OBJECTIVESTo investigate role of hypoxia inducible factor 1 (HIF-1) in the transcriptional activation of heat shock protein 70-2 (HSP70-2) in hepatocellular carcinoma (HCC) cells under hypoxic conditions.

METHODSHCC cells were exposed to reduced oxygen atmosphere (1% O2), or treated with YC-1 or HIF-1 alpha siRNA, the expression of HIF-1 alpha and HSP70-2 were detected by Western blot analysis. Serial deletions of the HSPA2 promoter were cloned in the reporter pGL3-Basic plasmid. These reporter plasmids were co-transfected with HIF-1 alpha siRNA, and the promoter activities were detected with the dual luciferase assay.

RESULTSWestern blot analysis showed that both HIF-1 alpha and HSP70-2 proteins were strongly increased after HCC cells were exposed to hypoxic conditions (1% O2) for 6 h, and the expression level of HSP70-2 was increased in a time-dependent manner. Treatment of HepG2 cells with YC-1 or HIF-1 alpha siRNA significantly inhibited the expression of HIF-1 alpha and HSP70-2. In silico analysis of the HSP70-2 promoter using the Gene2 Promoter software revealed the presence of two putative hypoxic response element (HRE) consensus at -446bp (HRE1) and -238bp (HRE2). Depletion of promoter sequence between -653 and -385 led to a dramatic reduction of promoter activity, whereas further deletion to position -201 did not reduce the activity further. These data suggested that HRE1 plays an important role in hypoxia-induced activation of the HSPA2 promoter. Site-directed mutagenesis further confirmed these results. Mutation of HRE1 but not of HRE2 abrogated the sensitivity of the HSP70-2 promoter to hypoxia.

CONCLUSIONSHSP70-2 expression is up-regulated in response to hypoxia and a HIF-1 binding site (HRE1) in the HSP70-2 promoter is involved in this response.