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Circulation Research. 2008;102:42-50
Published online before print October 25, 2007, doi: 10.1161/CIRCRESAHA.107.155143
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(Circulation Research. 2008;102:42.)
© 2008 American Heart Association, Inc.

Molecular Medicine

Hemin Upregulates Egr-1 Expression in Vascular Smooth Muscle Cells via Reactive Oxygen Species ERK-1/2–Elk-1 and NF-{kappa}B

Rukhsana N. Hasan, Andrew I. Schafer

From the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia. A.I.S. is currently at the Department of Medicine, Weill-Cornell Medical College, New York.

Correspondence to Andrew I. Schafer, Department of Medicine, Weill-Cornell Medical College, 525 East 68th St, Box-130, New York, NY 10021. E-mail ais2007{at}med.cornell.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reactive oxygen species (ROS) and oxidant stress are important mediators of cardiovascular pathologies including atherosclerosis. One source of ROS in the vasculature is free heme released from hemoglobin. Because Egr-1, the regulator of cell proliferation and apoptosis, is also induced by oxidant stress and is likewise implicated in atherosclerosis, we examined the regulation of Egr-1 by heme in vascular smooth muscle cells (SMCs). Hemin increased Egr-1 expression (mRNA, protein) within 30 minutes and ERK-1/2 phosphorylation and nuclear translocation within 5 minutes. Inhibiting hemin-induced ERK-1/2 activation by U0126 (MAPK-inhibitor), the antioxidant N-acetyl cysteine, the NADPH oxidase inhibitors apocynin and diphenyleneiodonium chloride, the superoxide scavenger tiron, or tricarbonyldichlororuthenium(II)-dimer (carbon-monoxide donor; CORM-2) blocked hemin-induced Egr-1 expression. Hemin activated Elk-1, SRF, and NF-{kappa}B and promoted their interaction with the Egr-1 promoter. Downregulating Elk-1 (via siRNA) or blocking NF-{kappa}B activation (via BAY-11-7082) abolished hemin induction of Egr-1. Finally, hemin-induced Egr-1 bound the promoters of tissue factor (TF), Plasminogen Activator Inhibitor (PAI)-1, and NGF-1A Binding (NAB)-2, upregulating their expression, and increased the biochemical activity of TF and PAI-1. Upregulation of Egr-1 and its target genes by heme-induced oxidant stress may be an important event in the initiation and progression of inflammatory vascular diseases such as atherosclerosis.


Key Words: hemin • reactive oxygen species • Egr-1 • Elk-1 • NF-{kappa}B


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Oxidative stress, caused by an increase in cellular reactive oxygen species (ROS) levels, is implicated in several vascular pathologies including atherosclerosis.1 Extracorpuscular or "free" heme (ferroprotoporphyrin IX), released from hemoglobin and other hemoproteins, is a potent prooxidant and proinflammatory agent by virtue of its ability to promote ROS formation. Free heme in the plasma is rapidly cleared by high-affinity heme-binding proteins and sequestered in the liver where it is enzymatically degraded by the heme oxygenases (HO) into iron (sequestered by ferritin), bilirubin (via the intermediate, biliverdin), and carbon monoxide (CO). Vascular tissue exposed to an acute increase in plasma free heme levels, for instance caused by injury-related hemorrhage or intravascular hemolysis, elicits an adaptive oxidative-stress response by increasing the expression of antioxidants and other detoxifying proteins, which sequester free heme and promote wound repair. However, localized accumulation of free heme in micromolar amounts, such as in a thrombus or an atherosclerotic lesion, overwhelms cellular detoxification systems provoking a more sustained oxidant stress and consequent cell and tissue damage.2–4 In addition to direct cellular damage via ROS-induced oxidation of lipids, proteins, and DNA, heme also triggers proinflammatory processes within the vasculature by catalyzing the oxidation of low-density lipoprotein (LDL) and covalent cross-linking of apolipoprotein B100, promoting foam cell formation and smooth muscle cell (SMC) proliferation, the hallmarks of vascular disease.

Heme is also a major regulator of redox-sensitive gene expression. Heme-regulated genes include stress-inducible proteins such as HO-1, ferritin, thioredoxin, Hsp70, and c-fos as well as chemokines and adhesion molecules.5–9 Modulation of gene expression by heme thus appears to play an important role in determining cell fate with regards to adaptation against disease when challenged with oxidative stress.

A major mediator of the cellular stress response is the immediate early gene (IEG) Early Growth Response (Egr)-1, itself induced by stress, injury, mitogens, and cytokines.10–11 Egr-1 binds to a GC-rich promoter motif to regulate the expression of multiple gene families including growth factors, cytokines, and transcription factors. As a key regulator of cellular proliferative and apoptotic pathways and a mediator of inflammation, Egr-1 is consistently implicated in vascular pathology.12 Indeed, sustained high-level expression of Egr-1 and its target genes was found in mouse and human atherosclerotic tissue, and Egr-1–deficient hyperlipidemic mice displayed decreased atherosclerosis.13,14 Egr-1 is readily upregulated by ischemia/reperfusion, hypoxia, hyperoxia, and hemorrhagic shock, all inducers of ROS-mediated signaling and inflammation.15–18 Transcriptional activation of Egr-1 and its downstream targets is therefore pivotal in coordinating the cellular events following oxidant stress or injury that result in inflammatory vascular damage.

Whereas ROS/oxidant stress–induced expression of several IEGs including Egr-1 has been previously described, regulation of Egr-1 by heme remains to be fully elucidated.9,19 The present study addressed the hypothesis that extracorpuscular free heme upregulates Egr-1 and its downstream targets in vascular tissue as part of the immediate cellular response to oxidative stress, the deregulation of which may contribute to inflammatory vascular diseases such as atherosclerosis. We examined the regulation of Egr-1 by hemin (ferriprotoporphyrin IX, the oxidized form of heme) in human vascular SMCs and present evidence that Egr-1 is upregulated by hemin in a redox-sensitive manner via the mitogen activated protein kinase (MAPK) ERK-1/2 and the transcription factors SRF, Elk-1, and NF-{kappa}B.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
For details, please see the Data Supplement available at http://circres.ahajournal.org. Briefly, human aortic SMCs were routinely treated with freshly prepared hemin (20µmol/L). Total RNA was subjected to either 2-step RT-PCR or 1-step real-time RT-PCR using human gene-specific primers/FAM-TAMRA dual-labeled TaqMan probes for respective genes. SDS-PAGE and Western blotting was performed following standard protocols. SMCs were transfected with pEgr1–699, a gift from Dr Shuko Harada of the University of Pennsylvania, or gene-specific siRNAs using Lipofectin/Plus-Reagent and Liopectamine RNAi-MAX, respectively. Intracellular ROS levels were determined by the CM-H2DCFDA assay. Nuclear localization of Egr-1, phospho-ERK-1/2, and NF-{kappa}B–p65 was detected by immunofluorescent staining. Transcription-Factor/DNA binding was detected by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA). TF and PAI-1 activity was measured using the respective assay kits (American Diagnostica).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Hemin Upregulates Egr-1 Rapidly and Transiently
Subconfluent quiescent SMCs were first treated with hemin in increasing concentrations for 30 minutes: 5 to 20 µmol/L hemin dose-dependently increased Egr-1 mRNA levels (1.3- to 2.5-fold; Figure 1A). Time-course analysis demonstrated that 20 µmol/L hemin induced Egr-1 mRNA and total protein (Figure 1B) within 30 minutes, which returned to baseline by 2 hours and were repressed thereafter. Consistent with this, hemin increased nuclear accumulation of Egr-1 within 15 minutes (Figure 1C) and a robust nuclear Egr-1 was detectable up to 2 hours later (not shown). These results demonstrate that hemin rapidly and transiently upregulates Egr-1 expression and induces its nuclear localization.


Figure 1
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  		Figure 1. Hemin induces Egr-1 expression in aortic SMCs. SMCs were treated with 0 to 20 µmol/L hemin for 30 minutes (A) or with 20 µmol/L hemin for 0 to 4 hours (B). Egr-1 and β-actin mRNA and protein levels were measured by RT-PCR and Western blot analysis (cell lysates), respectively. C, Immunofluorescent staining of hemin-induced nuclear localization of Egr-1 protein.

Hemin-Induced Egr-1 Expression Is Mediated via ERK-1/2
The 3 MAPKs—ERK-1/2, JNK, and p38—as well as PI3 kinase (PI3K) and Rho kinase (ROCK) associated with survival and growth, are activated by oxidative stress. Therefore, the effect of hemin on phopsphorylation of these kinases was first examined. Hemin markedly increased ERK-1/2 phosphorylation within 5 minutes, which returned to baseline by 15 minutes (Figure 2A). Phospho–ERK-1/2 translocated into the nucleus within 5 minutes and was detectable up to 30 minutes later (Figure 2B). Inhibition of ERK-1/2 activation with MAPK inhibitors U0126 (Figure 2C) or PD98059 (not shown) abolished not only hemin-induced Egr-1 mRNA increase but also the basal expression. Although hemin did increase phospho-JNK levels within 5 minutes, pharmacological inhibition of JNK activity as well as that of p38, PI3K, or ROCK failed to suppress hemin-induced Egr-1 expression (not shown). These results demonstrate that hemin increases Egr-1 expression via ERK-1/2.


Figure 2
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  		Figure 2. Hemin induces Egr-1 via ERK-1/2. A, SMCs were treated with hemin (0 to 60 minutes), and ERK-1/2 phosphorylation was detected in cell lysates by Western blot analysis. B, Nuclear localization of phospho-ERK-1/2 in hemin-treated SMCs (0 to 60 minutes) was examined by Western blot analysis of nuclear extracts (upper-panel) and immunofluorescent staining (lower-panel). C, SMCs were preteated with U0126 (10 µmol/L, 1 hour) and then hemin was added (30 minutes). Egr-1 and β-actin mRNA levels were measured by RT-PCR.

Hemin-Induced Cellular ROS Surge Causes ERK-1/2 Activation and Egr-1 Upregulation
Heme is a potent prooxidant. Therefore, we examined the effect of hemin on intracellular ROS levels in intact SMCs by the CM-H2DCFDA assay. The fluorescence generated by the oxidized product of the dye (DCF-fluorescence) is directly proportional to cellular oxidative activity, which is in turn indicative of cellular ROS levels. Addition of hemin to cells almost immediately increased DCF-fluorescence, which continued linearly over at least 1 hour (Figure 3A). At 30 minutes, hemin-induced DCF fluorescence had increased approximately 12-fold over basal level whereas it was approximately 6.5-fold for positive controls H2O2 (100 µmol/L) and glucose oxidase (GOX; 12 mU/mL, which steadily generates H2O2 equivalent to {approx}95 µmol/L; not shown).20 H2O2 did increase Egr-1 expression but it was modest and delayed (less than 2-fold at 1 to 2 hours; not shown). Pretreatment of cells with the antioxidant, N-acetyl cysteine (NAC), the superoxide scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid (tiron), the H2O2 scavenger PEG-catalase, the flavin inhibitor diphenyleneiodonium chloride (DPI), and the specific NADPH oxidase inhibitor apocynin, inhibited or significantly reduced hemin-induced increase in DCF-fluorescence. Tiron quenched the DCF-fluorescence down to only a third of that in untreated cells. NAC, tiron, DPI, and apocynin also inhibited hemin-induced ERK-1/2 phosphorylation (not shown) and Egr-1 mRNA increase (Figure 3B), with tiron consistently being the most effective. These data demonstrate that it is the hemin-induced surge in cellular ROS levels, in particular the superoxide generated most likely from NADPH oxidase activity, and consequent oxidative stress which mediates Egr-1 upregulation via ERK-1/2 activation.


Figure 3
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  		Figure 3. Hemin-induced Egr-1 is redox-regulated. A, CM-H2DCFDA assay: SMCs were pretreated for 3 hours with NAC (10 mmol/L), apocynin (100 µmol/L), DPI (20 µmol/L), or with tiron (10 mmol/L, 0.5 hour) and PEG-catalase (250 U/mL, 1 hour) followed by addition of hemin (1 hour) and measurement of DCF fluorescence. B, SMCs were pretreated with the antioxidants as above and then hemin was added (30 minutes). Egr-1 and β-actin mRNA was measured by RT-PCR (mean±SEM).

Hemin-Induced Egr-1 Expression Is Inhibited by CO
Hemin is a strong inducer of the major cellular stress response protein HO-1, which catalyzes the rate-limiting step of heme degradation into biliverdin, bilirubin, free iron, and CO. All of these but iron (sequestered via ferritin) are potent antioxidants and cytoprotectants. Increasing evidence supports a cytoprotective and antiproliferative role of CO, in particular in the vasculature, mainly because of its ability to block ROS/oxidative stress–mediated ERK-1/2 activation and Egr-1 expression.16,21–23 Therefore, we examined whether CO affected hemin-induced Egr-1 upregulation using as CO donor, the CO-releasing molecule (CORM) tricarbonyldichlororuthenium(II) dimer (CORM-2). RuCl3 was used as the noncarbonyl inert control. Preconditiong of SMCs with CORM-2, but not with RuCl3, dose-dependently inhibited both hemin-induced ERK-1/2 phosphorylation and Egr-1 mRNA increase (Figure 4). These results are consistent with previous reports in other systems and suggest that CO derived from CORM-2, like exogenously administered gaseous CO or that produced endogenously from HO-1 activity, may act as an antioxidant in an analogous manner in vascular SMCs by interrupting NADPH oxidase–dependent ROS generation to inhibit hemin/ROS/ERK-1/2–mediated Egr-1 expression.


Figure 4
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  		Figure 4. CORM-2 inhibits hemin-induced ERK-1/2 activation and Egr-1 expression. SMCs were preincubated with CORM-2 or RuCl3 (0 to 1 mmol/L, 3 hours) and then treated with hemin for indicated times. A, ERK-1/2 phosphorylation was detected in cell lysates by Western blot analysis. B, Egr-1 and GAPDH mRNA levels were measured by RT-PCR.

Hemin Activates Egr-1 Promoter and the Transcription Factors SRF, Elk-1, and NF-{kappa}B
IEGs are in general transcriptionally regulated. In reporter gene assays using the Egr-1 promoter-luciferase construct pEgr1–699, hemin dose-dependently increased Egr-1 promoter-driven luciferase activity suggesting that Egr-1 upregulation by hemin was mediated, at least partially, at the transcriptional level (Figure 5A). Stress-induced expression of IEGs, including Egr-1, primarily involves cooperative interaction between the serum response element (SRE), the serum response factor (SRF), and one or more of the ternary complex factors (TCFs; also called ETS-family proteins) Elk-1, Sap-1a, and Net. Stress/inflammation-induced regulators such as NF-{kappa}B, AP-1, and cAMP response element (CRE)-binding (CREB) protein may also be involved. Because phosphorylation of the TCFs typically precedes their interaction with SRF and because Elk-1 is the direct downstream target of ERK-1/2, we first examined the effect of hemin on SRF and Elk-1 activation. Hemin increased phospho-Elk-1 and phospho-SRF levels up to 4-fold at 5 and 20 minutes, respectively, which remained significantly elevated up to 30 minutes later (Figure 5B). The rapid increase in phosphorylation of Elk-1 and SRF by hemin may facilitate their interaction with each other and with the Egr-1-SREs. Hemin also induced nuclear translocation of the NF-{kappa}B subunits p50 and p65 within 30 minutes (Figure 5C) suggesting that NF-{kappa}B is also activated by hemin, quite likely via phosphorylation, which precedes p50/p65 nuclear translocation.


Figure 5
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  		Figure 5. Hemin activates Egr-1 promoter and the transcription factors SRF, Elk-1, and NF-{kappa}B. A, SMCs were transiently cotransfected with pEgr1–699 and phRL-TK(control-vector), treated with hemin (0 to 50 µmol/L, 4 hours) and lucifersae activity was measured (mean± SEM). B, SRF and Elk-1 phosphorylation was detected in nuclear extracts from hemin-treated SMCs (0 to 30 minutes) by Western blot analysis: upper-panel, representative blots; lower-panel, densitometric measurement of phospho- and total-protein levels (mean± SEM). C, Immunofluorescent staining of hemin-induced nuclear translocation of NF-{kappa}B subunits p50 and p65.

The human Egr-1 promoter contains 5 SREs, several TCF-binding (ETS) motifs, and multiple NF-{kappa}B, CRE, and AP-1 motifs. Therefore, we next assessed whether hemin influenced DNA-binding of these transcription factors to the Egr-1 promoter. Chromatin immunoprecipitation (ChIP) assays confirmed hemin-induced interaction of Elk-1, SRF, and NF-{kappa}B subunit p65 with the Egr-1 promoter (Figure 6). Whereas SRF occupied Egr-1 promoter in steady-state conditions and hemin further enhanced this interaction, chromatin-bound Elk-1 was immunoprecipitated only from hemin-treated cells, suggesting that SRF–SRE interaction may be required for basal Egr-1 expression, whereas ternary complex formation between SRF-SRE and Elk-1 may be critical to hemin-inducible Egr-1 expression. Interestingly, NF-{kappa}B subunit p65 also displayed very low-level steady-state promoter occupancy, which was significantly enhanced by hemin (within 30 minutes) at the proximal Egr-1 promoter (Figure 6), but not at the distal promoter (not shown). Hemin-inducible AP-1 and CREB binding to the Egr-1 promoter was not detected in ChIP assays, although hemin did increase phosphorylation of CREB-1 (not shown). These results were further validated by EMSA using as probe the distal-most ETS-SRE-ETS string of the Egr-1 promoter (SRE5). Consistent with ChIP data, SRE5-SRF binding was detected in steady-state and further strengthened by hemin within 5 minutes (supplemental Figure IA, lanes 1 to 6). The 2 SRE5-SRF-specific nucleoprotein complexes (arrows, A and B) disappeared when mutated SRE5 probe (mSRE5) or the specific competitor were used (lanes 7 to 8) and were completely upshifted by anti-SRF antibody (lane-9). Likewise, hemin increased DNA-binding of the oligonucleotide probe containing the NF-{kappa}B motif at –425 bp of the Egr-1 promoter (NFKB425) within 15 minutes (supplemental Figure IB, lanes 1 to 3). The hemin-inducible nucleoprotein complex failed to form with nuclear proteins derived from SMCs pretreated with the NF-{kappa}B inhibitor BAY-11-7082 (lane 4), or in the presence of the mutated probe mNFKB425 or the specific competitor (lanes 5 to 6). Both anti-p65 and anti-p50 antibodies generated a partial or complete supershift in the primary hemin-inducible nucleoprotein complex indicating the presence of both the NF-{kappa}B subunits therein (lanes 7 to 8).


Figure 6
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  		Figure 6. Hemin promotes SRF, Elk-1, and NF-{kappa}B interaction with the Egr-1 promoter (ChIP assays). Schematic representation of human Egr-1 promoter indicating the location of the SRE, ETS, and NF-{kappa}B motifs and PCR-primers (top). Purified nucleoprotein complexes from hemin-treated SMCs (0 to 60 minutes) were immunoprecipitated (IP) with the indicated antibodies and served as PCR-template (bottom). 1% of the total purified pre-IP chromatin was used to estimate input-DNA.

Furthermore, siRNA-mediated knockdown of Elk-1 or pharmacological inhibition of NF-{kappa}B activity via BAY-11-7082, which specifically inhibits stress-inducible phosphorylation and degradation of I{kappa}B{alpha} (a prerequisite for NF-{kappa}B activation) or via SN-50 (which inhibits NF-{kappa}B nuclear translocation; not shown), abolished hemin-induced Egr-1 expression (Figure 7), whereas it was only weakly attenuated by siRNA-mediated knockdown of SRF (not shown). Taken together, these data demonstrate that hemin activates SRF, Elk-1, and NF-{kappa}B and promotes their interaction with the Egr-1 promoter, and that Elk-1 and NF-{kappa}B are critical transcriptional activators of Egr-1 in hemin-induced oxidant stress.


Figure 7
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  		Figure 7. Elk-1 and NF-{kappa}B are critical regulators of hemin-induced Egr-1. SMCs were transfected with Elk-1-specific siRNA (20 nmol/L; A) or pretereated with BAY-11-7082 (5 to 10 µmol/L, 1 hour; B) and then treated with hemin (45 minutes). Egr-1, Elk-1, and β-actin were detected in cell lysates by Western blot analysis.

Hemin Increases Egr-1 DNA Binding and Target Gene Expression
Egr-1 is a major transcriptional regulator of multiple gene families including those implicated in vascular pathology. Therefore, we examined the effect of hemin on Egr-1–mediated gene expression. In EMSA using the oligonucleotide probes EGR1 and mEGR1 (containing the consensus and mutated Egr-1 motifs, respectively), hemin increased DNA-binding of the EGR1-probe within 60 minutes (supplemental Figure II, lanes 1 to 2). The EGR1-specific nucleoprotein complex disappeared in the presence of the mutated probe mEGR1 or specific competitor (lanes 3 to 4, respectively). Although anti–Egr-1 antibody could not generate a clear supershift, it significantly intensified the primary EGR1-specific complex suggesting an anti–Egr-1 antibody-specific interaction (lane-5).

Subsequently, we examined the effect of hemin on the expression of 3 stress/inflammation-induced Egr-1 responsive genes: Tissue Factor (TF), the key prothrombotic initiator of the coagulation cascade, Plasminogen Activator Inhibitor (PAI)-1, the primary regulator of fibrinolysis, and NGF-1-A Binding (NAB)-2, the major Egr-1 transcriptional corepressor.16,24,25 Hemin increased mRNA levels of all 3 genes within 2 hours as determined by semiquantitative RT-PCR and real-time RT-PCR analysis (Figure 8A). In the latter, mRNA fold-increase at 2 hours was 3.9±0.68 for TF and 2.7±0.32 for NAB-2, whereas PAI-1 peaked at 4 hours up to 4.8±0.69-fold. All 3 transcripts declined after 4 hours and returned to baseline by 8 hours (not shown). Promoters of the human TF and NAB-2 contain multiple Egr-1 binding motifs. Sequence analysis of the human PAI-promoter identified 2 putative Egr-1 motifs at –306 bp and –684 bp, although Egr-1 binding to neither has yet been demonstrated. Consistent with EMSA and RT-PCR, ChIP assays demonstrated that hemin promoted Egr-1 interaction not only with TF and NAB-2 promoters, but also with the proximal PAI-1 promoter, within 30 to 60 minutes (Figure 8B). That the distal Egr-1 motif in the PAI-1 promoter was constitutively occupied by Egr-1, with a slight decline in promoter occupancy at 120 minutes (Figure 8B), and that siRNA-mediated knockdown of Egr-1 significantly reduced basal PAI-1 expression (not shown) suggests that this motif may contribute to steady-state PAI-1 expression. To our knowledge, this is the first report demonstrating inducible (or steady-state) Egr-1 interaction with the human PAI-1 gene. Egr-1 interaction with the distal NAB-2 promoter was relatively stronger as compared with the proximal region, although chromatin-bound Egr-1 was immunoprecipitated within 30 minutes of addition of hemin in both cases.


Figure 8
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  		Figure 8. Hemin increases Egr-1–dependent gene expression and biochemical activity. A, SMCs were treated with hemin (0 to 4 hours), and mRNA levels of indicated genes were measured by conventional RT-PCR (left-panel) and 1-step real-time RT-PCR (right-panel; bars represent fold increase in mRNA levels after normalization with β-actin; n=2, mean±SEM). B, ChIP assays: Purified Egr-1–bound chromatin was immunoprecipitated from hemin-treated SMCs (0 to 120 minutes) and used as PCR template. Partial promoter regions of human TF, PAI-1, and NAB-2 genes, indicating the location of Egr-1 motifs and the PCR-primers (right), and PCR results (left) are shown. C, SMCs were transfected with Egr-1 siRNA (20 nmol/L) or left nontransfected (NTC), treated with hemin (H; 20 µmol/L, 2 hours). Procoagulant TF levels were determined in cell lysates whereas levels of active PAI-1 were measured by ELISA in conditioned medium.

Finally, using a chromogenic assay for measuring TF procoagulant activity and an enzyme-linked immunosorbant assay (ELISA) to detect active PAI-1, we examined whether hemin/Egr-1–mediated TF and PAI-1 upregulation translated into functional activation of the 2 proteins. Hemin transiently increased (within 2 hours) the levels of procoagulant TF and enzymatically active PAI-1 up to 2.02±0.27-fold and 4.14±0.4-fold, respectively (Figure 8C). The activity levels of both proteins declined progressively over the following 4 to 8 hours (not shown). siRNA-mediated knockdown of Egr-1 inhibited or significantly reduced hemin-induced increase in the procoagulant TF and active PAI-1. Taken together, these results demonstrate that hemin increases Egr-1–dependent expression of TF, PAI-1, NAB-2, and potentially of several other Egr-1 responsive genes implicated in the cellular response to oxidative stress and inflammation.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
ROS are mediators of vascular signal transduction but may provoke an oxidative stress when their levels exceed cellular detoxifying capacity. ROS-mediated oxidant stress and inflammation is increasingly implicated in cardiovascular disorders, including atherosclerosis.1–4 Free heme, released from hemoglobin at a site of vascular injury or hemolysis, promotes ROS formation and LDL oxidation. Egr-1, a major proatherogenic protein, is also induced by oxidative challenges including oxidized lipids.12 Because heme and Egr-1 are both important mediators of ROS-induced inflammatory signaling, the present study investigated the regulation of Egr-1 by free heme in human vascular SMCs.

The principal finding of this study is that hemin upregulates Egr-1 expression at both mRNA and protein levels in a dose- and time-dependent manner, increases Egr-1 nuclear accumulation, DNA-binding and downstream gene expression and biochemical activity of two key proatherogenic proteins, TF and PAI-1. Our results are in agreement with previous data that Egr-1 is upregulated by ROS/oxidant stress provoked by H2O2 as well as ischemia, hypoxia, hyperoxia, and hemorrhagic shock.15–18 However, our finding that Egr-1 is induced by hemin seems to contradict a recent report by Mishra et al that hemin inhibits hypoxic induction of Egr-1.16 This apparent inconsistency may be attributable to differences in cellular context (macrophages versus SMCs) and their observations made in macrophages over a much longer period (2 hours), by which time stress-induced Egr-1 expression is typically well past its peak induction and approaching steady-state level. In effect, data presented here shows the highly transient nature of hemin-induced Egr-1 expression in SMCs, which peaked at 30 minutes, returned to baseline by 2 hours and was repressed thereafter (Figure 1). This expression profile is typical of stress-induced IEGs. In fact, we also observed similar transient increases by hemin (within 30 to 60 minutes) in 3 other IEG transcripts: c-fos, c-Jun, and Fra-1 (not shown).

Hemin-induced Egr-1 expression was mediated by the MAPK pathway and ERK-1/2 because (1) hemin rapidly increased ERK-1/2 phosphorylation and nuclear translocation, and (2) pharmacological inhibition of ERK-1/2 activation abolished hemin-induced Egr-1 expression (Figure 2). Rapid and transient ERK-1/2 phosphorylation and nuclear translocation mediated by mitogen-derived ROS and oxidative stress is associated with cell proliferation and survival, as is hemin because of its potential to induce the cytoprotective HO-1/CO pathway.23,26,27

Hemin caused an almost immediate increase in cellular ROS levels (Figure 3A). At 30 minutes, H2O2 (100 µmol/L, native or generated via GOX) produced approximately 58% as much ROS-increase as hemin. This ROS-surge was inhibited by the antioxidant NAC and was dependent on cellular NADPH oxidase activity because inhibiting the latter with apocynin (the specific NADPH oxidase inhibitor) or the flavin inhibitor DPI inhibited the ROS-surge. Although the SOD mimetic tiron completely abolished hemin-induced ROS-increase, it was only partially reduced by the H2O2 scavenger PEG-catalase suggesting that the remaining ROS was primarily superoxide. Consistent with this, hemin-induced ERK-1/2 phosphorylation and subsequent Egr-1 upregulation were also inhibited by tiron, apocynin, DPI, and NAC (Figure 3B), indicating that increased ROS, notably superoxide derived from cellular NADPH oxidase activity, mediated hemin-induced Egr-1 expression. Taken together, these data support the hypothesis that chronic exposure to elevated heme/superoxide in the vasculature may overwhelm cellular antioxidant capacity resulting in elevated expression of key regulators including Egr-1, increased vascular inflammation, cell proliferation, and disease. Indeed, increasing evidence now implicates NADPH oxidase, the major source of superoxide, in the prooxidative and proinflammatory signaling leading to vascular cell migration and proliferation.28–30

CO, the byproduct of HO-1–mediated heme degradation, acts as an antiinflammatory and cytoprotective agent in tissues and vasculature challenged with oxidant stress and was recently shown to mediate these effects via inhibition of ROS formation by targeting NADPH oxidase and ERK/Egr-1 signaling.16,21–23 The dose-dependent attenuation by CO (from CORM-2) of hemin-induced ROS/ERK/Egr-1 signaling demonstrated in this study (Figure 4), and our observation that CORM-2 inhibits the hemin-induced ROS surge (not shown), could be mediated in an analogous manner. Because heme (and hemin) is itself an inducer of HO-1, the CO generated by HO-1 enzymatic activity may serve as a negative feedback regulator of the heme-activated pathway in vascular SMCs described here. CORM-2-derived CO, like gaseous and HO-1–derived CO, has consistently been shown to suppress stress/agonist-induced ROS generation and consequent downstream inflammatory events in various cell systems including SMCs.21,22–31,32 CORM-2 could therefore serve as a potential atheroprotective agent by virtue of its ability to release CO locally to counteract the inflammatory and proatherogenic effects of ROS/hemin-induced Egr-1.

Hemin-induction of Egr-1 was mediated at the transcriptional level because it increased (1) Egr-1 promoter activity, (2) phosphorylation of the transcription factors Elk-1 and SRF, nuclear translocation of NF-{kappa}B (Figure 5), and promoted their interaction with the Egr-1 promoter (Figure 6) and, (3) siRNA-mediated knockdown of Elk-1 or pharmacological inhibition of NF-{kappa}B activation abolished hemin-induced Egr-1 expression (Figure 7). Elk-1 and NF-{kappa}B therefore appear to be the primary transcriptional regulators of hemin-induced Egr-1. Although SRF is necessary for ternary complexing with Elk-1, siRNA-mediated knockdown of SRF only weakly attenuated hemin induction of Egr-1 (not shown). Some residual expression of the target-protein may occur in siRNA-mediated knockdown, which in case of SRF may be sufficient for ternary complex formation with Elk-1. Steady-state SRE-SRF interaction at the Egr-1 promoter, which was further enhanced by hemin, is consistent with its dual role in both basal and stress-inducible Egr-1 expression as described previously.33 The SRE2–5 cluster within the –473 bp region and the NF-{kappa}B motif located at –425 bp of the human Egr-1 promoter have been previously identified as mediators of stress-inducible Egr-1 expression involving ROS signaling.17,26,34,35 Further deletion and mutation analysis of the Egr-1 promoter will permit positive identification of the specific hemin-activated SRE(s) and NF-{kappa}B motif(s).

Agonist/stress-induced Egr-1 modulates the expression of multiple gene families including regulators of proliferation, apoptosis, and inflammation. Hemin transiently increased Egr-1–dependent expression of TF, PAI-1, and NAB-2 (Figure 8A and 8B) as well as that of NAB-1 (not shown), and significantly increased, in an Egr-1–dependent manner, the levels of procoagulant TF and of active PAI-1 in SMCs (Figure 8C). TF, now recognized as the major initiator of the blood coagulation cascade, and PAI-1, the key regulator of the fibrinolytic system, may both be critical in atherothrombotic complications after plaque rupture when the vessel wall is exposed to high levels of free heme. Indeed, elevated expression of these and several other Egr-1 responsive genes was reported in atherosclerotic lesions and in tissues subjected to ischemia and hypoxia.13,15,16 The induction by hemin of NAB-2, the major transcriptional repressor of Egr-1 and itself considered a delayed early gene, together with the corepressor NAB-1, suggests that the regulatory mechanisms ensuring the transient nature of stress-induced Egr-1 expression are also activated by hemin. The tight negative feedback loop between Egr-1 and the NAB proteins, as with CO, may contribute to extinguishing inducible Egr-1 expression rapidly, thus allowing transition into the damage-controlling adaptive phase of the stress response. However, extended exposure of cells to heme and resulting elevated ROS, such as in a vascular lesion or thrombus, may cause short-circuiting of the critical regulatory networks resulting in sustained Egr-1 expression and vascular inflammation.

Egr-1 is a major regulator of cell proliferation and growth whereas hemin, being a potent inducer of the HO-1/CO pathway, inhibits serum- and growth factor–induced proliferation in various cell types including SMCs.23 Hemin likewise inhibited serum-induced proliferation of vascular SMCs used in this study (not shown). Our preliminary observations further suggest that hemin/HO-1/CO–mediated inhibition of SMC proliferation is sensitive to variations in endogenous Egr-1 levels, because blocking Egr-1 expression (via knockdown of Egr-1 or Elk-1 or inhibition of upstream ERK-activation) halted serum-induced SMC growth and further accentuated the antiproliferative effect of hemin, whereas knockdown of NAB-2, which resulted in relatively sustained Egr-1 expression, caused SMCs to continue to proliferate despite an intact and activated heme/HO-1/CO pathway (not shown). Together, these data support the role of Egr-1 as a critical regulator of SMC proliferation and underscore the significance of a tightly regulated Egr-1 for SMCs to proliferate in a highly coordinated manner, such as in wound-repair and growth. In a pathological context, however, such as in a vascular lesion, disruption of the multi-layered feedback loops maintaining Egr-1 levels in check may lead to uncontrolled SMC proliferation and migration. The exact functional correlates of heme-related induction of Egr-1, such as its effects on SMC proliferation and procoagulant activity, are likely to be more complex, time-related, and modulated by physiological feedback mechanisms including, but not limited to, HO-mediated CO generation. These will require further investigation using both cell culture and animal models.

To summarize, we have examined the early molecular events underlying hemin-induced oxidative-stress response in vascular SMCs. We provide evidence that hemin transiently upregulates Egr-1 via the ROS/ERK/Elk-1 pathway and NF-{kappa}B, resulting in increased Egr-1–dependent gene expression and biological function. We propose that rapid induction by hemin of Egr-1 (and other IEGs) and downstream gene expression defines the immediate "phase-I" of the oxidative stress response, which is followed by the adaptive "phase-II" response characterized by an increase in endogenous antioxidants, cytoprotectants, and detoxifying molecules including HO-1 and CO. In a pathological context, however, where the vasculature may be persistently exposed to free heme, cellular homeostatic systems and antioxidant defense mechanisms may become insufficient to interrupt heme-induced ROS signaling, leading to sustained high level expression of Egr-1 and its downstream gene targets. This may result in increased inflammation, proliferation, and migration of SMCs, all of which contribute to the initiation and progression of inflammatory vascular pathologies such as atherosclerosis.


*    Acknowledgments
 
Sources of Funding

This study was supported by the NIH grant R01-HL36045 (to A.I.S.).

Disclosures

None.


*    Footnotes
 
Original received May 3, 2007; revision received October 5, 2007; accepted October 17, 2007.


*    References
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