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Cardiovasc Res. Author manuscript; available in PMC Oct 1, 2008.
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PMCID: PMC2094110
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Interleukin-1 β -induced Id2 gene expression is mediated by Egr-1 in vascular smooth muscle cells

Abstract

Objective

Id2 (inhibitor of DNA-binding 2), a member of the helix-loop-helix family of transcription regulators, plays important roles in cell proliferation and differentiation. Recent reports have documented that Id2 is up-regulated during vascular lesion formation and overexpression of Id2 promotes vascular smooth muscle cell (VSMC) proliferation. However, the transcriptional regulation of Id2 gene expression in VSMC remains unexplored.

Methods and Results

Using Northern- and Western-blot analyses, we documented that interleukin-1β (IL-1β) induced Id2 gene expression in VMSC in a time- and dose-dependent manner. Overexpression of early growth response factor-1 (Egr-1) in VSMC induced Id2 expression while IL-1β-induced Id2 expression was abrogated in VSMC by the Egr-1 repressor, NGFI-A binding protein 2 (NAB2), expressed from an adenovirus. Overexpression of Egr-1 transactivated the Id2 promoter in reporter assays dependent on the presence of intact putative Egr-1 binding sites as determined by mutagenesis. Finally, electrophoretic mobility shift assays (EMSA) demonstrated that the Egr-1 protein can bind the Egr-1 sites derived from the human Id2 promoter in vitro and chromatin immunoprecipitation identified the putative Egr-1 site between −723 to −712 as the functional Egr-1 binding site in vivo.

Conclusions

Our data demonstrate that IL-1β-induced Id2 expression in VSMC is mediated by the transcriptional factor Egr-1 in VSMC.

1. Introduction

In response to vascular injury, vascular smooth muscle cells (VSMC) show enhanced proliferation, migration and production of extracellular matrix thus lead to neointimal lesion formation. It is well established that growth factors and cytokines play critical roles during vascular lesion formation[1, 2]. Interleukin (IL)-1β has been shown to contribute to intimal hyperplasia and lesion progression in atherosclerosis by activating VSMC and monocytes as well as inducing expression of adhesion proteins in endothelial cells[3, 4]. Although it has been well documented that IL-1β treatment increases VSMC dedifferentiation and proliferation both in vitro and in vivo animal models[46], the molecular mechanisms involved are not fully understood.

Emerging data indicate that the helix-loop-helix (HLH), inhibitor of DNA-binding (Id) proteins regulate cell growth and differentiation in numerous cell types[7, 8]. In mammals, four distinct members of the Id proteins (Id1 to Id4) have been identified. Id proteins lack a basic DNA binding domain but contain a dimerization HLH domain, thus antagonizing the functions of basic helix-loop-helix (bHLH) transcription factors such as E2A through the formation of non-functional Id-bHLH heterodimers[9]. These heterodimers can not bind DNA and thus the Ids function in a dominant-negative manner to regulate cell differentiation and cell growth. To date, accumulating evidence suggests that Id2 enhances cell proliferation in a variety of cell types including VSMC[8, 10]. Overexpression of Id2 in VSMC results in a significant enhancement of cell growth via increased S-phase entry[10]. In addition, Id2 binds to the retinoblastoma protein and promote cell growth[11]. So far, the mechanisms regulating Id2 transcription in VSMC remain unexplored.

The early growth response (Egr)-1 transcription factor is a serum-inducible zinc finger protein that is a critical upstream regulator of cell proliferation, differentiation, and apoptosis[12]. Egr-1 gene expression in VSMC is rapidly induced by mitogens, hypoxia, shear stress, or mechanical injury[13]. Induction of Egr-1 by IL-1β has been demonstrated in a variety of cell types including VSMC[14]. During vascular lesion formation, Egr-1 is involved in transactivation of multiple genes including platelet derived growth factor (PDGF), tissue factor, and fibroblast growth factor(FGF)-2[13]. Two corepressors, NGFI-A-binding proteins 1 and 2 (NAB1 and NAB2) can markedly decrease Egr-1 transcriptional activity by binding to the inhibitory domain on Egr-1 [15]. In the present study, we documented for the first time that IL-1β induced Id2 expression and that this effect is mediated directly by Egr-1 binding to the Id2 promoter in VSMC, suggesting that the induction of Id2 expression in an Egr-1 dependent fashion contributes to the proliferative effect of IL-1β on VSMC.

2. Methods

2.1. Materials and Molecular Biology Techniques

Human recombinant IL-1β was obtained from Sigma (Saint Louis, MO). [γ-32P]ATP and [α-32P]d-CTP were obtained from Perkin-Elmer Life Sciences (Boston, MA). Northern and Western blot analysis were performed as previously described [14]. Rabbit anti-Id2 and anti-Egr-1 polyclonal antibodies (SC-489 and SC-189X, 1:500 and 1:10000 dilutions, respectively) and goat anti-β-actin polyclonal antibody( SC-47778, 1:1000 dilution )were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

2.2. Cell Culture

Human aortic smooth muscle cells (HASMC) were purchased from Bio-Whittaker and cultured in SmGM-2 (Cambrex) containing 5% FBS, 2 ng/ml human bFGF, 0.5 ng/ml human EGF, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, and 5 µg/ml bovine insulin. For all experiments, early passages (5–7) of HASMC were grown to 80–90% confluence and rendered quiescent by serum starvation in Opti-MEM (Invitrogen) for 24 hours. The A7r5 cell line was purchased from ATCC and cultured in DMEM-F/12 (Invitrogen) supplemented with 10% (v/v) FBS.

2.3. Plasmids and Adenoviral Recombinants

The pcDNA3.1-Egr-1* plasmid containing the constitutively active (NAB-insensitive) Egr-1, wild type Egr-1 as well as the adenoviruses AdEgr-1, AdEgr-1*, and AdNAB2 were obtained from Dr. Ehrengruber at the University of Zurich in Switzerland [16]. The human Id2 promoter luciferase reporter construct was generated in this study. A ~2.1 kb fragment of the Id2 promoter (nt −2130 to +25) was amplified by PCR and then cloned into a luciferase reporter plasmid pGL3-Basic (Promega) to generate pId2-Luc. The core sequences of the three putative Egr-1 binding sites in the 2.1 kb Id2 promoter were replaced by AT-rich nucleotides using the QuikChange Multi site-directed mutagenesis kit (Stratagene) as instructed by the manufacturer resulting in the pId2Mut-Luc plasmid. To generate luciferase reporter constructs containing putative Egr-1 binding sites, three-tandem repeats of the putative Egr-1 binding site (5′-gaatgcgtgcgtgggtggtttgtt-3’, nt −729 to nt −706 from the human Id2 promoter) or its mutated sequence (5′-gaatgcgtgcgtgaatagtttgtt-3′) were synthesized and inserted upstream of a TK-mini promoter-driven luciferase vector (Promega) resulting in the pEgr-1WT×3-TKLuc and pEgr-1Mut×3-TKLuc plasmids, respectively.

2.4. Electrophoretic Mobility Shift Assay (EMSA)

Nucleotide sequences for EMSA corresponding to the sense strand of the double-stranded oligonucleotides probes, with the putative Egr-1 binding sites underlined, were as follows: 1) Egr-1 binding site 1: 5’-gaatgcgtgcgtgggtggtttgtt-3’ (nt −729 to nt −706 of Id2 promoter); 2) the mutated version of Egr-1 binding site 1: 5’-gaatgcgtgcgtgAAtAgtttgtt-3’, with the mutated positions indicated by capital letters; 3) Egr-1 binding site 2: 5’-gctcgcgccccgcccaccccgcggggatt-3’ (nt −198 to nt −169 of Id2 promoter); 4)Egr-1 binding site 3: 5’-ggaagaaccaagcccacgccccgcgcccgc-3’ (nt −157 to nt −128 of Id promoter). The Egr-1 consensus nucleotide, 5’-ggatccagcgggggcgagcgggggcga-3’ that contains two Egr-1 binding sites was purchased from Santa Cruz. Nuclear extracts were isolated using NE-PER Nuclear and Cytoplasmic Extract Kit (Pierce). EMSA was performed as previously described[14].

2.5. Chromatin Immunoprecipitation (ChIP) Assay

The chromatin immunoprecipitation assay was performed with the kit from Upstate (cat#:17-295, Lake Placid, NY) followed by quantitative real-time PCR performed using the LightCycler 1.2 from Roche (Summerville, NJ) with SYBR Green JumpStart Taq ReadyMix from Sigma. Quiescent HASMC treated with 5 ng/ml of IL-1β or vehicle for 1.5 hours were used for the assay. Primer sequences (positions related to the transcriptional start site of Id2 gene) were as follow: 1) the upstream of Egr-1 binding sites: 5’- ccttacgggccggtctgtcg-3’ (nt −2710 to nt −2691 ) and 5’-tgggggaaaatcgtgtcggagcat-3’ (nt −2463 to nt −2486); 2) Egr-1 binding site 1: 5’-ggttgcaaaagcccacactaagc-3’(nt −760 to nt −738) and 5’-gttcccagaccaagccctacaca-3’ (nt −465 to nt −487); 3) Egr-1 binding sites 2 and 3: 5’-ccccgccagccccgcacttac-3’ (nt −248 to nt −228) and 5’-gagcttcccttcgtccccattg-3’( nt −54 to nt −75); 4) downstream of Egr-1 binding sites: 5’-cagtcccgtgaggtccgttag-3’ (nt 131 to nt 151) and 5’-ctgcaggtccaagatgtagtcg-3’ (nt 318 to nt 339). Values of the immunoprecipitated DNA at each promoter location, obtained from triplicate immunoprecipitations, were normalized with the corresponding values in the input at each Id2 promoter location.

2.6. Transient Transfection and Luciferase Assays

A7r5 cells grown to 90% confluence in DMEM-F/12 supplemented with 10% FBS were transiently transfected with reporter and expression plasmids as described in the corresponding sections using LipofectAMINE2000 (Invitrogen). The Green fluorescent protein (GFP) expression plasmid was co-transfected as the control for transfection efficiency. The total amount of transfected DNA was kept constant by using the corresponding empty vector. Twenty-four hours after transfection, cells were cultured for another 24 hours in Opti-MEM medium. Luciferase activity was measured by the luciferase assay system (Promega) using a TD20/20 luminometer (Turner Biosystems) and normalized by the corresponding GFP values.

2.7. Statistical Analysis

Each experiment was repeated a minimum of three times. Statistical analysis was performed by either AVOVA (for multiple comparisons) or Student’s t-test for comparing two means. For ANOVA, post-hoc mean comparisons were performed by the least significant difference (LSD) procedure. Data are presented as means ± SD. A value p<0.05 allows the differences to be considered significant.

3. Results

3.1. IL-1β induces Id2 gene expression in HASMC

Treatment of human aortic smooth muscle cells (HASMC) with IL-1β resulted in increased levels of expression of Id2, a known mediator of VSMC proliferation. In time dependent response studies, HASMC were treated with 5 ng/ml of IL-1β for 0, 0.5, 1, 2, 4, 8, 16, and 24 hours. The levels of Id2 mRNA significantly increased after 2 hours of IL-1β stimulation, reached a peak (~4.8 fold increase) at 8 hours and remained higher than baseline for at least 24 hours (Figure 1A). In addition, the Id2 mRNA was up-regulated by IL-1β stimulation in a dose-dependent manner with a significant increase at a concentration as low as 1 ng/ml and reached plateau at 2ng/mL (Figure 1B). Furthermore, Western blot analysis confirmed the induction of Id2 protein expression by IL-1β (Figure 1D). These results revealed that IL-1β stimulation induce Id2 gene expression in HASMC. Egr-1, a mediator of IL-1β effects, presented strong induction of mRNA expression levels in response to IL-1β stimulation of HASMC for 30 minutes and lasted for 1 hour (Figure 1A) while the Egr-1 protein increased and remained at high level even at 6 hours post-induction (Figure 1C), consistent with previous reports that indicate a pronounced stability of the Egr-1 protein[17]. This temporal pattern of Egr-1 gene expression suggested that Egr-1 may be a key mediator of IL-1β-induced Id2 gene expression in HASMC.

Fig. 1
IL-1β induces Id2 expression in HASMC. Quiescent human aortic smooth muscle cells (HASMC) were treated with 5 ng/ml of IL-1β for different time points (A) or for 8 hours with the indicated concentrations (B). Id2 and Egr-1 mRNA levels ...

3.2. Egr-1 mediates IL-2β dependent Id2 expression in HASMC

To define whether Egr-1 mediates the transcriptional regulation of Id2 gene expression, we used a well characterized adenoviral vector containing a constitutively active Egr-1 (referred to as Egr-1*) in which the corepressor (NAB) binding domain is mutated (I293F) rendering it NAB2-insensitive [14]. As shown in Figure 2A, Id2 mRNA levels in HASMC infected with AdEgr-1* were up-regulated in a concentration dependent manner and increased ~6 fold at 10 plaque-forming units(pfu)/cell.

Fig. 2
A, Overexpression of Egr-1 up-regulates Id2 gene expression in HASMC. Id2 mRNA levels upon infection with AdEgr-1* were determined by Northern blot (top panel), normalized by GAPDH levels are expressed in relative units to AdGFP treatment, set as 1 (lower ...

To further identify whether Egr-1 is the mediator of IL-1β-induced Id2 gene expression, HASMC infected with 5 pfu/ml of the adenovirus containing NAB2 (AdNAB2), a corepressor of Egr-1, were stimulated with IL-1β for 0, 4, 8, 16, and 24 hours respectively. The GFP adenovirus (AdGFP) was used as the negative control in this study. As shown in Figure 2B, overexpression of NAB2 abrogated IL-1β-induced Id2 expression in HASMC. These results strongly suggest that Egr-1 is a key mediator of IL-1β-induced Id2 expression in VSMC.

3.3. An Egr-1 binding element (site 1) mediates IL-1β-dependent Id2 expression

To explore the molecular mechanisms by which Egr-1 mediates the IL-1β-induced Id2 gene expression, the Id2 proximal promoter sequence was analyzed using TRANSFAC4.0[18]. Interestingly, we identified three putative Egr-1 binding sites within the proximal 2.1 kb of the Id2 promoter. To begin addressing the relevance of this finding, we first performed reporter assays to determine if Egr-1 could transactivate an artificial Egr-1 reporter construct in transient transfection experiments. Co-transfection of the wild-type reporter containing the three-tandem repeats of the putative Egr-1 binding site 1 (pEgr-1WT ×3- TKLuc) with a plasmid expressing the constitutive Egr-1* modestly but significantly increased luciferase activities when compared to the pcDNA3.1 control, whereas the mutant reporter (pEgr1Mut×3-TKLuc) failed to respond to Egr-1* (On line Supplement, Figure S1). Similarly, we generated reporter luciferase constructs containing the 2.1 kb proximal fragment of the Id2 promoter with the wild-type (pId2-Luc) or the three Egr-1 elements mutated (pId2Mut-Luc). As shown in Figure 3A, overexpression of wild type Egr-1 reproducibly and significantly transactivated wild-type Id2 promoter activity by 2-fold, whereas NAB2 abolished the Egr-1-dependent induction of the Id2 promoter activity. In addition, mutation of the three Egr-1 elements in the human Id2 promoter construct impaired both basal and Egr-1-induced transactivation.

Fig. 3Fig. 3Fig. 3
A, Id2 gene promoter activity is regulated by Egr-1. The pId2-Luc or pId2Mut -Luc reporter constructs were co-transfected with the wild-type Egr-1 or NAB2 expression plasmids as indicated in A7r5 cells. A GFP reporter plasmid was used as the control for ...

Consistent with those observations, electrophoretic mobility shift assays (EMSA) confirmed the ability of Egr-1 to bind all three putative Egr-1 elements in Id2 promoter and that this binding could be competed only by the Egr-1 consensus sequence (On line Supplement, Figure S2). Furthermore, we found that binding between endogenous levels of Egr-1 and the Egr-1 consensus element 1, identified as the physiologically relevant site by ChIP analysis (see below and figure 3C), could be enhanced after IL-1β stimulation. As shown in Figure 3B, nuclear extracts from HASMC treated with IL-1β for 2 hours showed enhanced complex formation with the probe corresponding to the Egr-1 element 1 (lanes 2 and 3). In addition, formation of this complex was abolished by adding 5-or 50-fold excess of cold probe (lanes 4 and 5) or the Egr-1 consensus sequence (lanes 8 and 9) but not by the probe containing the mutated (AT-rich) version of this Egr-1 site (lanes 6 and 7). The IL-1β induced binding was efficiently supershifted with an Egr-1 antibody (lane 11) whereas normal IgG had no effect on the complex (lane 10). These series of studies documented that Egr-1 specifically binds to the putative Egr-1 elements in the proximal Id2 promoter in vitro and that this is enhanced upon IL-1β treatment.

To further confirm the physiological relevance and functionality of Egr-1 through its putative binding sites in the Id2 promoter, chromatin domains at various locations in the Id2 promoter were scanned by ChIP analysis. As shown in Figure 3C, of the three putative Egr-1 sites in the Id2 promoter, Egr-1 only binds detectably the site located between nt −729 to nt −706, referred to as site 1, at baseline and that this Egr-1 binding was significantly stimulated by IL-1β treatment (~ 3 fold). No occupancy of any of the other two putative Egr-1 binding sites was observed by ChIP, even after IL-1β treatment. An isotypic IgG antibody was included as a negative immunoprecipitation control. Additional negative controls for the ChIP assay documented no binding of Egr-1 further upstream and downstream of the Egr-1 binding sites in the Id2 promoter. These data strongly suggest that in the context of chromatin, only the Egr-1 site 1 appears to be functional showing both basal binding and increased IL-1β stimulated binding, arguing for a functional role of this site in Egr-1-mediated IL-1β-dependent up-regulation of Id2 expression in VSMC.

4. Discussion

Given that IL-1β plays an important role in vascular lesion formation, we investigated the effect of IL-1β on Id2 gene expression in human aortic smooth muscle cells. In this present study, we documented for the first time that IL-1β induces Id2 gene expression in a time- and dose-dependent manner in VSMC. In addition, we identified Egr-1 as an essential factor that mediates both basal and IL-1β-induced Id2 transcription in VSMC through direct and specific interactions with the Id2 promoter region.

Id proteins are negative regulators of the basic helix-loop-helix (bHLH) transcription factors and they impair the binding of the bHLH to the E–box proteins. Previous studies have determined that the expression of various Id genes is down-regulated when cells terminally differentiate, and overexpression of some Ids impair differentiation and promote cell proliferation[19, 20]. To date, two mechanisms have been proposed to explain how Id proteins contribute to cell cycle entry. One mechanism suggests the down-regulation of cyclin-dependent kinase inhibitors at a transcriptional level, where Id proteins would interfere with bHLH-driven expression of p16Ink4a, p27Kip1, and p21Cip[2123]. Another proposed mechanism involves Id2 interaction with the tumor suppressor retinoblastoma protein (pRb). Id2 has been shown to bind the unphosphorylated pRb through interaction between the HLH region of Id2 and the pocket domain of pRB, resulting in the release of E2F[11]. In a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. Overexpression of Id2 resulted in a significant enhancement of VSMC growth via increased S-phase entry and downregulation of p21Cip1 expression32[8, 10], suggesting that Id2 may be a novel mediator of vascular lesion formation.

It has been well documented that IL-1β could promote intimal hyperplasia and lesion progression in atherosclerosis by several different mechanisms including activation of lymphocytes, induction of monocyte chemoattractant protein-1 expression, up-regulation of adhesion proteins in endothelial cells, and activation of VSMC[3]. Although the mitogenic effect of IL-1β on VSMC has been reported and attributed, at least in part, to the up-regulation of PDGF-A chain expression[24, 25], inhibition of expression of p21CIP1 and p27KIP1, and increase of pRb phosphorylation [26], the mechanism of IL-1β-induced VSMC activation has not been fully understood. Many growth factors and cytokines have been shown to induce expression of the pro-proliferative Id2 gene, including TNFα, BMP-2/4/7, TGFβ1, PDGF and IGF-I, through different signal pathways in different cell types[2730]. The effect of Id2 on the expression and function of growth factors like PDGF and how it is involved in the synergetic effect of IL-1β and PDGF in vascular lesion formation remain to be identified. In addition, we recently documented that anti-diabetic drugs, thiazolidinediones (TZD), inhibit Id2 gene expression in VSMC, suggesting that Id2 down-regulation may contribute to TZD-inhibited vascular lesion formation[31]. However, the transcriptional regulation of the Id2 gene in VSMC is poorly investigated. In the present study, we documented for the first time that IL-1β-induced Id2 expression in VSMC and that this is modulated by IL-1β-dependent changes in Egr-1 through an Egr-1 specific binding site in the Id2 promoter. Indeed, Id1 and Id3 expression increased under all-trans retinoic acid in normal human keratinocytes and enhanced binding of Egr-1 on the Id1 promoter was also observed[32]. Egr-1 also mediates Id1 expression in C2C12 muscle cells and Id3 expression in thymocytes[33, 34]. Taken together, these results reinforce the emerging notion that Egr-1 is an important transcriptional regulator of Id family members and establishes for the first time a direct mechanistic link between IL-1β, Egr-1 and Id2 expression and VSMC proliferation. Further investigation of the role of these interactions in vascular development, angiogenesis as well as in proliferative disorders and atherogenesis will provide new insights to understanding the essential role of these three factors in vascular proliferation.

Supplementary Material

01

Fig S1: Functional study of the putative Egr-1 element in the human Id2 promoter. Schematic diagram of pEgr-1WT×3–TKLuc (left top panel) and pEgr-1Mut×3–TKLuc (right top panel) reporter constructs. pEgr-1 WT×3 – TKLuc and pEgr-1Mut×3–TKLuc reporter constructs were cotransfected with an Egr-1* expression plasmid or the pcDNA3.1 control in A7r5 cells. A GFP reporter plasmid was used as the control for transfection efficiency. The luciferase activities (mean± SD, n = 6, *p<0.05) normalized by the GFP activity are expressed relative to pEgr-1WT×3-TKLuc plus pcDNA3.1 (left bottom panel) or pEgr-1Mut×3–TKLuc plus pcDNA3.1 (right bottom panel).

Fig S2: Three 30 bp probes corresponding to the three putative Egr-1 binding sites from the human Id2 promoter were radiolabeled with 32P and incubated with nuclear extracts (N.E.). For the competition assays 50-fold excess of the indicated cold probes were used.

Acknowledgements

This work was partially supported by National Natural Science Foundation of China (Project 30400223 to X.Z.), and by the NIH grant (HL68878 to Y.E.C.). M.B. was supported by a supplement to HL68878 (Y.E.C.). Y.L. was supported by a postdoctoral fellowship from the American Heart Association Southeast Affiliate 0225323B and M.T.G.B. was supported by the AHA Beginning-Grant-in-Aid 0465202B.

Footnotes

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