Proc. Natd. Acad. Sci. USAVol. 85, pp. 1524-1528, March 1988Cell Biology

Expression and developmental control of platelet-derived growthfactor A-chain and B-chain/Sis genes in rat aortic smoothmuscle cells

(gene family/RNA stability/arterial remodeling/atherosclerosis)

MARK W. MAJESKY*, EARL P. BENDITr, AND STEPHEN M. SCHWARTZDepartment of Pathology, SJ-60, University of Washington, Seattle, WA 98195

Contributed by Earl P. Benditt, October 8, 1987

ABSTRACT Cultured arterial smooth muscle cells (SMC)can produce platelet-derived growth factor (PDGF)-like mol-ecules. This property raises the possibility that SMC-derivedPDGFs function as autocrine/paracrine regulators in theformation and maintenance of the artery wall. In this study wehave asked if levels ofmRNAs directing synthesis of PDGF aremodulated in aortic SMC during postnatal development. Wereport here that genes encoding PDGF A- and B-chain pre-cursors are expressed at similar low levels in intact aortas fromnewborn and adult rats. Marked differences in regulation oftranscript abundance of these genes were revealed when aorticSMC were grown in cell culture. PDGF B-chain transcriptsaccumulated in passaged newborn rat SMC but not adult ratSMC, whereas PDGF A-chain RNA was found in comparableamounts in SMC from both age groups. Similarly, SMC fromnewborn rats secreted at least 60-fold more PDGF-like activityinto conditioned medium than did adult rat SMC. PDGFB-chain transcripts in newborn rat aortic SMC are short-livedand increased 5-fold by 3 hr after treatment with cyclohexi-mide. In contrast, PDGF A-chain transcripts are more stable,and their constitutive levels were generally unaffected bycycloheximide. These results show that PDGF A- and B-chaingenes are transcribed in the normal rat aorta and provideevidence for age-related change in the control of PDGFB-chain gene expression in aortic SMC. Independent regula-tion of transcript levels in cultured SMC leaves open thepossibility that PDGFs of different composition (AA, AB, BB)play different roles in normal function of the artery wall.

The discovery that arterial smooth muscle cells (SMC) inculture produce peptide growth factors thought to be re-quired for their own growth (1-4) provides new perspectiveson control of SMC proliferation in the intact artery wall.Previously, a role for platelet- and inflammatory cell-derivedmitogens in initiation of SMC growth has been emphasized(5), particularly for repair of arterial wounds. However,SMC replication in normal development (6), medial thicken-ing in hypertension (7), and certain focal proliferations in thearterial intima (8-10) occur in the absence of thrombosis orinflammatory cell responses. These forms of SMC prolifer-ation might be initiated by growth factors produced withinthe artery wall itself. Furthermore, SMC migration andreplication at sites of arterial injury or advancing atheroscle-rotic plaques might be enhanced by endogenously producedgrowth factors acting in concert with those released fromplatelets and adherent leukocytes.The concept that SMC replication in vivo depends upon

growth factors produced within the intact vessel wall issupported by the observations that in vitro both endothelialcells (11, 12) and SMC secrete molecules that structurally

and functionally resemble platelet-derived growth factor(PDGF) from human platelets. Seifert et al. (1) showed thataortic SMC from newborn rats released PDGF-like activityinto conditioned medium and had greatly reduced numbersof PDGF receptors on their surface. Nilsson et al. (3)reported that primary cultures of aortic SMC from adult ratsalso produce a PDGF-like molecule. However, they foundthat cells from adult animals, unlike SMC from newbornrats, did not maintain PDGF production during subsequentpassage. Likewise, significant production of PDGF-like mol-ecules by subcultured adult rat SMC was not observed bySeifert et al. (1). These data suggest that stable production ofPDGF by aortic SMC in culture reflects a change in smoothmuscle properties in vivo that occurs during normal postna-tal development.

Central among the questions raised by these observationsis whether aortic SMC in vivo transcribe the genes forPDGF. In the studies reported here, we have examinedexpression of the genes for PDGF A- and B-chain precursorsin intact aortas and passaged SMC from newborn and adultrats. We show that (i) PDGF A- and B-chain genes areexpressed at low levels in newborn and adult rat aortas, (ii)PDGF B-chain transcripts accumulate in subcultured SMCfrom newborn rats but not from adult rats, and (iii) PDGF A-and B-chain transcripts are differentially regulated at thelevel of RNA stability. The results suggest that cells of theintact rat aorta normally transcribe PDGF A- and B-chaingenes. Transcript levels for PDGF A- and B-chain precur-sors are independently controlled in rat aortic SMC, raisingthe possibility that distinct combinations of PDGF A and Bchains may be produced in response to different signals andmay play different roles in the formation and maintenance ofthe artery wall.

MATERIALS AND METHODSCell Culture. Thoracic aortas from 12-day-old (newborn)

or 3-month-old (adult) male Wistar rats were removed andstripped of endothelium and adventitia, and medial SMCwere obtained by collagenase and elastase digestion (13). Rataortic SMC were routinely grown in Waymouth's mediumsupplemented with 10%o adult bovine serum (HyClone, Lo-gan, UT) and were used between the fourth and twelfthpassage. Bovine aortic endothelial cells and human osteo-sarcoma cells (U-20S) were grown in Waymouth's mediumcontaining 5% fetal bovine serum (HyClone). For isolationof total cellular RNA, cells were grown in 125-cm2 dishes(Falcon) and harvested 1-3 days after reaching confluence.RNA Isolation. Total cellular RNA was isolated from rat

aortas by the NaDodSO4/proteinase K procedure (14). Ves-

Abbreviations: PDGF, platelet-derived growth factor; SMC, smoothmuscle cell(s).*To whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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sels were rapidly excised, rinsed with ice-cold phosphate-buffered saline (pH 7.4), stripped of endothelium and periad-ventitial tissues, and snap-frozen in liquid nitrogen. Frozentissue (0.2-0.5 g) was ground to a fine powder with a mortarand pestle cooled by liquid nitrogen, transferred to 3 ml ofisolation buffer [10 mM Tris-HCl, pH 7.4/0.15 M NaCl/1%(wt/vol) NaDodSO4/10 mM vanadyl-ribonucleoside com-plex/0.1% Antifoam A (Sigma)], and homogenized with aPolytron at high speed. Proteinase K digestion and RNApurification were carried out as described (14). RNA con-centrations were determined by spectrophotometry. DNAcomprised between 1% and 3% of the final nucleic acidsisolated, as determined by fluorimetric assay (15). Totalcellular RNA from cultured cells was purified by the guani-dinium isothiocyanate/LiCl procedure as described (16).Recovery of total cellular RNA by this method was between70%o and 80% as estimated by the amount of tritiatedribosomal RNA tracer recovered in the final precipitate.RNA Transfer Blot. RNA samples were electrophoresed

through 1.2% agarose gels submerged in 2.2 M formalde-hyde/10 mM sodium phosphate, pH 7.2; transferred to anylon membrane (Zeta Probe, BioRad) in 3 M NaCl/0.3 Msodium citrate; and baked at 800C for 2 hr. Membranes werehybridized in 50% (vol/vol) formamide/0.25 M sodium phos-phate, pH 7.2/7% NaDodSO4/0.25 M NaCl/1 mMEDTA/10% (wt/vol) polyethylene glycol (17) containing 100,ug of salmon sperm DNA per ml at 42°C for 24 hr with 1-2x 106 cpm of 32P-labeled nick-translated DNA probes perml. Blots were washed at 60°C in two changes of 0.045 MNaCl/0.0045 M sodium citrate, pH 7.0/0.1% NaDodSO4 for20 min each.DNA Probes. DNA probes used forRNA blot-hybridization

analysis were as follows: for PDGF A-chain, a 1.3-kilobase(kb) EcoRI human cDNA fragment released from plasmid D1and kindly provided for these studies by Betsholtz and Heldinand coworkers (18); for PDGF B-chain, a 1.2-kb Pst Ifiagment from plasmid pv-sis containing v-sis oncogene se-quences as described (19); for actin, a 1.3-kb Pst I bovinecDNA fragment released from pBA-1 (20) (it recognizessequences common to both muscle-specific and nonmuscleactins); for Myc, a 350-base-pair Pst I fragment (exon 2) of themouse Myc gene, generously provided by David Morris(MYC in human and Myc in rat gene nomenclature).PDGF Radioreceptor Assay. Levels of PDGF-like mole-

cules in SMC-conditioned medium were estimated by radio-receptor assay as described (21). Cultures of diploid humanfibroblasts were incubated with various amounts of condi-tioned medium (newborn SMC) or 10-fold concentratedconditioned medium (adult SMC) for 3 hr at 4°C, rinsed oncewith cold phosphate-buffered saline, and then incubatedwith 125"-labeled PDGF (125I-PDGF) at 0.5 ng/ml in bindingmedium for 1 hr at 4°C. The cultures were rinsed three timeswith cold binding rinse, and cell-bound 125I-PDGF wassolubilized with 2% Triton X-100. Purified human plateletPDGF (22) was used as the standard.

RESULTSCell Morphology. Cultured aortic SMC from newborn rats

were predominantly epithelioid in shape and formed a mono-layer of cells at confluence (Fig. 1) as described (13). Incontrast, aortic SMC from adult rats were more strap-like inshape, showed extensive overlapping, and organized intomultilayered "hills and valleys" at confluence. Because ofthese differences in morphology, the possibility that new-born aortic SMC cultures were contaminated with endo-thelial cells was considered. Newborn rat SMC cultureswere negative for the endothelial cell-specific marker proteinvon Willebrand factor by two criteria: (i) immunocytochem-ical staining using an antibody that recognized antigen on rat

FIG. 1. Morphology of rat aortic SMC in vitro. Newborn (Up-per) and adult (Lower) rat aortic SMC were grown to confluence andphotographed by phase-contrast light microscopy. (x 180.)

aortic endothelial cells and (ii) RNA-transfer-blot analysisusing a cloned von Willebrand factor cDNA fragment (23)that detected an 8.5-kb transcript in both rat and humanendothelial cells. In addition, newborn rat aortic SMC dis-played low but detectable levels of the 1.6-kb transcript formuscle-specific a-actin, similar to the low levels detected inpassaged adult rat SMC (data not shown).

Expression of PDGF A- and B-Chain Genes in Vivo and inCulture. Multiple transcripts homologous to PDGF A chainwere detected at similar, low levels in total cellular RNAisolated directly from newborn and adult rat aortas (Fig. 2A,lanes a and c). Somewhat higher levels of expression (about3-fold per jig of total RNA) were observed in subculturedaortic SMC derived from newborn and adult rats (Fig. 2A,lanes b and d). Consistently more intense signals wereobtained for the 2.3- and 2.9-kb bands in RNA from newbornrat SMCs (Fig. 2A, lane b), whereas the 1.7-kb band wasmore abundant in adult rat SMC cultures (Fig. 2A, lane d)when compared on a per-Iug-of-total-cellular-RNA basis.PDGF A-chain transcripts in cultured rat SMC, for compar-ison, were lower per ,ug of total cellular RNA by factors ofabout 6-8 than in the human osteosarcoma cell line U-20Sas estimated by scanning densitometry of autoradiographs ofRNA transfer blots and serial-dilution dot blots. Bovineaortic endothelial cell RNA produced low levels of PDGFA-chain hybridization, making quantitation of relative signalintensities unreliable.The PDGF B-chain gene was also expressed at low levels

in intact aortas of newborn and adult rats (Fig. 2B, lanes aand c). In contrast, levels of a single, 3.5-kb PDGF B-chaintranscript were at least 20-fold higher in cultured SMC fromnewborn rats compared with levels in the intact aorta (Fig.2B, lane b). Adult rat SMC cultures contained extremely lowor undetectable levels of PDGF B-chain RNA (Fig. 2B, lane

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A newborn adult0L- L

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FIG. 2. (A) RNA transfer blot analysis of PDGF A-chaintranscripts in rat aortic smooth muscle. Total cellular RNA (15 jug)isolated from aortas (aor) of newborn (lane a) and adult (lane c) ratsand from cultured (cltr) aortic SMC obtained from newborn (lane b)and adult (lane d) rats were analyzed. The RNA samples were runtogether on a 1.2% agarose gel, transferred to a single nylonmembrane, and hybridized with a 32P-labeled PDGF A-chain cDNAprobe. The membrane was exposed to film for 36 hr. For compari-son, 10 jig of total cellular RNA from confluent cultures of a humanosteosarcoma cell line (U-20S, lane e) and 15 jig of total RNA frombovine aortic endothelial cells (bae, lane f) were examined inparallel. The indicated sizes of PDGF A-chain transcripts wereestimated from the positions of 28S and 18S ribosomal RNA bands.(B) RNA transfer blot analysis of PDGF B-chain transcripts in rataortic smooth muscle. The same nylon membrane shown in A(except lane f) was rehybridized with a 32P-labeled PDGF B-chain(v-sis) probe and exposed to film for 36 hr. Total cellular RNA fromconfluent cultures of bovine aortic endothelial cells (10 ,ug) waselectrophoresed and hybridized as described above and exposed tofilm for 24 hr. The size of a single 3.5-kb PDGF B-chain rattranscript was estimated from the positions of the 28S and 18Sribosomal RNA bands and was about 200 bases shorter than thehuman and bovine transcripts.

d). Scanning densitometric analysis of RNA transfer blotsand serial-dilution dot blots indicated about 60-fold higherlevels of PDGF B-chain RNA in newborn versus adult ratSMC cultures. The abundance of PDGF B-chain transcriptsper ,ug of total cellular RNA in newborn rat SMC cultureswas =30-40% of that in bovine aortic endothelial cells (Fig.2B, lane f; note the different amounts of RNA loaded andexposure times) and 4- to 6-fold higher than in the humanosteosarcoma cell line U-20S (Fig. 2B, lane e). It should benoted that newborn rat SMC cultures examined had beenmultiply passaged. Primary cultures of aortic SMC fromnewborn rats contained lower levels of PDGF B-chaintranscripts than did subcultured cells by factors of 6-8 anddisplayed a mixture of epithelioid and spindle-shaped cells(13). Upon serial passage, PDGF B-chain RNA increased toreach the levels shown in Fig. 2B, lane b, and cultures moreuniformly displayed the epithelioid morphology. Among fouradditional isolates of passaged newborn rat SMC examined,PDGF B-chain RNA levels ranged from 1/2 to 1/8th thatshown in Fig. 2. Adult SMC were spindle-shaped andcontained no detectable PDGF B-chain RNA over the sameserial passage schedule.

Effects of Cycloheximide on PDGF B-Chain RNA. Toexamine the possibility that low levels of PDGF B-chainRNA in adult rat SMC were due to a short-lived negativeregulator, cells were treated with cycloheximide for 3 hrprior to isolation of total cellular RNA. No PDGF B-chaintranscripts were detected in the treated cultures (Fig. 3A,lanes d and e). The effectiveness of cycloheximide in this

B B S2.9.,-2.3l * -b1.7

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a b

FIG. 3. (A) Effect of cycloheximide and anisomycin on PDGFB-chain transcript levels in rat aortic SMC. Confluent cultures ofnewborn rat aortic SMC (lanes a-c) and adult rat aortic SMC (lanesd-f) were exposed to cycloheximide (10 ug/ml) (lanes b and e),anisomycin (10 ug/ml) (lanes c and f), or vehicle control (30 ,l ofWaymouth's medium) (lanes a and d) for 3 hr. Total cellular RNAwas isolated, electrophoresed (15 ug per lane), and transferred tonylon membrane, and replicate blots were hybridized with 32P-labeled PDGF B-chain (v-sis) (Upper) or Myc DNA (Lower) probes.Membranes were exposed to film overnight. The indicated tran-script sizes were estimated from the positions of the 28S and 18Sribosomal RNA bands. (B) Effect of cycloheximide on PDGFA-chain transcript levels in newborn rat aortic SMC. Confluentcultures of newborn rat SMC were exposed to cycloheximide (10j1g/ml) (lane b) or vehicle control (lane a) for 3 hr. Total cellularRNA (15 Ag) was electrophoresed, transferred to nylon membrane,and hybridized with 32P-labeled PDGF A chain (Upper) or MycDNA (Lower) probes.

experiment was indicated by 5- to 6-fold increases in MycRNA levels, similar to findings reported for a number ofother cell types (24).Cycloheximide treatment of newborn rat SMC cultures

produced 4- to 5-fold increases over basal levels of PDGFB-chain transcripts (Fig. 3A, lanes a and b). Similarly, MycRNA levels were increased 6-fold. Both anisomycin (Fig.3A, lanes c and f) and puromycin, inhibitors of proteinsynthesis structurally unrelated to cycloheximide, produced3- to 5-fold increases in PDGF B-chain transcripts in new-born rat SMC cultures. In contrast, constitutive levels ofPDGF A-chain RNA were not significantly altered in SMCof either donor age group after a 3-hr exposure to cyclohex-imide (Fig. 3B, lanes a and b).PDGF B-Chain RNA Is an Unstable Transcript. To exam-

ine the stability of PDGF B-chain transcripts in newborn rataortic SMC, RNA synthesis was inhibited with actinomycinD and total cellular RNA was isolated at subsequent inter-vals up to 4 hr. PDGF B-chain RNA rapidly disappearedafter addition of actinomycin D with an estimated half-life of40-50 min (Fig. 4). Simultaneous addition of both cyclohex-imide and actinomycin D prolonged the apparent half-life ofPDGF B-chain RNA to more than 2 hr in newborn rat aorticSMC (Fig. 4 Inset). In contrast, PDGF A-chain transcripts inthe presence of actinomycin D were considerably morestable with an apparent half-life of -4 hr (not shown).

Production of PDGF-Like Molecules. The striking differ-ence in PDGF B-chain transcript levels between newbornand adult rat aortic SMC in vitro (Fig. 2) was paralleled bysimilar differences in secretion of PDGF-like molecules.Culture medium conditioned by the same newborn and adultSMC isolates from which RNA was extracted and analyzedby RNA blot hybridization (Fig. 2) was examined for itsability to compete with '25I-PDGF for binding to PDGFreceptors. Medium conditioned by newborn rat aortic SMCcontained at least 60-fold more PDGF-like activity than didmedium conditioned by adult SMC (Table 1), similar topreviously published results (1). Thus, despite similar levelsof PDGF A-chain RNA, newborn and adult aortic SMCdiffer greatly in secretion of PDGF-like molecules detectedin the assay used. This suggests that either PDGF B chain isitself secreted or facilitates secretion of the A chain or that

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FIG. 4. Effect of actinomycin D on PDGF B-chain transcriptlevels in newborn rat aortic SMC. Confluent cultures of newborn rataortic SMC were exposed to actinomycin D (4 ,ug/ml) in the absence(-) or presence (+) of cycloheximide (10 Zg/ml) for the indicatedtimes. Total cellular RNA (15 ,ug) was electrophoresed, transferredto a nylon membrane, and hybridized with a 32P-labeled PDGFB-chain (v-sis) probe. The membrane was exposed to film for 24 hr.The autoradiograph shown in the insert was analyzed by scanningdensitometry and expressed in arbitrary units as a percentage of thesignal obtained at 0 min. Values shown are for SMC cultures treatedwith actinomycin D in the presence (o) and absence (e) of cyclo-heximide.

PDGF A-chain RNA is under translational or posttransla-tional control in adult rat SMC.

DISCUSSIONWe have asked if cells in the normal artery wall express thegenes for PDGF A- and B-chain precursors and have exam-ined certain features of the developmental pattern of thisexpression. We observed low levels of transcripts from bothgenes in RNA extracted directly from newborn and adult rataortas. In contrast, PDGF B-chain transcripts accumulatedin passaged newborn SMC in vitro. Under identical condi-tions in culture, however, adult aortic SMC contained unde-tectable levels of PDGF B-chain RNA. PDGF A-chaintranscripts were found at nearly equal levels in culturedSMC from both age groups. We have interpreted theseresults to indicate that capacity to express the PDGF B-chain gene, as evidenced by transcript accumulation incultured cells, is under developmental control in rat aorticSMC. Whether SMC differ at the PDGF B-chain gene itself,at controlling sequences elsewhere in the genome, in signaltransduction pathways responsive to cell culture conditions,or in other ways is not yet clear. Age-related differences inPDGF B-chain gene expression are consistent with previousobservations of secretion of PDGF-like activity by passagednewborn rat (but not adult rat) aortic SMC in vitro (1).However, PDGF A-chain transcript levels displayed no suchdevelopmental constraint. In addition, PDGF A- and B-chain transcripts differed significantly at the level of RNAstability in cultured SMC. Production ofPDGFs in the intactartery may play important roles in the formation, repair, and

Table 1. Production of PDGF-like molecules by rat aortic SMC

PDGF-like activity,Donor age ng per equiv.Newborn 0.77 ± 0.31Adult <0.014*

The concentration of PDGF-like molecules in culture mediumconditioned for 24 hr by newborn or adult rat aortic SMC wasdetermined by radioreceptor assay (21). One equivalent equals 1 mlof medium conditioned by 1 x 106 cells for 48 hr. Values shown aremeans (± SEM) of three determinations.*Adult values were below reliable limits of detection for the assay.

metabolic maintenance of normal vessel wall. Independentexpressions of PDGF A- and B-chain genes in aortic SMCraise the possibility that PDGFs of different subunit compo-sition may contribute in different ways to these activities.Developmental Regulation of PDGF B-Chain Gene Expres-

sion. Differences in expression of the PDGF B-chain gene, aswell as differences in morphology between SMC culturedfrom newborn and adult rat aortas, may be related to thespecial properties of rat aortic SMC in vivo during the firstfew weeks after birth. This period is one of active SMCproliferation, differentiation, and extensive arterial remod-eling (6). For example, during the first 14 days postpartum,aortic SMC number increases 2.5-fold, wall thickness dou-bles, collagen and elastin content increases >3-fold, andadditional elastic lamellae are formed (25, 26). Thesechanges in wall structure are regarded as adaptations torapidly increasing blood pressure (i.e., rising >50 mm Hg inthe first 2 weeks) (27). Aortic remodeling during this periodmay require many of the same abilities (e.g., migration,proliferation, synthesis of extracellular matrix molecules)needed at earlier stages of development, perhaps includingproduction of PDGF-like molecules.However, low levels of expression of PDGF A- and

B-chain genes in newborn rat aorta argue that the level of, orthe number of cells involved in, this activity in vivo is muchlower than would be predicted based on analysis of PDGFB-chain RNA levels and production of PDGF-like moleculesby newborn rat SMC in culture (1). This might mean thatlevels of PDGF B-chain RNA observed in passaged cellsrepresent stimulation of gene expression in vitro. Expressionof the PDGF B-chain gene in large-vessel endothelial cellshas previously been shown to be activated in response to cellculture conditions (28). Alternatively, accumulation ofPDGF B-chain RNA in passaged newborn rat SMC couldrepresent amplification of a subpopulation of cells engagedin production of PDGF in the developing aorta. Such cellsmight have a proliferative advantage over more fully differ-entiated SMC. Consistent with this possibility, we haveobserved that primary cultures of aortic SMC from newbornrats contain low levels of PDGF B-chain RNA, similar tointact aorta, and display a mixture of epithelioid and spindle-shaped cells as noted (13). Upon serial passage, the abun-dance of PDGF B-chain transcripts increased 6- to 8-fold(per 1Lg of total cellular RNA) to reach levels reported herefor passaged SMC, and cultures more uniformly displayedthe epithelioid morphology.PDGF A-chain RNA levels did not vary significantly be-

tween SMC from newborn (growing) and adult (nongrowing)aorta. Likewise, Sejersen et al. found that postmitotic skeletalmyotubes in vitro maintained expression of the PDGF A-chain gene and release of PDGF at levels only slightlydiminished from those in proliferating myoblasts (29). Thesefindings suggest additional, nonmitogenic functions for PDGFin normal arteries (30) and perhaps other tissues. The stimu-lation by PDGF of (i) transcription of a limited number ofgenes (31), (ii) polyribosome formation and protein synthesis(32), (iii) directed cell movement (33), and (iv) cell survival(34) may be particularly relevant in this respect.

Heterogeneity of SMC in the Adult Artery Wall. Formationof the adult artery wall may involve differentiation of amajority of SMC to mature myocytes that have lost thecapacity to express the PDGF B-chain gene together withretention of a population of SMC with fetal properties (35).Such reserve cells may be important for repair of arterialinjuries. It is intriguing that the luminal surface of regener-ated carotid neointima in adult rats is lined by altered SMCwith a "pseudoendothelial" morphology (36, 37). Perhapsincreased production of PDGF-like activity by carotid in-timal SMC in vitro, which are also epithelioid in shape (4), isdue to these specially adapted SMC. Although this scheme is

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hypothetical, we recently have found that adult carotidintimal SMC, like SMC from newborn rat aorta, transcribethe PDGF B-chain gene in culture, whereas SMC fromuninjured carotid arteries, like those from adult aorta, havevery low or undetectable levels ofPDGF B-chain RNA (38).What stimuli elicit PDGF B-chain gene expression in intimalSMC and if all or only a subpopulation of SMC in injuredarteries can respond to these stimuli are important questionsfor further study.

Rapid Turnover of PDGF B-Chain RNA in Aortic SMC. Ourconclusion that PDGF B-chain RNA is an unstable transcript inaortic SMC is based on the following observations: (i) cyclo-heximide treatment produced 3- to 5-fold increases in PDGFB-chain RNA levels within 3 hr, (it) PDGF B-chain transcriptlevels decayed rapidly after addition of actinomycin D with anapparent half-life of about 40-50 min, and (iii) the combinationof actinomycin D and cycloheximide almost completely pre-vented the decay of PDGF B-chain RNA. Shaw and Kamen(39) recently identified a conserved A + U-rich sequence in the3' untranslated region of human granulocyte-macrophage col-ony-stimulating factor RNA that signals rapid degradation ofthe transcript. They noted that a similar A + U-rich sequence ispresent in human SIS RNA and suggested that SIS transcriptswould be unstable in vivo. The results reported here concurwith this proposal and with the suggestion by Pantazis et al.that cycloheximide stabilizes SIS transcripts in HL-60 cellsinduced to differentiate by phorbol esters (40). However,PDGF A-chain RNA levels were unchanged or only slightlyincreased 3 hr after cycloheximide. Given that either chain canbe assembled into functional homodimers (41, 42) and that thetwo genes are expressed independently (18, 29, 38), it seemslikely that PDGFs of different composition (AA, AB, BB) areproduced by normal cells, including cells of the artery wall.Whether different PDGFs have distinct biological activities ashas been reported recently for the inhibins, a family ofpeptidessimilarly assembled into dimers of various composition bydifferent cell types (43), remains to be seen.

We thank Adam Evans and Isa Werny for technical assistancewith cell culture and RNA isolations, Ronald Seifert and DanielBowen-Pope for PDGF radioreceptor assays, Christer Betsholtz andCarl-Henrik Heldin for providing PDGF A-chain cDNA clone D-1,David Morris for helpful comments and advice, and Virginia Wejakfor preparation of this manuscript. This work was supported byNational Institutes of Health Grants HL-03174, IJL-18945, andHL-07312.

1. Seifert, R. A., Schwartz, S. M. & Bowen-Pope, D. F. (1984)Nature (London) 311, 669-671.

2. Clemmons, D. R. & van Wyk, J. J. (1985) J. Clin. Invest. 75,1914-1918.

3. Nilsson, J., Sjolund, M., Palmberg, L., Thyberg, J. & Heldin,C.-H. (1985) Proc. Nati. Acad. Sci. USA 82, 4418-4422.

4. Walker, L. N., Bowen-Pope, D. F., Ross, R. & Reidy, M. A.(1986) Proc. Nati. Acad. Sci. USA 83, 7311-7315.

5. Ross, R. (1981) Arteriosclerosis 1, 293-311.6. Gerrity, R. G. & Cliff, W. J. (1975) Lab. Invest. 32, 585-600.7. Schwartz, S. M., Campbell, G. R. & Campbell, J. H. (1986) Circ.

Res. 58, 427-444.8. Kim, D. N., Imai, H., Schmee, J., Lee, K. T. & Thomas, W. A.

(1985) Atherosclerosis 56, 169-188.

9. Velican, C. & Velican, D. (1976) Atherosclerosis 23, 345-355.10. Moss, N. S. & Benditt, E. P. (1970) Lab. Invest. 23, 231-245.11. DiCorleto, P. E. & Bowen-Pope, D. F. (1983) Proc. Natl. Acad.

Sci. USA 80, 1919-1923.12. Collins, T., Pober, J. S., Gimbrone, M. A., Jr., Hammacher, A.,

Betsholtz, C., Westermark, B. & Heldin, C.-H. (1987) Am. J.Pathol. 127, 7-12.

13. Gordon, D., Mohai, L. G. & Schwartz, S. M. (1986) Circ. Res. 59,633-644.

14. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory,Cold Spring Harbor, NY).

15. Labarca, C. & Paigen, K. (1980) Anal. Biochem. 102, 344-352.16. Cathala, G., Savouret, J. F., Mendez, B., West, B. L., Karin, M.,

Martial, J. A. & Baxter, J. D. (1983) DNA 2, 329-335.17. Amasino, R. M. (1986) Anal. Biochem. 152, 304-307.18. Betsholtz, C., Johnsson, A., Heldin, C.-H., Westermark, B., Lind,

P., Ureda, M. S., Eddy, R., Shows, T. B., Philpott, K., Mellor,A. L., Knott, T. J. & Scott, J. (1986) Nature (London) 320,695-699.

19. Robbins, K. C., Devare, S. G. & Aaronson, S. A. (1981) Proc.Natl. Acad. Sci. USA 78, 2918-2922.

20. Degen, J. L., Neubauer, M. G., Degen, S. J., Seyfried, C. E. &Morris, D. R. (1983) J. Biol. Chem. 258, 12153-12162.

21. Bowen-Pope, D. F. & Ross, R. (1986) Methods Enzymol. 109,69-100.

22. Raines, E. W. & Ross, R. (1982) J. Biol. Chem. 257, 5154-5160.23. Shelton-Inloes, B., Titani, K. & Sadler, J. E. (1986) Biochemistry

25, 3164-3171.24. Greenberg, M. E. & Ziff, E. B. (1984) Nature (London) 311,

433-438.25. Cliff, W. J. (1967) Lab. Invest. 17, 599-614.26. Olivetti, G., Anversa, P., Melissari, M. & Loud, A. V. (1980) Circ.

Res. 47, 417-424.27. Burlingame, P., Long, J. A. & Ogden, E. (1942) Am. J. Physiol.

134, 473-484.28. Barrett, T. B., Gajdusek, C. M., Schwartz, S. M., McDougall,

J. K. & Benditt, E. P. (1984) Proc. Natl. Acad. Sci. USA 81,6772-6774.

29. Sejersen, T., Betsholtz, C., Sjolund, M., Heldin, C.-H., Wester-mark, B. & Thyberg, J. (1986) Proc. Natl. Acad. Sci. USA 83,6844-6848.

30. Barrett, T. B. & Benditt, E. P. (1987) Proc. NatI. Acad. Sci. USA84, 1099-1103.

31. Cochran, B. H., Reffel, A. C. & Stiles, C. D. (1983) Cell 33,939-947.

32. Cochran, B. H., Lillquist, J. S. & Stiles, C. D. (1981) J. Cell.Physiol. 109, 429-438.

33. Grotendorst, G., Seppa, H. E. J., Kleinman, H. K. & Martin, G.(1981) Proc. Natl. Acad. Sci. USA 78, 3669-3672.

34. Scher, C. D., Young, S. A. & Locatell, K. L. (1982) J. Cell.Physiol. 113, 211-218.

35. Schwartz, S. M., Reidy, M. A. & Clowes, A. W. (1985) Ann. N. Y.Acad. Sci. 454, 292-304.

36. Schwartz, S. M., Stemerman, M. B. & Benditt, E. P. (1975) Am. J.Pathol. 81, 15-42.

37. Clowes, A. W., Clowes, M. M. & Reidy, M. A. (1986) Lab. Invest.54, 295-301.

38. Majesky, M. W., Reidy, M. A. & Schwartz, S. M. (1987) Fed.Proc. Fed. Am. Soc. Exp. Biol. 46, 976 (abstr.).

39. Shaw, G. & Kamen, R. (1986) Cell 46, 659-667.40. Pantazis, P., Sariban, E., Kufe, D. & Antoniades, H. N. (1986)

Proc. Natl. Acad. Sci. USA 83, 6455-6459.41. Heldin, C.-H., Johnsson, A., Wennergren, S., Wernstedt, C.,

Betsholtz, C. & Westermark, B. (1986) Nature (London) 319,511-514.

42. Stroobant, P. & Waterfield, M. D. (1984) EMBO J. 3, 2963-2967.43. Ling, N., Ying, S. Y., Ueno, N., Shimasaki, S., Esch, F., Hotta,

M. & Guillemin, R. (1986) Nature (London) 321, 779-782.

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