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Universitätskliniken des Saarlandes, Medizinische Klinik und Poliklinik, Innere Medizin III, 66424 Homburg/Saar, Germany
1Correspondence: Klinik und Poliklinik Innere Medizin III;, Universität des Saarlandes, 66424 Homburg, Germany. E-mail: nickenig{at}med-in.uni-sb.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: reactive oxygen species • VSMC • proliferation • gene expression
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. ROS exert direct cellular damage and mitogenicity, serve as intracellular second messengers, and scavenge vasoprotective nitric oxide (1
2
3
4
5)
. Among other factors, the induction of cellular growth in tumor cells and vascular smooth muscle cells (VSMC) by ROS is of fundamental relevance. O2. induces a wide array of second messengers typical of mitogens (3
, 6
, 7)
. In contrast, data concerning the role of OH. in cell proliferation are conflicting. Hypertrophic as well as apoptotic effects have been ascribed to OH., for example, in VSMC (8
9
10)
. Proliferation, growth arrest, and apoptosis are key steps in various states of atherogenesis and tumor progression; therefore, it is crucial to dissect not only the potentially differential influence of various ROS on cell growth but to clarify the molecular events that govern ROS-induced regulation of proliferation. We investigated potential differential effects of radicals on VSMC growth and monitored its influence on genes such as cyclin-dependent kinase inhibitors (11
12
13)
, which are involved in control of cell proliferation. To identify currently unknown gene expression alterations devoted to VSMC growth, we chose an unbiased gene hunting approach, the differential display technique. The latter enabled identification of two redox-sensitive genes engaged in proliferation pathways. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For cell counting, cells were treated with ROS for 12 h, removed from the culture dish by addition of trypsin, and pelleted. The pellet was resuspended in 1 mL DMEM and cells were counted in a Neubauer chamber.
Apoptosis
The rate of apoptosis in ROS-treated cells was assessed using the Cell Death Detection ELISAPLUS System (Roche Molecular Biochemicals). The test principle is based on determination of the amount of nucleosomes generated during the apoptotic fragmentation of cellular DNA. Cells were scraped and collected by centrifugation for 5 min at 1500 rpm (Heraeus Megafuge 1.0) and washed in 1 mL DMEM. The pellet was resuspended in 0.5 mL incubation buffer and left at 4°C for 30 min. After centrifugation for 10 min at 15,000 rpm and 4°C in a microcentrifuge, 200 µL of supernatant was diluted in 1.8 mL of incubation buffer and 100 µL of each sample was incubated in anti-histone-coated microtiter plate wells for 90 min, the wells were washed three times with incubation buffer and 100 µL of anti-DNA peroxidase-linked antibody was added, followed by further incubation for 90 min. After three washing steps with incubation buffer, 100 µL of ABTS® substrate solution for the peroxidase was added; after 10–20 min, the rate of apoptosis was determined by photometric measurement at 492 nm.
RNA isolation
Total RNA from ROS-treated and control cells was isolated using PEQGold RNAPure (PeqLab, Erlangen, Germany) according to the manufacturer’s protocol. To eliminate false positives generated from genomic DNA, RNA was treated with 10 U RNase-free DNaseI (Roche Molecular Biochemicals) for 30 min at 37°C and extracted with phenol/chloroform.
Differential display
Differential display of mRNA was carried out using the RNA image mRNA Differential Display System (GenHunter Corporation, Nashville). Two micrograms of purified total RNA was reverse transcribed using one of the three provided H-T11-M-Primers (M: A, T, or G) and cDNA representing 200 ng of RNA was submitted to the Differential Display PCR using the respective H-T11-M primer and one of eight arbitrary primers provided, 1 U of AmpliTaq DNA polymerase (Perkin-Elmer Corp., Ueberlingen, Germany) and 2 µCi 
-[33P]dATP (ICN Biomedicals, Eschwege, Germany) per reaction. PCR was carried out using 40 cycles of 94°C for 30 s, 42°C for 2 min, 72°C for 30 s, followed by a final extension step at 72°C for 5 min. PCR products were resolved on a 6% denaturing polyacrylamide gel in 1x TBE, followed by autoradiography. Reproducibly differentially expressed bands were cut out of the gel and the cDNA was eluted by boiling for 15 min. The gel debris was pelleted by centrifugation and the supernatant was transferred to a fresh microcentrifuge tube. The DNA was precipitated by addition of 100% ethanol, incubation on dry ice for 30 min, and centrifugation at 4°C in an Eppendorf microcentrifuge. The pellet was washed with 85% v/v ethanol, dried, and resuspended in ddH2O. Reamplification of the purified cDNA was performed using the same primer set and PCR conditions used for the differential display PCR. Reamplified cDNAs were either directly sequenced using the respective arbitrary primer or after cloning into the pCR2.2 vector (Invitrogen BV, Groningen, Netherlands) via the TA cloning method.
Northern blot and PCR
Fifteen micrograms of total RNA were electrophoresed on an 1.2% agarose/0.67% formaldehyde gel. After electrophoresis, RNA was transferred on Hybond N nylon membrane (Amersham Pharmacia Biotech, Freiburg, Germany). PCR fragments of 897 bp (ID3) and 1456 bp gut-enriched Kruppel-like factor (GKLF) were radiolabeled with -[32P]-dCTP (ICN Biomedicals, Eschwege, Germany) using the Prime-It® II Random Primer Labeling Kit (Stratagene, La Jolla, CA). Membranes were prehybridized in a solution containing 50% formamide, 6x SSC (standard saline citrate), 0.5% SDS, 5x Denhard’s solution, and 100 µg/mL salmon testes DNA (Sigma-Aldrich GmbH, Taufkirchen, Germany) for at least 30 min at 42°C. Hybridization was carried out in hybridization solution containing 50% formamide, 6x SSC, 0.5% SDS, 100 µg/mL salmon testes DNA, and the denatured radiolabeled probe in an overnight incubation at 42°C. Membranes were washed twice with 2x SSC and two to four times with 2x SSC/0.1% SDS at 50–65°C, sealed in a plastic bag, and submitted to autoradiography.
For semiquantitative RT-PCR, 2 µg of total RNA from ROS-treated and control cells was reverse transcribed using 100 pmol of p(dN6)-oligonucleotide-Primer (Roche Molecular Biochemicals) and 200 U of MMLV Reverse Transcriptase (Gibco BRL, Karlsruhe, Germany) in the supplied buffer for reverse transcription and 10 U RNasin (Promega, Heidelberg, Germany). One microliter of each reaction was used for PCR with 50 pmol each of the respective primers for amplification of Id3 (sense: 5'-CGACATGAACCACTGCTACTC-3', antisense: 5'-GGTCAGTGGCAAAAACTCCTC-3') or GKLF (sense: 5'-CAACGACCTCCTGGACCTAGA-3', antisense: 5'-TTCCTCGGGACTCAGTGTAGG-3') in red Taq PCR buffer, dNTP mix and 1.25 U of red Taq DNA polymerase (Sigma-Aldrich GmbH). PCR conditions were one 5 min cycle of 95°C, followed by 25 cycles (GAPDH: 23 cycles) of 94°C for 30 s, 57°C (Id3), or 60°C (GAPDH) for 45 s, 72°C for 45 s, and a final extension for 10 min at 72°C. 20–40 µL of each PCR reaction were analyzed on a 1% agarose-TAE gel, visualized by ethidium bromide-staining, and optical densities of the cDNA bands were quantified. The same cDNA samples were used for the amplification of a 452 nt fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5'-ACCACAGTCCATGCCATCAC-3', antisense: 5'-TCCACCACCCTGTTGCTGTA-3') to confirm that equal amounts of RNA were reverse transcribed.
Western blot
ROS-treated and control cells were washed twice with ice-cold PBS, scraped in 1 mL of ice-cold lysis buffer (100 mM TRIS pH6.8, 4% SDS, 20% glycerol, 0.1 mM PMSF, 1 µg/mL leupeptin, 1 µg/mL aprotinin), heated to 95°C for 5 min, and stored at -20°C until use. Aliquots (40 µg) of the cellular lysate were electrophoresed through an 0.1% SDS/10% polyacrylamide gel. Proteins were blotted to nitrocellulose membranes in a semidry blotting chamber (Pharmacia Biotech, Uppsala, Sweden). Blot membranes were stained with Ponceau red to verify appropriate protein transfer and equal loading for each lane. Immunoblotting was performed overnight at 4°C for all antibodies used. Antibody dilutions were p53 (Pab 240 mouse monoclonal IgG, sc-99, Santa Cruz Biotechnology Inc., Santa Cruz, CA), 1:300; p27 (F-8 mouse monoclonal IgG, sc-1641, Santa Cruz), 1:300; p21 (mouse mixed monoclonal IgG, #05–345, Upstate Biotechnology, Biomol, Hamburg, Germany), 1:600; Rb (M-153 rabbit polyclonal, sc-7905, Santa Cruz), 1:100; Id3 (C-20 rabbit polyclonal, sc-490, Santa Cruz), 1:100. Immunodetection was accomplished using the appropriate secondary antibody for 1 h at room temperature (1:20,000 dilution, Sigma Chemical, Deisenhofen, Germany) and the enhanced chemiluminescence kit (Amersham, Braunschweig, Germany). Autoradiography was performed at room temperature.
Transfection
Full-length cDNAs of Id3 were generated by PCR amplification of reverse transcribed RNA derived from VSMC (see above). Primers were for Id3: sense: 5'-CTCCAACCTCCAACATGAAGG-3', antisense: 5'-GTTAAAAATGGTTTATTATGCAAAATGTT-3'. PCR products were checked on a 1% TAE agarose gel and cloned into the pCR2.2 vector (Invitrogen BV, Groningen, Netherlands) via TA cloning. Orientation and validity of the insert was determined by automated sequencing, and Id3 sense and antisense constructs for electroporation were generated by cloning HindIII/NotI fragments into the pcDNA3 vector. For electroporation, VSMC grown at a confluent monolayer were removed from the culture dish by addition of trypsin and pelleted. The pellet was resuspended in 200 µL of Optimem I (Gibco BRL, Karlsruhe, Germany) and cells were counted. 106 cells per sample were incubated with 20 µg of the respective DNA in precooled cuvettes (Promega, Heidelberg. Germany) for 30 min on ice. After warming the cuvette to 37°C for 30 s in a water bath, electroporation was performed for 16 ms at 0.3 kV and 500 µF. After an additional incubation for 30 min at room temperature, cells were plated on the appropriate culture dishes or microtiter plates.
Luciferase assays
Binding of GKLF to putative binding sites within the Id3 promotor was investigated using a promotor-reporter construct with the luciferase gene driven by the Id3 promotor (luciferase assay system, Promega GmbH, Mannheim Germany). VSMC were cotransfected with GKLF, pGL2-Id3 (promotor-reporter construct) +pcDNA3, pGL2+pcDNA3, pGL2-Id3+GKLF, and pcDNA3 by electroporation. Afterward cells were plated on 10 cm plates and incubated for 24 h at 37°C until cell density was sufficient for the assessment of luciferase activity. After two washes with PBS, cells were lysed with 900 µL of the provided lysis buffer per plate, scraped, and transferred to a microcentrifuge tube. After vortexing for 10 s, the debris was pelleted by centrifugation for 2 min at 16,000 g and the supernatant was transferred to a new tube. For measurement of luciferase activity, an automatic luminometer (MicroLumat plus LB 96 V) was used. 20 µL of cellular lysate were incubated with 100 µL of luciferase-Assay-Reagent by automatic injection, luminescence was measured over a time course of 30 s
Electrophoretic mobility shift assay
Oligonucleotides containing the known binding site for GKLF (5'-G/AG/AGGC/TGC/T-3') 5'-ATGCAGGAGAAAGAAGGGCGTAGTATCTACTAG-3') (K-I) and oligonucleotide derived from the human Id3 promotor region in which a GKLF binding site putatively is localized, KII: 5'-ACTCCCCAGCATGAAGGCGCTGAGCCCGGTGCG-3', (Id3 promotor sequence 730bp-766bp) were purchased as single-stranded oligonucleotides (MWG Biotech, Ebersberg, Gemany) and annealed. The GKLF oligonucleotide was end-labeled with 
[32P]-ATP.
Nuclear extracts (2 µg) from VSMC were incubated in 10 µL binding buffer (20 mM HEPES, pH7.9 with KOH, 20% v/v glycerol, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, 1 mM DTT) and 2 µL EMSA buffer (150 mM HEPES, pH7.6 with KOH, 100 mM KCL, 2.5 mM DTT, 2 mg/mL bovine serum albumin, 20 mM MgCl2) containing 0.1 mM ZnCl2 and 1 µg POLY(dA-dT)·POLY(dA-dT) (Amersham Pharmacia Biotech) for 10 min at room temperature before addition of 50 fmol of 32P-end-labeled, double-stranded oligonucleotide specific for binding of GKLF. After incubation for 15 min, proteins were resolved on a nondenaturing 5% polyacrylamide gel. The gel was submitted to autoradiography. In experiments that contained competitor DNA, a 2- to 50-fold molar excess of unlabeled oligonucleotide over the labeled probe was included.
Vascular injury model
Carotid artery injury was performed in adult male C57Bl/6 mice. Using a dissecting microscope (MZ6, Leica, Heerbrugg, Switzerland), bifurcation of the left carotid artery was exposed via a midline incision of the ventral side of the neck. Two ligatures were placed proximally and distally around the external carotid artery. The distal ligature was tied off. After temporary occlusion of the internal and common carotid artery with ligatures, a transverse arteriotomy was performed between the ligatures of the external carotid artery to introduce a curved flexible wire (0.13 mm in diameter). The wire was passed along the common carotid artery in a rotating manner three times. After removal of the wire, the proximal ligature of the external carotid artery was tied off. Normal blood flow was reassured and the skin was closed with single sutures using 6/0 silk. Animals were allowed to recover and carotid arteries were harvested at various times.
Perfusion-fixed carotid arteries were embedded in Tissue Tek O.C.T. embedding medium (Miles Inc., Niles, MI), snap frozen, and stored at -80°C. Samples were sectioned on a Leica cryostat and placed on poly-L-lysine (Sigma) coated slides for immunohistochemical analysis.
Cryosections were assessed for endothelial cell markers (von Willebrand factor, Dako), MC (alpha smooth muscle actin, Sigma), or the Id3 antigen with a indirect immunoenzymatic method. Tissue cryosections were postfixed in 4% formaldehyde for 2 min. Slides were preincubated with 0.5% Igpal and 5% normal goat serum (Sigma) for 30 min each. The primary antibody (Id3 C20, Santa Cruz) was applied for 2–3 h at room temperature or at 4°C overnight, followed by application of the appropriate horseradish peroxidase-conjugated secondary antibody (Sigma) for 30 min. Id3 expression was visualized with a DAB chromogen substrate (Sigma). Sections were rinsed and mounted with Kaiser’s glycerin (Merck) for light microscopic analysis (Nikon E600).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A shows that X/XO caused a significant increase in DNA synthesis and cell proliferation whereas H2O2-Fe exerted a growth-arresting and proliferation-inhibiting effect. X/XO-induced mitogenicity (maximal at 100 µM) and H2O2-Fe-caused growth arrest (maximal at 100 µM) were concentration dependent (Fig. 1B
). In the absence of the Fenton reagent, H2O2 revealed no influence on cellular growth. Apoptosis in VSMC at concentrations of 100 µM X/XO or H2O2-Fe was excluded (Fig. 1C
).
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Gene expression analysis during oxidative stress
To identify concomitant changes in gene expression patterns, differential display was performed in VSMC incubated with either 100 µM X/XO or H2O2-Fe for 0, 0.5, 2, and 4 h. Serial analysis led to identification of two differentially regulated genes (Fig. 2
A). Reference methods such as Northern blotting and semiquantitative PCR demonstrated that the transcription factor GKLF was induced by H2O2-Fe and not by X/XO. Densitometric analysis of five separate experiments revealed that H2O2-Fe increased GKLF mRNA expression after a 2 h incubation to 177 ± 14% of control levels, P < 0.05. X/XO had no significant effect on GKLF expression (95±5%, 1 h incubation, n=4, p=ns), The dominant negative helix-loop-helix protein Id3 was induced by X/XO (maximal after 30 min to 170±6%, n=5, P<0.05) and decreased by H2O2-Fe (maximal after 4 h to 31±4, n=5, P<0.05) (Fig. 2B
). Western blot analysis confirmed that Id3 was up-regulated by X/XO and depressed by H2O2-Fe (Fig. 2C
).
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Influence of GKLF and Id3 on cell growth
To clarify if GKLF and Id3 mediate redox-sensitive growth processes, VSMC were transfected with sense/antisense GKLF and Id3 cDNAs were inserted in the expression vector pcDNA3. Overexpression or inhibition was confirmed by Western analysis (Id3 sense 204±12% of control, Id3 antisense 31±5% of control) (Fig. 3
A). VSMC were stimulated with 10% fetal calf serum (FCS), 100 µM X/XO, 100 µM H2O2-Fe, or H2O2-Fe and FCS. Control experiments were performed with an insertless vector. Overexpression of sense GKLF and antisense Id3 abrogated growth induction by FCS or X/XO (Fig. 3B
). Overexpression of antisense GKLF or sense Id3 increased X/XO-induced cell growth and diminished H2O2-Fe-caused growth arrest (Fig. 3B
). These findings demonstrate that induction of GKLF and depression of Id3 are decisively involved in H2O2-Fe-induced growth arrest, whereas induction of Id3 governs X/XO-caused VSMC proliferation.
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Effect of ROS, GKLF, and Id3 on p21WAF1/Cip1, p27KIP1, p53, and Rb
Cyclin-dependent kinase inhibitors are essential modulators of cellular proliferation (11
12
13)
. Therefore, VSMC were incubated for the indicated periods with X/XO or H2O2-Fe and protein expression of p21WAF1/Cip1, p27KIP1, and p53 was assessed by Western blotting. X/XO down-regulated expression of p21WAF1/Cip1 (23±7% of control level, n=3, P<0.05, 24 h incubation) and p53 (31±7% of control level, n=3, P<0.05, 24 h incubation), whereas H2O2-Fe led to the up-regulation of p21WAF1/Cip1 (421±123% of control level, n=3, P<0.05, 12 h incubation) and p53 (264±71% of control level, n=3, P<0.05, 24 h incubation). p27Kip1 expression was depressed by X/XO (75±9% of control level, n=3, P<0.05, 24 h incubation) and enhanced by H2O2-Fe (291±81% of control level, n=3, P<0.05, 24 h incubation). Hyperphosphorylation of the retinoblastoma gene product Rb is closely involved in the execution of mitogenic signals. Rb hyperphosphorylation was propagated by X/XO, whereas FCS-induced Rb hyperphosphorylation was diminished by H2O2-Fe (Fig. 4
A). Consistently, overexpression of sense GKLF and antisense Id3 caused an enhanced expression of p21WAF1/Cip1, p27Kip1, and p53 and hypophosphorylation of Rb (Fig. 4B
), suggesting that the cyclin-kinase inhibitors p21WAF1/Cip1 and p27Kip1, the tumor suppressor gene p53, and hypophosphorylation of Rb are induced by H2O2-Fe via induction of GKLF and depression of Id3. In contrast, overexpression of sense Id3 and antisense GKLF induces hyperphosphorylation of Rb. Therefore, GKLF as well as Id3 act upstream of these established growth-regulating genes.
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Interaction of GKLF and Id3
GKLF is up-regulated by OH. within 30 min; Id3 is down-regulated after 2–4 h. The Id3 promotor contains GKLF binding sites. To test whether GKLF regulation is a prerequisite of Id3 modulation, we incubated nuclear proteins isolated from VSMC, which were stimulated with 100 µM H2O2-Fe for 0–4 h with radioactively labeled oligonucleotides representing the GKLF binding site. Figure 5
A (left panel) shows the autoradiography of a gel shift experiment revealing that GKLF binding to its consensus sequence is profoundly induced by H2O2-Fe. Figure 5A
(right panel) shows that unlabeled oligonucleotides derived from regions of the Id3 promotor that contain a putative GKLF binding site inhibit the protein–DNA interaction demonstrating the specificity of the reaction. Therefore, GKLF binds to the Id3 promotor and this binding is increased by stimulation with H2O2-Fe. To clarify the functional relevance, VSMC were cotransfected with expression vectors harboring either the GKLF cDNA or/and a luciferase reporter gene fused to the Id3 promotor. Figure 5B
summarizes the data of four separate experiments. Transcription of the luciferase reporter gene is low without inserted promotor (pGL2). Activity is greatly enhanced on fusion to the Id3 promotor (Id3-pGL2). Cotransfection with GKLF as well as stimulation of cells with 100 µM H2O2-Fe for 2 h reduces significantly the luciferase activity, demonstrating that GKLF represses Id3 promotor activity and thereby Id3 gene transcription rate.
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Second messengers involved in GKLF and Id3 induction
Cells were incubated with 100 µM H2O2-Fe or 100 µM X/XO for 30 min with or without the following pharmacological inhibitors: 20 µmol/L PD98059 (p42/44 MAP kinase inhibitor), 1 µmol/L SB203580 (p38 MAP kinase inhibitor), 10 µmol/L genistein (tyrosin kinase inhibitor), 10 µmol/L wortmannin (PI-3 kinase inhibitor), 10 µmol/L N
-nitro-L-arginin (nitric oxide inhibitor), 20 µmol/L Bis-(2-amino-5-methylphenoxy)ethane-N, N, N‘, N'-tetraacetic acid tetraacetoxymethyl ester (MAPTAM, calcium chelator), 20 mmol/L mannitol (hydroxyl scavenger), 10 µg/mL cycloheximide (protein synthesis inhibitor), 200 U/mL PEG-catalase (CAT, H2O2 scavenger), and 100 U/mL PEG-superoxide dismutase (SOD, O2. scavenger), followed by RNA isolation and GKLF mRNA quantification by Northern blotting and PCR. The data show that GKLF induction by H2O2-Fe is dependent on OH. release, intracellular calcium, p38 MAP kinase, and protein synthesis (Fig. 6
A), whereas Id3 induction by X/XO is dependent on superoxide release, p38, as well as p42/44 MAP kinase and intracellular calcium.
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Effect of angiotensin II on Id3 and GKLF
Angiotensin II is known to release intracellular O2. and hydrogen peroxide upon activation of AT1 receptors. To test whether intracellular release of O2. and hydrogen peroxide could influence Id3 expression, cells were stimulated with 100 nmol/L angiotensin II for 0–2 h. Id3 mRNA was quantified by Northern analysis. Figure 6B
demonstrates that angiotensin II up-regulates Id3 expression within 1 h. This was completely inhibited in the presence of 10 µmol/L of the flavoprotein inhibitor diphenylene iodonium, which omits intracellular free radical release by angiotensin II. Superoxide dismutase, which specifically scavenges O2. but not catalase, a hydrogen peroxide scavenger, inhibited the effect of angiotensin II. GKLF expression was not influenced by angiotensin II (data not shown). Thus, angiotensin II, which acts as a VSMC mitogen via a membrane-bound receptor, modulates Id3 gene expression via intracellular superoxide release.
In vivo model
To extend our in vitro findings, we induced a vascular injury in arteria carotis of mice resulting in lesions characterized by neointima formation due to VSMC proliferation. The vessels were harvested and immunohistochemistry was performed in lesion segments. Figure 7
shows visualization of Id3 protein and VSMC-specific alpha-smooth muscle actin protein. Id3 is predominately expressed in VSMC of the media and the neointima but in the endothelium. Of note, Id3 expression is profoundly enhanced in the neointima that contains proliferating VSMC in comparison to media composed of mainly quiescent VSMC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Despite numerous experimental studies and interventional trials, the definitive molecular actions of ROS are poorly defined. Proliferation, growth arrest, and apoptosis are key events in atherogenesis and are observed in endothelial cells, macrophages, and VSMC within the vessel wall (11)
. ROS act as extra- and intracellular signaling molecules involved in cellular growth, apoptosis, and survival (9
, 10
, 12
, 13)
. Similar to growth factors and hormones, ROS interfere with various transduction pathways such as calcium, protein kinases, MAP kinase, and proto-oncogenes (3
, 14
15
16
17
18)
. The response elicited by ROS intermediaries is in part determined by the intracellular targets and is dependent on the species of oxidants produced (6
, 9
, 18
, 19)
.
Our data indicate that xanthin/xanthin oxidase and hydrogen peroxide/Fe exert differential effects on growth of VSMC. Concomitant application of specific radical scavengers such as mannitol, catalase and superoxide dismutase suggest that the effects of xanthin/xanthin oxidase are mediated through release of superoxide, whereas hydroxyl radicals seem to transfer the effects of hydrogen peroxide/Fe. It is concluded based on these observations that superoxide induces VSMC proliferation and that hydroxyl radicals cause growth arrest in the absence of detectable apoptosis. It is well established that superoxide induces second messenger pathways typical of mitogens, such as the mitogen-activated kinase family (14
15
16
17
18)
. However, the cascade of participating genes is less defined.
According to the data presented, induction of Id3 is a prerequisite of superoxide-caused proliferation. Inhibition of Id3 by transfection of antisense constructs inhibits superoxide and serum-induced growth. Moreover, superoxide-associated mitogenesis is enhanced by transfection of sense Id3 cDNA. The Id proteins 1–4 antagonize the function of DNA binding basic helix-loop-helix transcription factors such as E2A. The latter defines cellular differentiation and increased proliferation (20)
. Id3 has been implicated in the apoptosis of fibroblasts (21)
. However, the redox-sensitive properties of Id3 and its decisive role in regulation of VSMC growth have not been elucidated. Id3 expression is increased via receptor-mediated processes as exemplified with angiotensin II. This peptide propagates intracellular radical release and causes proliferation of VSMC (22
, 23)
. The fact that angiotensin II causes Id3 overexpression via radical release supports the concept of Id3-triggered growth events in VSMC. The second gene identified via differential display analysis, GKLF, acts in an opposite manner. GKLF expression is not altered by superoxide. Nevertheless, selective induction of GKLF abrogates completely superoxide- and serum-induced proliferation, whereas inhibition of GKLF enhances the mitogenic effects of superoxide.
GKLF has been shown to be expressed in epithelial cells of the gastrointestinal tract, skin, thymus, and endothelial cells (24
25
26
27
28)
. GKLF functions as transcription factor that interacts with a GC-rich consensus site residing in the promotor of various genes such as cytochrome P450IA1 and p21WAF1/Cip1 (29
, 30)
. In fibroblasts, induction of GKLF is associated with growth arrest potentially through interaction with p53 and p21WAF1/Cip1 (30)
.
The data demonstrate a differential impact of superoxide and hydroxyl radicals on VSMC growth and connect these effects to the novel redox-sensitive target genes, GKLF and Id3. GKLF expression is increased by hydroxyl radicals. Functional data reveal that the hydroxyl-caused growth arrest is mediated by the induction of GKLF. Transfection of GKLF antisense vectors completely abolishes the growth-arresting potential of hydroxyl radicals.
GKLF or inhibition of Id3 diminishes serum-elicited proliferation, suggesting that serum-associated mitogenesis is mediated by ROS or that GKLF and Id3 are generally involved in growth regulation pathways in VSMC.
It is not known by which signal transduction cascades GKLF and Id3 are activated or inhibited. The data reported here indicate that ROS play a pivotal role in the expression regulation of these genes. However, it cannot be excluded that other signaling pathways may also be involved. Our data suggest that hydroxyl radicals induce GKLF via the p38 subunit of MAP, which is a principal intracellular target of reactive oxygen species (14
15
16
17
18)
. Furthermore, intracellular calcium seems to participate in the hydroxyl-induced GKLF regulation. This agrees with previous reports of calcium-releasing properties of free radicals (31)
. Despite the rapid regulation of GKLF within 30 min, the process is protein synthesis dependent, suggesting that additional, so far unknown transcribed/translated factors mediate the hydroxyl effect on GKLF.
The time course of events of GKLF and Id3 regulation and the fact that the Id3 promotor contains putative GKLF binding sites led us to the hypothesis that GKLF my interact with the Id3 promotor and thereby influence Id3 gene transcription. According to the results presented, GKLF binds to its consensus sequence in the Id3 promotor. The cotransfection reporter assays clearly show that GKLF reduces Id3 promotor activity revealing a so far unknown interaction.
GKLF as well as Id3 have been associated with cell cycle factors such as p53 and p21WAF1/Cip1 (22)
. Our results show that superoxide, Id3, and inhibition of GKLF down-regulate the expression of p53 and p21WAF1/Cip1 and, to a lesser extent, the expression of p27Kip1. In contrast, hydroxyl radicals, GKLF overexpression, and inhibition of Id3 induce p53, p21WAF1/Cip1, and p27Kip1. Consequently, hyperphosphorylation of Rb, which is a well-established measure for cell cycle progress, is either propagated or abolished. Therefore, GKLF and Id3 interfere effectively with cell cycle-associated factors. This partly clarifies the intracellular targets of GKLF and Id3 with respect to their growth-regulating properties.
Pathological growth of vascular smooth muscle cells occurs especially during atherosclerotic disease and restenosis after angioplasty (32)
. To validate in vitro findings and at least speculate about a transfer of the cell culture results to atherosclerosis in humans, it is essential to prove the concept in an in vivo setting. Therefore, we applied a carotid injury model in mice. The induced lesion, the neointima, is predominately composed of proliferating vascular smooth muscle cells. The histological analysis showed that Id3 is expressed in vascular smooth muscle cells in vivo. Id3 expression is enhanced in neointimal, proliferating cells. These data may serve as a first proof of the concept. Of course, these data do not ultimately prove a causal relationship between Id3 overexpression and neointima formation, which would have to be tested in an in vivo gene transfer experiment.
GKLF and Id3 are of central relevance for ROS-induced growth regulation in VSMC via interaction with cyclin-dependent kinase inhibitors, p53, and Rb. It is speculated that these findings reveal general mechanisms of growth control that could lead to new therapeutic strategies for the treatment of atherosclerosis, coronary restenosis, and cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication October 25, 2001.
Revision received February 18, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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