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593 Transforming Growth Factor-/Jl Gene Activation and Growth of Smooth Muscle From Hypertensive Rats Alex Agrotis, John Saltis, Alex Bobik Abstract Cultured vascular smooth muscle cells derived from the spontaneously hypertensive rat (SHR) are known to replicate more rapidly than cells from the normotensive Wistar-Kyoto (WKY) rat. In this study we compared the responses of vascular smooth muscle cells from the two strains to transforming growth factor-01 (TGF-/31) and evaluated its potential to account for the different growth properties of these cells in response to a number of vascular-derived growth factors. TGF-/31 potentiated the proliferative effects of epider- mal growth factor, basic fibroblast growth factor, or the different isoforms of platelet-derived growth factor on vascular smooth muscle cells from SHR but inhibited growth factor- stimulated proliferation of vascular smooth muscle cells from WKY rats. These differential effects of TGF-01 on prolifera- tion could not be attributed to alterations in the expression of the type I, II, or III TGF-/3 receptors but appeared more V ascular hypertrophy is a major contributor to the increase in vascular resistance in sponta- neously hypertensive rats (SHR). 1 In these animals hypertrophy of the resistance vessels is, at least in part, attributed to a greater number of smooth muscle cells within the vessel wall. 2 It has been suggested that differences between the ability of vascular smooth mus- cle cells (VSMC) from SHR and normotensive Wistar- Kyoto (WKY) rats to respond to the mitogenic influ- ence of vascular-derived growth factors could account for this "proliferative hypertrophy" in genetic hyperten- sion. 36 One of these vascular-derived growth factors, transforming growth factor-^1 (TGF-/31), is a multi- functional protein that regulates the growth and differ- entiation of a wide variety of cells in culture. 78 In VSMC TGF-/31 can induce both cellular hypertrophy and polyploidy as well as stimulate and/or inhibit pro- liferation. 9 ' 10 For example, TGF-/31 has been shown to stimulate the proliferation of both sparse and dense cultures of human smooth muscle cells." Also, TGF-/31 inhibits serum-stimulated proliferation of rat VSMC, seeded at low densities; at high densities the opposite effect on proliferation is observed. 1013 These opposing effects of TGF-01 are elicited after its interaction with multiple receptors on the surface of smooth muscle cells. 7 Received May 13, 1993; accepted in revised form February 9, 1994. From the Baker Medical Research Institute and Alfred Hospi- tal, Prahran, Victoria, Australia. Correspondence to Dr Alex Agrotis, Baker Medical Research Institute, Commercial Rd, Prahran, 3181 Victoria, Australia. related to the ability of cells to autoinduce the TGF-/31 gene. TGF-/31 caused a time-dependent increase in its own mRNA levels in vascular smooth muscle cells of WKY rats but attenuated levels in vascular smooth muscle cells of SHR. This effect was specific to TGF-01 autoinduction since similar elevations in TGF-01 mRNA levels were observed when vascular smooth muscle cells from the two rat strains were exposed to phorbol myristate acetate, basic fibroblast growth factor, or platelet-derived growth factor-BB. These data sug- gest that the production of TGF-01 may contribute to the different growth properties of vascular smooth muscle cells from SHR and WKY rats through alterations in TGF-/31 signaling systems. (Hypertension. 1994;23:593-599.) Key Words transforming growth factor-/3 muscle, smooth, vascular • protein-tyrosine kinase • proliferation • rats, inbred SHR Several lines of evidence suggest that TGF-/31 may play an important role in the development of hyperten- sion. Initially, Sarzani et al 14 demonstrated elevated TGF-/31 mRNA expression in the aorta of rats whose hypertension was induced with deoxycorticosterone and salt. Others have shown increased expression of TGF-/31 mRNA levels in the aorta of SHR and WKY rats as their age increases from 5 to 40 weeks. 15 Elevated mRNA levels encoding TGF-/31 have also been re- ported in VSMC from SHR, compared with those from WKY rats, cultured in the presence of serum. 16 More recently it has been suggested that increased TGF-/31 expression and activation is associated with augmented proliferation of VSMC from SHR. 17 We have previously shown that TGF-/31 interacts with tyrosine kinase- activating growth factors such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) to potentiate their mitogenic activity in VSMC from the SHR. 18 The aim of the present study was twofold: (1) to investigate whether the ability of TGF-^1 to potentiate the effects of tyrosine kinase-activating growth factors was unique to VSMC from SHR and (2) to examine the relation between TGF-/31 and growth factor-stimulated proliferation of VSMC from SHR and WKY rats. Our results indicate that TGF-/31 inhibits growth factor- stimulated proliferation of VSMC from normotensive WKY rats, whereas under identical conditions there is potentiation of proliferation in VSMC from SHR. These differential effects appear to be due to alterations in intracellular signaling mechanisms stimulated by TGF-/31 and involved in the regulation of proliferation. In addition, the production of endogenous TGF-/31 by by guest on July 12, 2018 http://hyper.ahajournals.org/ Downloaded from

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593

Transforming Growth Factor-/Jl GeneActivation and Growth of SmoothMuscle From Hypertensive Rats

Alex Agrotis, John Saltis, Alex Bobik

Abstract Cultured vascular smooth muscle cells derivedfrom the spontaneously hypertensive rat (SHR) are known toreplicate more rapidly than cells from the normotensiveWistar-Kyoto (WKY) rat. In this study we compared theresponses of vascular smooth muscle cells from the two strainsto transforming growth factor-01 (TGF-/31) and evaluated itspotential to account for the different growth properties ofthese cells in response to a number of vascular-derived growthfactors. TGF-/31 potentiated the proliferative effects of epider-mal growth factor, basic fibroblast growth factor, or thedifferent isoforms of platelet-derived growth factor on vascularsmooth muscle cells from SHR but inhibited growth factor-stimulated proliferation of vascular smooth muscle cells fromWKY rats. These differential effects of TGF-01 on prolifera-tion could not be attributed to alterations in the expression ofthe type I, II, or III TGF-/3 receptors but appeared more

Vascular hypertrophy is a major contributor tothe increase in vascular resistance in sponta-neously hypertensive rats (SHR).1 In these

animals hypertrophy of the resistance vessels is, at leastin part, attributed to a greater number of smooth musclecells within the vessel wall.2 It has been suggested thatdifferences between the ability of vascular smooth mus-cle cells (VSMC) from SHR and normotensive Wistar-Kyoto (WKY) rats to respond to the mitogenic influ-ence of vascular-derived growth factors could accountfor this "proliferative hypertrophy" in genetic hyperten-sion.36 One of these vascular-derived growth factors,transforming growth factor-^1 (TGF-/31), is a multi-functional protein that regulates the growth and differ-entiation of a wide variety of cells in culture.78 InVSMC TGF-/31 can induce both cellular hypertrophyand polyploidy as well as stimulate and/or inhibit pro-liferation.9'10 For example, TGF-/31 has been shown tostimulate the proliferation of both sparse and densecultures of human smooth muscle cells." Also, TGF-/31inhibits serum-stimulated proliferation of rat VSMC,seeded at low densities; at high densities the oppositeeffect on proliferation is observed.1013 These opposingeffects of TGF-01 are elicited after its interaction withmultiple receptors on the surface of smooth musclecells.7

Received May 13, 1993; accepted in revised form February 9,1994.

From the Baker Medical Research Institute and Alfred Hospi-tal, Prahran, Victoria, Australia.

Correspondence to Dr Alex Agrotis, Baker Medical ResearchInstitute, Commercial Rd, Prahran, 3181 Victoria, Australia.

related to the ability of cells to autoinduce the TGF-/31 gene.TGF-/31 caused a time-dependent increase in its own mRNAlevels in vascular smooth muscle cells of WKY rats butattenuated levels in vascular smooth muscle cells of SHR. Thiseffect was specific to TGF-01 autoinduction since similarelevations in TGF-01 mRNA levels were observed whenvascular smooth muscle cells from the two rat strains wereexposed to phorbol myristate acetate, basic fibroblast growthfactor, or platelet-derived growth factor-BB. These data sug-gest that the production of TGF-01 may contribute to thedifferent growth properties of vascular smooth muscle cellsfrom SHR and WKY rats through alterations in TGF-/31signaling systems. (Hypertension. 1994;23:593-599.)

Key Words • transforming growth factor-/3 • muscle,smooth, vascular • protein-tyrosine kinase • proliferation• rats, inbred SHR

Several lines of evidence suggest that TGF-/31 mayplay an important role in the development of hyperten-sion. Initially, Sarzani et al14 demonstrated elevatedTGF-/31 mRNA expression in the aorta of rats whosehypertension was induced with deoxycorticosterone andsalt. Others have shown increased expression ofTGF-/31 mRNA levels in the aorta of SHR and WKYrats as their age increases from 5 to 40 weeks.15 ElevatedmRNA levels encoding TGF-/31 have also been re-ported in VSMC from SHR, compared with those fromWKY rats, cultured in the presence of serum.16 Morerecently it has been suggested that increased TGF-/31expression and activation is associated with augmentedproliferation of VSMC from SHR.17 We have previouslyshown that TGF-/31 interacts with tyrosine kinase-activating growth factors such as basic fibroblast growthfactor (bFGF), platelet-derived growth factor (PDGF),or epidermal growth factor (EGF) to potentiate theirmitogenic activity in VSMC from the SHR.18

The aim of the present study was twofold: (1) toinvestigate whether the ability of TGF-^1 to potentiatethe effects of tyrosine kinase-activating growth factorswas unique to VSMC from SHR and (2) to examine therelation between TGF-/31 and growth factor-stimulatedproliferation of VSMC from SHR and WKY rats. Ourresults indicate that TGF-/31 inhibits growth factor-stimulated proliferation of VSMC from normotensiveWKY rats, whereas under identical conditions there ispotentiation of proliferation in VSMC from SHR.These differential effects appear to be due to alterationsin intracellular signaling mechanisms stimulated byTGF-/31 and involved in the regulation of proliferation.In addition, the production of endogenous TGF-/31 by

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594 Hypertension Vol 23, No 5 May 1994

cells after stimulation with growth factors may accountfor the well-known differences in the growth propertiesof VSMC from the two rat strains.

MethodsAnimals

Male SHR and WKY rats (weight, 250 to 300 g) were bredat the Baker Medical Research Institute from stock originallysupplied by Y. Yamori. Immediately before excising the aortasfor dispersion into single cells (see "Methods"), the animalswere deeply anesthetized with halothane. This procedure wasapproved by the Baker Institute-Alfred Hospital AnimalsExperimentation Committee and conformed to the guidelinesof the Australian National Health and Medical ResearchCouncil.

MaterialsFetal calf serum (FCS), penicillin G, and Dulbecco's phos-

phate-buffered saline (PBS) were purchased from Common-wealth Serum Laboratories. We obtained Dulbecco's modifiedEagle's medium (DMEM) from FLOW Laboratories. Tissueculture dishes were purchased from Sterilin Ltd. Collagenasetype-I (C-0130), elastase (E-0258), EGF, phenylmethylsulfonylfluoride (PMSF), and leupeptin were obtained from SigmaChemical Co. Porcine transforming growth factor-/31 was ob-tained from British Bio-Technology Ltd. Human [123I]TGF-/31was purchased from Dupont Australia Ltd. bFGF and methyl-pHJthymidine were obtained from Amersham. PDGF-AA andPDGF-AB, bovine serum albumin (BSA), and the randompriming DNA labeling kit were obtained from Boehringer-Mannheim. PDGF-BB was obtained from Zymogenetics. A ratTGF-/31 cDNA probe consisting of a 1-kb fragment spanning themajor coding region of the TGF-01 precursor (Qian et al19) wasprovided by Dr A. Roberts, Laboratory of Chemoprevention,National Cancer Institute, National Institutes of Health, Be-thesda, Md. A 30-base oligonucleotide that hybridizes to the 18srRNA species was provided by Dr Z. Krozowski, MolecularHypertension Laboratory, Baker Medical Research Institute,Melbourne, Australia.

Isolation and Culture of AorticSmooth Muscle Cells

Primary cultures of VSMC were prepared by enzyme dis-persion of aortic media from 12- to 14-week-old SHR andWKY rats as previously described.6 Examination of the cul-tures by phase-contrast microscopy indicated a "hills andvalleys" pattern at confluency, a well-known characteristic ofVSMC in culture. The identity of smooth muscle cells in ourcultures was verified by immunocytochemical analysis usingantibodies for smooth muscle myosin.20 These "primary"smooth muscle cells were subsequently passaged every weekand grown in 10% FCS/DMEM using 90-mm tissue culturedishes. Cells between primary and fourth passages were usedin all experiments.

[•'HJThymidine Incorporationand Cell Proliferation

Cells were grown to confluency (1 to 2x 103 cells) in 24-welltissue culture dishes. At this time the medium was replacedwith 1.0 mL of DMEM, and cells were incubated for anadditional 48 to 72 hours. After 24 hours of serum deprivation,in excess of 86% of the VSMC were present in Go/G, assessedwith a fluorescent-activated cell sorter, the remainder werepresent in the S phase and G2-M phase.21 Growth factors, EGF(50 ng/mL), and/or TGF-/31 (1 ng/mL) (dissolved in DMEMcontaining 1% BSA) were then added to the quiescent cells, ina total volume of 0.5 mL of DMEM alone. At the indicatedtimes the medium was aspirated, and the cells were washedonce with 1.0 mL of DMEM and then incubated for anadditional 2 hours in 1.0 mL DMEM containing methyl-

[3H]thymidine (1 /xCi/mL). At the end of this period, themedium was removed and cells washed three times with 1.0ml, of ice-cold Dulbecco's PBS before the addition of 0.5 mLof ice-cold 10% trichloroacetic acid for 15 to 30 minutes. Afteran additional wash with 10% trichloroacetic acid, the cellswere solubilized in 1 mol/L NaOH, neutralized with 1 mol/LHC1, and radioactivity was then determined by scintillationspectrometry.

In the proliferation studies VSMC (^lxlO4) were platedinto 24-well tissue culture dishes in 1.0 mL of 10% FCS/DMEM. The next day the medium was replaced with 1.0 mLof DMEM, and cells were cultured for an additional 24 hours.Growth factors were then added (TGF-01 [1 ng/mL], EGF [50ng/mL], bFGF [25 ng/mL], PDGF-AA [400 ng/mL],PDGF-AB [200 ng/mL], and PDGF-BB [200 ng/mL]) in 0.5mL of DMEM containing insulin and transferrin (4%Monomed A, Commonwealth Serum Laboratories). Thegrowth factor-containing medium was replaced every 2 days,and cell number was then determined at indicated times usinga Coulter counter. We have previously demonstrated that theaforementioned concentrations of growth factors induce max-imal incorporation of [3H]thymidine into VSMC of SHR andWKY rats (Saltis et al).22

Affinity Labeling of Transforming GrowthFactor-^ Receptors

The identification of TGF-/3 receptors by affinity labelingwas performed essentially as described by Massagu6.23 Cellswere grown in DMEM containing 10% FCS to confluency("2X105 cells) in 12-well tissue culture dishes. They werewashed with 2.0 mL of binding buffer per well (DMEM, 1%BSA, 20 mmol/L HEPES [pH 7.4]) and then incubated in 2.0mL of this medium for 2 hours at 37°C. After additionalwashing of the cells, they were incubated for 3 to 4 hours at 4°Cin 0.5 mL binding buffer containing 150 pmol/L [12!I]TGF-/31,either alone or in the presence of a 100-fold excess ofunlabeled TGF-/31. The cells were then washed three timeswith 2 mL binding buffer, followed by an additional wash with2 mL of PBS buffer (without BSA). Disuccinimidyl suberate,freshly dissolved in dimethyl sulfoxide, was added to the cellsin 0.5 mL PBS to a final concentration of 0.5 mmol/L. After 15minutes at 4°C on an orbital shaker, the cells were washedtwice with 2.0 mL PBS containing 1 mmol/L PMSF and 10jig/mL leupeptin, scraped from the plastic surface, and col-lected by centrifugation at 10 000# for 10 minutes at 4°C.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electropho-resis sample buffer (50 to 100 jiL) was added to the pellets,and samples were boiled for 5 minutes, followed by centrifu-gation at lOOOOg for 10 minutes at 4°C. The supernatantsolutions were stored at -20°C for 1 to 2 days before electro-phoresis was performed on a 6.5% resolving gel under nonre-ducing conditions. Solubilized smooth muscle cells, adjusted tothe same cell number, were subjected to electrophoresis. Afterelectrophoresis, gels were fixed in 25% isopropanol/7% aceticacid and stained with 0.1% Coomassie brilliant blue. Autora-diography of dried gels was performed with Kodak X-OmatAR film using Dupont Lighting Plus screens.

RNA Isolation and AnalysisTotal RNA was extracted from confluent VSMC cultures

(60-mm tissue dishes) by the guanidinium thiocyanate methodof Chomczynski and Sacchi24 and then electrophoresed (5 to15 /xg) on 1% agarose/(2.2 mol/L) formaldehyde gels. Afterelectrophoresis, the gels were treated with 50 mmol/L NaOHfor 20 minutes, neutralized with a 0.5 mol/L Tris solution (pH7.4) containing 1.5 mol/L NaCl for 20 minutes, and thenequilibrated with 20x SSC for 20 to 40 minutes (20x SSC=3mol/L NaCl and 0.3 mol/L Na citrate [pH 7.0]). RNA wastransferred to 0.45-/im Biotrans Membranes (ICN), UV fixedfor 5 minutes, and then baked at 80°C for 1 to 2 hours.Prehybridization was carried out at 65°C for 1 to 4 hours in a

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Agrotis et al TGF-01 Regulation of Proliferation and Gene Expression 595

TGF-01 (ng/ml)

FIG 1. Une graph shows effects of transforming growth factor-/!}1(TGF-/J1) on r^HJtnymidlne incorporation in serum-deprived vascu-lar smooth muscle cells. Confluent and quiescent cells ( " I ^ x i o 5 )from spontaneously hypertensive rats (SHR) (v) and Wistar-Kyoto(WKY) rats (•) were exposed to increasing concentrations ofTGF-/31 for 24 hours; pUJthymidlne incorporation was then deter-mined as described in "Methods." The data points are mean±SEMvalues obtained from six experiments.

solution of 7% SDS, 1% BSA, 0.5 mol/L NaHPO4> and 1mmol/L EDTA. Hybridization was carried out at 65°C for 15hours in the same buffer containing ^-labeled cDNA probe(1 to 1.5 xlO6 cpm/mL). The TGF-01 probe was preparedusing the random priming method as previously described23

(109 cpm//ig DNA). The 18s rRNA probe was prepared byend-labeling. Filters were subsequently washed three times at65°C (1/2 hour each wash) in 1% SDS, 40 mmol/L NaHPO4,and 1 mmol/L EDTA before being sealed in plastic andexposed to Kodak X-Omat AR film with intensifying screensfor 2 days at -70°C. The resulting autoradiographs wereanalyzed by laser densitometry at 600 nm.

Statistical AnalysisThe significance of differences within and between strains

was assessed by one-way ANOVA.26 Results have been ex-pressed as mean±SEM of three to seven experiments.

ResultsTransforming Growth Factor-01 and[3H]Thymidine Incorporation in DNA

Initially we compared the effects of increasing con-centrations of TGF-/J1 alone on [3H]thymidine incorpo-ration into VSMC from SHR and WKY rats. Whenconfluent and serum-deprived VSMC from SHR wereexposed to TGF-/31 for 24 hours, they increased theirincorporation of [3H]thymidine into DNA. This stimu-latory effect of TGF-/31 became apparent at concentra-tions above 0.05 ng/mL and was maximal at 1 ng/mL; atthis concentration the increase in [3H]thymidine incor-poration was approximately 200% (Fig 1). Under thesame conditions TGF-/31 attenuated, rather than in-creased, [3H]thymidine incorporation into VSMC fromWKY rats. At 1 ng/mL of TGF-/31, [3H]thymidineincorporation into these cells was reduced by approxi-mately 25% (Fig 1).

These differential effects of TGF-01 on [3H]thymi-dine incorporation into VSMC from SHR and WKYrats were also observed in the presence of growthfactors such as EGF. In VSMC from SHR, TGF-01potentiated maximal rates of EGF-stimulated [3H]thy-midine incorporation; the extent of potentiation medi-ated by TGF-01 varied, ranging from 30% to 100% (Fig

2B and Reference 18). The enhancing effect of TGF-/31on growth factor-stimulated DNA synthesis in VSMCfrom the SHR was evident over the concentration range0.1 to 1.0 ng/mL (Fig 2D), similar to that seen on[3H]thymidine incorporation in serum-deprived cells(Fig 1). This action of TGF-/31 on [3H]thymidine incor-poration became evident approximately 18 hours afterexposing the cells to the two growth factors and per-sisted for the ensuing 30 hours (Fig 2B). In contrast tothese results, TGF-/31 inhibited by 10% to 50% EGF-stimulated DNA synthesis in VSMC from WKY rats(Fig 2A). This effect of TGF-/31 was not simply aconsequence of any prolongation of the Gi phase butrather appeared to be the consequence of a smallernumber of cells being stimulated by EGF to enter the Sphase. This inhibitory effect of TGF-/J1 on EGF-stim-ulated [3H]thymidine incorporation was observed atconcentrations approximately 10-fold lower than itsenhancing effects on growth factor-induced mitogenesisin VSMC from SHR (Fig 2C). Even when TGF-01concentrations were increased in VSMC from WKYrats to those that caused maximum enhancement ofEGF-induced DNA synthesis in VSMC from SHR,there was still no enhancement of mitogenesis.

Transforming Growth Factor-01and VSMC Proliferation

The effects of TGF-01 on the proliferation of sparsecultures of VSMC from SHR and WKY rats exposed toEGF, bFGF, or the isoforms of PDGF (PDGF-AA,-AB, -BB) are shown in Fig 3. In VSMC from WKY rats,TGF-/31 inhibited by 70% to 95% mitogen-stimulatedproliferation (Fig 3A). This inhibition by TGF-01 wasevident at concentrations that inhibited the incorpo-ration of [3H]thymidine into these cells (Fig 2). Incontrast, TGF-/31 enhanced growth factor-stimulatedproliferation of VSMC from SHR (Fig 3B). The mag-nitude of this stimulation varied with the differentgrowth factors and ranged from 12% to 90%. Noinhibition of proliferation by TGF-)31 was observed inVSMC from SHR.

We have previously demonstrated that differentmechanisms appear to be responsible for the regulationof growth rates of VSMC and the density at which thesecells became refractory to growth factor stimuli.6

Hence, it was also of interest to examine whetherTGF-01 differentially affected the density at whichquiescence was attained by growth factor-stimulatedVSMC from SHR and WKY rats. When proliferatingVSMC were exposed to high concentrations (1 ng/mL)of TGF-/31, the density at which VSMC became refrac-tory to the proliferative effects of EGF was lowered inWKY cultures and increased in SHR cultures (Fig 4). InVSMC from WKY rats this reduction in cell density wasapproximately 60% (P<.01), whereas in the culturesfrom the genetically hypertensive rats the increase wasgreater than 200% (P<.01) (Fig 4).

Transforming Growth Factor-/31 Gene ExpressionBecause the effects of TGF-01 on proliferation and

the density at which proliferating VSMC from SHR andWKY rats became refractory to growth factor stimulidiffered markedly, it was also of interest to examinewhether other TGF-01 responses might also be differ-entially affected. Thus, we compared the relative abili-

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596 Hypertension Vol 23, No 5 May 1994

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Fe 2. A and B, Line graphs show timecourse of effects of transforming growth fac-tor-01 (TGF-01) on epidermal growth factor(EGF)-stimulated fHJthymidine incorpo-ration. Confluent and quiescent cells (<»1-2x10=) from Wistar-Kyoto (WKY) rats (A) andspontaneously hypertensive rats (SHR) (B)were incubated in the presence of Dulbecco'smodified Eagle's medium (•), 1 ng/mLTGF-01 (v), 50 ng/mL EGF (T), or both agentstogether (•) . At the indicated times, pH]thy-midine incorporation was determined as de-scribed in "Methods." The data points aremean±SEM values obtained from five toseven experiments. C and D, Graphs showconcentration-response curves for TGF-/31 onEGF-stimulated pH]thymidine incorporation.Confluent and quiescent vascular smoothmuscle cells (VSMC) (>» 1-2x10*) were stimu-lated with EGF (50 ng/mL) either alone or inthe presence of increasing concentrations ofTGF-01. For VSMC from WKY rats (C), EGF-stimulated cells were incubated for 16 hoursin the presence of increasing concentrationsof TGF-01. Results are expressed as percentinhibition of maximal rates of phflthymidineincorporation observed in the presence ofEGF alone. VSMC from SHR (D) were incu-bated for 16 hours in the presence of EGFalone and for 24 hours in the presence of bothagents. Results are expressed as percentstimulation of maximal rates of p-fjthymldineincorporation observed in the presence ofboth EGF and TGF-01. The data points arethe mean±SEM values obtained from sixexperiments.

ties of TGF-01 to activate TGF-/31 gene transcription inVSMC from the two strains. Previously, Van Ob-berghen-Schilling and coworkers27 demonstrated thatTGF-/31 induces its own mRNA levels in a number ofcell lines.

When quiescent VSMC from WKY rats were exposedto TGF-/31, there was a time-dependent increase inTGF-/31 mRNA levels (major transcript, 2.5 kb; minortranscript, 1.9 kb); maximal induction (twofold to three-fold) occurred after 24 hours (Fig 5A). In contrast,when VSMC from SHR were exposed to TGF-/31, therewas an approximately 20% to 50% reduction in mRNAlevels for TGF-/31 after 24 hours compared with levelsin quiescent cells. To examine whether this differentialexpression of TGF-01 mRNA between the strains wasdue to alterations in the promoter region of the TGF-/31gene,28-29 quiescent VSMC were also exposed to phorbolmyristate acetate (PMA). This phorbol ester has previ-ously been shown to stimulate TGF-/31 gene expressionin a manner similar to that of TGF-/31 via discretepromoter DNA sequences that bind the transcriptionfactors AP-1 and Spl.29 Similar levels of TGF-01mRNA were attained in VSMC from SHR and WKYrats after 16 hours of exposure to this activator ofprotein kinase C (Fig 5B). Furthermore, when VSMCfrom SHR were exposed to either bFGF or PDGF-BB,the mRNA levels for TGF-/31 were elevated to the sameextent as observed in VSMC from WKY rats (Fig 6).Increases in TGF-/31 mRNA were already apparent 6hours after exposure to these growth factors; maximalinduction occurred between 6 and 16 hours.

Receptor Subtypes for TransformingGrowth Factor-01

Although we have previously shown that the totalnumber of receptor sites for TGF-/31 does not differ onVSMC from SHR and WKY rats,6 differences in theexpression of receptor subtypes could be responsible forits differential affects on VSMC growth, as has beenreported by Boyd and Massague'30 in mink lung epithe-lial cells; it could also contribute to differences inTGF-/31 autoinduction. When [125I]TGF-/31, bound toserum-deprived VSMC from SHR and WKY rats, wascovalently cross-linked with disuccinimidyl suberate andthe ligand-receptor complexes subjected to SDS-poly-acrylamide gel electrophoresis and autoradiogTaphy,three major bands were detected: Mr =53 kd, =75 kd,and =300 kd, corresponding to type I, type II, and typeIII receptor subtypes, respectively (Fig 7). At the con-centrations of [ I]TGF-£1 used in the binding studies(150 pmoI/L), cross-linking was greatest to the high-molecular-weight type III proteoglycan TGF-/3 bindingprotein and least to the type II TGF-/3 receptor. Therewas no apparent difference in the electrophoretic mo-bilities of the three TGF-0 receptor subtypes.

DiscussionThis study demonstrates that TGF-/31 differentially

regulates growth factor-stimulated proliferation ofVSMC from SHR and WKY rats. Both the rate ofproliferation and cell density at which quiescence isachieved by VSMC from WKY rats are reduced by thepresence of TGF-/31, whereas in VSMC cultures fromSHR enhancement in both growth parameters occurs.

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Agrotis et al TGF-/31 Regulation of Proliferation and Gene Expression 597

WKY

FIG 3. Bar graphs show effects of transforming growth factor-/31(TGF-01) on growth factor-stimulated vascular smooth musclecell proliferation. Cells ( " I x i o 4 ) from Wistar-Kyoto (WKY) rats(A) or spontaneously hypertensive rats (SHR) (B) were stimu-lated with the individual growth factors, epidermal growth factor(EGF; 50 ng/mL), basic fibroblast growth factor (bFGF; 25ng/mL), platelet-derived growth factor (PDGF)-AA (400 ng/mL),PDGF-AB (200 ng/mL), or PDGF-BB (200 ng/mL) either alone(closed bars) or In the presence (hatched bars) of TGF-/31 (1ng/mL). Cell numbers were then determined after 7 days usinga Coulter counter. Results represent mean±SEM values ob-tained from three to six experiments.

These differential effects of TGF-/31 on VSMC from thetwo strains are not apparently a consequence of differ-ences in TGF-/3 receptor subtype expression; rather, itwould appear that an alteration in intracellular signal-ing mechanisms could account for these opposing ac-tions of TGF-^1. These observations, together with ourfinding that tyrosine kinase-activating growth factorsare equipotent in their ability to elevate TGF-/31mRNA levels, suggest that the observed differences inthe growth properties of VSMC from SHR and WKYrats may be mediated through the production of endog-enous TGF-/31.

Despite intensive investigation, the mechanisms bywhich TGF-/31 elicits its diverse array of biologicalresponses remain unknown.7-8 The effects of TGF-/31appear to be mediated by three cell surface receptorstermed types I, II, and III with apparent molecularweights of =55 kd, -15 kd, and =280 kd, respectively.31

Selective loss of the type I and/or type II TGF-/3receptors has been associated with the inability ofTGF-/31 to inhibit the proliferation of some cell types.32

Such a mechanism does not account for the lack ofTGF-/31 action to inhibit growth factor-stimulated pro-liferation of VSMC from SHR. We have previouslydemonstrated that VSMC from SHR and WKY ratspossess similar numbers of high-affinity TGF-/3 recep-tors.6 They also express all three TGF-/3 receptor sub-

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FIG 4. Line graphs show time course of action of transforminggrowth factor-01 (TGF-/91) on epidermal growth factor (EGF)-stimulated vascular smooth muscle cell proliferation. Vascularsmooth muscle cells from Wlstar-Kyoto (WKY) rats (A) or spon-taneously hypertensive rats (SHR) (B) were exposed to 50ng/mL EGF alone (T) or in the presence of 1 ng/mL TGF-01 (a)for 22 days. Cell numbers were determined at the indicatedtimes using a Coulter counter. The data points are themean±SEM values obtained from three experiments.

types in roughly similar proportions, as determined byaffinity cross-linking studies. Most probably alterationsin events distal to TGF-/3 binding to its receptorsaccount for the differential ability of this growth factorto affect the proliferation of VSMC from SHR andWKY rats. It is possible that key intracellular proteins,such as the retinoblastoma gene product33 or the p34cdc2

kinase,34 thought to be involved in TGF-/3-mediatedinhibition of proliferation, may differ in VSMC fromSHR and WKY rats. Recently it has been demonstratedthat the concentration of G proteins, in particular someof the G,a subtypes, is increased in VSMC from SHR35;enhanced Q{ax has also been associated with increased[3H]thymidine incorporation stimulated by TGF-^1.3*Whether one or more of these processes account for theobserved differences in TGF-01 responses we observedrequires further study.

A general response of many cells in culture afterstimulation with TGF-01 is an increase in TGF-/31mRNA expression.27 Similarly, in VSMC from WKYrats TGF-01 positively regulates its own expression.However, TGF-/31 is ineffective in activating its owngene in VSMC from the SHR. This inability of VSMCfrom SHR to elevate TGF-/31 mRNA levels is specific forTGF-/31 because other growth factors, such as PDGF-BB, bFGF, and PMA, all increase TGF-01 mRNAlevels to a similar extent in VSMC from SHR and WKYrats. Because both PMA and TGF-01 elevate TGF-/31mRNA levels by stimulating gene expression throughthe AP-1 binding site in the TGF-01 gene promoterregion,28 it would appear that signals before stimulation

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598 Hypertension Vol 23, No 5 May 1994

Control 2h 4h 8h 16h 24h

A

bFGF

Q 2 6 16 24 Q 2 6 16 24

TGF-01

IISP D Q F

BB

CONTROL

w s

PMAieh

W 8

TGF-01

IIS

FIG 5. A, Blot shows transforming growth factor-01 (TGF-01)mRNA expression In vascular smooth muscle cells (VSMC) fromspontaneously hypertensive rats (SHR) and Wlstar-Kyoto (WKY)rats. Total RNA was isolated from control, serum-deprived cellsfrom WKY rats and SHR as well as from VSMC incubated withTGF-/31 (1 ng/mL) for periods of 2 to 24 hours. Northern blotanalysis was performed using a specific mP-labeled cDNA probefor TGF-01, as described in "Methods." W and S indicate VSMCfrom WKY and SHR rats, respectively. B, Blot shows TGF-01mRNA levels in VSMC after exposure to phorbol myristateacetate (PMA). TGF-01 mRNA levels were assessed after expos-ing VSMC of WKY (W) rats and SHR (S) to 1 ^niol/L PMA for 16hours. Panels beneath autoradlographs in both A and B repre-sent hybridization to an 18s rflNA probe, indicating similar RNAloading.

through the AP-1 site are responsible for the impairedability of TGF-01 to elevate TGF-/31 mRNA in VSMCfrom SHR. Whether the same intracellular mechanismsalso contribute to the differential ability of TGF-/31 tomodulate cell proliferation in VSMC of SHR and WKYrats remains to be determined.

Exaggerated expression of TGF-^1 mRNA has re-cently been associated with enhanced growth of serum-stimulated VSMC from SHR.16 Our observations sug-gest that the enhanced proliferative ability of these cellsis more likely to be the consequence of the enhancedresponsiveness of the cells to mitogenic stimulation inthe presence of TGF-01. Stimulation of VSMC fromSHR and WKY rats to enter the mitotic cell cycle byPDGF-BB or bFGF was associated with similar in-creases in TGF-/31 mRNA levels. Despite the essen-tially similar elevations in TGF-/31 mRNA levels inthese circumstances, enhanced replication of VSMCfrom SHR compared with those of WKY rats was stillobserved in the presence of these growth factors. Ourobservations raise the possibility that endogenouslybiosynthesized and activated TGF-^1 may be responsi-ble for the different growth properties of VSMC fromthe two rat strains. Replicating VSMC are known tosecrete a variety of proteases, including plasminogen

FIG 6. Blots show growth factor-stimulated transforming growthfactor-01 (TGF-/J1) mRNA expression in vascular smooth musclecells. Control and quiescent cells (Q) (^SxiO6) from Wistar-Kyoto (WKY) rats or spontaneously hypertensive rats (SHR) wereincubated with either (A) basic fibroblast growth factor (bFGF; 25ng/mL) or (B) platelet-derived growth factor (PDGF)-BB (200ng/mL) for the indicated times in hours; total RNA was thenextracted and Northern blot analysis performed using a specificMP-labeled cDNA probe for TGF-^1. Panels beneath autoradio-graphs in both A and B represent hybridization to an 18s rRNAprobe, indicating similar RNA loading.

activator37 and collagenase.38 Furthermore, in serum-containing medium both proteinase inhibitors and anantisense oligonucleotide to TGF-/31 have recently beenshown to attenuate DNA synthesis in VSMC fromSHR.17 In this instance serum-derived plasmin mayhave also contributed to the activation of latent TGF-)31.39 In the future it will be of interest to determinewhether a significant proportion of the latent TGF-/31that accumulates when VSMC are cultured in serum-free medium is activated during exposure to growthfactors.

In summary, our findings indicate that TGF-/31 dif-ferentially affects the proliferation of vascular smoothmuscle from SHR and WKY rats. Our observation thatTGF-/31 mRNA levels are elevated to a similar extent

SHR WKY

HI - ^ 300kda

Type II

Type I

UrdabeOedTGF-01 — ~-

FIG 7. Autoradiograph shows transforming growth factor-/3(TGF-0) receptor expression on vascular smooth muscle cells.TGF-/3 receptor subtypes on vascular smooth muscle cells wereestimated after [ia1]TGF-/51 binding and cross-linking with disuc-cinimidyl suberate to membrane proteins. Receptor-ligand com-plexes were separated by sodium dodecyl sulfate-polyacrylamldegel electrophoresis and visualized by autoradiography as de-scribed in "Methods." A representative autoradiograph is shown,indicating the presence of type I, type II, and type III TGF-/3receptors. SHR indicates spontaneously hypertensive rats; WKY,Wlstar-Kyoto rats.

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Agrotis et al TGF-01 Regulation of Proliferation and Gene Expression 599

when VSMC from SHR and WKY rats are stimulatedby growth factors raises the possibility that alterations inTGF-/31 signaling pathways account for the differencesin growth properties of VSMC from the two rat strains.

AcknowledgmentsThis study was supported by a grant-in-aid from the Na-

tional Heart Foundation of Australia. We thank Zymogeneticsfor their generous gift of PDGF-BB. We also thank JulieSimpson for assisting with the preparation of this manuscript.

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A Agrotis, J Saltis and A Bobikhypertensive rats.

Transforming growth factor-beta 1 gene activation and growth of smooth muscle from

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