title: Hypoxia inducible factor-1α directly regulates nuclear clusterin transcription by interacting with hypoxia response elements in the clusterin promoter.
abstract: Differential transcription of the clusterin (CLU) gene yields two CLU isoforms, a nuclear form (nCLU) and a secretory form (sCLU), which play crucial roles in prostate tumorigenesis. Pro-apoptotic nCLU and anti-apoptotic sCLU have opposite effects and are differentially expressed in normal and cancer cells; however, their regulatory mechanisms at the transcriptional level are not yet known. Here, we examined the transcriptional regulation of nCLU in response to hypoxia. We identified three putative hypoxia response elements (HREs) in the human CLU promoter between positions -806 and +51 bp. Using a luciferase reporter, electrophoretic gel mobility shift, and chromatin immunoprecipitation assays, we further showed that hypoxia-inducible factor-1α (HIF-1α) bound directly to these sites and activated transcription. Exposure to the hypoxiamimetic compound CoCl₂, incubation under 1% O₂ conditions, or overexpression of HIF-1α enhanced nCLU expression and induced apoptosis in human prostate cancer PC3M cells. However, LNCaP prostate cancer cells were resistant to hypoxia-induced cell death. Methylation-specific PCR analysis revealed that the CLU promoter in PC3M cells was not methylated; in contrast, the CLU promoter in LNCap cells was methylated. Co-treatment of LNCaP cells with CoCl₂ and a demethylating agent promoted apoptotic cell death through the induction of nCLU. We conclude that nCLU expression is regulated by direct binding of HIF-1α to HRE sites and is epigenetically controlled by methylation of its promoter region.
authors.affiliation: Department of Anatomy and Neurobiology, Institute of Health Science, Medical Research Center for Neural Dysfunction, School of Medicine, Gyeong-sang National University, Jinju 660-290, Korea ; Department of Food & Nutrition, College of Natural Sciences, Gyeong-sang National University, Jinju 660-290, Korea.
authors.forename: Jeongsook
authors.initials: J
authors.lastname: Park
authors.forename: So Yun
authors.initials: SY
authors.lastname: Park
authors.forename: Eunkyung
authors.initials: E
authors.lastname: Shin
authors.forename: Sun Hee
authors.initials: SH
authors.lastname: Lee
authors.forename: Yoon Sook
authors.initials: YS
authors.lastname: Kim
authors.forename: Dong Hoon
authors.initials: DH
authors.lastname: Lee
authors.forename: Gu Seob
authors.initials: GS
authors.lastname: Roh
authors.forename: Hyun Joon
authors.initials: HJ
authors.lastname: Kim
authors.forename: Sang Soo
authors.initials: SS
authors.lastname: Kang
authors.forename: Gyeong Jae
authors.initials: GJ
authors.lastname: Cho
authors.forename: Bo-Young
authors.initials: BY
authors.lastname: Jeong
authors.forename: Hwajin
authors.initials: H
authors.lastname: Kim
authors.forename: Wan Sung
authors.initials: WS
authors.lastname: Choi
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia / Q000187:drug effects; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D051152:Clusterin / Q000235:genetics* / Q000378:metabolism; D003035:Cobalt / Q000494:pharmacology; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008297:Male; D010100:Oxygen / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism
chemical_list: C495242:CLU protein, human; D051152:Clusterin; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D020033:Protein Isoforms; D003035:Cobalt; C018021:cobaltous chloride; D010100:Oxygen