Transcriptional regulation of the heme oxygenase 1 gene by pyrrolidine dithiocarbamate.

Heme oxygenase 1 (HO-1), a stress response protein, is highly induced in response to various agents causing oxidative stress including ultraviolet irradiation, sodium arsenite, hyperoxia, and glutathione depletors. We recently characterized the induction of HO-1 gene expression by nitric oxide (NO) and postulated that the addition of an antioxidant, such as pyrrolidine dithiocarbamate (PDTC), would attenuate HO-1 induction in response to NO. Surprisingly, PDTC was a very potent inducer of HO-1 gene expression, causing increases in the steady-state level of HO-1 mRNA in rat aortic vascular smooth muscle (aVSM) cells in a time- and concentration-dependent manner. PDTC-induced HO-1 gene expression correlated with a rise in protein levels and was dependent on both increased gene transcription and mRNA stability. Deletional analyses of the proximal promoter and the entire 5' distal upstream region 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 transcription site, respectively. Plasmid vectors containing various fragments of this region were linked to a chloramphenicol acetyl transferase (CAT) reporter gene, stably transfected into RAW 264.7 cells, and transfectants were assayed 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 transcription. Mutational analyses of this enhancer showed that the activator protein 1 (AP-1) regulatory element is crucial for PDTC-induced HO-1 gene transcription. Electrophoretic mobility shift assays supported this data, demonstrating increased AP-1 DNA binding activity after PDTC treatment. Taken together, our data demonstrate that the antioxidant PDTC enhances HO-1 gene transcription and that the induction appears to be mediated by AP-1 activation of regulatory elements specific to the distal enhancer AB1.