TRANSCRIPTIONAL ACTIVATION OF THE H0-1 GENE BY LIPOPOLYSACCHARIDE IS MEDIATED BY 5'-DISTAL ENHANCERS - ROLE OF REACTIVE OXYGEN INTERMEDIATES AND AP-1

Heme oxygenase-1 (HO-1) is a stress-response protein, the expression o f which is transcriptionally regulated by agents that cause oxidative stress. We have previously shown that lipopolysaccharide (LPS)-induced HO-1 gene transcription in RAW 264.7 macrophage cells is mediated by a distal enhancer called SX2, located 4 kb upstream from the HO-1 tran scription initiation site (Am. J, Respir. Cell Mol. Biol. 1995:13:387- 398). We have recently identified a second distal enhancer, called AB1 , located 6 kb upstream from the SX2 distal enhancer (J. Biol. Chem. 1 995;270: 11977-11984). Here we report the extension of our studies to investigate whether the AB1 distal enhancer and/or other potential reg ulatory elements in the entire 5' distal flanking sequences (11-kb reg ion) of the HO-1 gene may also mediate HO-1 gene transcription in resp onse to LPS. Using deletional analysis, we found that the AB1 enhancer also mediates LPS-induced HO-1 gene transcription. Mutational analysi s of the AB1 enhancer and electrophoretic-mobility-shift assays of nuc lear extracts from LPS-treated cells further demonstrated that the tra nscription factor activator protein-1 (AP-1) is critical for AB1-media ted HO-1 gene activation by LPS. We also found increased expression of AP-1 family members c-fos and c-jun by Northern blot analyses after t reatment with LPS. Further, we observed that LPS-treated RAW 264.7 cel ls produced high levels of reactive oxygen intermediates (ROI) as meas ured through flow-cytometric analysis of dichlorofluoroscein (DCF)-sta ined cells. Treatment of cells with the antioxidants N-acetyl-L-cystei ne (NAC) and dimethyl sulfoxide (DMSO) not only blunts LPS-induced pro duction of ROI, but also significantly attenuates LPS-induced HO-1 mes senger RNA (mRNA) expression and gene transcription Taken together, th ese data suggest that LPS regulates HO-1 gene transcription in part by inducing the production of ROI, which initiate signal-transduction pa thway(s) leading to the activation of AP-1-dependent HO-1 gene transcr iption.