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Heme Oxygenase-1 Gene Activation by the NAD(P)H Oxidase Inhibitor 4-(2-Aminoethyl) Benzenesulfonyl Fluoride via a Protein Kinase B, p38-dependent Signaling Pathway in Monocytes*

  • Nastiti Wijayanti
    Affiliations
    Institut für Klinische Immunologie und Transfusionsmedizin, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
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  • Thomas Kietzmann
    Affiliations
    Fachbereich Chemie/Biochemie, Universität Kaiserslautern, D-67663 Kaiserslautern, Germany
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  • Stephan Immenschuh
    Correspondence
    To whom correspondence should be addressed: Institut für Klinische Immunologie und Transfusionsmedizin, Justus-Liebig-Universität Giessen, Langhansstr. 7; 35392 Giessen, Germany. Tel.: 49-641-99-41521; Fax: 49-641-99-41529;
    Affiliations
    Institut für Klinische Immunologie und Transfusionsmedizin, Justus-Liebig-Universität Giessen, D-35392 Giessen, Germany
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  • Author Footnotes
    * This work was supported by Grants SFB 402 A8 (to S. I.) and SFB402 A1 (to T. K.) from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Heme oxygenase (HO)-1 is the inducible isoform of the rate-limiting enzyme of heme degradation and modulates the inflammatory immune response. Because HO-1 is up-regulated by NAD(P)H oxidase activators such as lipopolysaccharide and 12-O-tetradecanoylphorbol-13-acetate in monocytic cells, we investigated the gene regulation of HO-1 by the chemical NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF). Unexpectedly, AEBSF induced endogenous gene expression and promoter activity of HO-1 in cell cultures of human and mouse monocytes. Inhibition of the phosphatidylinositol 3-kinase/protein kinase B (PKB) pathway by pharmacological inhibitors and cotransfection of an expression vector for a dominant negative mutant of PKB reduced the AEBSF-dependent induction of HO-1 gene transcription. Accordingly, overexpressed constitutively active PKB markedly up-regulated HO-1 promoter activity. AEBSF activated the mitogen-activated protein kinases (MAPK) JNK and p38. Inhibition of p38α and p38β, but not that of JNK or p38γ and p38δ, 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. Finally, AEBSF- and PKB-dependent induction of HO-1 promoter activity was reduced by simultaneous mutation of an E-box motif (–47/–42) and a cAMP response element/AP-1 element (–664/–657) of the proximal HO-1 gene promoter. Overexpression of the basic helix-loop-helix transcription factor USF2 and coactivator p300 enhanced the AEBSF-dependent response of the HO-1 promoter. The data suggest that the transcriptional induction of HO-1 gene expression by AEBSF is mediated via activation of a PKB, p38 MAPK signaling pathway.
      Heme oxygenase (HO
      The abbreviations used are: HO, heme oxygenase; AEBSA, 4-(2-aminoethyl) benzenesulfonamide; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; CRE, cAMP response element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PBMC, peripheral blood monocytes; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; TF, transcription factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; USF, upstream stimulatory factor.
      1The abbreviations used are: HO, heme oxygenase; AEBSA, 4-(2-aminoethyl) benzenesulfonamide; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride; CRE, cAMP response element; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor-κB; PBMC, peripheral blood monocytes; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; TF, transcription factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; USF, upstream stimulatory factor.
      )-1 is the inducible isoform of HO that catalyzes the first and rate-limiting step of heme degradation (
      • Tenhunen R.
      • Marver H.S.
      • Schmid R.
      ). The homeostasis of cellular heme levels is controlled tightly, because heme is the prosthetic group of key enzymes such as inducible nitric-oxide synthase, cytochrome P450s, or soluble guanylate cyclase (
      • Wijayanti N.
      • Katz N.
      • Immenschuh S.
      ). On the other hand excess heme can cause cellular injury in its “free,” non-protein bound form (
      • Ryter S.W.
      • Tyrrell R.M.
      ). More recently, the HO products carbon monoxide and biliverdin have been recognized to be of physiological significance (
      • Maines M.D.
      ). Carbon monoxide is an important signaling gas (
      • Otterbein L.E.
      • Bach F.H.
      • Alam J.
      • Soares M.
      • Tao Lu H.
      • Wysk M.
      • Davis R.J.
      • Flavell R.A.
      • Choi A.M.
      ), and biliverdin, which is converted into bilirubin via the biliverdin reductase, is a potent antioxidant (
      • Stocker R.
      • Yamamoto Y.
      • McDonagh A.F.
      • Glazer A.N.
      • Ames B.N.
      ). Gene expression of HO-1 is induced by multiple stimuli and is primarily regulated at the transcriptional level (
      • Choi A.M.K.
      • Alam J.
      ,
      • Alam J.
      • Igarashi K.
      • Immenschuh S.
      • Shibahara S.
      • Tyrrell R.M.
      ). Overexpression of the HO-1 gene not only protects cells and tissues against oxidative damage (
      • Abraham N.G.
      • Lin J.H.
      • Schwartzman M.L.
      • Levere R.D.
      • Shibahara S.
      ) but also has anti-inflammatory effects (
      • Otterbein L.E.
      • Soares M.P.
      • Yamashita K.
      • Bach F.H.
      ,
      • Wagener F.A.
      • Volk H.D.
      • Willis D.
      • Abraham N.G.
      • Soares M.P.
      • Adema G.J.
      • Figdor C.G.
      ). The anti-inflammatory effect of HO-1 has initially been shown in a model of acute complement-dependent pleurisy (
      • Willis D.
      • Moore A.R.
      • Frederick R.
      • Willoughby D.A.
      ) and has essentially been confirmed in a HO-1 knock-out mouse model and a case of genetic human HO-1 deficiency (
      • Poss K.D.
      • Tonegawa S.
      ,
      • Poss K.D.
      • Tonegawa S.
      ,
      • Yachie A.
      • Niida Y.
      • Wada T.
      • Igarashi N.
      • Kaneda H.
      • Toma T.
      • Ohta K.
      • Kasahara Y.
      • Koizumi S.
      ). HO-1-deficient mice develop a chronic inflammatory state and are highly susceptible to organ damage by the prototypical inflammatory mediator lipopolysaccharide (LPS) (
      • Poss K.D.
      • Tonegawa S.
      ).
      In mononuclear phagocytes HO-1 gene expression is induced by LPS (
      • Camhi S.L.
      • Alam J.
      • Otterbein L.
      • Sylvester S.L.
      • Choi A.M.K.
      ) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (
      • Muraosa Y.
      • Shibahara S.
      ) both of which increase the cellular production of reactive oxygen species via activation of the plasma membrane-associated NAD(P)H oxidase. LPS-dependent activation of NAD(P)H oxidase is mediated by direct interaction of Toll-like receptor 4 with this enzyme (
      • Park H.S.
      • Jung H.Y.
      • Park E.Y.
      • Kim J.
      • Lee W.J.
      • Bae Y.S.
      ), and TPA is a prototypical activator of NAD(P)H oxidase in monocytes (
      • Babior B.M.
      ). More recently, gene expression of NAD(P)H oxidase and HO-1 has been demonstrated to be simultaneously increased in monocytes of diabetic patients (
      • Avogaro A.
      • Pagnin E.
      • Calo L.
      ). To investigate the potential regulatory role of NAD(P)H oxidase for the induction of HO-1 gene expression by LPS and TPA we applied the pharmacological NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) to monocytes. AEBSF, also known as Pefabloc, chemically modifies cytochrome b559 activity (
      • Diatchuk V.
      • Lotan O.
      • Koshkin V.
      • Wikstroem P.
      • Pick E.
      ) and is a serine protease inhibitor that regulates the differentiation of promyelocytic cells (
      • Bestilny L.J.
      • Riabowol K.T.
      ) and F-actin distribution in pancreatic cells (
      • Singh V.P.
      • Saluja A.K.
      • Bhagat L.
      • Hietaranta A.J.
      • Song A.
      • Mykoniatis A.
      • Van Acker G.J.
      • Steer M.L.
      ).
      Unexpectedly, we observed that AEBSF induces HO-1 gene expression in cell cultures of human peripheral blood monocytes (PBMC) and the mouse monocytic cell line RAW264.7. It is reported that the AEBSF-dependent induction of the HO-1 gene expression is regulated via activation of a phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signaling pathway as determined by the application of pharmacological inhibitors and overexpressed mutant isoforms of PKB. Moreover, activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway is required in a PKB-dependent manner for the AEBSF-dependent induction of HO-1 gene expression. An E-box motif and a cAMP response element (CRE)/AP-1 element of the proximal rat HO-1 gene promoter region are involved in the regulation of HO-1 gene expression by AEBSF.

      EXPERIMENTAL PROCEDURES

      Materials—Dulbecco's modified Eagle's medium and RPMI 1640 was obtained from Invitrogen, fetal bovine serum was from Biochrom (Berlin, Germany), Ficoll-Paque was from Pharmacia (Freiburg, Germany), CD14+ immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and polyvinylidene difluoride membranes were from Millipore. Falcon tissue culture dishes were from BD Biosciences. All other chemicals were purchased from Sigma and Roche Applied Science, unless otherwise indicated.
      Generation of Monocytes and Cell Culture—Human PBMC were isolated from buffy coats of healthy blood donors by Ficoll-Paque density gradient centrifugation. CD14+ monocytes were purified (>95%) using CD14+ immunomagnetic microbeads, and 3 × 106 cells were cultured in six-well flat bottom plates in 3 ml of RPMI 1640 with l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, sodium pyruvate, nonessential amino acids, and 10% fetal bovine serum. After 2 days, 50% of the supernatant was replaced with medium containing either AEBSF (250 μm), LPS (1 μg/ml), or TPA (0.5 μm). The studies with human blood samples were approved by the Institutional Review Board. RAW264.7 cells were from American Type Culture Collection (Manassas, VA) and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Cell cultures were kept under air/CO2 (19:1) at 100% humidity. Treatment of cells with AEBSF was performed with serum-free medium at 250 μm, unless otherwise indicated. The specific inhibitors of PI3K LY294002 (Calbiochem), PKB SH-5 (Alexis Biochemicals), MAPK/extracellular signal-regulated kinase (ERK) kinase PD98059 (Calbiochem), of c-Jun N-terminal kinase (JNK) SP600125 (Biomol) and of p38 SB203580 or SB202190 (Calbiochem) were added to the culture medium at the indicated concentrations.
      Western Blot Analysis—After washing cell cultures twice with 0.9% NaCl, 300 μl of lysis buffer (2% sodium dodecyl sulfate, 10% glycerol, bromphenol blue, 0.4 mol/liter dithiothreitol, 4% protease inhibitors) were added, and cells were scraped from culture dishes and then homogenized by passage through a 25-gauge needle (
      • Hess A.
      • Wijayanti N.
      • Neuschafer-Rube A.P.
      • Katz N.
      • Kietzmann T.
      • Immenschuh S.
      ). The homogenate was incubated for 3 min at 95 °C, and the protein content was determined in the supernatant by the Bradford method (
      • Hess A.
      • Wijayanti N.
      • Neuschafer-Rube A.P.
      • Katz N.
      • Kietzmann T.
      • Immenschuh S.
      ). Total protein (60 μg) was loaded onto a 12% SDS-polyacrylamide gel and blotted onto polyvinylidene difluoride membranes after electrophoresis. Membranes were blocked with Tris-buffered saline containing 5% dry milk or 5% bovine serum albumin, 50 mm Tris/HCl (pH 7.5), 150 mm NaCl, and 0.1% Tween 20 for 1 h at room temperature. The primary antibodies against HO-1 (Stressgen, Victoria, BC, Canada), cyclooxygenase-2 (Alexis Biochemicals), inducible nitric-oxide synthase (Santa Cruz Biotechnology), and glyceraldehyde-3-phosphate dehydrogenase (HyTest, Turku, Finland) were used at 1:1000 dilution. The primary antibodies for the detection of phosphorylated and total MAPKs, phosphorylated PKB (Thr308) and total PKB, were from Cell Signaling and were applied at the concentrations recommended by the manufacturer. The secondary antibodies were goat anti-rabbit IgG and anti-mouse IgG (Acris Antibodies, Hiddenhausen, Germany) and used at 1:10,000. A commercial detection system (Lumi-Light, Roche Applied Science) was used for detecting chemiluminescence signals according to the manufacturer's instructions. The obtained chemiluminescent autoradiographic signals were scanned by videodensitometry and quantitated with Imagequant software (
      • Kietzmann T.
      • Samoylenko A.
      • Immenschuh S.
      ).
      Plasmid Constructs—The luciferase reporter gene constructs pHO-1338, pHO-754, pHO-754Am, pHO-754Em, and pHO-754Am/Em have been described previously (
      • Kietzmann T.
      • Samoylenko A.
      • Immenschuh S.
      ,
      • Immenschuh S.
      • Hinke V.
      • Katz N.
      • Kietzmann T.
      ,
      • Wijayanti N.
      • Huber S.
      • Samoylenko A.
      • Kietzmann T.
      • Immenschuh S.
      ). The reporter gene plasmid pNF-κB with four tandem copies of the nuclear factor-κB (NF-κB) consensus sequence was obtained from Clontech, the plasmid pAP-1 with three AP-1 repeats in front of a minimal fos promoter was a gift from Dr. Craig A. Hauser (Burnham Institute, La Jolla, CA) (
      • Galang C.K.
      • Der C.J.
      • Hauser C.A.
      ), and pGal4-luc with five copies of the yeast Gal4 binding element was from Stratagene. Expression vectors for dominant negative PKB, constitutively active PKB (myrPKB), and wild type p300 have been described previously (
      • Roth U.
      • Curth K.
      • Unterman T.G.
      • Kietzmann T.
      ). Plasmid pFA-CHOP with the transactivation domain of the TF CHOP fused with the DNA-binding domain of yeast Gal4 and the empty control vector pFC2-dbd were from Stratagene. Expression vectors containing dominant negative mutants (AF) of p38α, β, γ, and δ isoforms were from Dr. Jiahuai Han (Scripps Research Institute, La Jolla, CA). The expression vectors for USF2 wild type and mutant (ΔbTDU1) were from Dr. Axel Kahn (Institut Cochin, Paris, France) (
      • Lefrancois-Martinez A.M.
      • Martinez A.
      • Antoine B.
      • Raymondjean M.
      • Kahn A.
      ).
      Transfection and Luciferase Assay—After growth for 24 h, transfection of plasmid DNA into RAW264.7 cells was performed by the liposome method using FuGENE (Roche Applied Science) as described previously (
      • Hess A.
      • Wijayanti N.
      • Neuschafer-Rube A.P.
      • Katz N.
      • Kietzmann T.
      • Immenschuh S.
      ). Unless otherwise stated cells were transfected with 0.5–1 μg of reporter plasmid, and in cotransfection experiments with 0.1–1.5 μg of the indicated expression vectors. Cells were lysed with luciferase lysis reagent (Promega), and luciferase activity was determined with a commercial luciferase assay system (Promega) according to the manufacturer's instructions. Cells were either harvested 24 h after transfection or treated for another 18 h with AEBSF or other reagents, as indicated. Relative light units of luciferase activity were normalized with sample protein.

      RESULTS

      Induction of HO-1 Gene Expression by the NAD(P)H Oxidase Inhibitor AEBSF in Cell Cultures of Human PBMC and Mouse RAW264.7 Cells—HO-1 gene expression has previously been shown to be up-regulated by the inflammatory mediator LPS (
      • Camhi S.L.
      • Alam J.
      • Otterbein L.
      • Sylvester S.L.
      • Choi A.M.K.
      ) and the phorbol ester TPA in monocytic cells (
      • Muraosa Y.
      • Shibahara S.
      ). Because LPS and TPA are activators of the monocyte NAD(P)H oxidase, we postulated that the chemical NAD(P)H oxidase inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF) would decrease the induction of HO-1 gene expression by these compounds. Unexpectedly, AEBSF treatment alone of both human PBMC and the mouse monocytic cell line RAW264.7 led to a marked induction of endogenous HO-1 gene expression as determined by Western blot analysis (Fig. 1). The magnitude of endogenous HO-1 gene induction by AEBSF was in the same range as that observed for LPS in human PBMC and that for LPS and TPA in RAW264.7 cells (Fig. 1, A and B). For a comparison, we also determined the effects of these stimuli on cyclooxygenase-2 gene expression, which has previously been shown to be induced by LPS and TPA in monocytes (
      • Smith W.L.
      • DeWitt D.L.
      • Garavito R.M.
      ). In contrast to HO-1, cyclooxygenase-2 gene expression was markedly up-regulated by TPA and LPS but not by AEBSF in RAW264.7 cells (Fig. 1B). A similar observation was made for the regulation of inducible nitric-oxide synthase gene expression which was not affected by treatment with AEBSF but was strongly up-regulated by the known inducer LPS (data not shown).
      Figure thumbnail gr1
      Fig. 1Induction of HO-1 gene expression by AEBSF in cell cultures of human PBMC and mouse RAW264.7 cells. Human PBMC and mouse monocytic RAW264.7 cells were cultured as described under “Experimental Procedures.” PBMC (A) and RAW264.7 cells (B–D) were treated with TPA (0.5 μm), LPS (1 μg/ml), AEBSF (250 μm), AEBSA (250 μm), combinations of AEBSF plus LPS, AEBSA plus LPS, AEBSF plus TPA, or control (Ctrl) medium for 18 h, as indicated, or (D) in the presence of AEBSF (250 μm) for the times indicated. Total protein (30–60 μg) was subjected to Western blot analysis and probed sequentially with antibodies against rat HO-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In addition, a cyclooxygenase-2 (COX-2) antibody was applied in B. Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry. Values ± S.E. represent the -fold induction of HO-1 or cyclooxygenase-2 normalized to glyceraldehyde-3-phosphate dehydrogenase from at least three independent experiments. Student's t test for paired values: *, significant differences AEBSF versus control, p ≤ 0.05.
      When added in combination with LPS or TPA, AEBSF had an additive effect on the LPS-dependent induction of HO-1 protein levels and caused no major alteration of HO-1 induction by TPA (Fig. 1C). Treatment with 4-(2-aminoethyl)benzenesulfonamide (AEBSA), a homologue of AEBSF in which the fluoride of AEBSF is replaced by an amide group and has no inhibitory effect on NAD(P)H oxidase activity (
      • Diatchuk V.
      • Lotan O.
      • Koshkin V.
      • Wikstroem P.
      • Pick E.
      ), did not affect the basal or LPS-induced HO-1 gene expression (Fig. 1C). The increase of HO-1 protein expression by AEBSF occurred in a time-dependent manner with a maximum after 8 h (Fig. 1D). The specificity of the AEBSF-dependent induction of HO-1 was confirmed by dose-dependence experiments. The minimum concentration that reproducibly induced HO-1 gene expression was higher than 50 μm, and a maximum was observed at concentrations between 250 μm and 500 μm (data not shown). Concentrations higher than 1 mm appeared to be toxic in RAW264.7 cells. An increase of HO-1 gene expression by AEBSF was also observed in human umbilical cord endothelial cells but not in cell cultures of porcine kidney epithelial cells (LLC-PK1) or human cervix epithelial cells (HeLa cells), suggesting a cell-specific mode of HO-1 gene regulation by this compound (data not shown). The data indicate that AEBSF is a potent inducer of endogenous HO-1 gene expression in monocytic cells.
      Induction of HO-1 Promoter Activity by AEBSF—HO-1 gene regulation by most stimuli occurs at the transcriptional level (
      • Choi A.M.K.
      • Alam J.
      ,
      • Alam J.
      • Igarashi K.
      • Immenschuh S.
      • Shibahara S.
      • Tyrrell R.M.
      ). To probe into the mechanism of AEBSF-dependent HO-1 gene induction, RAW264.7 cells were transiently transfected with a luciferase reporter gene construct containing the proximal 1338 bp of the rat HO-1 gene promoter (pHO-1338), which was markedly up-regulated by treatment with AEBSF. The level of AEBSF-dependent induction of HO-1 promoter activity was in a similar range as that observed for LPS, but lower than that observed for TPA (Fig. 2A). Simultaneous treatment with AEBSF plus LPS had an additive effect on HO-1 promoter activity. For a comparison, the effect of AEBSF was also examined in RAW264.7 cells transfected with reporter gene constructs containing multiple copies of the consensus recognition sequences for either NF-κB (pNF-κB) or AP-1 (pAP-1). Both NF-κB and AP-1 have previously been shown to be key TFs for LPS- and TPA-mediated gene regulation in mononuclear phagocytes (
      • Guha M.
      • Mackman N.
      ). No induction by AEBSF was observed for luciferase activity of pAP-1 and pNF-κB (Fig. 2A), but LPS and TPA markedly induced luciferase activity of these reporter gene constructs (Fig. 2A). Remarkably, LPS-dependent induction of constructs pNF-κB and pAP-1 was reduced by simultaneous treatment with AEBSF (Fig. 2A, lower panel). Similarly, the TPA-dependent induction of pNF-κB, but not that of pAP-1, was inhibited by the presence of AEBSF (Fig. 2A, lower panel). No regulatory effect on the promoter activity of pHO-1338 was observed for the AEBSF homologue AEBSA (Fig. 2B), which agrees with the regulation of endogenous HO-1 gene expression (Fig. 1C). The data suggest that the AEBSF-dependent induction of HO-1 gene expression is mediated via a transcriptional mechanism which seems to be independent of NF-κB and AP-1.
      Figure thumbnail gr2
      Fig. 2Induction of rat HO-1 promoter activity by AEBSF in transiently transfected RAW264.7 cells. A, RAW264.7 cells were transfected with reporter gene constructs containing the proximal 1338 bp of the rat HO-1 gene promoter region (pHO-1338), four copies of the consensus sequence of NF-κB (pNFκB), or three copies of the AP-1 consensus motif (pAP-1) and empty control vector pGL3basic. 24 h after transfection cells were treated for 18 h with or without AEBSF (250 μm), LPS (1 μg/ml), TPA (0.5 μm), or combinations of AEBSF plus LPS and AEBSF plus TPA, as indicated. B, cells were transfected with pHO-1338, and 24 h after transfection cells were treated for 18 h with or without AEBSF (250 μm) and AEBSA (250 μm), as indicated. Cell extracts were assayed for luciferase activity and the -fold induction in each experiment relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences treatment versus control; **, AEBSF + TPA versus TPA and AEBSF + LPS versus LPS; p ≤ 0.05. Ctrl, control.
      Regulatory Role of the PI3K/PKB Pathway for AEBSF-dependent Activation of HO-1 Gene Expression—The PI3K/PKB signaling pathway has recently been demonstrated to be involved in the induction of HO-1 gene expression by carnosol (
      • Martin D.
      • Rojo A.I.
      • Salinas M.
      • Diaz R.
      • Gallardo G.
      • Alam J.
      • De Galarreta C.M.
      • Cuadrado A.
      ) and the 3-hydroxy 3-methylglutaryl-coenzyme A reductase inhibitor simvastatin (
      • Lee T.S.
      • Chang C.C.
      • Zhu Y.
      • Shyy J.Y.
      ). To evaluate the regulatory role of this pathway for the AEBSF-dependent induction of HO-1 expression various pharmacological inhibitors were used for pretreatment of RAW264.7 cells. Up-regulation of endogenous HO-1 expression and that of HO-1 promoter activity by AEBSF was markedly reduced by pretreatment with the PI3K inhibitor wortmannin (Fig. 3, A and B). Moreover, the PI3K inhibitor LY294002 and the PKB inhibitor SH-5 caused a significant reduction of HO-1 promoter induction by AEBSF (Fig. 3B). To verify that AEBSF is an activator of PKB, cell extracts of AEBSF-treated RAW264.7 cells were analyzed for phosphorylated and total PKB. A marked increase of Thr308-phosphorylated PKB was observed upon AEBSF treatment (Fig. 3C).
      Figure thumbnail gr3
      Fig. 3Role of the PI3K/PKB pathway for AEBSF-dependent induction of HO-1 gene expression. A, RAW264.7 cells were pretreated for 1 h with wortmannin (wort, 500 nm) before incubation with AEBSF (250 μm). Total protein (40 μg) was subjected to Western blot analysis and probed sequentially with antibodies against rat HO-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Autoradiographic signals were obtained by chemiluminescence and scanned by videodensitometry. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences treatment versus control; **, wortmannin + AEBSF versus AEBSF; p ≤ 0.05. B, cells were transiently transfected with reporter gene construct pHO-1338. 24 h after transfection cells were treated for 1 h with the PI3K inhibitor wortmannin (500 nm), LY294002 (LY, 10 μm), or the PKB inhibitor SH-5 (10 μm) before incubation with AEBSF (250 μm) or control medium. After 18 h cell extracts were assayed for luciferase activity and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences AEBSF versus control; **, wortmannin + AEBSF versus AEBSF, LY + AEBSF versus AEBSF, SH-5 + AEBSF versus AEBSF; p ≤ 0.05. C, RAW264.7 cells were treated for 1 h with AEBSF and total protein (60 μg) was subjected to Western blot analysis and probed with an antibody for phosphorylated p-PKB (Thr308). Subsequently, the membrane was stripped and reprobed with an antibody against total PKB. Similar results were obtained in three independent experiments and a representative autoradiogram is shown. Ctrl, control. D, RAW264.7 cells were cotransfected with pHO-1338 and an expression vector for dominant negative (dn) PKB or empty control vector, as indicated. 24 h after transfection cells were treated with or without AEBSF (250 μm) for another 18 h. Cell extracts were assayed for luciferase activity and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant difference AEBSF + empty vector versus control; **, AEBSF + PKBdn versus AEBSF + empty vector; p ≤ 0.05. E, RAW264.7 cells were cotransfected with pHO-1338 and empty expression vector (ev) or an expression vector for constitutively active (ca) PKB, as indicated. 24 h after transfection cell extracts were assayed for luciferase activity and the -fold induction was determined relative to the control. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences PKBca versus control; p ≤ 0.05.
      To confirm the functional relevance of PKB activation for AEBSF-dependent induction of HO-1 gene expression, we also determined the effects of overexpressed dominant negative and constitutively active mutants of PKB. Overexpression of dominant negative PKB slightly increased basal HO-1 promoter activity, but abolished the AEBSF-dependent induction of HO-1 promoter activity (Fig. 3D). Overexpressed dominant negative phosphoinositide-dependent protein kinase-1, an effector molecule of PI3K that targets PKB, also inhibited the AEBSF-dependent activation of HO-1 promoter activity (data not shown). Finally, the specificity of HO-1 gene expression via the PKB pathway was examined by cotransfection of a constitutively active mutant of PKB (myrPKB), which led to a significant up-regulation of HO-1 gene promoter activity (Fig. 3E). The data indicate that the PI3K/PKB signaling cascade is crucially involved in the AEBSF-dependent regulation of HO-1 gene expression.
      Role of p38 MAPK for HO-1 Gene Regulation by AEBSF—To determine the potential regulatory role of MAPKs for the induction of HO-1 gene expression by AEBSF, the phosphorylation of MAPKs was analyzed by Western blotting in RAW264.7 cells. Phosphorylation of JNK and p38 was increased in AEBSF-treated cells with a maximum after 30 min. By contrast, no effect was observed for the level of phosphorylated ERK1/2 in AEBSF-treated cells (Fig. 4A). As a control, treatment with LPS led to phosphorylation of ERK1/2, JNK, and p38 in RAW 264.7 cells (data not shown). Next, we examined the effects of specific MAPK inhibitors on the AEBSF-dependent regulation of the endogenous HO-1 gene expression. The p38 inhibitor SB203580, but not the ERK inhibitor PD98059 nor the JNK inhibitor SP600125, reduced the AEBSF-dependent induction of HO-1 protein expression (Fig. 4, B–D).
      Figure thumbnail gr4
      Fig. 4Activation of MAPKs by AEBSF and effect of MAPK inhibitors on AEBSF-dependent induction of HO-1 gene expression. A, RAW264.7 cells were cultured as described under “Experimental Procedures” and were treated with AEBSF (250 μm) for the times indicated. Total protein (60 μg) was subjected to Western blot analysis and probed with antibodies for various MAPKs. Membranes were initially used to detect phosphorylated MAPKs then stripped and probed with antibodies against total MAPKs. B–D, RAW264.7 cells were pretreated for 1 h with inhibitors of MAPKs at the indicated concentrations. Thereafter, cells were incubated for another 18 h in the absence or presence of AEBSF (250 μm), as indicated. Total protein (60 μg) was analyzed by Western blot analysis with antibodies against rat HO-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar results were obtained in three independent experiments. Autoradiograms of a representative experiment are shown, respectively. Student's t test for paired values: *, significant differences treatment versus control; p ≤ 0.05. p-ERK, phospho-ERK; p-JNK, phospho-JNK; p-p38, phospho-p38.
      To investigate the role of MAPKs for the transcriptional regulation of the HO-1 gene by AEBSF, the effects of MAPK inhibitors on HO-1 promoter activity were determined. Although inhibitors of the ERK and JNK pathways, PD98059 and SP600125, respectively, had no effect (Fig. 5A), the p38 inhibitors SB203580 and SB202190 abolished AEBSF-dependent induction of HO-1 promoter activity (Fig. 5B). The regulatory role of p38 for AEBSF-dependent HO-1 gene induction was further delineated by cotransfection of expression vectors with dominant negative mutants (AF) of the p38α, -β, -γ, and -δ isoforms. Overexpression of dominant negative p38α and p38β significantly reduced the induction of HO-1 promoter activity by AEBSF, but dominant negative forms of p38γ and p38δ had only minor inhibitory effects (Table I). Taken together, these data suggest that p38α and p38β play a major regulatory role for the induction of HO-1 gene expression by AEBSF.
      Figure thumbnail gr5
      Fig. 5Role of MAPKs for AEBSF-dependent induction of HO-1 promoter activity. RAW264.7 cells were transfected with the HO-1 reporter gene construct pHO-1338. 24 h after transfection cells were pretreated with the MAPKs inhibitors PD98059 (4 μm) or SP600125 (10 μm) (A) or SB203580 and SB202190 (B) at the indicated concentrations for 1 h. Then, cells were incubated for another 18 h in the absence or presence of AEBSF (250 μm). Cell extracts were assayed for luciferase activity and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences AEBSF versus control; **, SB203580 + AEBSF versus AEBSF, SB202190 + AEBSF versus AEBSF; p ≤ 0.05.
      Table IComparative effects of overexpressed dominant negative p38 isoforms on AEBSF-dependent induction of HO-1 promoter activity
      Contransfected plasmid-Fold induction of luciferase activity by AEBSF
      Empty vector4.5 ± 0.5
      p38α dn1.3 ± 0.2
      Significant differences p38α dn + AEBSF versus empty vector + AEBSF, p38β dn + AEBSF versus empty vector + AEBSF; p ≤ 0.05. Student's t test for paired values.
      p38β dn1.2 ± 0.1
      Significant differences p38α dn + AEBSF versus empty vector + AEBSF, p38β dn + AEBSF versus empty vector + AEBSF; p ≤ 0.05. Student's t test for paired values.
      p38γ dn2.5 ± 0.2
      p38δ dn3.2 ± 0.4
      a Significant differences p38α dn + AEBSF versus empty vector + AEBSF, p38β dn + AEBSF versus empty vector + AEBSF; p ≤ 0.05. Student's t test for paired values.
      p38 Is a Downstream Target of AEBSF-dependent PKB Activation—To find out whether PKB is required for AEBSF-dependent stimulation of p38 MAPK, p38 activity was determined with a fusion plasmid containing the transactivation domain of the TF CHOP and the DNA-binding domain of yeast Gal4 (pFA-CHOP). Transactivation via pFA-CHOP is specifically controlled by p38-dependent phosphorylation of two adjacent regulatory serine residues of the CHOP transactivation domain (
      • Wang X.Z.
      • Ron D.
      ), which can be monitored via a cotransfected Gal4 luciferase reporter gene construct (pGal4-luc). Treatment with AEBSF strongly induced activity of pFA-CHOP in a similar range as the known p38 activator TPA (Fig. 6). AEBSF-dependent induction of pFA-CHOP was reduced by pretreatment with the PKB inhibitor SH-5 and overexpression of dominant negative PKB. As expected, the up-regulation of pFA-CHOP-mediated luciferase activity was inhibited by the p38 inhibitor SB202190. The data suggest that in RAW264.7 cells AEBSF-dependent activation of PKB is required for activation of p38.
      Figure thumbnail gr6
      Fig. 6Regulation of AEBSF-dependent induction of CHOP transactivity by inhibition of PKB. RAW264.7 cells were cotransfected with luciferase reporter gene construct pGal4-luc, pFC2-dbd, pFA-CHOP, and an expression vector for dominant negative (dn) PKB, as indicated. 24 h after transfection cells were treated with AEBSF (250 μm), TPA (0.5 μm), SB202190 (2.5 μm), or PKB inhibitor SH-5 (20 μm), as indicated. Cell extracts were assayed for luciferase activity, and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences treatment versus control; **, SB202190 + AEBSF versus AEBSF, SH-5 + AEBSF versus AEBSF, SB202190 + TPA versus TPA, dominant negative PKB + AEBSF versus AEBSF; p ≤ 0.05.
      Role of the E-box and CRE/AP-1 Elements for AEBSF- and PKB-dependent Induction of HO-1 Promoter Activity—To identify the cis-acting regulatory element(s) that mediate(s) the AEBSF-dependent induction of HO-1 gene expression, various HO-1 promoter gene constructs were transiently transfected into RAW264.7 cells. Deletion of the rat HO-1 gene promoter sequence from –1338 to –755 did not affect the responsiveness of reporter gene activity by AEBSF (pHO-754; Fig. 7A). Neither of two reporter gene constructs with a mutation of the E-box site (–47 to –42; pHO-754Em) or the CRE/AP-1 site (–664 to –657; pHO-754Am) showed a lower responsiveness to AEBSF. By contrast, simultaneous mutation of the E-box site and the CRE/AP-1 element (pHO-754Am/Em) led to a marked reduction of AEBSF-mediated induction of reporter gene activity (Fig. 7A). The regulation of these HO-1 reporter gene constructs was also examined in cells that were cotransfected with an expression vector for constitutively active PKB. Similar to the regulatory pattern of reporter gene activity by AEBSF, the overexpression of a constitutively active PKB mutant induced luciferase activity of wild type pHO-754, but not that of pHO-754Am/Em (Fig. 7B). No appreciable reduction of PKB-mediated responsiveness was observed for the HO-1 promoter gene constructs with either a mutation of the E-box or the CRE/AP-1 site (data not shown). These findings suggest that the AEBSF- and PKB-dependent activation of HO-1 gene expression is mediated via a transcriptional mechanism that involves the E-box and the CRE/AP-1 element of the proximal HO-1 promoter region.
      Figure thumbnail gr7
      Fig. 7Regulation of various rat HO-1 promoter gene constructs by AEBSF and overexpressed constitutively active PKB. A, HO-1 reporter gene constructs with the indicated mutations of the E-box and the CRE/AP-1 element of the rat HO-1 promoter were transfected into RAW264.7 cells. 24 h after transfection cells were treated for 18 h with control (Ctrl) medium or medium supplemented with AEBSF (250 μm). Cell extracts were assayed for luciferase activity, and the -fold induction was determined relative to the control. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences treatment versus control; **, pHO-754Am/Em + AEBSF versus pHO-754 + AEBSF; p ≤ 0.05. B, cells were cotransfected with the indicated HO-1 reporter gene constructs and empty expression vector (ev) or an expression vector for constitutively active (ca) PKB, as indicated. 24 h after transfection cell extracts were assayed for luciferase activity, and the -fold induction was determined relative to the control. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences constitutively active PKB versus control; **, pHO-754Am/Em + constitutively active PKB versus pHO-754 + constitutively active PKB; p ≤ 0.05.
      Role of USF2 and Coactivator p300 for the AEBSF-dependent Induction of HO-1 Promoter Activity—The identification of a functional E-box motif in the HO-1 promoter, along with the previous findings that basic helix-loop-helix TFs may serve as nuclear targets for p38 (
      • Galibert M.D.
      • Carreira S.
      • Goding C.R.
      ), may suggest that USF2 is involved in the AEBSF-dependent induction of HO-1 gene expression. Therefore, we performed cotransfection experiments with expression vectors for wild type USF2 and a dominant negative mutant of USF (ΔbTDU1), which dimerizes with USF proteins but lacks the DNA-binding and transactivation domain (
      • Lefrancois-Martinez A.M.
      • Martinez A.
      • Antoine B.
      • Raymondjean M.
      • Kahn A.
      ). The AEBSF-dependent responsiveness of HO-1 reporter gene activity was enhanced by overexpressed wild type USF2 and was reduced by overexpressed ΔbTDU1 mutant (Fig. 8A).
      Figure thumbnail gr8
      Fig. 8Effect of overexpressed USF2 and p300 on AEBSF-dependent induction of HO-1 promoter activity. A, RAW264.7 cells were cotransfected with pHO-1338 and expression vectors for either wild type USF2 or dominant negative USF2 (ΔbTDU1) or an empty control expression vector (ev), as indicated. 24 h after transfection cells were treated for another 18 h with or without AEBSF (250 μm). Cell extracts were assayed for luciferase activity, and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences AEBSF versus control (Ctrl); **, ΔbTDU1 + AEBSF versus control + AEBSF; p ≤ 0.05. B, RAW264.7 cells were cotransfected with reporter gene constructs pHO-754, pHO-754Am/Em and an expression vector for p300, or empty expression vector. 24 h after transfection cells were treated for 18 h with or without AEBSF (250 μm). Cell extracts were assayed for luciferase activity, and the -fold induction relative to the control was determined. Values are means ± S.E. from at least three independent experiments with duplicates of each point. Student's t test for paired values: *, significant differences AEBSF versus control; **, p300 versus empty vector; p ≤ 0.05.
      USF2 has recently been found to recruit the transcriptional coactivator p300/CRE-binding protein (
      • Goueli B.S.
      • Janknecht R.
      ,
      • Blobel G.A.
      ). Therefore, we examined the effect of a cotransfected expression vector for p300 on reporter gene activity of wild type pHO-754 and that of pHO-754Am/Em with targeted mutations of the E-box and the CRE/AP-1 sites in RAW264.7 cells. Overexpression of p300 strongly increased the basal and AEBSF-dependent induction of pHO-754 promoter activity (Fig. 8B, upper panel). In contrast, overexpression of p300 had only a minor effect on the basal and AEBSF-augmented promoter activity of pHO-754Am/Em. No effect of cotransfected p300 was observed for luciferase activity of the control vector pGL3basic (Fig. 8B). Taken together, these data suggest that USF2 and p300 are involved in the transcriptional induction of HO-1 gene expression by AEBSF in RAW264.7 cells.

      DISCUSSION

      The HO-1 gene codes for the first and rate-limiting enzyme of heme degradation and is induced by multiple stress stimuli. Overexpression of the HO-1 gene not only plays a protective role against oxidant damage of cells and tissues but also modulates the inflammatory immune response. The major findings of the present study are that the NAD(P)H-oxidase inhibitor AEBSF induces HO-1 gene expression in cell cultures of human and mouse monocytes by a transcriptional mechanism that involves an E-box motif and a CRE/AP-1 site of the rat HO-1 gene promoter. The AEBSF-dependent induction of the HO-1 gene is mediated via activation of a PKB, p38 MAPK signaling cascade.
      Transcriptional Induction of HO-1 Gene Expression by the NAD(P)H Oxidase Inhibitor AEBSF—The NAD(P)H-oxidase inhibitor AEBSF which is also known to be a serine protease inhibitor (
      • Diatchuk V.
      • Lotan O.
      • Koshkin V.
      • Wikstroem P.
      • Pick E.
      ) is a robust inducer of HO-1 gene expression in cell cultures of human monocytes and the mouse monocytic cell line RAW264.7 (Fig. 1). The magnitude of endogenous HO-1 gene induction by AEBSF in these cells was similar to that elicited by LPS (Fig. 1A) and, in RAW264.7 cells, also to that by the phorbol ester TPA (Fig. 1B). Both LPS and TPA are known to activate NAD(P)H oxidase in professional phagocytes, which generates the prooxidant superoxide (
      • Park H.S.
      • Jung H.Y.
      • Park E.Y.
      • Kim J.
      • Lee W.J.
      • Bae Y.S.
      ,
      • Babior B.M.
      ). The AEBSF-dependent induction of HO-1 in monocytes was unexpected, because we initially postulated that AEBSF may prevent the up-regulation of HO-1 gene expression by LPS and TPA. This unexpected observation is similar to the up-regulation of HO-1 gene expression by the antioxidant compounds pyrrolidine dithiocarbamate, caffeic acid phenethyl ester, or carnosol (
      • Martin D.
      • Rojo A.I.
      • Salinas M.
      • Diaz R.
      • Gallardo G.
      • Alam J.
      • De Galarreta C.M.
      • Cuadrado A.
      ,
      • Hartsfield C.
      • Alam J.
      • Choi A.M.K.
      ,
      • Scapagnini G.
      • Foresti R.
      • Calabrese V.
      • Giuffrida Stella A.M.
      • Green C.J.
      • Motterlini R.
      ). The antioxidant and metal chelator pyrrolidine dithiocarbamate has been demonstrated previously to be a potent inducer of HO-1 gene expression in monocytes (
      • Hartsfield C.
      • Alam J.
      • Choi A.M.K.
      ). More recently, caffeic acid phenethyl ester and the herb-derived diterpene carnosol were shown to induce HO-1 gene expression in astrocytes and rat pheochromocytoma cells (
      • Martin D.
      • Rojo A.I.
      • Salinas M.
      • Diaz R.
      • Gallardo G.
      • Alam J.
      • De Galarreta C.M.
      • Cuadrado A.
      ,
      • Scapagnini G.
      • Foresti R.
      • Calabrese V.
      • Giuffrida Stella A.M.
      • Green C.J.
      • Motterlini R.
      ), respectively.
      The present data indicate that the AEBSF-dependent upregulation of HO-1 gene expression in monocytes occurs on the transcriptional level which is mediated via an E-box motif (–47 to –42) and a CRE/AP-1 site (–664 to –657) of the proximal rat HO-1 promoter region (Figs. 2 and 7). Remarkably, reporter gene constructs with target sequences for the TFs NF-κB and AP-1, both of which are known to be involved in gene regulation by LPS and TPA (
      • Guha M.
      • Mackman N.
      ), did not respond to treatment with AEBSF (Fig. 2). The basic helix-loop-helix TF USF2 may be involved in the AEBSF-dependent induction of HO-1 promoter activity via interaction with the proximal HO-1 E-box motif (Fig. 8A). In accordance with such a conclusion the proximal E-box motif of the human HO-1 gene promoter has been shown previously to play a major regulatory role for the phorbol ester-dependent HO-1 gene regulation in the monocytic cell line THP-1 (
      • Muraosa Y.
      • Shibahara S.
      ). More recently, Hock et al. (
      • Hock T.D.
      • Nick H.S.
      • Agarwal A.
      ) reported that USF2 mediated basal and inducible HO-1 gene expression in renal proximal tubular cells via a physical interaction with the proximal E-box of the human HO-1 gene. The data of the present study strongly suggest that p300, which is synonymous with CRE-binding protein, is involved in AEBSF-dependent HO-1 gene induction. Because p300 mediates cooperation between transcriptional regulators by protein-protein contacts and links TFs with components of the basal transcription machinery (
      • Blobel G.A.
      ), it is conceivable that an interaction of p300 and USF2 may participate in this regulatory mechanism.
      Signaling Pathways of AEBSF-dependent HO-1 Gene Activation—To identify the signaling pathways involved in AEBSF-dependent HO-1 up-regulation, the effects of various pharmacological inhibitors and dominant negative expression vectors on AEBSF-stimulated HO-1 transcription were measured. A schematic summary of our findings is shown in Fig. 9. The reduction of AEBSF-dependent HO-1 gene induction by pharmacological inhibitors of the PI3K/PKB pathway and overexpression of dominant negative mutants of PKB indicated that the serine/threonine kinase PKB plays a major role for the AEBSF-dependent activation of the HO-1 gene in monocytes (Fig. 4). The significance of PKB activation for HO-1 gene regulation was confirmed by the findings that HO-1 promoter activity was highly induced by overexpressed constitutively activated PKB (Fig. 3E). A regulatory role of the PKB pathway for the up-regulation of HO-1 gene expression has recently been demonstrated in other cell culture systems. PKB has been shown to mediate the HO-1 gene induction by the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor simvastatin in human and rat vascular smooth muscle cells (
      • Lee T.S.
      • Chang C.C.
      • Zhu Y.
      • Shyy J.Y.
      ). Independently, the phytochemical carnosol has been shown to activate HO-1 gene expression in a PKB-dependent manner in PC12 pheochromocytoma cells (
      • Martin D.
      • Rojo A.I.
      • Salinas M.
      • Diaz R.
      • Gallardo G.
      • Alam J.
      • De Galarreta C.M.
      • Cuadrado A.
      ). The downstream targets of PKB, however, remained elusive in these earlier reports. Our present study shows that PKB is required for the activation of p38 MAPK by AEBSF (Fig. 6). It is demonstrated in a Gal4 luciferase assay that the AEBSF-dependent induction of a specific p38 target construct Gal4-CHOP (
      • Wang X.Z.
      • Ron D.
      ) is inhibited by treatment with the PKB inhibitor SH-5 and by overexpressed dominant negative PKB (Fig. 6). Therefore, p38 MAPK appears to be a downstream target of PKB in RAW264.7 monocytes. The inhibitory effects of specific pharmacological MAPK inhibitors and various isoforms of dominant negative mutants of p38 indicated that p38α and p38β, but not ERK and JNK, are involved in AEBSF-dependent induction of HO-1 gene expression (Figs. 4 and 5). Contradictory findings on cross-talk between the PKB and p38 signaling pathways have previously been reported by others. In agreement with our present findings, Lee et al. (
      • Lee H.M.
      • Jin H.S.
      • Park J.W.
      • Park S.M.
      • Jeon H.K.
      • Lee T.H.
      ) have demonstrated that the stimulation of p38 MAPK by anisomycin was attenuated by the inhibition of the PKB pathway in a follicular dendritic cell line, suggesting that PKB is necessary for the activation of p38 MAPK. In contrast, others have shown that p38 was necessary for the MAPK-activated protein kinase-2-dependent phosphorylation of PKB in human neutrophils concluding that p38 is a functional phosphoinositide-dependent kinase-2 for PKB (
      • Rane M.J.
      • Coxon P.Y.
      • Powell D.W.
      • Webster R.
      • Klein J.B.
      • Pierce W.
      • Ping P.
      • McLeish K.R.
      ). In accordance with these findings Taniyama et al. (
      • Taniyama Y.
      • Ushio-Fukai M.
      • Hitomi H.
      • Rocic P.
      • Kingsley M.J.
      • Pfahnl C.
      • Weber D.S.
      • Alexander R.W.
      • Griendling K.K.
      ) have demonstrated in vascular smooth muscle cells that the p38 MAPK pathway mediated angiotensin-dependent activation of PKB. More recently, it has been demonstrated in a model of mouse myoblast differentiation that the p38 and PI3K/PKB pathways are functional in a reciprocal manner. In this elegant study it was shown that the inhibition of p38 reduced PKB activity and that the down-regulation of PI3K/PKB decreased p38 MAPK activity (
      • Gonzalez I.
      • Tripathi G.
      • Carter E.J.
      • Cobb L.J.
      • Salih D.A.M.
      • Lovett F.A.
      • Holding C.
      • Pell J.M.
      ).
      Figure thumbnail gr9
      Fig. 9Schematic summary of signal transduction pathways by AEBSF. Activating steps of AEBSF-dependent induction of HO-1 up-regulation in monocytes as demonstrated in this study. dn, dominant negative.
      Physiological Significance of AEBSF-dependent Induction of HO-1 Gene Expression in Monocytic Cells—The major functions of HO enzyme activity are the degradation of the prooxidant heme and production of the antioxidant bilirubin, which provides cellular protection against oxidative stress (
      • Abraham N.G.
      • Lin J.H.
      • Schwartzman M.L.
      • Levere R.D.
      • Shibahara S.
      ). More recently, accumulating evidence indicates that HO-1 is an important modulator of the inflammatory response possibly via the generation of the second messenger gas, carbon monoxide (
      • Otterbein L.E.
      • Bach F.H.
      • Alam J.
      • Soares M.
      • Tao Lu H.
      • Wysk M.
      • Davis R.J.
      • Flavell R.A.
      • Choi A.M.
      ,
      • Otterbein L.E.
      • Soares M.P.
      • Yamashita K.
      • Bach F.H.
      ,
      • Kapturczak M.H.
      • Wasserfall C.
      • Brusko T.
      • Campbell-Thompson M.
      • Ellis T.M.
      • Atkinson M.A.
      • Agarwal A.
      ). An anti-inflammatory function of HO-1 has previously been shown in models of acute complement-dependent pleurisy and heme-induced inflammation of various mouse organs (
      • Willis D.
      • Moore A.R.
      • Frederick R.
      • Willoughby D.A.
      ,
      • Wagener F.A.
      • Eggert A.
      • Boerman O.C.
      • Oyen W.J.
      • Verhofstad A.
      • Abraham N.G.
      • Adema G.
      • van Kooyk Y.
      • de Witte T.
      • Figdor C.G.
      ). In addition, HO-1-deficient mice have been reported to be highly susceptible to endotoxin-mediated toxicity and to exhibit an immune phenotype that is associated with an exaggerated activation of mononuclear phagocytes (
      • Poss K.D.
      • Tonegawa S.
      ,
      • Kapturczak M.H.
      • Wasserfall C.
      • Brusko T.
      • Campbell-Thompson M.
      • Ellis T.M.
      • Atkinson M.A.
      • Agarwal A.
      ). These findings suggest a general proinflammatory tendency in genetic HO-1 deficiency. Moreover, the specific up-regulation of the HO-1 gene in monocytes by compounds such as AEBSF may provide novel strategies for the pharmacological therapy of inflammatory diseases (
      • Immenschuh S.
      • Ramadori G.
      ). In summary, we have shown that the induction of HO-1 gene expression by the NAD(P)H oxidase inhibitor AEBSF is mediated by a transcriptional mechanism that involves activation of a PKB, p38 MAPK signaling pathway.

      Acknowledgments

      We thank E. Welzel and D. Brusius for excellent technical assistance, Dr. J. Han, Dr. C. Hauser, and Dr. A. Kahn for the supply of plasmids, and Dr. K. Kissel and Dr. H. Hackstein for the preparation of human monocytes.

      References

        • Tenhunen R.
        • Marver H.S.
        • Schmid R.
        Proc. Natl. Acad. Sci. U. S. A. 1968; 61: 748-755
        • Wijayanti N.
        • Katz N.
        • Immenschuh S.
        Curr. Med. Chem. 2004; 11: 981-986
        • Ryter S.W.
        • Tyrrell R.M.
        Free Radic. Biol. Med. 2000; 28: 289-309
        • Maines M.D.
        Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554
        • Otterbein L.E.
        • Bach F.H.
        • Alam J.
        • Soares M.
        • Tao Lu H.
        • Wysk M.
        • Davis R.J.
        • Flavell R.A.
        • Choi A.M.
        Nat. Med. 2000; 6: 422-428
        • Stocker R.
        • Yamamoto Y.
        • McDonagh A.F.
        • Glazer A.N.
        • Ames B.N.
        Science. 1987; 235: 1043-1046
        • Choi A.M.K.
        • Alam J.
        Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19
        • Alam J.
        • Igarashi K.
        • Immenschuh S.
        • Shibahara S.
        • Tyrrell R.M.
        Antioxid. Redox Signal. 2004; 6: 924-933
        • Abraham N.G.
        • Lin J.H.
        • Schwartzman M.L.
        • Levere R.D.
        • Shibahara S.
        Int. J. Biochem. 1988; 20: 543-558
        • Otterbein L.E.
        • Soares M.P.
        • Yamashita K.
        • Bach F.H.
        Trends Immunol. 2003; 24: 449-455
        • Wagener F.A.
        • Volk H.D.
        • Willis D.
        • Abraham N.G.
        • Soares M.P.
        • Adema G.J.
        • Figdor C.G.
        Pharmacol. Rev. 2003; 55: 551-571
        • Willis D.
        • Moore A.R.
        • Frederick R.
        • Willoughby D.A.
        Nat. Med. 1996; 2: 87-90
        • Poss K.D.
        • Tonegawa S.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10919-10924
        • Poss K.D.
        • Tonegawa S.
        Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10925-10930
        • Yachie A.
        • Niida Y.
        • Wada T.
        • Igarashi N.
        • Kaneda H.
        • Toma T.
        • Ohta K.
        • Kasahara Y.
        • Koizumi S.
        J. Clin. Investig. 1999; 103: 129-135
        • Camhi S.L.
        • Alam J.
        • Otterbein L.
        • Sylvester S.L.
        • Choi A.M.K.
        Am. J. Respir. Cell Mol. Biol. 1995; 13: 387-398
        • Muraosa Y.
        • Shibahara S.
        Mol. Cell. Biol. 1993; 13: 7881-7991
        • Park H.S.
        • Jung H.Y.
        • Park E.Y.
        • Kim J.
        • Lee W.J.
        • Bae Y.S.
        J. Immunol. 2004; 173: 3589-3593
        • Babior B.M.
        Blood. 1999; 93: 1464-1476
        • Avogaro A.
        • Pagnin E.
        • Calo L.
        J. Clin. Endocrinol. Metab. 2003; 88: 1753-1759
        • Diatchuk V.
        • Lotan O.
        • Koshkin V.
        • Wikstroem P.
        • Pick E.
        J. Biol. Chem. 1997; 272: 13292-13301
        • Bestilny L.J.
        • Riabowol K.T.
        Exp. Cell Res. 2000; 256: 264-271
        • Singh V.P.
        • Saluja A.K.
        • Bhagat L.
        • Hietaranta A.J.
        • Song A.
        • Mykoniatis A.
        • Van Acker G.J.
        • Steer M.L.
        Gastroenterology. 2001; 120: 1818-1827
        • Hess A.
        • Wijayanti N.
        • Neuschafer-Rube A.P.
        • Katz N.
        • Kietzmann T.
        • Immenschuh S.
        J. Biol. Chem. 2003; 278: 45419-45434
        • Kietzmann T.
        • Samoylenko A.
        • Immenschuh S.
        J. Biol. Chem. 2003; 278: 17927-17936
        • Immenschuh S.
        • Hinke V.
        • Katz N.
        • Kietzmann T.
        Mol. Pharmacol. 2000; 57: 610-618
        • Wijayanti N.
        • Huber S.
        • Samoylenko A.
        • Kietzmann T.
        • Immenschuh S.
        Antioxid. Redox Signal. 2004; 6: 802-810
        • Galang C.K.
        • Der C.J.
        • Hauser C.A.
        Oncogene. 1994; 9: 2913-2921
        • Roth U.
        • Curth K.
        • Unterman T.G.
        • Kietzmann T.
        J. Biol. Chem. 2004; 279: 2623-2631
        • Lefrancois-Martinez A.M.
        • Martinez A.
        • Antoine B.
        • Raymondjean M.
        • Kahn A.
        J. Biol. Chem. 1995; 270: 2640-2643
        • Smith W.L.
        • DeWitt D.L.
        • Garavito R.M.
        Annu. Rev. Biochem. 2000; 69: 145-182
        • Guha M.
        • Mackman N.
        Cell. Signal. 2001; 13: 85-94
        • Martin D.
        • Rojo A.I.
        • Salinas M.
        • Diaz R.
        • Gallardo G.
        • Alam J.
        • De Galarreta C.M.
        • Cuadrado A.
        J. Biol. Chem. 2004; 279: 8919-8929
        • Lee T.S.
        • Chang C.C.
        • Zhu Y.
        • Shyy J.Y.
        Circulation. 2004; 110: 1296-1302
        • Wang X.Z.
        • Ron D.
        Science. 1996; 272: 1347-1349
        • Galibert M.D.
        • Carreira S.
        • Goding C.R.
        EMBO J. 2001; 20: 5022-5031
        • Goueli B.S.
        • Janknecht R.
        Oncogene. 2003; 22: 8042-8047
        • Blobel G.A.
        J. Leukocyte Biol. 2002; 71: 545-556
        • Hartsfield C.
        • Alam J.
        • Choi A.M.K.
        FASEB J. 1998; 12: 1675-1682
        • Scapagnini G.
        • Foresti R.
        • Calabrese V.
        • Giuffrida Stella A.M.
        • Green C.J.
        • Motterlini R.
        Mol. Pharmacol. 2002; 61: 554-561
        • Hock T.D.
        • Nick H.S.
        • Agarwal A.
        Biochem. J. 2004; 383: 209-218
        • Lee H.M.
        • Jin H.S.
        • Park J.W.
        • Park S.M.
        • Jeon H.K.
        • Lee T.H.
        FEBS Lett. 2003; 549: 110-114
        • Rane M.J.
        • Coxon P.Y.
        • Powell D.W.
        • Webster R.
        • Klein J.B.
        • Pierce W.
        • Ping P.
        • McLeish K.R.
        J. Biol. Chem. 2001; 276: 3517-3523
        • Taniyama Y.
        • Ushio-Fukai M.
        • Hitomi H.
        • Rocic P.
        • Kingsley M.J.
        • Pfahnl C.
        • Weber D.S.
        • Alexander R.W.
        • Griendling K.K.
        Am. J. Physiol. 2004; 287: C494-C499
        • Gonzalez I.
        • Tripathi G.
        • Carter E.J.
        • Cobb L.J.
        • Salih D.A.M.
        • Lovett F.A.
        • Holding C.
        • Pell J.M.
        Mol. Cell. Biol. 2004; 24: 3607-3622
        • Kapturczak M.H.
        • Wasserfall C.
        • Brusko T.
        • Campbell-Thompson M.
        • Ellis T.M.
        • Atkinson M.A.
        • Agarwal A.
        Am. J. Pathol. 2004; 165: 1045-1053
        • Wagener F.A.
        • Eggert A.
        • Boerman O.C.
        • Oyen W.J.
        • Verhofstad A.
        • Abraham N.G.
        • Adema G.
        • van Kooyk Y.
        • de Witte T.
        • Figdor C.G.
        Blood. 2001; 98: 1802-1811
        • Immenschuh S.
        • Ramadori G.
        Biochem. Pharmacol. 2000; 60: 1121-1128