The inconsistent of responses of IRP1 and HIF-1 alpha to
hypoxia and the similar tendencies in the changes of IRP1 and pCREB contents led us to hypothesize that pCREB might be involved in the regulation of IRP1 under
hypoxia. Here, we investigated the role of pCREB in IRP1 expression in HepG2 cells under
hypoxia using quantitative PCR, western blot, immunofluorescence, electrophoretic mobility shift assay (EMSA) and
chromatin immunoprecipitation (ChIP). We demonstrated that 1)
Hypoxia increased pCREB levels inside of the nucleus; 2) Putative CREs were found in the IRP1 gene; 3) Nuclear extracts of HepG2 cells treated with
hypoxia could bind to CRE1 and CRE3, and 100-fold competitor of putative CREs could abolish the binding activity to varying degrees; 4) pCREB was found in the CRE1 and CRE3
DNA-
protein complexes of EMSA; 5) CRE1 and CRE3 binding activity of IRP1 depended on CREB activation but not on HIF-1; 6) Increased IRP1 expression under
hypoxia could be prevented by
LY294002; 7) ChIP assays demonstrated that pCREB binds to IRP1 promoter; and 8) HIF-1 and/or HIF-2
siRNA had no effect on the expression of pCREB and IRP1
proteins in cells treated with
hypoxia for 8 hours. Our findings evidenced for the involvement of pCREB in IRP1 expression and revealed a dominant role of PI3K/Akt pathway in CREB activation under
hypoxia and also suggested that dual-regulation of IRP1 expression by HIF-1 and pCERB or other
transcription factor(s) under
hypoxia might be a common mechanism in most if not all of
hypoxia-inducible genes.