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The Journal of Immunology, 2006, 177: 1894-1903.
Copyright © 2006 by The American Association of Immunologists

The Antiapoptotic Effect of Heme Oxygenase-1 in Endothelial Cells Involves the Degradation of p38{alpha} MAPK Isoform1

Gabriela Silva2, Andreia Cunha, Isabel Pombo Grégoire, Mark P. Seldon and Miguel P. Soares2

Instituto Gulbenkian de Ciência, Oeiras, Portugal


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Heme oxygenase-1 (HO-1) protects endothelial cells (EC) from undergoing apoptosis. This effect is mimicked by CO, generated via the catabolism of heme by HO-1. The antiapoptotic effect of CO in EC was abrogated when activation of the p38{alpha} and p38beta MAPKs was inhibited by the pyridinyl imidazole SB202190. Using small interfering RNA, p38beta was found to be cytoprotective in EC, whereas p38{alpha} was not. When overexpressed in EC, HO-1 targeted specifically the p38{alpha} but not the p38beta MAPK isoform for degradation by the 26S proteasome, an effect reversed by the 26S proteasome inhibitors MG-132 or lactacystin. Inhibition of p38{alpha} expression was also observed when HO-1 was induced physiologically by iron protoporphyrin IX (hemin). Inhibition of p38{alpha} no longer occurred when HO activity was inhibited by tin protoporphyrin IX, suggesting that p38{alpha} degradation was mediated by an end product of heme catabolism. Exogenous CO inhibited p38{alpha} expression in EC, suggesting that CO is the end product that mediates this effect. The antiapoptotic effect of HO-1 was impaired when p38{alpha} expression was restored ectopically or when its degradation by the 26S proteasome was inhibited by MG-132. Furthermore, the antiapoptotic effect of HO-1 was lost when p38beta expression was targeted by a specific p38beta small interfering RNA. In conclusion, the antiapoptotic effect of HO-1 in EC is dependent on the degradation of p38{alpha} by the 26S proteasome and on the expression of p38beta.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of the stress-responsive gene heme oxygenase-1 (HO-1)3 is part of a vascular response to injury that prevents the development of chronic inflammatory lesions such as those involved in the pathogenesis of atherosclerosis or the rejection of transplanted organs (reviewed in Refs. 1, 2). HO-1 cleaves the mesocarbon of heme, yielding equimolar amounts of CO, iron (Fe), and biliverdin (3), the latter being reduced into bilirubin by biliverdin reductase (reviewed in Ref. 4). Heme-derived iron induces the expression of H chain ferritin, by which it is subsequently sequestered (5).

HO-1 exerts salutary actions in endothelial cells (EC) where it acts in an antiapoptotic manner (6, 7). The antiapoptotic effect of HO-1 can be mimicked by exogenously applied CO (7, 8), suggesting that heme-derived CO mediates to a large extent this effect (7, 8, 9, 10). The antiapoptotic effect of CO is abrogated by SB203580, a pyridinyl imidazole that targets the ATP binding site on p38 MAPK, blocking their kinase activity (8, 10). This suggested that the antiapoptotic effect of CO depends on the activation of the p38 MAPK signal transduction pathway (8, 10).

The p38 MAPK family of proteins regroups four distinct kinases, encoded by different genes, i.e., p38{alpha} (p38{alpha}/CSBP-1, CSBP-2, Mxi2, and Exip; 38 kDa) (11), p38beta (p38beta/p38beta1 and p38beta2; 39 kDa) (12), p38{gamma} (ERK6/SAPK3; 43 kDa) (13), and p38{delta} (SAPK4; 40 kDa) (Ref. 14 and reviewed in Ref. 15). These share a sequence homology ranging from 74% (p38{alpha} vs p38beta) to 98% (p38beta vs p38beta2) and a canonical dual phosphorylation site (Thr-Gly-Tyr) (reviewed in Ref. 15). Pyridinyl imidazoles, such as SB203580, inhibit the activity of p38{alpha} and p38beta but spare p38{gamma} and p38{delta} (16), thus suggesting that the antiapoptotic effect of CO, abrogated by SB203580, acts via the p38{alpha} and/or the p38beta MAPK isoforms.

Mice genetically deficient in p38{alpha} (17) but not p38beta (18) are embryonic lethal, showing unequivocally that the biological actions of p38{alpha} and p38beta are not overlapping. Cardiomyocytes and fibroblasts derived from p38{alpha}-deficient mice are less susceptible to undergo apoptosis, suggesting that p38{alpha} is proapoptotic (19). In support of this notion, p38{alpha} activation promotes apoptosis (20) in L929 fibroblasts (21), myocytes (22), HeLa cells (23), and Jurkat T cells (24). In contrast, p38beta activation is antiapoptotic in these cells (21, 22, 23, 24). This suggests that p38{alpha} and p38beta have antagonistic effects in controlling apoptosis, i.e., p38{alpha} being pro- and p38beta antiapoptotic (reviewed in Ref. 25). Based on these studies, as well as the growing body of evidence that CO acts via p38 MAPK to prevent EC from undergoing apoptosis, we hypothesized that HO-1/CO might avoid signaling via the cytotoxic p38{alpha} isoform, signaling preferentially via the cytoprotective p38beta isoform. Our present data provide a mechanism to explain how this occurs, i.e., HO-1 targets specifically p38{alpha} for degradation by the 26S proteasome sparing p38beta. This finding is in keeping with the recent observation that the antiapoptotic effect of CO in EC requires the expression of p38beta (26).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Primary HUVEC (Cambrex) and bovine aortic EC (BAEC; Cell Systems) were cultured as previously described (8). HeLa cells were obtained from American Type Culture Collection.

Plasmid constructs

Wild-type (WT) hemagglutinin (HA)-tagged human p38{alpha} (HA-CSBP2) cDNA (provided by Dr. J. Anrather, Cornell University, Ithaca, NY) was expressed in pcDNA3 (Invitrogen Life Technologies) (8). WTp38{alpha} (FLAG-CSBP2) expression plasmid was generated by ligating BamHI/XbaI-excised CSBP2 with the FLAG epitope sequence and cloned into HindIII/XbaI-digested pcDNA3 (Invitrogen Life Technologies). Expression plasmids encoding the WT human FLAG-tagged p38beta, p38{gamma}, and p38{delta} cDNAs (a gift from Dr. R. J. Davis, University of Massachusetts, Worcester, MA) were expressed in pcDNA3 (13, 14, 27). Rat HO-1 cDNA was expressed under the control of the beta-actin (beta-actin/HO-1) or the CMV (pcDNA3/HO-1) enhancers/promoters (8). The pcDNA3/HO-1 vector contains 97 bp from the rat HO-1 promoter region. LacZ cDNA was expressed under the simian virus 40 early promoter and enhancer (pRSV-beta-galactosidase; Promega). Firefly luciferase cDNA was expressed under the control of the simian virus 40 promoter (pGL3-Control; Promega). PCAGGS-AFP, a vector containing the gene coding for the firefly GFP, was expressed under the control of the chicken beta-actin promoter (a gift from Dr. T. Momose, Himeji Institute of Technology, Hyogo, Japan) (28). Human p38{alpha} and p38beta small interfering RNA (siRNA) were expressed under the control of the polymerase III promoter H1 using the pSuper expression vector (29). The sequences used to target human p38{alpha} and p38beta MAPK were: p38{alpha} sense, 5'-CCAGTGGCCGATCCTTATGTTCAAGAGACATAAGGATCGGCCACTG-3'; antisense, 5'-CCAGTGGCCGATCCTTATGTCTCTTGAACATAAGGATCGGCCACTGG-3'; p38beta sense, 5'-GTACATCCACTCGGCCGGGTTCAAGAGACCCGGCCGAGTGGATGTAC-3'; and antisense, 5'-GTACATCCACTCGGCCGGGTCTCTTGAACCCGGCCGAGTGGATGTAC-3'; p38{alpha} and p38beta siRNA vectors were amplified in Escherichia coli (ATCC 35691 cells). DNAs were purified using the HiSpeed Plasmid Maxi kit (Qiagen).

Cell treatments and reagents

Actinomycin D (ActD; Sigma-Aldrich) in PBS was added to the culture medium 24 h after transfection (0.1 µg/ml). Human rTNF-{alpha} (R&D Systems) in PBS/0.1% BSA was added to the culture medium (10–50 ng/ml) 24 h after transfection. The 26S proteasome inhibitors carbobenzoxyl-Leu-Leu-Leucimal (MG-132) and lactacystin (Calbiochem-Novabiochem) in DMSO (Sigma-Aldrich) were added to the culture medium (0–20 µM) 22 h after transfection until the end of the experiment (4 h thereafter). Fe protoporphyrin IX (hemin/FePPIX) and tin protoporphyrin IX (SnPPIX; Frontier Scientific) were prepared as described in Ref. 8 . Hemin was used to induce HO-1 expression in cultured BAEC, as described by Balla et al. (30). Briefly, EC were washed once in HBSS containing Ca2+ and Mg2+ (Invitrogen Life Technologies) and exposed to hemin in HBSS (2.5 and 5 µM, 2 h). Hemin/HBSS was removed and HUVEC or BAEC were incubated in culture medium for an additional period of 12 or 24 h, respectively. Alternatively, hemin was added to the culture medium (10–20 µM) 6 h after transfection until the end of the experiment (24 h thereafter). SnPPIX was added (10–20 µM) 6 h after transfection for 24 h. The p38 MAPK inhibitor pyridinyl imidazole SB202190 (Calbiochem-Novabiochem) was dissolved in DMSO and added to the culture medium (30 µM) 30 min before induction of apoptosis. HO-1 siRNA (sc-35554; Santa Cruz Biotechnology) was used at 30 pmol/well of a 6-well plate.

RT-PCR

Total RNA was isolated using TRIzol (Invitrogen Life Technologies), genomic DNA was removed using RNase-free DNase I with RNase inhibitor RNase Out (Invitrogen Life Technologies), and purified RNA (2–5 µg) was reverse transcribed using SuperScriptII RNase H-reverse transcriptase and random hexamer primers (Invitrogen Life Technologies). mRNA encoding specific p38 MAPK isoforms was detected by PCR using specific primers for p38{alpha}: sense, 5'-AACCTGTCTCCAGTGGGCTCT-3' and antisense, 5'-AGCTTCTTAACTGCCACACG-3'; for p38beta: sense, 5'-GGCCACGTCCATCGAGGAC-3' and antisense, 5'-CGCCTGGCACTTGACGATG-3'; for p38{gamma}: sense, 5'-CGCCTCCGGCTGAGTTT-3' and antisense, 5'-GCTTGCATTGGTCAGGATAGA-3'; and for p38{delta}: sense, 5'-GTGCTCGGCCATCGACA-3' and antisense, 5'-CGGTAAGCGCGCTTGGC-3'. HO-1 cDNA was amplified using sense, 5'-TCTCAGGGGGTCAGGTC-3' and antisense, 5'-GGAGCGGTGTCTGGGATG-3. beta-tubulin (loading control) with sense 5'-GGCAAATATGTTCCTCGTGC–3' and antisense 5'-CCCAGTGAGTGGGTCAGC-3'. Conditions for cDNA amplification were 94°C (5 min), followed by 25 cycles of 94°C (1 min), 65°C (30 s), and 72°C (1 min). Relative levels of mRNA were quantified by densitometry using the ImageJ software version 1.29 (National Institute of Health).

Transient transfections

Cells were transfected at 60–75% confluence using LipofectAMINE 2000 (Invitrogen Life Technologies). LipofectAMINE reagent was used to examine the effect of HO-1 in TNF-{alpha}-induced apoptosis. The total amount of DNA used per well of a 6-well plate was 3 µg for BAEC, 1.5–2.5 µg for HUVEC, and 2.5 µg for HeLa cells. The total amount of DNA was kept constant in each transfection using pcDNA3 or pSuper vectors. Transfection efficiency was determined by flow cytometry or by fluorescence microscopy in cells cotransfected with PCAGGS-AFP. Transfection efficiency was typically 20–40%, 5–20%, and 40–60% for BAEC, HUVEC, and HeLa cells, respectively. HO-1 siRNA was transfected using oligofectamine (Invitrogen Life Technologies). Transfection efficiency was typically 90–100%.

Cell extracts and Western blot analysis

Whole cell extracts were resolved by electrophoresis using 10% polyacrylamide gels under denaturing conditions and transferred into polyvinyldifluoridine membranes (Bio-Rad) as described elsewhere (31). HO-1 was detected using an anti-human HO-1 rabbit polyclonal Ab (SPA-895; StressGen Biotechnologies). HA-tagged proteins were detected using a rabbit anti-HA polyclonal Ab (Santa Cruz Biotechnology) or rat anti-HA polyclonal Ab (Roche). FLAG-tagged proteins were detected using a mouse anti-FLAG monoclonal M2 Ab (Sigma-Aldrich). Total and phosphorylated p38 MAPK were detected with rabbit polyclonal Abs directed against the total or phosphorylated forms of these MAPK, respectively (Cell Signaling Technology). p38{alpha} and p38beta were detected using a rabbit anti-human p38{alpha} or p38beta polyclonal Ab, respectively (a gift from Dr. C. Fearns, The Scripps Research Institute, La Jolla, CA) (32). To ascertain equivalent sample, loading membranes were stripped and reprobed with a mouse anti-human {alpha}-tubulin mAb (Sigma-Aldrich). Primary Abs were detected using HRP-conjugated donkey anti-rabbit, goat anti-mouse, or goat anti-rat IgG secondary Abs (Pierce). Peroxidase activity was visualized using the SuperSignal West Pico Chemiluminescent substrate (Pierce). Signals were stored as photoradiographs (Kodak Biomax Light Film; Eastman Kodak). Digital images were obtained using an image scanner equipped with Adobe Photoshop software. Relative level of protein expression was quantified using ImageJ 1.29 Software (National Institutes of Health).

Immunoprecipitation

Cells were lysed in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM PMSF (Sigma-Aldrich). Soluble fractions were incubated with protein G-Sepharose beads/Ab complex in borate-buffered solution and 1% Triton X-100 (4 h or overnight at 4°C). Endogenous p38{alpha} and p38beta were immunoprecipitated (IP) with anti-human p38{alpha} or p38beta mAbs (2–5 µg), respectively (32). For overexpressed proteins, rat anti-HA polyclonal (5 µg; Roche) and mouse anti-FLAG M2 monoclonal (5 µg; Sigma-Aldrich) Abs were used. Proteins were washed in lysis buffer as above, resuspended in 2x sample buffer, heated to 100°C for 5 min, resolved by SDS-PAGE, and subjected to Western blotting.

Immunofluorescence

Confluent BAEC were fixed in 3.7% paraformaldehyde and stained using rabbit anti-human p38{alpha} or p38beta polyclonal Abs for the detection of endogenous proteins. Mouse anti-FLAG M2 mAb (5 µg; Sigma-Aldrich) was used for the detection of overexpressed p38{alpha} or p38beta. Rabbit anti-human p38{alpha} and anti-human p38beta Abs were raised in rabbits immunized with the SFVPPPLDQEEMES and SFKPPEPPKPPGSLEIEQ peptides, respectively (provided by Dr. L. Otterbein, Harvard Medical School, Boston, MA). Primary Abs were detected using a FITC-labeled goat anti-rabbit polyclonal Ab (Pierce). Nuclear (i.e., DNA) and cytoplasmic (i.e., F-actin) compartments were stained using 4',6'-diamidino-2-phenylindole (DAPI, 20 ng/ml; Sigma-Aldrich) and tetramethylrhodamine isothiocyanate (TRITC)-labeled phalloidin (50 ng/ml; Sigma-Aldrich), respectively. Cells were analyzed under a fluorescence microscope (Leica DMRA2). Fluorescence was acquired at {lambda}Ex = 480/40 nm and {lambda}Em = 527/30 nm for FITC, {lambda}Ex = 535/50 nm and {lambda}Em = 610/75 nm for TRITC and {lambda}Ex = 360/40 nm and {lambda}Em = 470/40 nm for DAPI using Metamorph v4.6r5 software (Universal Imaging Corporation) and treated with ImageJ software.

Flow cytometry

Cells were harvested by trypsin/EDTA digestion and resuspended in cold PBS/10% FCS. Fluorescent labeling was evaluated using a FACS equipped with CellQuest software (BD Biosciences). GFP expression was evaluated by comparison of fluorescent labeling of GFP-transfected vs GFP-nontransfected cells.

Cell viability assays

HUVEC were either cotransfected with p38{alpha} or p38beta siRNA expression vectors (1.5 µg/well of a 6-well plate) plus luciferase (pGL3-Control) or beta-galactosidase (pRSV-beta-galactosidase) reporters (0.25 µg/well of a 6-well plate). Viability was assessed 72 h after transfection according to luciferase or beta-galactosidase activities as assayed using a luciferase assay system (Promega) or Galacto-Light (Applied Biosystems), respectively. Cells were washed once in cold PBS under agitation (5 min; 200 rpm at room temperature) in a basic orbital shaker (KS 260; IKA-Works) and once in cold PBS without further agitation before lysis (Promega). Luciferase and beta-galactosidase activities were measured using a Microlumat Plus luminometer (LB96V; Berthold Technologies). When indicated, apoptosis was induced by TNF-{alpha} (50 ng/ml) in the presence of the transcription inhibitor ActD (10 µg/ml) for 8 h. To assess the effect of HO-1 in TNF-{alpha}-induced apoptosis, BAEC were cotransfected with beta-actin/HO-1 (0.5–0.7 µg/well of a 6-well plate). To evaluate whether reconstitution of the cellular pool of p38{alpha} reverted the protective effect of HO-1, BAEC were cotransfected with HO-1 plus FLAG-p38{alpha}. FLAG-p38beta was used as a control. To evaluate the effect of p38beta on HO-1 cytoprotection, HUVEC were cotransfected with HO-1 alone or HO-1 plus p38beta siRNA vectors. HUVEC were serum-starved (1% FBS) 24 h after transfection and exposed to cycloheximide (CHX; 10 µg/ml) alone or CHX plus TNF-{alpha} (50 ng/ml) for 8 h. Transfections were done in triplicate or in quadruplicate in at least three independent experiments. Relative percentage of cell viability were normalized for each transfection to control EC treated with ActD or CHX without TNF-{alpha} (100%) for BAEC and HUVEC, respectively.

Apoptosis assays

HUVEC were either cotransfected with p38{alpha} siRNA or p38beta siRNA vectors (1.5 µg/well) plus pCAGGS-AFP reporter (50 ng/well). Alexa Fluor 647-conjugated annexin V (Molecular Probes) was used to detect apoptosis in GFP-positive cells by flow cytometry, according to the manufacturer’s instructions.

CO exposure

BAEC were exposed to synthetic air or to 10,000 parts per million (ppm) CO in synthetic air, both supplemented with 5% CO2 for 24 h, as previously described (31).

Statistical analysis

The standard experimental design used consisted of having at least three independent assays for every condition tested. Each assay was taken as a unique experiment independent of other assays. For viability/apoptosis assays, each experiment was performed in triplicate or quadruplicate. Statistical analysis was performed using Student’s t test. Significance was inferred after the Bonferroni correction for {alpha} = 0.05/k, where k is the number of tests performed within the same set of experiments. Data are shown relative to control conditions as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The antiapoptotic effect of HO-1 is abrogated under inhibition of the p38{alpha} and p38beta activity

When transiently overexpressed in EC, HO-1 prevented TNF-{alpha} plus ActD-mediated apoptosis, as assessed by monitoring the expression of a coexpressed luciferase reporter (Fig. 1A) (8, 10). Similar results were obtained when GFP or beta-galactosidase reporters were used to monitor EC viability (data not shown). The protective effect of HO-1 was abrogated by SB202190 (Fig. 1A) (8, 10), a pyridinyl imidazole that inhibits the activation of both the p38{alpha} and p38beta, but not that of the p38{gamma} or p38{delta} MAPK isoforms (16). Expression of mRNA encoding p38{alpha} and p38beta was detected in quiescent EC by RT-PCR (Fig. 1B). Specificity of the oligonucleotides used was confirmed using human p38{alpha}, p38beta, p38{gamma}, or p38{delta} cDNA expression vectors (data not shown). Expression of p38{alpha} and p38beta in EC was further assessed at the protein level by Western blot (Fig. 1C) and immunofluorescence (Fig. 1D). Specificity of the Abs used was confirmed by Western blot using whole cell lysates from EC transiently transfected with human p38{alpha}, p38beta, p38{gamma}, or p38{delta} cDNA expression vectors (data not shown). Both endogenous and transiently overexpressed p38{alpha} and p38beta localized in the nuclei and only to a lesser extent in the cytoplasm of quiescent EC, as assessed by immunostaining (Fig. 1D). These data show that quiescent EC express both the p38{alpha} and p38beta MAPK isoforms (32) and that when overexpressed these p38 isoforms localize in similar cellular compartments to those of the endogenous isoforms.


Figure 1
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  		FIGURE 1. Inhibition of p38{alpha} and p38beta MAPK abrogate HO-1-mediated cytoprotection in EC. A, BAEC were transiently transfected with a RSV-driven luciferase reporter with or without beta-actin/HO-1 expression vector. When indicated, p38 MAPK activity was inhibited by SB202190. Apoptosis was induced by TNF-{alpha} plus ActD. Cellular viability (percent viability) was assessed according to luciferase activity, as described in Materials and Methods. Results are expressed as mean ± SD from three independent experiments, each done in triplicate (*, p < 0.001 vs ActD treated alone). B, Expression of p38{alpha} and p38beta mRNA was detected by RT-PCR in quiescent HUVEC. Purified pcDNA3/CSBP2 (p38{alpha}) or pcDNA3/p38beta (p38beta) cDNAs were used as positive controls. Purified pcDNA3 was used as negative control. C, p38{alpha} and p38beta were IP from quiescent HUVEC and detected by Western blot (WB) using isoform-specific polyclonal Abs. Ig indicates control Ig used in IP. D, Endogenous p38{alpha} (a) and p38beta (b) were detected in confluent BAEC using isoform-specific polyclonal Abs. Background staining was established using rabbit serum in naive BAEC (c). Overexpressed p38{alpha} (d) and p38beta (e) were detected in BAEC transiently transfected with FLAG-p38{alpha} (d) or FLAG-p38beta (e) using an anti-FLAG Ab. Background staining was established using anti-FLAG Ab in BAEC transiently transfected with pcDNA3 (f). In all panels, DNA was stained with DAPI (blue) and F-actin with TRITC-labeled phalloidin (red). Arrows indicate p38{alpha} and p38beta staining (green).

 
The p38beta but not p38{alpha} isoform is cytoprotective in EC

The p38{alpha} and p38beta MAPK isoforms have antagonistic effects in controlling cellular viability in a variety of cell types, i.e., p38{alpha} is proapoptotic, whereas p38beta is antiapoptotic (21, 22, 23, 24). We asked whether this would also be the case in EC. Inhibition of p38{alpha} using a p38{alpha} siRNA expression vector increased EC viability, as compared with EC transfected with a control siRNA (Fig. 2A). This observation suggests that endogenous p38{alpha} is cytotoxic in EC. On the contrary, when the expression of p38beta was targeted using a p38beta siRNA, EC viability was decreased, as compared with EC transfected with a control siRNA (Fig. 2A). This observation suggests that endogenous p38beta is cytoprotective in EC.


Figure 2
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  		FIGURE 2. p38beta but not p38{alpha} is cytoprotective in EC. A, HUVEC were transiently transfected with the RSV-driven luciferase reporter plus p38{alpha}, p38beta, or control (c) siRNA (pSuper) expression vectors. Cellular viability (percent viability) was assessed according to luciferase activity as described in Materials and Methods. Relative percentage of viability is shown as the mean ± SD from seven independent experiments done in triplicate (*, p < 0.005 vs control (c)). B, HUVEC were transiently transfected as in A and apoptosis was monitored by flow cytometry according to annexin V and propidium iodide staining in GFP-positive (transfected) cells. C, BAEC were transiently transfected with HA-tagged p38{alpha} (CSBP2) plus p38{alpha} siRNA (0, 1500, and 3000 ng per 3 x 105 cells). HA-p38{alpha} and {alpha}-tubulin were detected by Western blot using anti-HA and anti-{alpha}-tubulin Abs, respectively. D, BAEC were transiently transfected with FLAG-tagged p38beta-2 and p38beta siRNA (0, 1500, and 3000 ng per 3 x 105 cells). The rest of the procedure was conducted as in C using an anti-FLAG Ab for the detection of overexpressed p38beta. One representative gel of three independent experiments is shown in all panels. E, HeLa cells were transiently transfected with p38{alpha} siRNA (0, 500, and 1000 ng of DNA per 3 x 105 cells). Endogenous p38{alpha}, p38beta, and beta-tubulin mRNAs were detected by RT-PCR using specific primers. F, HeLa cells were transiently transfected with p38beta siRNA (0, 500, and 1500 ng per 3 x 105 cells) and the rest of the procedure was conducted as in E.

 
Transient overexpression of p38beta siRNA with a GFP reporter revealed that transfected EC expressed the early apoptotic marker phosphatidylserine V at the cell surface (~40–50% of GFP+ EC), as detected by flow cytometry (Fig. 2B). This suggests that inhibition of endogenous p38beta expression triggers EC to undergo apoptosis.

Transient cotransfection of EC with p38{alpha} plus p38{alpha} siRNAs or p38beta plus p38beta siRNA expression vectors resulted in suppression of the targeted p38 isoforms. Namely, expression of HA-p38{alpha} (Fig. 2C) or FLAG-p38beta (Fig. 2D) was decreased in a dose-response manner by the p38{alpha} and p38beta siRNAs, respectively. Specificity of the siRNA for the targeted p38 isoforms was confirmed in transiently transfected HeLa cells (Fig. 2, E and F). The p38{alpha} siRNA inhibited endogenous p38{alpha} but not p38beta mRNA expression (Fig. 2E). The p38beta siRNA inhibited endogenous p38beta but not p38{alpha} mRNA expression (Fig. 2F). This data demonstrate that the p38{alpha} and p38beta siRNAs are specific for the targeted p38 isoforms. Expression of p38{alpha} or p38beta mRNA was not decreased to undetectable levels (Fig. 2, E and F), probably because HeLa transfection efficiency was ~50%, as assessed by flow cytometry using a GFP reporter (data not shown).

Effect of HO-1 on p38{alpha} and p38beta activation

Given that in EC p38beta is cytoprotective whereas p38{alpha} is not (Fig. 2, A and B), we asked whether HO-1 would inhibit specifically the activation of p38{alpha} and/or induce that of p38beta. EC were transiently cotransfected with p38{alpha} or p38beta with or without HO-1 expression vectors and exposed to TNF-{alpha} (50 ng/ml, 5 min). Activation of p38{alpha} and p38beta was monitored by the relative level of phosphorylation, as assessed by Western blot after isoform-specific immunoprecipitation. Overexpression of HO-1 resulted in a relative decrease of TNF-{alpha}-mediated p38{alpha} phosphorylation, as compared with control EC that did not express HO-1 (Fig. 3A). However, this effect was due to a decrease in p38{alpha} expression, because when normalized to the total level of p38{alpha}, the relative level of phosphorylated p38{alpha} remained unchanged in EC overexpressing HO-1, as compared with control EC (Fig. 3, A and C). Overexpression of HO-1 failed to modulate p38beta phosphorylation (Fig. 3, B and C). These data suggest that HO-1 does not interfere with the signal transduction pathway triggered by TNF-{alpha} and leading to p38{alpha} or p38beta MAPK activation.


Figure 3
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  		FIGURE 3. Activation of p38{alpha} or p38beta by TNF-{alpha} is not modulated by HO-1. A, BAEC were transiently transfected with HA-p38{alpha} with or without pcDNA3/HO-1 and exposed to TNF-{alpha} (50 ng/ml, 5 min). HA-p38{alpha} was IP with anti-HA Ab. Phosphorylated or total HA-p38{alpha} (p-p38{alpha} and p38{alpha}, respectively) was detected by Western blot. HO-1 and {alpha}-tubulin were detected in whole cell lysates (WCL) by Western blot. B, BAEC were transiently transfected with FLAG-p38beta with or without pcDNA3/HO-1 and exposed to TNF-{alpha} as in A. FLAG-p38beta was IP with anti-FLAG Ab. Phosphorylated or total FLAG-p38beta (p-p38beta and p38beta, respectively) was detected by Western blot. HO-1 and {alpha}-tubulin were detected in whole cell lysates by Western blot. C, Relative levels of p38{alpha} and p38beta phosphorylation were quantified and normalized to total p38{alpha} or p38beta as described in Materials and Methods. Relative values shown as arbitrary units (AU) were normalized to EC, not transfected with HO-1 and exposed to TNF-{alpha} (= 1). Values from four independent experiments are shown as mean ± SD.

 
HO-1 inhibits specifically the expression of the p38{alpha} isoform in EC

Given that the amount of IP p38{alpha} was decreased in EC overexpressing HO-1, as compared with control EC (Fig. 3A), we asked whether HO-1 would decrease p38{alpha} expression in EC. Transient coexpression of HO-1 with HA-p38{alpha} plus FLAG-p38beta in EC resulted in significant inhibition of p38{alpha} but not of p38beta protein expression, as compared with control EC that did not express HO-1 (Fig. 4, A and B). The ability of HO-1 to inhibit p38{alpha} expression was dose dependent in that increasing levels of HO-1 resulted in decreasing levels of p38{alpha} protein expression, as assessed by Western blot (Fig. 4, C and D). This effect was specific to p38{alpha}, since HO-1 did not modulate p38beta expression (Fig. 4, E and F). Transient coexpression of HO-1 with FLAG-p38{alpha} also resulted in inhibition of p38{alpha} protein expression, as compared with control EC that did not overexpress HO-1 (data not shown). This data demonstrate that the ability of HO-1 to suppress p38{alpha} expression is not linked to the epitope used to detect p38{alpha} in this assay, i.e., HA vs FLAG.


Figure 4
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  		FIGURE 4. HO-1 decreases specifically p38{alpha} protein expression in EC. A, BAEC were transiently transfected with HA-p38{alpha} and FLAG-p38beta with or without pcDNA3/HO-1 expression vectors. HA-p38{alpha}, FLAG-p38beta, HO-1, and {alpha}-tubulin were detected by Western blot in whole cell lysates. B, Relative levels of p38{alpha} and p38beta were quantified, normalized to {alpha}-tubulin, and percentage expression calculated taking values in EC not transfected with HO-1 as 100%. C, BAEC were transiently transfected with HA-p38{alpha} and increasing amounts of pcDNA3/HO-1 (0.5, 50, 100, 200, 400, 600, 800, and 1000 ng of DNA per 3 x 105 cells). HA-p38{alpha}, HO-1, and {alpha}-tubulin were detected by Western blot. D, Relative levels of p38{alpha} were quantified as in B. E, BAEC were transiently transfected with FLAG-p38beta and increasing amounts of pcDNA3/HO-1 as in C. FLAG-p38beta, HO-1, and {alpha}-tubulin were detected by Western blot. F, Relative levels of p38beta were quantified as in B. A, C, and E, Immunoblots are representative of three independent experiments. B, D, and F, Quantifications are the mean ± SD from these three independent experiments. (**, p = 0.007 vs control; *, p < 0.05 vs control).

 
To ascertain whether inhibition of p38{alpha} can occur under physiological conditions, we tested whether hemin, an inducer of endogenous HO-1 expression, would inhibit p38{alpha} in EC. As expected (30), hemin induced high levels of HO-1 expression in EC (Fig. 5, A, C, and D). This was associated with reduced expression of transiently overexpressed p38{alpha}, i.e., 40–80% reduction, as compared with control EC not exposed to hemin (Fig. 5, A and B). Hemin had no significant effect on transiently overexpressed p38beta, as assessed by Western blot (Fig. 5, A and B). We then asked whether hemin would also inhibit endogenous p38{alpha} and/or p38beta expression. Endogenous p38{alpha} was reduced by 70–80% in EC exposed to hemin, as compared with control EC (Fig. 5, C and E). Hemin failed to reduce the expression of endogenous p38beta, suggesting that its effect is specific to p38{alpha} (Fig. 5, D and E). To ascertain that HO-1 mediated the inhibition of p38{alpha} expression observed in EC exposed to hemin, we assessed whether this effect was ablated when HO-1 expression was targeted by a HO-1 siRNA. Indeed, hemin failed to inhibit p38{alpha} expression when HO-1 expression was suppressed by a HO-1 siRNA (Fig. 5F). These data demonstrate that under physiological conditions, i.e., exposure to 2–5 µM hemin, HO-1 inhibits specifically the expression of the p38{alpha} MAPK isoform in EC.


Figure 5
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  		FIGURE 5. Induction of HO-1 expression by hemin inhibits p38{alpha} expression in EC. A, BAEC were transiently transfected with HA-p38{alpha} plus FLAG-p38beta expression vectors and exposed to hemin as specified. HA-p38{alpha}, FLAG-p38beta, HO-1, and {alpha}-tubulin were detected by Western blot in whole EC lysates. B, Relative levels of p38{alpha} and p38beta proteins were quantified and normalized to 100% of p38{alpha} or p38beta expression in EC not exposed to hemin. Shown is the mean ± SD of three independent experiments (*, p = 0.01 vs untreated cells). C and D, HUVEC were exposed to hemin (2.5 µM), endogenous p38{alpha} (C), and p38beta (D) were IP and detected by Western blot. HO-1 and {alpha}-tubulin were detected in whole cell lysates (WCL) as in A. E, Relative levels of endogenous p38{alpha} and p38beta were quantified as in B and are shown as mean ± SD from three independent experiments (*, p = 0.0003 vs untreated cells). F, HUVEC were either not transfected or transfected with HO-1 siRNA and exposed 48 h thereafter to hemin (2.5 µM). Endogenous p38{alpha} was IP and detected by Western blot. HO-1 and {alpha}-tubulin were detected in whole cell lysates as in A. A, C, and D, Immunoblots are representative of three independent experiments used for quantification.

 
CO inhibits p38{alpha} expression in EC

We have previously shown that CO is antiapoptotic in EC (8). We reasoned that if inhibition of p38{alpha} was required to sustain this antiapoptotic effect then CO should inhibit p38{alpha} expression. We first tested whether HO-1 enzymatic activity, which generates CO, was required to inhibit p38{alpha} expression. We found this to be the case as the ability of transiently transfected HO-1 to inhibit the expression of cotransfected p38{alpha} was impaired when HO activity was repressed by tin protoporphyrin IX (SnPPIX) (33) (Fig. 6, A and B). In contrast, the inhibition of p38{alpha} expression was enhanced when EC were exposed to hemin (Fig. 6, A and B), the natural substrate of HO-1 enzymatic activity (30). In the absence of detectable HO-1 expression, exogenous CO (10,000 ppm) mimicked the effect of transiently overexpressed HO-1, inhibiting p38{alpha} expression (Fig. 6, C and D). These observations suggest that HO-1 inhibits p38{alpha} expression in EC via the generation of CO.


Figure 6
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  		FIGURE 6. CO inhibits p38{alpha} expression in EC. A, BAEC were transiently transfected with HA-p38{alpha} with or without pcDNA3/HO-1 and exposed to either hemin or SnPPIX. Proteins in whole cell extracts were detected by Western blot. B, Relative levels of p38{alpha} were quantified, normalized to {alpha}-tubulin, and normalized again to the level of p38{alpha} expressed in EC not transfected with HO-1 (100%). Results shown are the mean ± SD from three independent experiments using 10 µM hemin or SnPPIX (*, p = 0.01 vs control). C, BAEC were transiently transfected with HA-p38{alpha} with or without pcDNA3/HO-1 and when indicated exposed to CO (10,000 ppm). Proteins were detected by Western blot as in A. D, Relative levels of p38{alpha} were quantified as in B and values expressed relative to control EC that did not express HO-1 and not exposed to CO (100%). Values shown are the mean ± SD from three independent experiments (*, p = 0.01 vs control, **, p = 0.008 vs control). Immunoblots are representative of the three independent experiments.

 
The antiapoptotic effect of HO-1 is impaired upon reconstitution of the cellular pool of p38{alpha}

To determine whether the antiapoptotic effect of HO-1 was functionally linked to its ability to inhibit specifically the expression of p38{alpha} (Figs. 4–6), we assessed whether the antiapoptotic effect of HO-1 was impaired when p38{alpha} expression was restored ectopically. We found that this is the case. Transient overexpression of p38{alpha} with HO-1 impaired the antiapoptotic effect of HO-1 (Fig. 7A). This did not occur when p38beta was transiently overexpressed with HO-1 (Fig. 7A). To ensure that p38{alpha} and p38beta were coexpressed at similar levels, their relative levels of expression were compared by Western blot using an anti-FLAG Ab recognizing both tagged isoforms. This proved to be the case (Fig. 7B).


Figure 7
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  		FIGURE 7. The antiapoptotic effect of HO-1 is impaired by ectopic expression of p38{alpha} or by inhibition of p38beta. A, BAEC were transiently transfected with a RSV-luciferase reporter with or without beta-actin/HO-1 expression vector. When indicated, EC were cotransfected with FLAG-tagged p38{alpha} (p38{alpha}) or p38beta (p38beta) expression vectors. Apoptosis was induced by TNF-{alpha} plus ActD and percentage of cell viability (percent viability) was assessed by luciferase activity as described in Materials and Methods. Relative percent viability was normalized in each transfection to control EC treated with ActD without TNF-{alpha} (100%). Results are expressed as the mean ± SD from three independent experiments, each done in triplicate or quadruplicate (*, p < 0.001 vs ActD treated alone). B, BAEC were transiently transfected with FLAG-tagged p38{alpha} or p38beta with or without pcDNA3/HO-1. Overexpressed p38{alpha} and p38beta were detected in whole cell lysates by Western blot using anti-FLAG Ab. HO-1 and {alpha}-tubulin were detected in a similar manner using specific Abs. C, HUVEC were transiently transfected as in A. When indicated, EC were cotransfected with p38beta siRNA vectors. Apoptosis was induced by TNF-{alpha} plus CHX and percentage of cell viability (percent viability) was assessed by luciferase activity as described in Materials and Methods. Relative percent viability was normalized to control EC treated with CHX without TNF-{alpha} (100%). Results are expressed as the mean ± SD from two independent experiments, each done in triplicate (*, p < 0.0001 and (**, p = 0.0002).

 
To assess whether the cytoprotection afforded by HO-1 was dependent on the expression of p38beta, EC were transiently cotransfected with HO-1 with or without a p38beta siRNA expression vector. When coexpressed with HO-1, p38beta siRNA decreased EC viability, as compared with control EC that overexpressed HO-1 but not the p38beta siRNA (Fig. 7C). TNF-{alpha} plus CHX failed to induce significant EC apoptosis when HO-1 was overexpressed (Fig. 7 C) (8). However, this was no longer the case when EC overexpressed HO-1 plus p38beta siRNA (Fig. 7 C), indicating that p38beta expression is required to sustain the cytoprotective effect of HO-1 against TNF-{alpha}-mediated apoptosis.

HO-1 targets p38{alpha} for degradation by the 26S proteasome

Because HO-1 suppressed the expression of p38{alpha} but not p38beta when their cDNAs were overexpressed under the control of the same promoter, i.e., minimal CMV (Fig. 4A), HO-1 should not target the transcription of these p38 MAPK isoforms. Therefore, we tested whether HO-1 would act posttranscriptionally, i.e., targeting mRNA or protein stability, to inhibit specifically the expression of p38{alpha}. When transiently p38{alpha} or p38beta were coexpressed with HO-1, the expression of p38{alpha} or p38beta mRNAs was not modulated, as compared with control EC that did not express HO-1 (Fig. 8, A and B). This excluded not only the possibility that HO-1 would target the transcription of these p38 isoforms but also that it would modulate the stability (half-life) of their mRNA. We then asked whether HO-1 would act directly on the p38{alpha} protein to inhibit its expression, such as by targeting it for proteolytic degradation by the 26S proteasome. We found this to be the case, as suppression of 26S proteasome activity by MG-132 impaired the decrease of p38{alpha} protein expression observed in EC that overexpress HO-1 (Fig. 8, C and D). This effect was dose dependent in that increasing concentrations of MG-132, i.e., 0.5–20 µM, resulted in increasing levels of p38{alpha} protein expression (Fig. 8, C and D). The involvement of the 26S proteasome pathway in p38{alpha} degradation was confirmed using lactacystin, an inhibitor of the 26S proteasome pathway that is more specific than MG-132 (Fig. 8E). This data suggest that the ability of HO-1 to inhibit the expression of p38{alpha} is strictly dependent on the presence of a fully active 26S proteasome pathway.


Figure 8
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  		FIGURE 8. The antiapoptotic effect of HO-1 requires p38{alpha} degradation via the 26S proteasome. A, BAEC were transiently transfected with HA-p38{alpha} and FLAG-p38beta with or without pcDNA3/HO-1. Overexpressed p38{alpha}, p38beta, and HO-1, as well as endogenous beta-tubulin mRNAs were detected by RT-PCR. B, Relative levels of p38{alpha} and p38beta mRNA were normalized to beta-tubulin and are presented as percentage of EC not transfected with pcDNA3/HO-1 (100%). Mean ± SD from three independent experiments is shown. C, BAEC were transiently transfected with p38{alpha} plus pcDNA3/HO-1 and exposed to increasing concentrations of the 26S proteasome inhibitor MG-132. Proteins were detected by Western blot in whole cell lysates. D, Relative levels of p38{alpha} were quantified as in B and normalized to EC that did not express HO-1 (100%). Values from three independent experiments are shown as mean ± SD (*, p < 0.001 vs control). Blots presented in A and C are representative of the three independent experiments used for quantification. E, BAEC were transfected as in C and exposed to increasing concentrations of the 26S proteasome inhibitors lactacystin or MG-132 (5 µM) as specified. Proteins were detected by Western blot in whole cell lysates. Immunoblot shown is representative of three independent experiments. F, BAEC were transiently transfected with a RSV-luciferase reporter with or without beta-actin/HO-1 and when indicated the 26S proteasome pathway was inhibited with MG-132 (5 µM). Apoptosis was induced by TNF-{alpha} plus ActD and cell viability (percent viability) was assessed according to luciferase activity as described in Materials and Methods. Percentage of viability was normalized in each transfection to control EC treated with ActD without TNF-{alpha} (100%). Results are expressed as the mean ± SD from three independent experiments, each done in triplicate (*, p < 0.0001 vs ActD treated alone).

 
Inhibition of p38{alpha} degradation impairs the antiapoptotic effect of HO-1

If the ability of HO-1 to trigger p38{alpha} degradation via the 26S proteasome was required to support its antiapoptotic effect, then inhibition of the 26S proteasome activity should impair the antiapoptotic effect of HO-1. Indeed, the 26S proteasome inhibitor MG-132 reverted the antiapoptotic effect of HO-1 in EC (Fig. 8F). This was associated with complete restoration of p38{alpha} expression, despite the overexpression of HO-1 (Fig. 8C), suggesting that degradation of p38{alpha} by the 26S proteasome is a prerequisite for the antiapoptotic effect of HO-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Expression of HO-1 plays a critical role in the control of inflammatory reactions such as those involved in the pathogenesis of several inflammatory diseases (34), including atherosclerosis (reviewed in Refs. 1, 35). The salutary effects of HO-1 were originally associated with its ability to confer cytoprotection against a broad spectrum of pro-oxidant stimuli. In keeping with this notion, we found that HO-1-derived CO is antiapoptotic in EC (6, 8), an effect that should contribute to the overall salutary effects of HO-1, since EC apoptosis is a highly proinflammatory event per se (36, 37). The finding that HO-1-derived CO modulates the proinflammatory response of activated monocyte/macrophages (38) suggests that its protective effects might also be exerted via this action (39).

Both the anti-inflammatory (38) and antiapoptotic (8, 10, 40) effects of HO-1-derived CO are exerted via the p38 MAPK signal transduction pathway (reviewed in Ref. 1). At first, this seemed paradoxical, since p38 MAPK can promote the expression of proinflammatory cytokines in monocyte/macrophages, e.g., TNF-{alpha} (11), as well as apoptosis (25). This was, therefore, difficult to conciliate with the observation that CO exerts the exact opposite effects via the same signal transduction pathway. We reasoned that CO might signal via a specific subset of p38 MAPK isoforms that would act essentially in an anti-inflammatory and/or antiapoptotic manner. We tested this hypothesis in the context of EC apoptosis, restricting our analysis to the p38{alpha} and p38beta isoforms, because SB203580, a compound which inhibits only the activity of these two isoforms (16) suppresses the antiapoptotic effects of HO-1 and/or CO in EC (8).

We have confirmed that both p38{alpha} and p38beta MAPK isoforms were expressed in EC and that the antiapoptotic effect of HO-1 was impaired when the activation of these p38 isoforms was inhibited (Fig. 1). As for other cell types (21, 22, 23, 24), p38{alpha} and p38beta were found to have opposite effects in sustaining EC viability. Expression of p38{alpha} was essentially cytotoxic, whereas p38beta was required to sustain EC viability in vitro (Fig. 2, A and B). We reasoned that to prevent EC from undergoing apoptosis, HO-1 must avoid signaling via the cytotoxic p38{alpha} isoform and probably in this manner promote signaling via the cytoprotective p38beta isoform. Since HO-1 failed to modulate the activation of either one of these p38 isoforms (Fig. 3), we reasoned that there must be another mechanism via which this can be accomplished. The observation that HO-1 decreased the cellular pool of p38{alpha} but not that of p38beta (Figs. 4 and 5) suggested that this might be the mechanism via which HO-1 avoids the cytotoxic action of p38{alpha}, thus favoring signaling via the cytoprotective p38beta isoform. This effect was found to be strictly dependent on the enzymatic activity of HO-1 (Fig. 6, A and B) and to be mimicked in the absence of detectable HO-1 expression by exogenously applied CO (Fig. 6, C and D), suggesting that it is CO that inhibits p38{alpha} expression in EC.

Given the above, it appears that the ability of HO-1 to modulate signaling via the p38 MAPK does not involve upstream kinases or phosphatases targeting specifically one or the other of these p38 isoforms. Instead, HO-1 acts in a cytoprotective manner by inhibiting specifically the expression of p38{alpha}, thus directing signals emanating from upstream kinases toward the cytoprotective p38beta isoform. That p38beta sustains the antiapoptotic effect of HO-1 was demonstrated by the observation that the cytoprotective effect of HO-1 was ablated when p38beta expression was suppressed (Fig. 7C). This finding is in keeping with the recent observation that the cytoprotective effect of one of the products of heme degradation by HO-1, i.e., CO, is also lost when EC fail to express p38beta, such as in EC isolated from p38beta-deficient mice (26). Our present data add significantly to these findings in that we demonstrate not only that the antiapoptotic effect of HO-1 requires the expression of p38beta (Fig. 7C), but also that it requires the inhibition of p38{alpha} expression. This notion is supported by the observation that reconstitution of p38{alpha} expression negated the antiapoptotic effect of HO-1 in EC (Fig. 7A). This set of observations should solve the somewhat paradoxical finding that HO-1-derived CO signals via the p38 MAPK signal transduction pathway to exert its antiapoptotic effects (8).

The inhibition of p38{alpha} expression by HO-1 occurred under physiological conditions such as when endogenous HO-1 expression was induced by hemin (Fig. 5, A–C). This effect was lost when HO-1 expression was suppressed by a HO-1 siRNA, confirming that the ability of hemin to reduce p38{alpha} expression was mediated by HO-1 (Fig. 4F). Based on these observations, it is reasonable to assume that under the experimental conditions used overexpressed HO-1 mimics the physiological effect of endogenous HO-1 in inhibiting specifically the expression of p38{alpha} in EC.

Our present data also reveal that HO-1 targets specifically the p38{alpha} protein for degradation by the 26S proteasome, an effect that was blocked by MG-132 and lactacystin, two specific inhibitors of the 26S proteasome (Fig. 8, C, D, and E). That degradation of p38{alpha} by the 26S proteasome is required to sustain the antiapoptotic effect of HO-1 is suggested by the observation that MG-132 negated the antiapoptotic effect of HO-1 (Fig. 8F).

Based on the data reported here, there are at least two possible explanations as to the mechanism underlying the involvement of p38{alpha} and p38beta in the antiapoptotic effect of HO-1. One is that p38{alpha} degradation is sufficient per se to mediate this effect. This possibility should probably be disregarded based on the observation that p38beta expression is required to sustain the antiapoptotic effect of HO-1 (Fig. 7C) (26). An alternative explanation would be that HO-1 alters the ratio of cytotoxic p38{alpha} vs cytoprotective p38beta, simply allowing in this manner the action of p38beta to predominate over p38{alpha}. As argued above, our data support this as being the mechanism involved in the antiapoptotic effect of HO-1 in EC.

In conclusion, HO-1-derived CO modulates signaling via the p38 MAPK signal transduction pathway in a manner that is essentially antiapoptotic in EC. CO inhibits the cytotoxic action of p38{alpha}, probably promoting that of the cytoprotective p38beta isoform. This occurs via specific degradation of p38{alpha} by the 26S proteasome, an effect shown hereby to be critical for the antiapoptotic action of HO-1 in EC. Whereas it is likely that a similar mechanism may be involved in other biological activities of CO, such as its anti-inflammatory effects in monocyte/macrophages, this remains to be demonstrated.


   Acknowledgments
 
We thank Josef Anrather (Cornell University, NY) and Roger Davis (University of Massachusetts, Amherst, MA) for providing several cDNA constructs, and Colleen Fearns (The Scripps Research Institute) for providing Abs against p38 isoforms. We also thank Henrique Teotónio for valuable assistance with statistical analysis (Instituto Gulbenkian de Ciência), as well as António Jacinto, Sukalyan Chatterjee, and Sérgio Dias (Instituto Gulbenkian de Ciência) for critical reading of the manuscript.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was partially supported by National Institutes of Health Grant RO1 HL67040, European Community 5th Framework Grant QLK3-CT-2001-00422 (to M.P.S.), Fundação para a Ciência e a Tecnologia Grant POCTI/MGI/37296/2001 (to M.P.S.), Fundação para a Ciência e a Tecnologia Postdoctoral Fellowships SFRH/BPD/6303/2001 (to G.S.), SFRH/BPD/21072/2004 (to G.S.), and SFRH/BPD/9380/2002 (to I.P.G.), and Fundação para a Ciência e a Tecnologia PhD Fellowships SFRH/BD/21558/2005 (to A.C.) and SFRH/BD/2990/2000 (to M.P.S.). Back

2 Address correspondence and reprint requests to Dr. Miguel P. Soares or Dr. Gabriela Silva, Instituto Gulbenkian de Ciência, Apartado 14, 2781-901, Oeiras, Portugal. E-mail addresses: mpsoares{at}igc.gulbenkian.pt and gsilva{at}igc.gulbenkian.pt, respectively. Back

3 Abbreviations used in this paper: HO-1, heme oxygenase-1; EC, endothelial cell; WT, wild type; ActD, actinomycin D; BAEC, bovine aortic EC; DAPI, 4',6'-diamidino-2-phenylindole; TRITC, tetramethylrhodamine isothiocyanate; siRNA, small interfering RNA; ppm, parts per million; IP, immunoprecipitated; HA, hemagglutinin; CHX, cycloheximide. Back

Received for publication January 26, 2006. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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