Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate

Heme oxygenase 1 (HO-1), a stress response protein, is highly induced in re sponse tn various agents causing oxidative stress including ultraviolet irr adiation, sodium arsenite, hyperoxia, and glutathione depletors, We recentl y characterized the induction of HO-1 gene expression by nitric oxide (NO) and postulated that the addition of an antioxidant, such as pyrrolidine dit hiocarbamate (PDTC), would attenuate HO-1 induction in response to NO, Surp risingly, PDTC was a very potent inducer of HO-1 gene expression, causing i ncreases in the steady-state level of HO-1 mRNA in rat aortic vascular smoo th muscle (aVSM) cells in a time- and concentration-dependent manner, PDTC- induced HO-1 gene expression correlated with a rise in protein levels and w as dependent on both increased gene transcription and mRNA stability, Delet ional analyses of the proximal promoter and the entire 5' distal upstream r egion of the HO-1 gene (11 kbp) were performed including the two 5' distal enhancers, SX2 and AB1, located 4 kbp and 10 kbp upstream of the transcript ion site, respectively. Plasmid vectors containing various fragments of thi s region were linked to a chloramphenicol acetyl transferase (CAT) reporter gene, stably transfected into RAW 264.7 cells, and transfectants were assa yed for CAT activity after treatment with PDTC, We show that the AB1 distal enhancer plays an important role in mediating PDTC-induced HO-1 gene trans cription, Mutational analyses of this enhancer showed that the activator pr otein 1 (AP-1) regulatory element is crucial for PDTC-induced HO-1 gene tra nscription, Electrophoretic mobility shift assays supported this data, demo nstrating increased AP-1 DNA binding activity after PDTC treatment, Taken t ogether, our data demonstrate that the antioxidant PDTC enhances HO-1 gene transcription and that the induction appears to be mediated by AP-1 activat ion of regulatory elements specific to the distal enhancer AB1.