Figure 5.

Hypoxia induces HIF-2α binding to the CXCL12 promoter in LP-1 cells. (A) Human CXCL12 locus containing two HIF binding site (HBS) sequences and transcriptional start site. Hypoxic induction of CXCL12 is primarily mediated via HBS1. (B) LP-1 cells were transiently transfected with a luciferase plasmid containing the proximal CXCL12 promoter (top, pGL3b-CXCL12). Twenty-four hours post-transfection, cells were cultured for 48 h under normoxic (white bars) or hypoxic (black bars) conditions, and luciferase assays performed. Columns, mean (n=3); bars, SEM. *P<0.05, compared to normoxia. (C) The pGL3b-CXCL12 construct (top) was transiently transfected into the HIF-over-expressing LP-1 cell lines and luciferase assays performed. Columns, mean (n=3); bars, SEM. *P<0.05, compared to vector control. (D) LP-1 cells were cultured under normoxic (N, lane 1) or hypoxic (H, lane 2) conditions for 48 h and DNA binding activity to HBS1 examined by electromobility shift assay. To determine the contribution of HIF-2 to the hypoxia-inducible complex formation, extracts were pre-incubated with HIF-2α antibody (lanes 3 and 4). (E) Chromatin immunoprecipitation was performed on LP-1 cells cultured under normoxic (white bars) or hypoxic (black bars) conditions for 48 h, and the level of HIF-2α binding to HBS1 of the CXCL12 promoter assessed by PCR. Columns, mean; bars, SEM. *P<0.05, compared to normoxia.