© 1996 American Heart Association, Inc.
Articles |
Heparin-Binding Epidermal Growth Factor-Like Growth Factor Regulates Fibroblast Growth Factor-2 Expression in Aortic Smooth Muscle Cells
the Department of Molecular Biology (K.A.P., G.F.A., D.K.W.H., S.-L.Y.F., J.A.W.), Holland Laboratory, American Red Cross, Rockville, Md, and the Department of Biochemistry and Molecular Biology (J.A.W.), George Washington University Medical Center, Washington, DC.
Correspondence to Dr Jeffrey A. Winkles, Department of Molecular Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail winkles@hlsun.red-cross.org.
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Abstract |
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Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is a vascular smooth muscle cell (SMC) mitogen and chemotactic factor that is expressed by endothelial cells, SMCs, monocytes/macrophages, and T lymphocytes. Both the membrane-anchored HB-EGF precursor and the secreted mature HB-EGF protein are biologically active; thus, HB-EGF may stimulate SMC growth via autocrine, paracrine, and juxtacrine mechanisms. In the present study, we report that HB-EGF treatment of serum-starved rat aortic SMCs can induce fibroblast growth factor (FGF)-2 (basic FGF) gene expression but not FGF-1 (acidic FGF) gene expression. Increased FGF-2 mRNA expression is first detectable at 1 hour after HB-EGF addition, and maximal FGF-2 mRNA levels, corresponding to an
46-fold level of induction, are present at 4 hours. The effect of HB-EGF on FGF-2 mRNA levels appears to be mediated primarily by a transcriptional mechanism and requires de novo synthesized proteins. HB-EGF induction of FGF-2 mRNA levels can be inhibited by treating cells with the anti-inflammatory glucocorticoid dexamethasone or the glycosaminoglycan heparin. Finally, Western blot analyses indicate that HB-EGF–treated SMCs also produce an increased amount of FGF-2 protein. These results indicate that HB-EGF expressed at sites of vascular injury or inflammation in vivo may upregulate FGF-2 production by SMCs.
Key Words: heparin-binding epidermal growth factor–like growth factor • smooth muscle cell • fibroblast growth factor • dexamethasone • heparin
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Introduction |
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Heparin-binding epidermal growth factor–like growth factor was initially identified as an
22-kD polypeptide mitogen secreted by human macrophages1 and was subsequently purified to homogeneity from U-937 cell–conditioned medium.2 Sequence analysis of human HB-EGF cDNA clones indicated that HB-EGF was (1) a member of the EGF family of structurally related proteins and (2) derived from a membrane-bound precursor.2 This 208-residue precursor molecule consisted of signal peptide, propeptide, mature protein, transmembrane, and cytoplasmic domains.2 3 4 Recent studies have demonstrated that membrane-anchored HB-EGF is the receptor for diphtheria toxin5 6 and can function in a juxtacrine manner to stimulate cell growth.7 Mature secreted HB-EGF, which is generally 75 to 86 amino acids in length, contains an amino-terminal heparin-binding region8 9 and a carboxy-terminal EGF-like region.2 It is a mitogen for various cell types, including murine BALB/c 3T3 cells,1 2 4 rat mesangial cells,10 and human keratinocytes.11 These effects are mediated via binding to and activation of the EGF receptor tyrosine kinase.2 3 12 13 The interaction of HB-EGF with cell surface HSPG is required for optimal binding to the high-affinity EGF receptor.8 9 12 13 14 HB-EGF has several biological properties indicating that it may play a role in the excessive SMC accumulation that occurs during atherogenesis and in response to arterial injury.15 16 17 First, HB-EGF is expressed by endothelial cells,18 19 SMCs,20 21 22 23 monocytes/macrophages,1 2 24 25 and T lymphocytes.26 27 Second, it has been reported that HB-EGF is a mitogen1 2 4 13 23 27 and chemotactic factor4 14 for vascular SMCs. Third, increased levels of both HB-EGF and EGF receptor protein are present in human atherosclerotic plaques compared with nonatherosclerotic arteries.28 In plaques, HB-EGF is expressed primarily by SMCs and monocyte-derived macrophages, whereas EGF receptor expression is restricted to SMCs. Fourth, HB-EGF transcripts are expressed in rat arteries,23 and after balloon injury, there is an increase in the number of EGF (and thus also HB-EGF) binding sites on the neointimal SMC surface.29 Furthermore, local or systemic delivery of an EGF receptor–targeted cytotoxin after balloon injury inhibits SMC proliferation and neointimal thickening.29 Taken together, these results indicate that HB-EGF, acting via autocrine, paracrine, and/or juxtacrine mechanisms, may be an important mediator of SMC proliferation and migration in vivo.
In consideration of the potential role of HB-EGF in vascular SMC growth control, we investigated whether HB-EGF could regulate FGF-1 (acidic FGF) or FGF-2 (basic FGF) expression. FGF-1 and FGF-2, two members of the FGF family of structurally related heparin-binding proteins,30 31 are angiogenic factors32 33 34 and potent mitogens for many cell types, including vascular SMCs.32 33 35 36 37 There is evidence that these two proteins may be involved in atherogenesis38 39 40 as well as postangioplasty restenosis.41 42 Since the overexpression of FGF-1 or FGF-2 in transfected cell lines can result in autocrine growth stimulation,43 44 45 46 cellular production of these proteins in vivo must be tightly controlled. Previous studies have shown that several different vasoactive hormones,47 48 49 50 cytokines,51 and polypeptide growth factors52 53 54 55 56 can elevate FGF-1 and/or FGF-2 production by SMCs. In the present study, we demonstrate that HB-EGF–treated rat aortic SMCs express increased levels of FGF-2 mRNA and protein. In addition, HB-EGF induction of FGF-2 mRNA expression can be inhibited by treating cells with dexamethasone or heparin. These results, in combination with previous studies demonstrating HB-EGF autoinduction,22 FGF-2 autoinduction,55 and FGF-2 induction of HB-EGF expression22 in vascular SMCs, indicate that both autoregulatory and cross-regulatory mechanisms may control HB-EGF and FGF-2 levels within the vessel wall.
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Materials and Methods |
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Cell Culture
Adult rat (Wistar-Kyoto) thoracic aorta SMCs were kindly provided by M. Majesky, Baylor College of Medicine, Houston, Tex. The cells were cultured at 37°C in a 1:1 mixture of DMEM and Ham's F-12 medium (Mediatech) supplemented with 5% FBS (Hyclone Laboratories, Inc), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B (JRH Biosciences). Rat SMC cultures were fed every 48 hours and expanded by trypsin-EDTA (JRH Biosciences) treatment and subculturing at a 1:5 split ratio. Cells were incubated for
72 hours in normal growth medium without FBS but containing 5 µg/mL insulin, 5 µg/mL transferrin, and 5 ng/mL selenious acid (ITS, Collaborative Biomedical Products) to obtain a relatively quiescent SMC population.
RNA Isolation and Northern Blot Hybridization
Serum-starved SMCs were treated with the following agents either added alone or in various combinations: actinomycin D (2 µg/mL, Sigma Chemical Co), cycloheximide (10 µg/mL, Sigma), recombinant human HB-EGF (residues 63 to 148 of the HB-EGF precursor4 purified from baculovirus-infected insect cell–conditioned medium, 10 ng/mL unless otherwise noted; either obtained commercially [R&D Systems] or from M. Klagsbrun, Children's Hospital and Harvard Medical School, Boston, Mass), recombinant human EGF (10 ng/mL, Upstate Biotechnology), dexamethasone (1, 10, or 100 nmol/L, Sigma), and heparin (10, 30, or 100 µg/mL, Upjohn). In the heparin treatment experiments, either HB-EGF or EGF was preincubated with heparin for 5 minutes and then added to cells. Cells were harvested, and total RNA was isolated using RNA-Stat 60 (Tel-Test "B" Inc) according to the manufacturer's instructions. Each sample (10 µg) was denatured, loaded onto 1.2% agarose gels containing 2.2 mol/L formaldehyde, and subjected to electrophoresis. The gels were stained with 0.3 µg/mL ethidium bromide (Sigma) to confirm that the RNA samples were undegraded and that similar amounts were present in each lane. RNA was transferred onto Zetabind nylon membranes (Cuno Inc) by electrophoresis in 0.3x TEA (12 mmol/L Tris-HCl, 0.5 mmol/L EDTA, 6 mmol/L CH3COONa, and 8.88 mmol/L CH3COOH) and then cross-linked to the membrane by UV irradiation. The blots were prehybridized at 65°C for at least 2 hours in 1% BSA, 7% SDS, 0.5 mol/L NaH2PO4 (pH 7.0), 1 mmol/L EDTA, 100 µg/mL denatured salmon sperm DNA (Sigma), and 20% (vol/vol) formamide (Fluka Chemicals). The prehybridization solution was then discarded, and the blots were placed in the same solution as described above, except a 32P-labeled cDNA probe was included. The cDNA probes used were (1) rat FGF-1, 517-bp EcoRI fragment of pRSV-Neo-HBGF-1 (kind gift of W. McKeehan, Albert B. Alkek Institute of Biosciences and Technology, Texas A&M University, Houston), (2) rat FGF-2, 477-bp Xho I–Nco I fragment of RObFGF.477 (kind gift of A. Baird, Scripps Research Institute, La Jolla, Calif), and (3) human GAPDH, 800-bp Pst I–Xba I fragment of pHcGAP (American Type Culture Collection). The DNA fragments were labeled to high specific activity with [32P]dCTP (3000 Ci/mmol, DuPont/NEN) using a random primer labeling kit (Boehringer Mannheim). The blots were hybridized for 18 hours at 65°C in a shaking water bath and then washed sequentially with wash buffer A (0.5% BSA, 5% SDS, 40 mmol/L NaH2PO4 [pH 7.0], and 1 mmol/L EDTA), wash buffer B (1% SDS, 40 mmol/L NaH2PO4 [pH 7.0], and 1 mmol/L EDTA), and 0.1x SSPE (18 mmol/L NaCl, 0.1 mmol/L EDTA, and 1 mmol/L NaH2PO4 [pH 7.0]). The blots were then air-dried and exposed to Kodak X-Omat AR x-ray film. Autoradiographic signals or photographic images were analyzed by densitometry using the BioImage whole band analyzer (Millipore). FGF-2 mRNA signal intensities were calculated after normalization to either GAPDH mRNA or 18S rRNA levels. FGF-2 mRNA half-lives in the absence or presence of HB-EGF were calculated as described previously.57 All of the best-fit lines had correlation coefficients (R2) of >.929; thus, the data obtained were consistent with a first-order decay model.
Western Blot Analysis
Serum-starved SMCs were either left untreated or treated with HB-EGF (10 ng/mL) for 12 hours. The conditioned medium was then collected and frozen at -70°C. Cells were washed twice with PBS (Mediatech) and then with 2 mol/L NaCl in PBS. The NaCl wash fraction was then collected, diluted with H2O to 0.5 mol/L NaCl, and frozen at -70°C. Cells were lysed in HNTG buffer (20 mmol/L HEPES [pH 7.4], 150 mmol/L NaCl, 10% [vol/vol] glycerol, 1% [vol/vol] Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L EGTA, 0.01 mg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride). HNTG lysates were incubated on ice for 10 minutes and then spun at 10 000g for 10 minutes to remove insoluble material and nuclei. The amount of protein in each clarified lysate was determined using the BCA assay (Pierce Chemical Co). Each lysate (100 µg) and appropriate volumes (normalized according to cell protein amounts) of the conditioned medium and the 2 mol/L NaCl wash samples were diluted with 1/10 volume of 10x binding buffer (0.5 mol/L Tris-HCl [pH 7.5] and 100 mmol/L EDTA). As a control, a duplicate sample of conditioned medium from untreated SMCs was mixed with 100 ng of recombinant human FGF-2 protein (18-kD isoform, Bachem) and treated identically to the other samples. Heparin-Sepharose CL-6B (Pharmacia) was added, and the mixture was incubated (rocking) at 4°C for 18 hours. The resin and bound proteins were pelleted, washed three times with 50 mmol/L Tris-HCl (pH 7.5), 0.5 mol/L NaCl, and 10 mmol/L EDTA, and combined with an equivalent volume of sample buffer (125 mmol/L Tris-HCl [pH 6.8], 4% SDS, 20% [vol/vol] glycerol, and 10% [vol/vol] 2-mercaptoethanol). The samples were denatured at 95°C for 4 minutes and subjected to electrophoresis on a 15% polyacrylamide-SDS slab gel. Recombinant human FGF-1 (2 ng, kind gift of W. Burgess, American Red Cross, Rockville, Md), recombinant human FGF-2 (2 ng, Bachem), and low-molecular-weight markers (Bio-Rad) were also loaded onto the gel. Proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell, Inc) by electrophoresis in 25 mmol/L Tris, 192 mmol/L glycine, 0.02% SDS, and 20% (vol/vol) methanol. The membrane was stained with Ponceau S (Sigma) to verify that each lane contained equivalent amounts of protein, blocked with TBST (50 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, and 0.5% [vol/vol] Tween 20) containing 5% nonfat dry milk and incubated at 4°C for 18 hours in TBST containing a 1:250 dilution of an anti–FGF-2 monoclonal antibody (Transduction Labs). The membrane was then washed with TBST and incubated with a horseradish peroxidase–conjugated anti-mouse IgG secondary antibody (1:20 000 dilution, Bio-Rad). Bound secondary antibody was visualized using an enhanced chemiluminescence kit (Amersham). Film exposure and densitometry were performed as described above for Northern blots.
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Rat aortic SMCs are particularly well suited for studies examining HB-EGF regulation of gene expression since they (1) are relatively easy to culture, (2) express cell surface EGF receptors,13 58 59 60 61 and (3) proliferate in response to HB-EGF13 or EGF13 58 60 62 stimulation. Therefore, these cells were used in all of the experiments described in this report. We first determined whether HB-EGF treatment of serum-starved rat aortic SMCs altered FGF-1 or FGF-2 mRNA levels. Cells were either left untreated or treated with HB-EGF for 4 hours, RNA was isolated, and Northern blot hybridization analysis was performed. FGF-1 and FGF-2 transcripts of
4.4 kb and 6.0 kb in size, respectively, were coexpressed in rat SMCs (Fig 1
). HB-EGF treatment had no detectable effect on FGF-1 mRNA levels but significantly increased FGF-2 mRNA levels.
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The kinetics of HB-EGF–induced FGF-2 mRNA expression were then investigated by Northern blot analysis using RNA isolated from SMCs treated with HB-EGF for different periods of time. HB-EGF induced FGF-2 mRNA levels in a time-dependent manner (Fig 2A). Reprobing of this Northern blot with an FGF-1 cDNA clone demonstrated no increase in FGF-1 mRNA levels (data not shown). Increased FGF-2 mRNA expression was first detected at 1 hour after HB-EGF addition; as estimated by densitometry, FGF-2 mRNA levels were elevated 3.4-fold. The maximal level of FGF-2 mRNA was apparent after 4 hours of stimulation, representing a 45.6-fold induction. FGF-2 mRNA levels were still elevated at 12 hours (14.7-fold induction), the latest time point examined.
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We also determined whether HB-EGF caused a concentration-dependent increase in FGF-2 mRNA levels. Serum-starved SMCs were either left untreated or treated for 4 hours with increasing concentrations of HB-EGF, RNA was isolated, and Northern blot analysis was performed. HB-EGF increased FGF-2 mRNA levels in a dose-dependent manner (Fig 2B
); in contrast, reprobing of this blot with an FGF-1 cDNA clone revealed that FGF-1 mRNA levels were unaffected (data not shown). Elevated FGF-2 mRNA expression was first apparent when cells were stimulated with 3 ng/mL HB-EGF, and maximal induction occurred at an HB-EGF concentration of 10 ng/mL.
We next investigated whether de novo RNA and protein synthesis were required for HB-EGF induction of FGF-2 mRNA levels. SMCs were either left untreated or treated for 4 hours with HB-EGF in the absence or presence of either actinomycin D, an RNA synthesis inhibitor, or cycloheximide, a protein synthesis inhibitor. SMCs were also treated with each of the inhibitors alone. Cells were collected, RNA was isolated, and Northern blot hybridization analysis was performed. Actinomycin D treatment prevented HB-EGF induction of FGF-2 mRNA levels (Fig 3); thus, the increase in FGF-2 mRNA expression after HB-EGF addition is likely to be due to transcriptional activation of the FGF-2 gene. Actinomycin D also decreased the basal level of FGF-2 mRNA expression in nonstimulated cells, indicating that FGF-2 transcripts have a relatively short half-life (see below). Cycloheximide treatment partially inhibited the ability of HB-EGF to elevate FGF-2 mRNA expression; thus, the synthesis of intermediary proteins is required for maximal induction of FGF-2 mRNA expression.
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FGF-2 mRNA stability measurements provided additional evidence in support of a transcriptional control mechanism for HB-EGF induction of FGF-2 mRNA levels. Serum-starved SMCs were either left untreated or treated for different periods of time with actinomycin D in either the absence or presence of HB-EGF. RNA was isolated, and FGF-2 mRNA levels were assayed by Northern blot hybridization (Fig 4). Autoradiographic signals were quantified using scanning laser densitometry and FGF-2 mRNA levels normalized to GAPDH mRNA levels. The natural logarithm of percent RNA remaining was plotted against time after actinomycin D addition, and half-lives were calculated by linear regression analysis. FGF-2 mRNA decayed with first-order kinetics and had a slightly longer half-life in HB-EGF–treated SMCs (2.9 hours) than in serum-starved SMCs (2.4 hours). This minor difference is unlikely to account for the large and rapid increase in FGF-2 mRNA levels observed after HB-EGF treatment.
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The effect of glucocorticoid treatment on HB-EGF regulation of FGF-2 mRNA expression was then examined. Glucocorticoids are potent anti-inflammatory and immunosuppressive agents.63 Dexamethasone, a synthetic glucocorticoid, can inhibit SMC proliferation in vitro64 65 and suppress neointimal formation after balloon angioplasty of rabbit66 67 or rat68 arteries. In a previous study, we demonstrated that dexamethasone inhibits interleukin-1–induced FGF-2 mRNA expression in human SMCs.51 To test whether dexamethasone could inhibit HB-EGF–induced FGF-2 mRNA expression in rat SMCs, serum-starved cells were either left untreated or treated with HB-EGF for 4 hours in either the absence or presence of 1, 10, or 100 nmol/L dexamethasone. Cells were collected, RNA was isolated, and Northern blot hybridization was performed. Dexamethasone inhibited HB-EGF induction of FGF-2 mRNA levels in a dose-dependent manner (Fig 5); as estimated by densitometry, 1 nmol/L dexamethasone inhibited induction by 38%, whereas concentrations of either 10 or 100 nmol/L had a similar potency, inhibiting induction by 70% and 66%, respectively. The addition of dexamethasone alone to serum-starved nonstimulated SMCs did not alter the basal level of FGF-2 mRNA expression (data not shown).
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We also investigated whether the glycosaminoglycan heparin could inhibit HB-EGF–induced FGF-2 mRNA expression. As mentioned previously, HB-EGF can bind to both immobilized heparin1 2 3 and to cell surface HSPG.9 14 Recent studies have indicated that the interaction with cell surface HSPG is essential for optimal binding to high-affinity EGF receptors.8 9 12 13 14 For example, treatment of bovine SMCs with either heparitinase (which degrades heparan sulfate chains), chlorate (which inhibits sulfation of glycosaminoglycans), or a synthetic peptide corresponding to the heparin-binding domain of HB-EGF inhibits HB-EGF binding to the EGF receptor as well as HB-EGF–stimulated cell migration.14 In consideration of these findings, we tested whether exogenously added heparin could inhibit HB-EGF induction of FGF-2 mRNA expression. Serum-starved SMCs were either left untreated or treated for 4 hours with HB-EGF in either the absence or presence of 10, 30, or 100 µg/mL heparin. As a control, we also examined the effect of heparin on EGF-induced FGF-2 mRNA expression. EGF does not bind heparin-affinity columns69 or cell surface HSPG12 14 ; thus, EGF-mediated EGF receptor activation should be heparin independent. After the various treatments, the SMCs were collected, RNA was isolated, and Northern blot hybridization was performed. Heparin inhibited HB-EGF–induced but not EGF-induced FGF-2 mRNA expression (Fig 6). The extent of inhibition varied in a dose-dependent manner; as estimated by densitometry, 10 µg/mL heparin inhibited induction by 47%, and either 30 or 100 µg/mL heparin had a similar effect, inhibiting induction by 66% and 62%, respectively. Heparin treatment alone did not alter the basal level of FGF-2 mRNA expression in serum-starved SMCs (data not shown).
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Finally, Western blot analysis was performed to determine whether HB-EGF stimulation of rat SMCs resulted in elevated FGF-2 production. Serum-starved cells were either left untreated or treated with HB-EGF for 12 hours. Although FGF-2 lacks a conventional signal peptide sequence required for secretion via the endoplasmic reticulum/Golgi pathway, it can be released from at least some cell types by an alternative mechanism.70 71 Accordingly, FGF-2 has been found in conditioned medium46 71 and in association with the extracellular matrix.72 73 74 Therefore, we examined FGF-2 protein levels in conditioned medium (to assay soluble secreted FGF-2), 2 mol/L NaCl washes of the cell monolayers (to assay cell surface/extracellular matrix–associated FGF-272 ), and HNTG lysates (to assay cytoplasmic FGF-2). Heparin-Sepharose bound proteins were analyzed by SDS-PAGE and immunoblotting with an anti–FGF-2 monoclonal antibody. This antibody does not recognize FGF-1, the FGF family member with the greatest sequence identity to FGF-2 (Fig 7A). Three immunoreactive proteins with approximate molecular masses consistent with the predicted rat FGF-2 isoforms (18, 21.5, and 22 kD)75 were detected in the SMC lysates (Fig 7B). The expression level of all three species increased after HB-EGF treatment; as estimated by densitometry, FGF-2 protein levels were elevated 1.9-fold at 12 hours after stimulation. We were unable to detect FGF-2 in either the 2 mol/L NaCl wash fractions or the conditioned medium samples. This is not likely to reflect ineffective binding of FGF-2 to the heparin-Sepharose resin, since FGF-2 that had been added to conditioned medium was readily recovered under our experimental conditions (Fig 7C).
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Discussion |
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The results of the present study demonstrate that the polypeptide mitogen HB-EGF can induce FGF-2 mRNA levels in rat aortic SMCs in a time- and dose-dependent manner. The increase in FGF-2 mRNA abundance noted after HB-EGF treatment (1) does not occur if transcription is inhibited, (2) does not reflect FGF-2 mRNA stabilization, (3) requires de novo protein synthesis, and (4) results in an approximately twofold elevation of FGF-2 protein expression. Furthermore, both dexamethasone (an anti-inflammatory glucocorticoid) and heparin (a glycosaminoglycan) can suppress HB-EGF induction of FGF-2 mRNA expression.
There have been relatively few previous reports describing the ability of HB-EGF to regulate the expression of specific gene products. In the one study using vascular cells, Dluz et al22 found that HB-EGF–stimulated fetal human SMCs expressed elevated levels of HB-EGF mRNA. HB-EGF autoinduction has also been noted in human pancreatic cancer cells,76 human keratinocytes,11 and rat intestinal epithelial cells.77 In the latter cell type, HB-EGF can also increase the expression of several genes encoding other members of the EGF family, including transforming growth factor-
, amphiregulin, and betacellulin.77 Indeed, many members of the EGF family of structurally related mitogens are capable of autoinduction and cross induction.11 76 77
HB-EGF does not induce FGF-2 mRNA levels to the maximal extent if RNA synthesis is inhibited using the drug actinomycin D. This suggests that HB-EGF treatment is causing an increase in FGF-2 gene transcription. This mechanism of action is also supported by the FGF-2 mRNA decay measurements, indicating that HB-EGF does not significantly alter FGF-2 mRNA stability. However, it is also possible that actinomycin D is preventing the synthesis of an HB-EGF–inducible protein that stabilizes FGF-2 mRNA. However, this seems unlikely in consideration of the rapid kinetics of FGF-2 mRNA accumulation following HB-EGF addition.
HB-EGF also cannot induce maximal levels of FGF-2 mRNA if added in combination with the protein synthesis inhibitor cycloheximide. Similar results were obtained in our previous experiments investigating the molecular basis for serum,52 interleukin-1,51 or FGF-255 regulation of FGF-2 mRNA expression in vascular SMCs. We have recently found that angiotensin II induction of FGF-2 mRNA expression in rat SMCs also requires de novo protein synthesis (authors' unpublished data, 1996). Taken together, these studies indicate that FGF-2 is a delayed early-response gene regulated by serum factors, cytokines, polypeptide mitogens, and vasoactive hormones.
We found that the potent anti-inflammatory glucocorticoid dexamethasone could suppress HB-EGF–induced FGF-2 mRNA expression. It is presently unknown whether this effect is due to a decrease in the rate of FGF-2 gene transcription and/or to increased FGF-2 mRNA turnover. We reported previously that this compound could also inhibit interleukin-1–induced FGF-2 gene expression in human saphenous vein SMCs.51 Additionally, Nakano et al21 demonstrated that similar concentrations of dexamethasone could decrease both constitutive and thrombin-induced PDGF A-chain and HB-EGF gene expression in fetal human SMCs. Dexamethasone inhibits SMC growth in vitro64 65 and can decrease neointima formation following balloon catheter–induced arterial injury.66 67 68 These biological effects may reflect, at least in part, the ability of dexamethasone to decrease autocrine growth factor production by SMCs.
Several studies have demonstrated that HB-EGF, but not EGF, requires a low-affinity interaction with cell surface HSPG for efficient binding to and activation of the EGF receptor tyrosine kinase.8 9 12 13 14 Additional evidence in support of a "dual receptor system" was obtained in our experiments, which revealed that exogenous heparin can suppress HB-EGF–induced FGF-2 mRNA expression. This heparin effect cannot be attributed to a general inhibition of SMC gene activity since (1) EGF-induced FGF-2 mRNA expression was not effected by heparin, (2) heparin treatment of unstimulated SMCs did not alter the basal level of FGF-2 mRNA expression, and (3) GAPDH mRNA levels were not decreased by heparin treatment. Heparin did not completely inhibit HB-EGF activity, which is consistent with a report by Besner et al8 indicating that similar concentrations of heparin could only partially block HB-EGF binding to EGF receptors on human endometrial carcinoma cells. Thus, it appears that HB-EGF can also bind EGF receptors in a HSPG-independent manner.
We were able to detect an HB-EGF–mediated increase in the cytoplasmic levels of FGF-2 by Western blot analysis. However, we could not detect FGF-2 in the extracellular matrix or in the conditioned medium before or after HB-EGF treatment. The increase in intracellular FGF-2 levels was significantly less than one would predict from the mRNA expression data. These findings are very similar to those reported in our previous study analyzing FGF-2 induction of FGF-2 mRNA and protein expression in rat SMCs.55 The relatively low level of FGF-2 protein induction noted in these experiments indicates that FGF-2 synthesis in rat SMCs may be regulated at the translational level. This possibility is supported by recent studies examining FGF-2 mRNA translational efficiency in vitro and in vivo.78 79
In summary, we have demonstrated that HB-EGF treatment of rat aortic SMCs induces FGF-2 gene expression. We reported previously that FGF-1 and FGF-2 could also regulate FGF-2 expression in this same cell type.55 Dluz et al22 found that HB-EGF– or FGF-2–stimulated fetal human SMCs expressed elevated levels of HB-EGF mRNA. Taken together, these results indicate that both autoregulatory and cross-regulatory mechanisms may control HB-EGF and FGF-2 production by SMCs in vivo. It should be emphasized that the abundance of HB-EGF or FGF-2 within the vessel wall is likely to reflect the relative expression and activity of both positive and negative regulatory factors. For example, although HB-EGF expression is elevated in human atherosclerotic plaques,28 FGF-2 expression is not.39
The biological significance of our findings in the context of vascular SMC growth regulation in vivo is presently unknown. HB-EGF and FGF-2 released by SMCs themselves could independently stimulate SMC proliferation via both autocrine and paracrine mechanisms. In developing atherosclerotic lesions, HB-EGF and FGF-2 derived from macrophages28 38 39 and/or T lymphocytes27 could act on neighboring SMCs via a paracrine mechanism or, in the case of membrane-anchored HB-EGF, via a juxtacrine mechanism. Elevated levels of FGF-2 within the vessel wall may not only promote SMC accumulation but could also stimulate reendothelialization and the growth of microvessels. We conclude that interactions between individual members of the EGF or FGF families of structurally related mitogens could play a role in the pathogenesis of vascular disease.
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Acknowledgments |
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This study was supported in part by National Institutes of Health research grant HL-39727 (Dr Winkles). Portions of this work were performed by K.A. Peifley in partial fulfillment of the requirements for a Master's degree in Biomedical Sciences at Hood College, Frederick, Md. Dr Hsu was supported in part by a training grant from the National Institutes of Health (HL-07698). We thank the five investigators mentioned in "Materials and Methods" for generously providing reagents. We are also grateful to K. Wawzinski for excellent secretarial assistance.
Received September 6, 1995; accepted May 6, 1996.
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