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.

Transcriptome‐Based Analysis of Molecular Pathways for Clusterin Functions in Kidney Cells

“…The hypoxia‐inducible factor (HIF) 1, a heterodimer of HIF‐1β (constitutively expressed), and HIF‐1α (O 2 ‐regulated), is a primary factor for these adapting mechanisms (Ke and Costa, ). Hypoxia up‐regulates CLU expression in kidney cells (Alnasser et al, ) or tenocytes (Millar et al, ), and HIF‐1 binding hypoxia response elements are identified in CLU gene promoter (Park et al, ), suggesting that up‐regulation of CLU may be part of survival mechanisms of cells in hypoxia. Indeed, our previous study has demonstrated that CLU expression is required for activation of prosurvival autophagy in the kidneys with IRI, and in cultured kidney cells in hypoxia that is associated with UPR (Alnasser et al, ).…”

Regulation of fatty acid binding proteins by hypoxia inducible factors 1α and 2α in the placenta: Relevance to pre-eclampsia

“…2A). CoCl 2 is a well-described hypoxiamimetic agent [7,12,14] but also activates the nuclear transcription factor NF-κB [15]. There are two putative κB elements within the FABP3 gene at À 834 and À 825 bp that are presumably responsible for this increase in luciferase activity (data not shown).…”

“…We concluded that the region from À 1315 to À 648 bp of FABP1 responds predominantly to HIF-1α. In other studies the number of functional HRE within the gene being tested is proportional to the intensity of the luciferase activity [12].…”

Proteomic Profiling of the Retinas in a Neonatal Rat Model of Oxygen-Induced Retinopathy with a Reproducible Ion-Current-Based MS1 Approach

“…58 Recent studies showed that nuclear clusterin transcription could be directly regulated by HIF-1α. 59 Our previous study showed that reactive oxygen species (ROS) such as H 2 O 2 level in choroid of OIR is significantly higher ( p < 0.01) than that in controls and may lead to high H 2 O 2 level in retinas of OIR. 17 Thus, the elevated STAT1 here may play a similar role of response to intracellular ROS in retinas of OIR as these in human diseases.…”