypoxia; OATP; OATP1B3; Pancreatic cancer; RT-PCR; Splicing variants; WT; electrophoretic mobility shift assay; hypoxia inducible factor-1; hypoxia response element; organic anion transporting polypeptide; reverse transcription-polymerase chain reaction; wildtype