Placental growth factor mediated transcriptional and posttranscriptional regulation of hemeoxygenase-1

Hemeoxygenase-1 (HO-1) is a detoxifying enzyme produced by cells in response to oxidative stress. It is up-regulated in Sickle Cell Disease patients due to RBC lysis and release of free heme from hemoglobin (I. T. Lee et al., 2009).In SCD patients, the level of the Placental Growth factor (PlGF) is elevated (Perelman et al., 2003). However, there was no direct link between levels of PlGF to expression of cytoprotective enzymes such as hemeoxygenase-1.;Our studies showed PlGF augmented HO-1 mRNA and protein expression. Also, our studies showed PlGF induced signaling involved activation of PI-3- kinase, JNK kinase, p38MAP kinase, NADPH-oxidase and HIF-1α, as demonstrated by use of pharmacological inhibitors specific for each kinase and others in the signaling pathway.;We studied the role of HIF-1α in the regulation of HO-1 expression, as most inducers up-regulate the expression of HO-1 by activation of Nrf2. Our studies showed that shRNA for HIF-1α attenuated the expression of HO-1 mRNA. Furthermore, transfection of shRNA for HIF-1α attenuated HO-1 promoter luciferase activity.;Additionally, mutation of HRE sites in HO-1 promoter (∼4.5Kb) reduced PlGF mediated luciferase activity. These results showed that PlGF mediated HO-1 expression involved HIF-1α. The role of HIF-1α in HO-1 expression was further supported by chromatin immunoprecipitation analysis (ChIP), wherein chromatin immunoprecipitated with HIF-1α antibody showed increased recruitment of HIF-1α to HRE-2 and HRE-3 sites in the HO-1 promoter, utilizing primers spanning across these HRE sites i.e. -669/-666 and -862/-859. Next we examined the post-transcriptional regulation of HO-1 mediated by miRNAs. Our studies showed that levels of miR-518, among other miRNAs, were significantly reduced in response to PlGF treatment. Transfection of miR-518 reduced the mRNA level of HO-1. Conversely, anti-miR-518 increased HO-1 mRNA level, indicating miR518 regulated HO-1 mRNA levels. Next, we determined whether miR-518 bound to the 3'UTR of HO-1 mRNA to exert its effect. We observed that miR-518 reduced 3'UTR-HO-1 luciferase reporter activity in response to PlGF. Our studies showed that PlGF-mediated HO-1 transcription is mediated by HIF-1α, and post-transcriptionally regulated by miR-518.