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Originally published In Press as doi:10.1074/jbc.M304103200 on September 30, 2003

J. Biol. Chem., Vol. 278, Issue 51, 51006-51014, December 19, 2003
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Oxidized Phospholipids Induce Expression of Human Heme Oxygenase-1 Involving Activation of cAMP-responsive Element-binding Protein*

Gerhard Krönke, Valery N. Bochkov, Joakim Huber, Florian Gruber, Stefan Blüml, Alexander Fürnkranz, Alexandra Kadl, Bernd R. Binder, and Norbert Leitinger{ddagger}

From the Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna A-1090, Austria

Received for publication, April 18, 2003 , and in revised form, August 4, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-{alpha} and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heme oxygenase-1 (HO-1)1 is the rate-limiting enzyme of heme catabolism, catalyzing the breakdown of heme into biliverdin, iron, and carbon monoxide (1). The phenotype of humans and mice deficient of HO-1 suggests an important physiological role of HO-1 in the regulation of the inflammatory process (2-5). Indeed, HO-1 is highly up-regulated in acute and chronic inflammation (6, 7). HO-1 not only provides protection against oxidative stress (4, 8, 9) but also possesses potent anti-inflammatory properties (10-12), which seem to be mediated by its products biliverdin (13, 14) and carbon monoxide (11, 15-19). Carbon monoxide inhibits the expression of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and, in addition, increases the expression of anti-inflammatory cytokines (11). In this respect HO-1 seems to play a key role in the resolution of inflammation (10, 20-22), which is pivotal to limit tissue damage resulting from oxidation of DNA, proteins, and membrane lipids by free radicals generated by inflammatory cells (23-27).

Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC), which is an integral part of cellular membranes and lipoproteins, leads to the generation of intermediate oxidation products (OxPAPC), some of which are potent bioactive substances (28). We have shown that OxPAPC profoundly modulates the fate of an inflammatory response by limiting LPS-induced inflammation (29). In endothelial cells OxPAPC induces the adhesion of monocytes (28) and activates, probably via yet non-identified G-protein-coupled receptors (30), protein kinases A (PKA) (30) and C (PKC) (31), as well as the ERK pathway (31). OxPAPC induces genes like tissue factor (31), monocyte chemoattractant protein-1, and interleukin-8 (32) via transcription factors such as peroxisome proliferator-activated receptor (PPAR)-{alpha} (33), nuclear factor of activated T cells (NFAT), and early growth response factor-1 (Egr-1) (31). It has been shown recently that OxPAPC is also an inducer of HO-1 gene transcription in vitro (13) and in vivo (34).

The induction of HO-1 expression is controlled primarily at the transcriptional level and has been studied extensively in the mouse promoter (35). DNA motifs alternatively termed as Maf recognition elements (MAREs), NF-E2 sites, stress responsive elements, or antioxidant-responsive elements have been shown to be crucial for the activation of mouse HO-1 gene expression in response to numerous stimuli (36-41) and are also present in the human promoter (42). Different members of the basic-leucine zipper (bZIP) family of transcription factors, with Nrf2 as a central regulator, are able to bind to this element (36, 38-41). Nevertheless the mechanism of the OxPAPC-mediated induction of HO-1 gene expression remains elusive.

In the present study, we investigated in detail the mechanism of OxPAPC-induced HO-1 gene expression in human umbilical vein endothelial cells (HUVEC). We show that the HO-1 induction by OxPAPC depends on the cAMP-responsive element-binding protein (CREB) and is mediated via signaling pathways including PKA, PKC, p38MAPK, and ERK1/2, which promote the phosphorylation of CREB. By using luciferase promoter reporter constructs and mutational analysis, we demonstrate that a regulatory region, located 4 kb upstream the transcription start site, is responsible for the induction of the HO-1 transcription by OxPAPC and that a cAMP-responsive element (CRE) and a Maf recognition element, both of which are present in this region, are involved responsive elements. Finally we show in gel shift assays that OxPAPC increases binding of several nuclear complexes, some of which include CREB, to the human HO-1 promoter.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—TNF-{alpha} was purchased from Genzyme (Cambridge, MA), and L-{alpha}-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and M199 were from Sigma. PD98059, SB203580, H89, and bisindolylmaleimide I (BisI) were obtained from Calbiochem. CDC (cinnamoyl-3,4-dihydroxy-a-cyanocinnamate) and AACOCF3 (arachidonyltrifluoromethyl ketone) were from Biomol. Polyclonal antibodies against HO-1 were from Stressgen. Antibodies against NFATc, CREB/ATF1, Nrf1, Nrf2, MafF/G/K, and c-Fos were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against phospho-CREB was obtained from New England Biolabs (Beverly, MA). Peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences.

Cell Culture—HUVEC were cultured at 37 °C and 5% CO2 in M199 containing 20% supplemented calf serum (SCS), 1 unit/ml heparin, 50 µg/ml bovine endothelial cell growth supplement (Technoclone, Vienna, Austria), 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. Experiments were performed using cells at passage 4. In all experiments, before addition of the stimulus, HUVEC were incubated in M199 containing 1% SCS for 4 h, except in the experiment shown in Fig. 1D, where medium containing 5% SCS was used to provide HUVEC with enough serum-derived soluble CD14 to respond to LPS.



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  		FIG. 1.
OxPAPC induces HO-1 mRNA and protein. A, HUVEC were incubated with the indicated concentrations of OxPAPC or 100 µg/ml PAPC, and the induction of HO-1 mRNA was measured after 4 h by quantitative real-time RT-PCR. The insert shows HO-1 protein levels as determined by Western blot after 6 h of treatment with different concentrations of OxPAPC or 100 µg/ml PAPC, respectively. B, 100 µg/ml of OxPAPC were used to stimulate HUVEC for the indicated times, and levels of HO-1 mRNA and protein (insert) were assessed by quantitative real-time RT-PCR and by Western blot respectively. C, HUVEC were transfected with hHO4.9luc and incubated with indicated concentrations of OxPAPC or PAPC. After 14 h induction of relative luciferase activity was determined. D, HUVEC were stimulated for 4 h with 100 µg/ml OxPAPC, 100 units/ml TNF-{alpha}, 400 µM H2O2, or 300 ng/ml LPS. HO-1 and E-selectin mRNA levels were assessed using quantitative real-time RT-PCR. Data represent means ± S.D.

 
Transfection and Enzyme Assays—Cells were seeded in 6-well plates and transfected after 48 h at ~80% confluence. Transient transfections were performed using the LipofectAMINE Plus reagent (Invitrogen) according to the protocol provided by the manufacturer. Cells were incubated with a transfection mixture containing 1.5 µg of total DNA (including 0.25 µg of PRL-SV40 vector as transfection-efficiency control), 6 µl of Plus reagent, and 4 µl of LipofectAMINE in a total volume of 1 ml of M199 for 130 min. After 48 h, cells were starved for 4 h and stimulated for, unless otherwise stated, 14 h in M199 containing 1% SCS. Luciferase activity of the cell lysates was determined using a Dual-Luciferase Reporter Assay system (Promega). Firefly activity was then normalized to SV40 Renilla activity.

Plasmids—A bacterial artificial chromosome clone containing the human HO-1 gene and the complete 5'regulatory region was a kind gift from Dr. Markus Exner (Clinical Institute of Medical and Chemical Laboratory Diagnostics, General Hospital of Vienna, Vienna, Austria). A 4.9-kb SacI-XhoI fragment containing the human HO-1 promoter including the previously described cadmium-responsive elements (43) was cloned into the pGL3 basic vector (Promega) to obtain hHO4.9luc. Plasmid hHO4.9luc was simultaneously digested at the SacI site and at a series of restriction endonuclease sites located in the promoter, blunt-ended, and re-circularized to generate a set of 5'-nested deletions. The end points of the deletion constructs and the enzymes used were as follows: -3870 (XbaI), -2245 (HindIII), -1435 (EcoRI), and -302 (PstI). The vector over-expressing the dominant-negative CREB (pCMV-CREB133) was purchased from Clontech (Palo Alto, CA).

Lipid Oxidation—PAPC was oxidized by exposure of dry lipid to air for 72 h. The extent of oxidation was monitored by positive ion electrospray mass spectrometry as described previously (28).

Quantitative Real-time RT-PCR—RNA was isolated using Trizol reagent (Invitrogen). Nine hundred ng of total RNA were reverse-transcribed with murine leukemia virus reverse transcriptase using the Gene Amp RNA PCR kit (Applied Biosystems, Foster City, CA) and oligo(dT) (16) primers. The mRNA sequences of the investigated genes were obtained from GenBankTM. The primers for {beta}-2 microglobulin were described previously (44). The primers for E-selectin and HO-1 were designed using the PRIMER3 software from the Whitehead Institute for Biomedical Research (Cambridge, MA). The following forward (F) and reverse (R) primers were used: HO-1: F, 5'-AAGATTGCCCAGAAAGCCCTGGAC-3'; R, 5'-AACTGTCGCCACCAGAAAGCTGAG-3'; E-selectin: F, 5'-GGTTTGGTGAGGTGTGCTC-3'; R, 5'-TGATCTTTCCCGGAACTGC-3'. Quantitative real-time RT-PCR was performed using LightCycler technology (Roche Diagnostics) and the Fast Start SYBR Green I kit for amplification and detection. In all assays, cDNA was amplified using a standardized program (10-min denaturing step; 55 cycles of 5 s at 95 °C, 15 s at 65 °C, and 15 s at 72 °C; melting point analysis in 0.1 °C steps; final cooling step). Each LightCycler capillary was loaded with 1.5 µl of DNA Master Mix, 1.8 µl of MgCl2 (25 mM), 10.1 µl of H2O, 0.4 µl of 10 µM stock of each primer. The final amount of cDNA per reaction corresponded to 2.5 ng of total RNA used for reverse transcription. Quantification of target gene expression was performed using a mathematical model by Pfaffl (45). The expression of the target molecule was normalized to the expression of {beta}-2 microglobulin.

Electrophoretic Mobility Shift Assay—Nuclear extracts from HUVEC were prepared as described previously (46), except that phosphatase inhibitor mixture 1 + 2 (Sigma) were added. The protein concentration was determined using the Coomassie protein assay reagent (Pierce) with bovine serum albumin as a standard. To prepare probes for the electrophoretic mobility shift assay, 100 pmol of each complementary single-stranded oligonucleotide (VBC-Genomics, Vienna, Austria) were annealed in 20 µl of H2O to obtain a double-stranded oligonucleotide with the sequences 5'-GCTGCATTTCTGCTGCGTCATGTTTGGGAG-3' (HO-CRE) or 5'-CTAGATTTTGCTGAGTCACCAGTGC-3' (MARE). 1 µg of nuclear extracts were incubated initially for 10 min at room temperature in 20 µl containing 18 mM HEPES-KOH (pH 7.9), 80 mM KCl, 2 mM MgCl2, 10 mM dithiothreitol, 10% glycerol, 0.2 mg/ml bovine serum albumin, and 1.5 µg of poly(dI-dC). The mixture was then incubated for an additional 20 min after adding 105 cpm of the [{gamma}-32P]ATP-labeled probe, with or without an unlabeled competitor or antibody for supershift. In antibody supershift assays, 2 µl (4 µg) of antibody were added to the reaction mixture. The mixture was electrophoresed on a 6.5% polyacrylamide gel in a 0.5% TBE buffer. The gel was dried, and the radioactivity was visualized and quantified using a PhosphorImager and ImageQuant software (Amersham Biosciences).

Western Blot Analysis—After stimulation, HUVEC were lysed in Laemmli buffer, and proteins were separated by electrophoresis in 12% SDS-polyacrylamide gels. Proteins were blotted onto polyvinylidene difluoride membranes and, after blocking with 5% dry milk/0.1% Tween 20, incubated with primary antibodies in the same solution. Bound antibodies were detected by anti-IgG conjugated with peroxidase and subsequent chemiluminescent detection.

Site-directed Mutagenesis—The CRE and MARE of the HO-1 promoter were mutated by a PCR-based technique using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene) according to the protocol provided by the manufacturer. For each mutation two complementary primers containing the mutation were synthesized (VBC-Genomics). The sequences of the primers used to mutate the HO-CRE site were F, 5'-CCCTCGTGCAGCTGCATTTCTGCGTAGATCTGTTTGGGAGGGGGG-3' and R, 5'-CCCCCCTCCCAAACAGATCTACGCAGAAATGCAGCTGCACGAGGG-3'. To mutate the MARE in the HO-1 promoter, primers with the sequences F, 5'-GGCGGATTTTGCTAGATTTTGCGTAGGTACCAGTGCCTCCTCAGC-3' and R, 5'-GCTGAGGAGGCACTGGTACCTACGCAAAATCTAGCAAAATCCGCC-3' were used.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of HO-1 in HUVEC Is Induced by OxPAPC but Not by TNF-{alpha} and LPS—HO-1 mRNA expression and HO-1 protein levels after OxPAPC stimulation were determined by quantitative real time RT-PCR and by Western blot, respectively. OxPAPC increased levels of HO-1 mRNA and protein in HUVEC in a concentration- and time-dependent manner starting at 25 µg/ml (Fig. 1, A and B). In contrast to OxPAPC, native PAPC did not affect HO-1 expression.

To investigate the effect of OxPAPC on the human HO-1 promoter activity, we cloned the proximal 4.9 kb of the human HO-1 promoter into the PGL-3 vector to obtain a HO-1 promoter-driven luciferase reporter construct (hHO4.9luc). hHO4.9luc activity was induced by OxPAPC in a concentration-dependent manner (Fig. 1C), whereas native PAPC had no effect.

Among potential HO-1-stimulating agents that are involved in inflammation are substances like TNF{alpha}, LPS, or H2O2 (4, 47, 48). To compare the effect of these agents on the induction of HO-1 gene expression in endothelial cells, we stimulated HUVEC with OxPAPC, TNF-{alpha}, H2O2, or LPS. Then mRNA levels of HO-1 and E-selectin, which is known to be strongly induced by TNF-{alpha} and LPS, were determined by quantitative real time RT-PCR. Treatment of HUVEC with OxPAPC increased levels of HO-1 mRNA 17-fold (Fig. 1D). At concentrations where TNF-{alpha} and LPS strongly induced E-selectin expression, we did not detect a significant rise in HO-1 mRNA levels, whereas 400 µM H2O2 increased the expression of HO-1 mRNA ~1.5-fold. Neither OxPAPC nor H2O2 induced a significant elevation of E-selectin mRNA levels (Fig. 1D). These results indicate the activation of distinct signaling pathways by OxPAPC and the pro-inflammatory stimuli TNF-{alpha} and LPS in endothelial cells. In accordance with these findings, we have shown previously that OxPAPC does not activate the NF-{kappa}B pathway in endothelial cells (31), although OxPAPC shares a set of target genes with TNF-{alpha} and LPS, such as tissue factor, interleukin-8, and monocyte chemoattractant protein-1 (31, 32, 49).

Induction of HO-1 Expression by OxPAPC Is Mediated by PKA, PKC, p38MAPK, and ERK—To address the role of individual signaling pathways in HO-1 gene regulation by OxPAPC, we examined the effect of the PKA inhibitor H89, the PKC inhibitor BisI, the p38MAPK inhibitor SB203580, and the MAP/ERK kinase 1/2 inhibitor PD98059 on OxPAPC-mediated HO-1 induction. H89, BisI, SB203580, and PD98059 all inhibited the induction of HO-1 mRNA levels and of HO-1 promoter-driven luciferase activity by OxPAPC (Fig. 2, A and B). In addition we examined the influence of the inhibitors on HO-1 protein levels after OxPAPC treatment (Fig. 2C). Although PD98059 did not significantly affect the induction of HO-1 protein by OxPAPC, SB203580, BisI, and H89 all inhibited HO-1 protein induction. Thus protein kinases A and C, as well as the MAPK pathways p38 and ERK, seem to be involved in OxPAPC-mediated induction of HO-1 expression.



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  		FIG. 2.
Induction of HO-1 expression by OxPAPC involves PKA, PKC, p38MAPK, and ERK. A, HUVEC were pre-treated with 20 µM H89, 10 µM BisI, 10 µM PD98059 (PD), 10 µM SB203580 (SB), or vehicle for 30 min and then incubated with 100 µg/ml OxPAPC or medium for 1.5 h in the presence of the indicated inhibitors. HO-1 mRNA levels were determined by quantitative real-time RT-PCR (A), and HO-1 protein levels were assessed by Western blot after 6 h (C). B, HUVEC were transfected with hHO4.9luc and stimulated with 100 µg/ml OxPAPC or medium in the presence of 20 µM H89, 10 µM BisI, 10 µM PD98059, 10 µM SB203580, or vehicle. After 14 h cells were harvested, and relative luciferase activity was measured. The data represent means ± S.D.

 
OxPAPC Induces Phosphorylation of CREB via PKA, PKC, p38MAPK, and ERK—A potential common downstream target of the above-mentioned signaling pathways is the transcription factor CREB (50), which plays a central role in cellular signaling and promotes activation of a large set of genes. The capability of CREB to activate gene transcription is determined by its phosphorylation state, which, in turn, regulates its association with adapter proteins like CREB-binding protein or p300, both responsible for trans-activation of the transcriptional machinery. To determine whether OxPAPC induces phosphorylation of CREB, the levels of phospho-CREB were analyzed using an antibody against CREB phosphorylated at serine 133. Ox-PAPC induced phosphorylation of CREB in a dose-dependent manner, which was inhibited in the presence of the PKA inhibitor H89 (Fig. 3A). In addition to H89, the specific inhibitors BisI, SB203580, and PD98059 all reduced phosphorylation of CREB after OxPAPC treatment (Fig. 3B), demonstrating an involvement of PKC, p38MAPK, and the ERK pathway, in addition to PKA, in CREB phosphorylation induced by OxPAPC. Indomethacin, a cyclooxygenase inhibitor, CDC, an inhibitor of lipooxygenase, and AACOCF3, an inhibitor of cytosolic phospholipase A2, all inhibitors of enzymes that have been implicated in OxPAPC-induced signaling (51), did not affect the OxPAPC-induced phosphorylation of CREB (Fig. 3B).



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  		FIG. 3.
CREB phosphorylation is induced by OxPAPC via PKA, PKC, p38MAPK, and ERK1/2. A, after HUVEC were pre-treated with 20 µM H89 or vehicle for 30 min, increasing amounts of OxPAPC were added. Cells were harvested after additional 30 min, and the levels of total CREB, as well as of serine 133-phosphorylated CREB, were determined by Western blotting. B, HUVEC were stimulated with 100 µg/ml OxPAPC or medium for 30 min following 30 min of pre-incubation with 10 µM SB203580 (SB), 10 µM PD98059 (PD), 10 µM Indomethacin (Indo), 10 µM CDC, 20 µM H89, 10 µM BisI, 10 µM AACOCF3 (AA), or vehicle (co). Afterwards, the amount of Ser-133-phosphorylated CREB was determined by Western blotting.

 
A Dominant-negative Mutant of CREB Inhibits OxPAPC-induced HO-1 Reporter Activity—To determine whether phosphorylation and activation of CREB is necessary for HO-1 induction by OxPAPC, we co-transfected the hHO4.9luc vector with a dominant-negative CREB (dn-CREB) expression vector. The dn-CREB, which carries a serine to alanine mutation at serine 133 and therefore cannot be phosphorylated, significantly inhibited OxPAPC-induced hHO4.9luc activity (Fig. 4), indicating a central role of CREB in induction of HO-1 promoter activity by OxPAPC. Luciferase reporter activity induced by forskolin, a known activator of adenylate cyclase and HO-1 inducer (52), was also inhibited by the dn-CREB (Fig. 4, inset).



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  		FIG. 4.
A dominant-negative mutant of CREB inhibits OxPAPC- and forskolin-induced hHO4.9luc reporter activity. HUVEC were co-transfected with hHO4.9luc and a vector expressing dn-CREB or a control vector (vector). Each well was transfected with 0.25 µg of PRL-SV40 vector, 0.75 µg of hHO4.9luc, and 0.25 µg of dn-CREB vector or 0.25 µg of control vector. Transfected cells were stimulated for 14 h with vehicle, 100 µg/ml OxPAPC, or 10 µM forskolin (inset). Relative luciferase activity was measured as described under "Experimental Procedures." The data represent means ± S.D.

 
A Distal CRE and a MARE Mediate the OxPAPC Effect on the Human HO-1 Promoter—To further examine the transcriptional events involved in the activation of the HO-1 promoter by OxPAPC, we cloned luciferase reporter gene constructs containing different lengths of the human HO-1 promoter. The induction of luciferase activity by OxPAPC was dependent on a distal 1.1-kb fragment, located between 3.8 and 4.9 kb upstream the transcription start site (Fig. 5A). This region contains a described functional MARE (42) and a Cadmium responsive element (43). Furthermore, our analyses of this 1.1-kb fragment revealed a putative CRE half-site located 5' of the MARE (HO-CRE). Full-length HO-1 promoter-reporter constructs carrying a mutation within the HO-CRE (hHO4.9_M1luc) showed a 50% decrease in OxPAPC-induced relative luciferase activity. Mutation of the MARE (hHO4.9_M2luc) also caused a 50% reduction of the OxPAPC-mediated induction of the HO-1 promoter-reporter activity. The introduction of mutations into both responsive elements (hHO4.9_M1 + 2luc) further inhibited the OxPAPC effect (Fig. 5B).



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  		FIG. 5.
OxPAPC activates the HO-1 promoter via a CRE half-site (HO-CRE) and a MARE. A, HUVEC were transfected with HO-1 promoter fragments of various lengths, which were fused to a firefly luciferase reporter gene. Transfected cells were treated with vehicle or 100 µg/ml OxPAPC, and induction of relative luciferase activity was determined. B, a construct carrying a mutation within the HO-CRE (hHO4.9_M1luc), the MARE (hHO4.9_M2luc), or both responsive elements (hHO4.9_M1 + 2luc) were used to transfect HUVEC. The core sequences in the responsive elements are marked with boxes. Base changes within the responsive elements are shown in bold letters. After treatment with vehicle or 100 µg/ml OxPAPC, the induction of relative luciferase activity was determined. HUVEC transfected with the promoter constructs carrying the point mutations in the HO-CRE (C) and MARE (D) (hHO4.9_M1luc and hH4.9_M2luc), respectively, were treated with 100 µg/ml OxPAPC or medium in the presence of 20 µM H89, 10 µM BisI, 10 µM PD98059 (PD), SB203580 (SB), or vehicle. After 14 h induction of relative luciferase activity was measured. The data represent means ± S.D.

 
To elucidate the role of PKA, PKC, ERK, and p38MAPK in the activation of the HO-CRE and the MARE, we measured the OxPAPC-induced HO-1 promoter activity of the constructs carrying the mutations within the respective elements after inhibition of these kinases by H89, BisI, PD98059, and SB203580, respectively. After mutation of the HO-CRE (hHO4.9_M1luc) (Fig. 5C) or the MARE (hHO4.9_M2luc) (Fig. 5D), blockade of PKA and PKC still reduced the induction of promoter activity by OxPAPC, although this reduction was less pronounced as compared with the wild type construct (Fig. 2B). These results indicate a role of PKA and PKC in the activation of the HO-CRE and the MARE. On the other hand, inhibition of the ERK pathway did not significantly affect the mutated constructs, indicating that mutation of both the HO-CRE and the MARE made the HO-1 promoter unresponsive to the ERK pathway. Inhibition of p38MAPK still reduced the induction of promoter activity if the MARE was mutated (Fig. 5D) but not if a mutation was introduced in the HO-CRE (Fig. 5C). These data suggest that transcription via the HO-CRE, but not via the MARE, is induced by p38MAPK.

OxPAPC Induces the Binding of CREB to the Human HO-1 Promoter—To identify transcription factors mediating the signals induced by OxPAPC, nuclear extracts from HUVEC that had been treated with OxPAPC or medium were analyzed by gel shift assays. OxPAPC induced binding of several complexes to the HO-CRE (Fig. 6A). Competition experiments demonstrated two slow migrating complexes (A+B) to be specific, because competition with a cold wild -type, but not with a cold mutated oligonucleotide (M1), eliminated these complexes. In addition, we performed competition experiments with the palindromic CRE of the somatostatin promoter, resembling a consensus-binding site for CREB (53). Competition with this CRE specifically eliminated complexes A and B (Fig. 6A), demonstrating the presence of CRE-binding proteins within this two complexes. Treatment of HUVEC with OxPAPC also increased binding of a specific nuclear complex (X) to the MARE (Fig. 6B). The complex disappeared after competition with a wild type but not a mutated oligonucleotide (M2) proving its specificity. To verify binding of CREB to the HO-CRE, we applied several specific antibodies to the binding reactions. An antibody against CREB/ATF1 eliminated complex A and reduced the intensity of complex B, whereas a slower migrating supershifted new complex C appeared. These results clearly demonstrate the binding of CREB/ATF1 family members to the HO-CRE site of the HO-1 promoter. Antibodies against MafF/G/K, Nrf1, Nrf2, and c-Fos, as well as NFAT, did not affect any of the observed complexes (Fig. 6C). The identity of complex X, which binds to the MARE, remains elusive, because addition of antibodies against CREB/ATF1, MafF/G/K, Nrf1, Nrf2, and c-Fos to the binding reaction had no significant effect (data not shown).



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  		FIG. 6.
OxPAPC induces specific binding of CREB to the CRE half-site of the human HO-1 promoter. Electrophoretic mobility shift assay binding reactions were carried out with nuclear extracts isolated from HUVEC, which had been treated with 100 µg/ml OxPAPC or medium for 1.5 h. HO-CRE (A) or the MARE (B) were used as probes in the binding reaction. To verify specificity of binding, a 100-fold excess of different cold competitor oligonucleotides was added as indicated. The M1 oligonucleotide carries a mutation within the CRE half-site, whereas the M2 oligonucleotide represents a mutated MARE, as shown in Fig. 5B. Specific bands are marked with arrows. C, different specific antibodies were added to the electrophoretic mobility shift assay binding reaction, which included nuclear extracts from HUVEC treated with 100 µg/ml OxPAPC for 1.5 h and the HO-CRE as labeled probe. Supershifted complexes are marked by asterisks. HUVEC were treated with 20 µM H89, 10 µM BisI, 10 µM PD98059 (PD), 10 µM SB203580 (SB), or vehicle for 30 min and then stimulated for 1.5 h in the presence of the inhibitors. Nuclear extracts were isolated, and electrophoretic mobility shift assay binding reactions were performed using the HO-CRE (D) and the MARE (E) as probes.

 
Although it is generally accepted that phosphorylation of CREB at Ser-133 enhances its transactivation potential, conflicting evidence exists as to whether this phosphorylation influences the binding of CREB to its responsive element (50). Because PKA, PKC, ERK, and p38MAPK mediated OxPAPC-induced phosphorylation of CREB, we were interested which of these kinases would be involved in the formation of the complexes A and B at the HO-CRE. Interestingly, only inhibition of p38MAPK, but not inhibition of the other kinases, diminished the OxPAPC-induced binding of the two complexes (Fig. 6D). Because also PKA, PKC, and the ERK pathway were involved in the OxPAPC-induced phosphorylation of CREB at Ser-133 (Fig. 3), these results indicate that p38MAPK activation by OxPAPC, but not the phosphorylation of Ser-133 per se, increases binding of CREB to the HO-CRE. On the other hand, the formation of complex X at the MARE was reduced by inhibition of PKC after OxPAPC treatment, whereas inhibition of other kinases had no discernable effect (Fig. 6E).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Inflammatory disorders such as atherosclerosis, sepsis, ischemia/reperfusion injury, and acute and chronic allograft rejection all share increased oxidative stress as a common feature (54-56). As a consequence oxidative modification of proteins and lipids occurs (27, 57), and generated oxidized lipids act as important modulators of the inflammatory response. HO-1, whose expression is increased during these disease states (6, 7), represents a major protective factor (15, 58-65) because of its potent anti-oxidative (4, 8, 9) and anti-inflammatory properties (10, 12). Furthermore HO-1 is potently induced by phospholipid oxidation products in vitro (13) and in vivo (34), as well as by other lipid oxidation products (66).

The endothelium, which serves as the major interface between the circulation and tissue, coordinates the inflammatory process by controlling cellular adhesion and transmigration. It is therefore a major target for pro- and anti-inflammatory stimuli. Here we demonstrate that in HUVEC, HO-1 mRNA, and protein expression is potently induced by OxPAPC.

TNF-{alpha} and LPS, both of which are major factors promoting inflammation, have also been shown to induce HO-1 in several cell types and tissues (47, 48, 67-70). However, in HUVEC we did not observe an induction of HO-1 mRNA after TNF-{alpha} or LPS treatment. Nevertheless E-selectin mRNA expression was potently induced by both agents. This observation is consistent with some, but not all previous studies (71-73), which might be explained by cell type-specific signaling or differences in used LPS and TNF-{alpha} concentrations. Our data implicates distinct signal transduction pathways activated by OxPAPC and TNF-{alpha}/LPS in HUVEC and points out oxidized phospholipids as important inducers of HO-1 in endothelial cells during inflammation.

In endothelial cells, OxPAPC signals via pathways involving cAMP/PKA (30), PKC, Ca2+/NFAT, ERK/Egr-1 (31), and PPAR{alpha} (32, 33). Several of these signaling pathways, including PKA, PKC, different MAPKs pathways (70), Egr-1 (74), and PPARs (75) have also been connected to the regulation of HO-1 gene transcription. Accordingly, we observed reduced OxPAPC-mediated HO-1 induction after inhibition of PKA, PKC, the ERK pathway, and p38MAPK, suggesting an involvement of these signaling pathways in the regulation of HO-1 transcription by OxPAPC.

Moreover, the activation of the cAMP/PKA pathway by OxPAPC has been demonstrated (30), although no direct downstream effectors have been identified. The transcription factor CREB is phosphorylated not only in response to PKA, but also after PKC, p38MAPK, and ERK activation (50) and was therefore a candidate transcription factor capable of mediating the OxPAPC effects at the transcriptional level. It has been reported previously that dn-CREB blocked arsenite-induced activation of the mouse HO-1 promoter (39), although in this study CREs in the mouse HO-1 promoter have not been described.

Here we demonstrate that OxPAPC induces phosphorylation of CREB at serine 133 in a PKA, PKC, ERK, and p38MAPK-dependent manner. In addition, over-expression of a dn-CREB significantly reduced the induction of a human HO-1 luciferase reporter construct by OxPAPC. Forskolin-induced reporter activity was abrogated by the dn-CREB. In our experiments, forskolin induced a 2-fold increase in HO-1 luciferase reporter activity, compared with a 12-fold increase after OxPAPC treatment (Fig. 4). An explanation for this difference might be the specific activation of PKA in forskolin-stimulated cells, whereas OxPAPC activates additional pathways that would result in an enhanced response.

Such additional signaling pathways may induce the binding of transcription factors to the MARE, which is also necessary for the complete induction of HO-1 by OxPAPC. In addition, other transcription factors such as PPARs or Egr-1, both of which have been shown to be involved in OxPAPC-induced signaling (31-33), as well as in the induction of HO-1 (74, 75), may be involved.

Most studies on the transcriptional control of HO-1 focused on the mouse promoter, although the regulation of the human and the mouse promoter differs in some respects (42). We investigated the transcriptional events induced by OxPAPC at the human promoter. By deleting promoter fragments and by using mutational analysis, we identified a CRE half-site (HO-CRE) and a MARE, both located 4 kb upstream the HO-1 transcription start site, as responsible for the activation of the human HO-1 promoter by OxPAPC.

Interestingly, in the rat HO-1 promoter a proximal CRE/AP1 site has been described that is necessary for the HO-1 induction by cGMP (76) and arsenite (77), although c-Jun seems to be the critical binding factor in this case. MAREs have also been described in the mouse HO-1 promoter where different members of the basic-leucine zipper (bZIP) family of transcription factors including the Nrf (NF-E2-related factor) 2, which forms heterodimers with small Maf proteins, binds to MAREs and mediates the induction of HO-1 by different agents including heavy metals (36, 38-40). We show that OxPAPC induces binding of a nuclear complex to the MARE of the human promoter, and identification of the involved transcription factors is the focus of current research.

Neither a mutation of the HO-CRE nor of the MARE rendered the constructs insensitive to an inhibition of PKA and PKC, although the extent of inhibition was reduced by both mutations (Fig. 5, C and D). Thus PKA and PKC seem to be involved in the induction of transcription via the HO-CRE and the MARE. Both PKA and PKC are known to induce phosphorylation of CREB and hence increase its potential to trans-activate the transcriptional machinery. Although we can just speculate about the factors binding to the MARE in response to OxPAPC, it is known that MARE-binding transcription factors like p45 and Nrf2 are targets of PKA (78) and PKC (79), respectively. p38MAPK, on the other hand, seems to specifically activate OxPAPC-induced transcription via CREB, because only mutation of the HO-CRE, but not of the MARE, abolished the inhibitory effect of SB203580. Interestingly after mutation of the HO-CRE or the MARE, inhibition of the ERK pathway did no longer affect the induction of promoter activity by OxPAPC (Fig. 5, A, C, and D). This implicates that both elements are required to mediate the ERK signal. The ERK pathway has been implicated in the phosphorylation of CREB (50), as well as in the activation of the MARE binding factor Nrf2 (80). The fact that blocking the ERK pathway inhibits CREB phosphorylation, HO-1 mRNA induction, and induction of the wild type promoter by OxPAPC but has little or no effect on the HO-1 protein level after OxPAPC treatment raises the question about the exact role of ERK in HO-1 transcriptional regulation. Recent data from our laboratory shows that inhibition of the ERK pathway mainly affects the early phase of HO-1 mRNA induction by OxPAPC, whereas later stages seem to be unaffected (data not shown). However this subject requires further investigation.

With most described CREs located in close proximity to the TATA box (50), it was surprising to find a functional CRE in an enhancer element positioned 4 kb upstream the transcription start site. Interestingly it has been shown that a functional CRE element is co-localized with a functional MARE also in the Ucp1 promoter (81) within an enhancer region 2.5 kb upstream of the transcription start site. MARE binding factors like NF-E2 are able to access the DNA in the repressive state (82) and mediate histone acetylation (83), which, in turn, increases the accessibility of nucleosomal DNA for other transcription factors. Therefore this co-localization of a MARE with a CRE might be a prerequisite for the binding of CREB to far upstream enhancer regions.

Although we did not observe binding of Nrf2, MafF/G/K, or AP1 proteins, all of which have been implicated previously in the transcriptional regulation of HO-1 (36, 38-40), we cannot exclude a possible heterodimerization of CREB with additional, not-identified transcription factors. The influence of Ser-133 phosphorylation on CREB binding affinity is a subject of discussion (50) and seems to differ between individual CREs (84, 85). Although the binding of CREB to the HO-CRE is induced by OxPAPC (Fig. 6A), this does not seem to be dependent on Ser-133 phosphorylation of CREB, because H89, BisI, and PD98059 all inhibited CREB phosphorylation (Fig. 3B) and consequently the induction of HO-1 (Fig. 2) but had no influence on the DNA binding of CREB (Fig. 6D). In contrast p38MAPK, which specifically activates the HO-CRE and not the MARE, does not only induce phosphorylation and activation but also binding of CREB. This might involve direct phosphorylation of CREB, as well as of additional regulatory factors that facilitate the binding of CREB. Because the HO-CRE resembles a CRE half-site it is also possible that p38MAPK facilitates the binding of a dimerization partner of CREB.

The binding of complex X to the MARE does not seem to be regulated by p38 but involves activation of PKC (Fig. 6E). Here it is of interest that Nrf2, a MARE binding factor that has been implicated in HO-1 regulation, is known to be specifically phosphorylated by PKC and hence translocated into the nucleus to drive transcription (79).

Taken together, we show that OxPAPC induces phosphorylation of CREB via PKA, PKC, p38MAPK, and ERK and that CREB itself promotes HO-1 gene transcription via a CRE half-site located in the HO-1 promoter. These data suggest a novel role for CREB in OxPAPC-mediated signaling events and provide further insight into transcriptional mechanisms in inflammatory diseases accompanied by lipid oxidation such as atherosclerosis and sepsis.


   FOOTNOTES
 
* This work was supported by a grant from the österreichische National Bank and by Fonds zur Förderung wissenschaftlicher Forschung Grant P16086 [GenBank] . 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. Back

{ddagger} To whom correspondence should be addressed: Dept. of Vascular Biology and Thrombosis Research, University of Vienna, Schwarzspanierstr. 17, Vienna A-1090, Austria. Tel.: 43-1-4277-62507; Fax: 43-1-4277-9625; E-mail: norbert.leitinger{at}univie.ac.at.

1 The abbreviations used are: HO-1, heme oxygenase-1; LPS, lipopolysaccharide; PAPC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine; OxPAPC, oxidized PAPC; PKA, protein kinase A; PKC, protein kinase C; ERK, extracellular signal-regulated kinase; PPAR, peroxisome proliferator-activated receptor; NFAT, nuclear factor of activated T cells; Egr-1, early growth response factor-1; MARE, Maf recognition element; HUVEC, human umbilical vein endothelial cells; CREB, cAMP-responsive element-binding protein; MAPK, mitogen-activated protein kinase; CRE, cAMP-responsive element; BisI, bisindolylmaleimide I; CDC, cinnamoyl-3,4-dihydroxy-a-cyanocinnamate; SCS, supplemented calf serum; RT, reverse transcriptase; dnCREB, dominant-negative CREB. Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Exner for providing the bacterial artificial chromosome clone including the HO-1 promoter, R. Beckmann and Z. Zhegu for preparation and cultivation of HUVEC, and Thomas Nardelli for assistance in preparation of the figures.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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