Signaling pathways of heme oxygenase-1 gene activation by lipopolysaccharide and NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzensulfonyl fluorid in monocytes
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Heme oxygenase-1 (HO-1) is the inducible isoform of the first and rate-limiting enzyme of heme degradation and is up-regulated by a host of stress stimuli. Activation of the HO-1 gene not only protects cells and tissues against oxidative damage, but also modulates the inflammatory immune response. Lipopolysaccharide (LPS) is a prototypical mediator of inflammation and is known to activate the phagocyte NAD(P)H oxidase. To further understand the regulatory role of HO-1 in mononuclear phagocytes during inflammation, the HO-1 gene regulation by LPS and by the NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) was investigated in the monocytic cell line RAW264.7. HO-1 gene expression was markedly induced by LPS and, unexpectedly, also by AEBSF in these cells. To determine the molecular mechanisms and signaling pathways of HO-1 gene expression by these compounds, reporter gene constructs with proximal HO-1 promoter gene sequences were examined in transiently transfected RAW264.7 cells. Up-regulation of HO-1 promoter activity by LPS was decreased by pharmacological NF-kappaB inhibitors and by overexpression of dominant negative mutants of NF-kappaB inducing kinase, inhibitor of NF-kappaB (IkappaB) kinase beta and IkappaB alpha. The p38 MAPK inhibitor SB203580 and overexpressed dominant negative p38 beta decreased, whereas p38 delta increased, LPS-dependent induction of HO-1 gene expression. Deletion and mutation analysis with transfected HO-1 promoter gene constructs indicated that a CRE/AP-1 site (-668/-654), but not an E-box motif (-47/-42), was involved in LPS-dependent HO-1 gene regulation. AEBSF-dependent induction of endogenous HO-1 gene expression and promoter activity was abolished by treatment with chemical inhibitors of the phosphatidyl inositol 3-kinase/ protein kinase B (PKB) pathway and overexpression of dominant negative mutants of PKB. Accordingly, cotransfected constitutive active PKB markedly up-regulated HO-1 promoter activity. Inhibition of p38 alpha and p38 beta prevented the induction of HO-1 gene expression by AEBSF. p38 was stimulated by AEBSF in a PKB-dependent manner as demonstrated by a luciferase assay with a Gal4-CHOP fusion protein. Deletion and mutation analysis indicated that both, the E-box and the CRE/AP-1 element, were essential for mediating the full response of HO-1 promoter activity to AEBSF. Cotransfection of the coactivator p300 and the basic helix-loop-helix transcription factor USF2 enhanced the AEBSF-dependent response of the HO-1 promoter activity. Taken together, the data indicate that the NF-kappaB, PKB and p38 signaling pathways play an important regulatory role for the induction of HO-1 gene expression by LPS and the NAD(P)H oxidase inhibitor AEBSF in mononuclear phagocytes.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Gießen : Köhler 2005 (Antioxid. Redox Signal. 6 (2004), 802 810; J. Biol. Chem. 280 (2005), 21820-21829)