P120	Oxidative stress and thrombin induce HIF-1α via activation of nuclear factor κ B.
1S.Bonello, 1Chr.Zähringer, 1R.S.Belaiba, 1T.Djordjevic, 2T.Kietzmann, 3C.Michiels, 1J.Hess, 1A.Görlach
1Experimentelle Kinderkardiologie, Deutsches Herzzentrum München an der TU München, München, DE; 2Fachbereich Chemie, Technische Universität Kaiserslautern, Kaiserslautern, DE; 3Laboratory of Biochemistry and Cellular Biology, University of Namur, Namur, BE.

Chronic hypoxia and thrombotic activity are associated with pulmonary hypertension (PH) and promote vascular remodeling. Reactive oxygen species have been suggested to contribute to this disease, although the underlying mechanisms are not resolved. Whereas the hypoxia-inducible transcription factor HIF-1alpha contributes to the pathogenesis of hypoxia-mediated PH, the role of the oxidative stress-responsive transcription factor nuclear factor-kappa B (NFkB) is not well known. We therefore investigated whether NFkB and HIF-1alpha are involved in the response to thrombin and oxidative stress in pulmonary artery smooth muscle cells (PASMC). Thrombin and H2O2 increased HIF-1 and NFkB transcriptional activity using reporter gene assays. Immunofluorescence confirmed nuclear translocation of HIF-1alpha and of NFkB p50 by these stimuli. HIF-1alpha protein and mRNA levels were induced by thrombin and H2O2, and this response was diminished by pretreatment with antioxidants. Expression of an IkB superrepressor, which inhibits NFkB activation, attenuated HIF-1alpha induction by these stimuli. Transfection of p50/p65 mimicked the response to thrombin and H2O2. HIF-1alpha upregulation was abrogated by actinomycin-D, indicating the involvement of a transcriptional mechanism. Consistently, activity of a construct where the luciferase gene was fused to a 1.2 kb sequence 5 to the HIF-1alpha coding sequence (p12) was increased by thrombin and H2O2. Sequential deletion of the HIF-1alpha promoter showed reduced activity of a construct lacking a putative NFkB binding site (p3). Coexpression of p50/p65 enhanced p12, but not p3 mediated luciferase activity. Expression of the IkB mutant inhibited thrombin and H2O2 stimulated reporter gene activity. Mutation of the NFkB binding site prevented activation of this construct and gel shift analysis confirmed that NFkB is able to bind to this consensus sequence in the HIF-1alpha promoter. Taken together, these findings provide a novel mechanism whereby the redox-sensitive transcription factor NFkB is required for transcriptional upregulation of HIF-1alpha by thrombin and oxidative stress. Since inhibition of NFkB and HIF-1alpha reduced VEGF and PAI-1 expression by thrombin and H2O2, induction of HIF-1alpha by NFkB may play an important role in promoting pulmonary remodeling processes and thrombosis in PH.

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