Hypoxia Inducible Factor-1 α And DDAH1 Regulation

Objection: The purpose of this to investigate was to determine the effects of CoCl2 onDDAH1 expression, and the role of HIF-1αin DDAH1 gene regulation.Method and Results: Wistar rat were pretreated with CoCl2 (30mg/kg) or saline for 8hours, significant up regulation of DDAH1 protein could be seen in CoCl2 mimic hypoxiastimulation. HEK293T and HepG2 cell were treated with CoCl2, obvious up-regulation ofHIF-1 and DDAH1 protein was obverted after CoCl2 mimic hypoxia. Inhibition of HIF-1αby HIF-1αsiRNA inhibited CoCl2 induced HIF-1αprotein accumulation and DDAH1protein elevation. Sequence analysis found two possible hypoxia response elements atregions of 5'to the proximal 2000-bp fragment of rat DDAH1promoter relative to thetranscription start site. Four reporter gene plasmids were constructed after promotertruncation analysis, and then transiently transfected into HepG2 and HEK293T cells. After8 hours treated with CoCl2, promoter activity was detected and showed HIF consensusbinding may spanning bp-370~-387 relative to the transcription start site were functionalfor DDAH1 promoter activity induction by hypoxia. The HRE motif in the above plasmidwere mutated, promoter activity showed mutated DDAH1 promoter fragment was notstimulated in HIF-1αover expressed cells.Conclusion: We demonstrate that DDAH1 is a hypoxia inducible gene, whose transcriptionis stimulated through HIF-1αinteraction with HRE sites located at -370~-387 of theDDAH1 promoter in rat.