Fig. 4.

HIF-2α overexpression increases SP-1 activity. (A) SP-1 transcription factor binding site found in the promoter of IL-8 using the Alibaba 2.1 program. (B) Cells were cotransfected with the pSP-1-luc vector containing the SP-1 binding site driving luciferase expression (500 ng) and a plasmid containing the LacZ gene (100 ng) as an internal control and after 24 h transduced with AdHIF-1α, AdHIF-2α, or AdGFP for the next 48 h. As shown by measurement of luciferase activity, HIF-2α augmented SP-1 activity. (C) Real-time PCR was performed to examine the mRNA level of a TP gene containing binding sites for SP-1 after 48 h transduction with AdHIF-1α, AdHIF-2α, or AdGFP. TP mRNA was upregulated only by HIF-2α (a similar tendency of increase in TP was noted in three independent experiments). (D) Real-time PCR and (E) ELISA were performed to examine the mRNA and protein level of IL-8, respectively, after 48 h transduction with AdHIF-2α or AdGFP together with stimulation with 10 μM SP-1 inhibitor, mithramycin A. Note that the effect of HIF-2α on IL-8 was partially reversed by mithramycin A, particularly at the mRNA level. (F) Cells were transfected for 48 h with the pCMV-SP-1 vector (500 ng) or control Bluescript plasmid (100 ng). As shown by ELISA, SP-1 overexpression tended to increase IL-8 production. (G) SP-1 transcription factor binding site found in the promoter of IL-8 was mutated at guanine residues (in red in (A)). Such mutation did not change HIF-2α-induced IL-8 promoter activity. Each bar represents the mean ± SD of three to six independent experiments performed in duplicate. *p < 0.05, comparing AdGFP vs AdHIF-1α or AdHIF-2α; #p < 0.05, comparing AdGFP control vs AdGFP stimulated or AdHIF-2α control vs AdHIF-2α stimulated.