Vol. 2, 1 4 5 1 54. 51,1r(h I 991 cell Gross’th & Ditlerentiation 145

Received 10/1/90.1 To svhotii requests for reprints should 1)5’ addressed.

Epidermal Growth Factor Receptor, Platelet-derivedGrowth Factor Receptor, and c-erbB-2 ReceptorActivation All Promote Growth but HaveDistinctive Effects upon Mouse MammaryEpithelial Cell Differentiation

Daniela Taverna,1 Bernd Groner, and Nancy E. Hynes

Friedrk h Mk’s her Institute, P.O. Box 2543, 4002 Basel, Switzerland

Abstract

Three different receptor tyrosine kinases, epidermalgrowth factor (EGF), c-erbB-2/neu, and platelet-derivedgrowth factor (PDGF) receptors, have been found to bepresent in the mouse mammary epithelial cell lineHC1 1. We have investigated the consequences ofreceptor activation on the growth and differentiation ofHC1 1 cells. HC1 1 cells are normal epithelial cellswhich maintain differentiation-specific functions.Treatment of the cells with the lactogenic hormonesglucocorticoids and prolactin leads to the expression ofthe milk protein fl-casein. Activation of EGF receptorhas a positive effect on cell growth and causes the cellsto become competent for the lactogenic hormoneresponse. HC1 1 cells respond optimally to thelactogenic hormone mixture and synthesize high levelsof $-casein only if they have been kept previously in amedium containing EGF. Transfection of HC1 1 cellswith the activated rat neuT receptor results in theacquisition of competence to respond to the lactogenichormones even if the cells are grown in the absence ofEGF. The activation of PDGF receptor, through PDGF-BB, also stimulates the growth of HC1 1 cells. Cells keptonly in PDGF do not become competent for lactogenichormone induction. The results show that activation ofthe structurally related EGF and c-erbB-2/neureceptors, but not the PDGF receptor, allows the HC1 1cells to subsequently respond optimally to lactogenichormones.

Introduction

A number of polypeptides function as growth or differ-entiation factors and niediate their action through cellsurface receptors that have intrinsic tyrosine kinase activ-ity. Different members of the receptor tyrosine kinasefamily have been identified and, based upon sequenceand structural similarities, have been grouped into foursubclasses (1). Although all receptors have intrinsic tyro-sine kinase activity, it is assumed that activation of eachwill ultimately result in distinct biological effects. Muchof our current knowledge of the intracellular events thatfollow ligand binding and receptor activation is based onstudies of in vitro cultured fibroblasts. Different growth

factor receptors are present on fibroblasts, and in mostcases their activation leads to DNA replication (2). Thesestudies have proved useful to identify cellular eventsactivated by a particular receptor and their importancefor the mitogenic stimulus. Cell culture systems in whichadditional biological consequences of receptor kinaseactivation can be measured may prove useful for adeeper understanding of the various intracellular signaltransduction pathways. Epithelial cells that have the abil-ity to differentiate provide a useful alternative to studieswith uibroblasts. The activation of a particular receptormay lead to alterations in DNA synthesis as well asaffecting the differentiation process.

HC1 1 mammary epithelial cells are clonal derivativesfrom the COMMA-D line, obtained from midpregnantBALB/c mouse mammary gland tissue (3). These cellshave a variety of receptor tyrosine kinases and provide

us with an in vitro culture system in which the conse-quences of activation of different growth factor receptorscan be studied. The mammary gland undergoes a com-plex pattern ofgrowth and differentiation, which is underthe control of many steroid hormones and polypeptidegrowth factors. In vitro studies indicate that 1 7fl-estradiol,

progesterone, insulin, EGF,2 prolactin, aldosterone, andhydrocortisone are necessary to achieve full lobuloalveo-lar development of the mammary gland (4-17). HC11cells have retained important characteristics of mammaryepithelial cells. Following stimulation with lactogenic hor-mones, the cells differentiate and synthesize the milkprotein f3-casein (18). HC11 cells have been used to studythe action of the lactogenic hormones glucocorticoid andprolactin at the molecular level (19). In this paper, wehave used HC1 1 cells to characterize the role of receptortyrosine kinases in mammary epithelial cells. The recep-tors examined were the FGF-R, c-erbB-2/neu-R, andPDGF-R.

Three distinct consequences of receptor activation onHC1 1 cells were investigated: (a) effects upon cellgrowth, (b) effects upon the acquisition of competenceto become responsive to lactogenic hormone action, and(c) responsiveness to lactogenic hormones, i.e., inductionof �3-casein synthesis. We show here that activation ofeach receptor leads to a distinct phenotype. The stimu-lation of all three receptors has positive effects upon cell

2 The abbreviations used are: EGF, epidermal growth las br; PDGF,

platelet-derived growth factor; -R, receptor; -AA and -BB, A and B type

homodiniers; poly(A)#{176}, polyadenylated; kb, kilobase(s); 51)5, sodium do-decyl sulfate; BrdUrd, 5-bromo.2 ‘ -deoxyuridine; F ITC, fluorescein so-thiocyanate; DIP, dexamethasone, insulin, and prolactin; Ins, insulin; FCS,

fetal calf serum; PBS, phosphate-buffere(l saline; BSA, bovine serumalbumin; DMEM, Dulheco’s modified Eagle’s medium.

1A-‘20i-PDGF-BB BOUND pg/mi

lB200

‘5#{176}

100

no

//

,//, , ,128i-PDGF-BB ADDED ng/mi

B/F0.0

fig. 1. 25l-EGF and ‘25IPDGF

BB bind Is) HC1 1 cells. Specificbinding is shown using differentconcentrations of mouse 1251

EGF (A) (specific activity, 68.4

MCi/pig) and human recombinant‘251-PDGF-BB (B) (specific activ-

ity, 3 uCi/�zg). Nonspecific bind-ing of 2 and 50%, respectively,has been subtracted. This wascalculated using a 100-fold ex-

cess of cold ligand. Scatchardplots are shown for EGF (C) andPDGF-BB (0) binding to HC1 1cells. Binding parameters were

computed using the Ligand pro-gram (44).

BOUND pM

146 Growth Factor Re eptors in Mammary Epithelial c’lls

3 N. E. Hynes, unpublished observations.

��#{176}#{176}ee3FBOUND pg/mi

EE.. -

20L /� ,/

‘#{176}#{176}i-EGFADDED ng/mi

B/F _________ic #{176}#{176}‘

0.00e� �

0.004� �

BOUND pM

100.025

0.02

0.0�0

0.01

0.000

growth. Only activation of the EGF and c-erbB-2/neu

receptors and not of the PDGF-R in undifferentiated

HC1 1 cells causes them to subsequently become com-

petent to respond to lactogenic hormones. We havepreviously demonstrated (20) that simultaneous activa-

tion of the EGF receptor during the lactogenic hormone

induction phase has a negative effect upon 13-casein

promoter transcription. In contrast, c-erbB-2/neu and

PDGF receptor stimulation do not interfere with �-caseinsynthesis. These results suggest that signal transduction

pathways can be distinguished that distinctly affect the

growth and differentiation status of mammary epithelial

cells.

Results

HC1 1 Mammary Epithelial Cells Express EGF, c-erbB-2,and PDGF Receptors. Primary mammary epithelial cells

possess a number of different peptide growth factor and

steroid hormone receptors (11, 15, 21). Since the mam-mary epithelial cell line HC1 1 maintains important fea-

tures of epithelial cells in vivo, we have analyzed these

cells for the expression of three growth factor receptorsof the tyrosine kinase family: the EGF-R, c-erbB-2-R, andPDGF-R.

Table 1 Binding parameters for the EGF and PDGF receptors on mam-mary epithelial cells

The values repogram (44).

rted here have be en obtained us ing the Ligand pro-

Cell line.

Ligand Kd pM)No. of

sites/cell

HdllHdll

31ENOG-8

EGFPDGF-BBPDGF-AAPDGF-BBPDGF-BB

211190

33

28,0003,000

9,000

EGF and PDGF receptor expression was quantitatedin a binding assay using iodinated ligands. The results areshown in Fig. 1 and summarized in Table 1. HC11 cellsexpress approximately 28,000 high affinity EGF bindingsites/cell with a dissociation constant of 211 �M (Fig. 1,A and C). Approximately 3,000 high affinity PDGF recep-tors/cell could be detected, and the Kd was 190 �M (Fig.1, B and D). The cells were tested with both iodinatedPDGF-AA and PDGF-BB homodimers but bound specif-ically only to the BB homodimers. Therefore, HC1 1 cellsexpress PDGF B-type receptors. The presence of PDGFreceptors in mammary epithelial cells has not been re-ported previously. Therefore, we tested two other mam-mary epithelial cell lines, 31E (22) and NOG-8 (23, 24),for the presence of PDGF receptors. 31 F cells, whencocultured with fibroblasts, are able to differentiate andproduce �-casein (22), whereas NOG-8 cells do notrespond to lactogenic hormones.1 31 F cells specificallybind #{176}51-PDGF-BBand express approximately 9,000PDGF receptors with a dissociation constant of 33 �M

(Table 1). The binding assay was performed only with#{176}5I-PDGF-BBhomodimers; therefore, we cannot ex-dude the presence of A-type PDGF receptors. However,results presented below suggest that most, if not all, ofthe receptor present on 31 F cells is of the B-type. NOG-8 cells do not bind #{176}5l-PDGF-BBhomodimers and there-fore do not express either the A- or B-type PDGFreceptor.

HC1 1 cells express a moderate amount of c-erbB-2/neu protein.3 Since a ligand for the c-erbB2/neu receptoris not yet available, it is not possible to carry out theequivalent binding analysis. This also precludes experi-ments in which unstimulated and stimulated cells aredirectly compared. The c-erbB-2 receptor, however, canbe constitutively activated by introduction of a pointmutation into its transmembrane domain (25). We haveexploited this constitutive activation for our functional

78910

�.0 �

(‘ll (�riiss th �s, I )i(tereriti,itiiin 147

�% � ‘<,Kb _

5.3-

I ‘,t�. .2 Northern blot ,lri,ilVsjS iii PDGF-B receptor niRNA. Poly(A(’ RNA(B �g/l,ine( ss is (r,ls tiiinat(’(( un a d(’naturing formaldehyde gel, transterred

(i .1 �(‘ii(’ s( reen ili(’mhr,lne, ,lnd hybridized to a 2P-labeled oligo(iirrespiinding tii anlino aci(l pi)siti(ins 32 -48 of the niouse B-type PDGF

r(’( (‘l)tOr. RNA ss ,is isi)l,lt(’d from the in(ii(,lted ( (‘II lines. RNA hand at5. 3 kh, d ((lull of the nouse PDGF re eptor. Band running at approxi-

ni.itelv 5. 1 kh, I)ackgroun(1 hvllridiz,ati(in to ribosonial RNA. I IC I 1 lines.RNA isol,it(’d iriinl ((‘115 grown in the pr(’sellce of insulin and ECF (right)

or iiiil� insulin ( Iett

studies (see below) and have stably introduced an on-cogene variant of the c-erbB-2/neu gene into HC1 1 cells(20).

mRNA Analysis of PDGF Receptor. PDGF receptormRNA present in the nianiniary epithelial cells was char-

acterized. RNA was prepared from HC11, NOG-8, and31E niiammary epithelial cells. NIH 3T3 fibroblast cellRNA served as a positive control. PoIy(AY� RNA wasanalyzed for the presence of PDGF receptor-specific

transcripts using a 50-nucleotide probe specific formouse PDGF B-type receptor. The results are shown inFig. 2. A PDGF receptor-specific mRNA of 5.3 kb wasdetected in RNA from HC11, 31E, and NIH 3T3 cells. No

PDGF receptor transcripts were detected in NOG-8 cells.These results confirm our binding studies with ‘251-PDGF.

The HC1 1 and 31 E cells which were positive for bindingcontain the expected PDGF receptor mRNA, whereasthe NOG-8 cells which displayed no binding to PDGFdo not contain the 5.3-kb transcript.

Immunoprecipitation and Autophosphorylation of thePDGF Receptor. Enzymatic activity of the PDGF receptor

was analyzed by an in vitro kinase assay. PDGF-R wasininiunioprecipitated from lysates of HC1 1, NOG-8, 31E,

and NIH 3T3 cells with a rabbit polyclonal antibody(PDGFR-B3) specific for the PDGF B-type receptor (26).The precipitates were incubated with [‘y-12P]ATP, and

the in vitro labeled proteins were analyzed on SDS-polyacrylaniide gels. The results are presented in Fig. 3.A i2pIaI)eled PDGF receptor, migrating at M, 190,000,was specifically immunoprecipitated from HC1 1 (Lanes1-4 and 9), NIH 3T3 (Lanes 7and 8), and 31E (Lane 10)cell extracts. The PDGF receptor from HC1 1 cells pre-

treated for 10 nun in vivo with PDGF-BB displayed ele-

vated in vitro kinase activity compared to the receptor

from untreated cells (Lane 1 versus Lane 2). When theantibody was incubated with the immunizing peptidebefore addition to the lysates from HC1 1 and NIH 3T3cells, the M, 190,000 band was no longer visible (Lanes 3

and 4 and Lane 8. respectively). In accordance with theprevious results, fl() PDGF receptor protein was precipi-tated from NOG-8 cells (Lane 6).

Kd 123456

190-

I ig. .3. lmmunol)recil)itation ,ind in t (rn kinast’ .issav i if th(’ PDC Freceptor. L�-sates troni different cells were inlnlun(ipr(’( ipit.st(’(I with .i

rabl)it polyclonal antibody against the PDGF B-type recel)tiir. An in t itro

kinase assay ssas carried out as described in “Materials and Niethods.

Extracts are from: HC 1 1 cells stimulated 1 3) nun hetiire (�sis with 4(3 ng/tail o PDGF-BB I Lines I and I I or 1ii)nstiiiiul,st(’(I ( Lint’s 2, 4, 111(1 �

NOG-8 ((‘llS I Lane 6 (, N I H 3T � cells I Lines ‘ Old B (, an(I I 1 F s (‘lls I Line

ILl). In L,ines .1. 4, ,snd 8, the antiserum was pr(’in( uI).lted with in ex ‘ssof pepti(i(’, against xvhi h the PDGF-R ,lntiserum sv,is d(’ri(ecI, heRire

ad(iing ((‘II lysates. Lane 5, results using a )reinlniun(’ seruili ��ith ( (C 1 1

c(’ll (ysate.

The PDGF receptor that can be precipitated fromHC1 1 and 3 1 E mammary epithelial cells has the same

characteristics in an in vitro kinase assay as the receptorpresent in NIH 3T3 fibroblasts. In addition, HC1 1 cells

that were stimulated in vivo for 10 mm with PDGF displaya receptor with elevated autophosphorylation activity.This suggests that the PDGF receptor present in thesecells is enzymatically active.

Growth Response of HC1 1 Cells to Activated EGF,PDGF, and c-erbB-2/neu Receptors. Two growth param-

eters were analyzed in HC1 1 cells following activation ofthe EGF, PDGF, and c-erbB-2/neu receptors: DNA syn-thesis and determination of the final cell density. First,DNA synthesis was measured in cultures of HC1 1 cellstreated with growth factors. Parallel cultures of serum-starved cells were stimulated for 24 h with insulin, EGF,or PDGF-BB, and then the cells were labeled for 90 mmwith BrdUrd. The thymidine analogue incorporated incellular DNA was detected by a specific antibody againstBrdUrd conjugated with FITC. The results are shown in

Fig. 4 and summarized in Table 2. Insulin increased DNAsynthesis slightly over background (12% versus 7%).

HC1 1 cells treated with EGF or PDGF-BB displayed,respectively, 43 and 49% labeled nuclei. Higher concen-trations of PDGF-BB (up to 20 ng/mI) had no furthereffect. This suggests that EGF and PDGF-BB act as niito-gens for the HC1 1 cells.

The second growth parameter measured was the finalcell density reached by HC1 1 cells following the additionofgrowth factors. The results are shown in Table 3. HC1 1cells grown in EGF reach a high density. This is notdependent upon serum, since the same density wasreached in the presence or absence of serum. HC1 1 cellsexpressing the transfected neuT reach an equivalentdensity in the absence or presence of EGF. Cells express-ing the control neuN protein behave like nontransfectedHC1 1 cells. Compared to EGF-treated cultures, HC1 1cells grown in insulin reach approximately a 3-5-foldlower final cell density. Cells grown in PDGF-BB reach a

2-3-fold lower final cell density compared to EGF-treated

148 (;ri)sxth 1,istiir Ret eptiirs in M,iiiini,irv F )itheli.il (c’lls

I ig. 4. Stiniulation of DNA syn-

thesis in FtC 11 ells by different

growth factors. H�i i cells weregrown for 24 h with: no growth

factor ,ldditic)n ( .-\ and B ), PDG F -

BB (C arlcl 1)), or EGF (I. and I).Ten �iii BrdUrd were added to

the medium for the final 1 .5 h.B, 1). .in(I 1. ,lnti-Br(IUr(I-FIT( an-

tibody staining; .\, C. and F, l)r�-pidiuiii iodidc’-stained icic lei.

cultures.These results suggest that activation of the EGF and c-

erbB-2 receptors has an equivalent effect upon the finalcell density of HC1 1 cells. ni contrast, PDGF-BB is equiv-

alenit to EGF in stmniulating DNA synthesis in resting HC1 1

cells, l)Llt it cannot fully substitute for EGF when (‘eli

density is nieasured. The final cell density reflects not

T,il)le 2 L)NA ss it hesis in ( ((‘ 1 1 i ells in r�’spiins�’ lii gri isst(i u (or

,i((((itiiii)

Si’runi-st,lr\(’(l (IC 1 1 ( (‘115 svere s( iniul,ited 24 (a (iv ,idulitiiiil of v,iriii(is

griisxth f.i (tirs. T(’ii pM Brc(LJrd sseo’ in(lu(1(’(I ri tile Fi1(’dilinl for th(’ final

1 . S h . I )NA synthesis ssas .tk ul,iti’il is a l)’r c’nt,ig(’ of tIle cells staiil(’(I

hs Bril F Jrd -F I T( ( i iiiip.irt’i( Ii i the Ii (il ii uiii( or i if (‘110, ,i5 detc’riliirlecl

1)5 pr�pi((iun1 iii(Iidt’ st.iiiliog ( i( ouile,ir L)NA. Results are the av(’rag(’ of(ss,ii (‘\l)(’riill(’llts svht’rt’ at lent 1 (3 .ire.is/pl.ite ss’re ,lil,llVt(’d.

(;riissth (ii tiirs �o of i ells ni iirpiiriting

.1(((((’(( OrdLlrd

Ins 12[CF 41

P[)GF1313 4i)

Niine 7

only DNA synthesis but also the shape of the cells. Fig.5 shows that HC1 1 cells grown in insulin (A) or PDGF(C) are flatter and more spread out than those grown inEGF (B). neuT-expressing HC1 1 cells display a morphol-ogy siniiilar to that of EGF-treated cells (data not shown).This is reflected in the high final cell density that theyachieve in medium lacking EGF.

EGF-treated or neuT-expressing HC1 1 Cells BecomeCompetent to Respond to Lactogenic Hormones. HC1 1cells respond to the lactogenic hormones dexametha-

sone, insulin, and prolactin and synthesize the milk pro-tein /3-casein. Only confluent cultures of HC1 1 cells canbe induced by the DIP-containing medium (19). We

investigated which growth conditions optimally predis-

�JO5E� HC1 1 cells to respond to the lactogenic hormonemixture. For this purpose, HC1 1 cells were grown toconfluency in medium containing Ins, Ins plus EGF, or

Ins plus PDGF-BB. HC1 1 cells transfected with neuT orneuN expression plasmids were used to analyze theeffects of an activated c-erbB-2/neu receptor. The cellswere niaintained at confluency for 2-4 days and thenswitched to DIP-containing medium for 4 days. Cellextracts were prepared, and the expression of �-caseinwas analyzed using a protein blotting technique.

-0

s’,,

‘�

�( � : � 1::: � �:i�i� I � �‘ �

#�, --. , . “ ��‘.‘;‘_,-_�__“ �

. � � . � �

. ��L’ ‘ .- � ‘( � � �, ,�,

L � i_, . ,, .4

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results in the same eflect upon suI)seqlierit �1-casein

synthesis. Insulin or PDGF-BB cannot substitute for EGF,whereas activation of the c-erbB-2/neu receptor niimics

ligand activation of the EGF receptor.

The Competence Effect of [GE Cannot Be Attributedto Cell Density. HC1 1 cells grown in EGF or transfected

with neuT reach a higher final cell density than cultures

grown in insulin or PDGF. It is conceivable that the highercell nunil)er could l)e causal for the response to lacto-

genic hormones. To test this possii)ility, cells were grown

Cell Griisvt (1 iS. 1)i((erent iatiiiii 149

1,il)li’ 1 Find ( (‘Il (((‘iisit\ /( iii.? ( X 1 (C( re,i( (l(’(l hs Fi( 1 1 ( ells in re�

� iil�(’ ti i (.�ri isx( 6 (,ii1i ir 51 ni u lit ion

1 lie v,ilut’s ire tl)(’ .iV(’r.ig(’ ii( tsvii (‘xp(’r inleots. N I ), ii ut det(’rnlined.

(;riiss ili iii tiirs

.iddt’il., (C 1 1 �

iis’ciN”( 1( 1 1

ns’uT5FIC 1 1

Noni’ NI) 1.3 ± (1.11) NI) NE)

lii’. 1 .9 ± I). 18 1 .9 ± I). 1 2 0.8 ± I). 1 6 4.6 ± 0.42

In’. + F Cl -,.-, ± O.’i7 6.5 ± 1)84 6.5 ± 0.61 5.4 ± 0.83

Ins + (#{176}E)(F-BI3 3.4 ± 0.Si) 2.5 ± 0. 3.1 ND NI)

-, ,(i(i.(iO() ( elk ss i’i(’ Pl,it(’i( in 6-c in i(ishes, ,silii (ic’ (io,il c elI c(ensity/cnl2

55 i’ (li’ti’rililiii’(l tIter 4 d,is s. The ( ills sx ere griissri in I lani’s F 1 2/DMEM

(5 abut sersiiii ,iddiliiiil.0 1(8),(iii0 ( ills ss (re I)l.it(’(l iii (,-( i-il (iisii(’S, ,ln(i the (io,ll ( elI density/ nl

57, iS ih’i(’riliiii(’il dies B i(.iss. The (‘115 ss(’re griissn in RPMI 1640

i iiill.iiiiiilg B#{176}oF(S

Fig. 6’\ shOWs tIi�it cells grown and maintained at(onilluenicy in insulin-containing mediuni synthesize no

OF barely detectable levels of f�-casein in response to

latogeni horniionies (Lanes 7 and 8). Cells grown in Ins

PILlS EGF respond optimally and express high levels of �1-(aseini (conipare Lanes 7 and 8 to Lanes 3 and 4, respec-lively). Cells grown in EGF-containinig medium show thesaniie results as those grown in Ins l)Ius EGF (data notsliovi,’ti). When EGF was oniiitted during the growth phaseof the HC 1 1 cells (La,ies 9- 16 ) i)ut transiently addeddurinig the (onfluen(y l)Iiase for 1 -4 (lays, it was possible

to restore the ability of the cells to respond to lactogenichorniones. Two (lays ot EGF treatnient caused an in-crease in the level of 3-caseiri induction (Lane.s 13 and14 (, arid the niaximal level was achieved after 3-4 (lays

of EGF treatnienit (Lanes 9-12) prior to DIP induction.Fig. 613 ( Lane 3) shows the results obtained with cells

grown and niiainitainie(l at coriflLiency in insulin plusPDGF-BB-containinig niediuni. These cells are not able

to respond to lactogenic horniones arid do not synthesize(1-(asein. Tue results oi)tainied with parallel control cLil-tures grown with Iris alone or Ins F)lus EGF are shown inFig. 6B, Lanes 1 and 2, respectively.

The results obtained with the neuT- (activated neiiallele) arid nieuN- (wild-type, niona(tivated allele) trans-tected HC1 1 cells are shown in Fig. 6C. Cells expressingthe neoN )rotein i)eliave like norniial unitransfectedHC1 1 (ellS, i.e., Fells grosvn arid maintained at confluency

iii irisulin-(ontaininig niediunii do riot respond to thelaitogeniic iiorniionie niixture (Lanes 1 5 arid 16 ). Cellstreated with EG F respond optimally ( Lanes 1 1 and 12).In (ontrast, neuT-transfected cells synthesize equivalent

aniounits of (1-(aseini following DI P induction irresf)ectiveof the presence or absence of EGF in the growth niedrum(Lanes � and 4 c�’rctis 7 arid 8).

Fig. 61) shows that the effect of EGF upon nondiffer-etitiated HC1 1 cells is long lasting. The HC1 1 cells weregrown to confluenicy in niediuni containing insulin andEGF, arid then EGF �s’as removed for different periodsranging fronii 1 to 5 days. The cells were then treatedwith the DIP niiixture for 2 days. The confluent cellsreniioved froni EGF for 1 -3 (lays still responded optimallyto [)lP. Following reniioval for 4-5 days, there is a slightdrop in the �-casemn level.

These results show not only that the effect of EGFF1I)�ni HC 1 1 cells is long lasting but also that the addition(if EGF (luring the growth phase (Fig. 6D) or the con-flLieri-y phase (Fig. 6/\) of nondifferentiated HC1 1 cells

, �. 1 � ,

.‘ . I. ‘� . � �

i:-’ t�::�e� �‘“i� I � : , �

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�,ie’ .

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,

I ig. 5. Psitirphiiliigs iii F-((’ 1 1 ( elk griisx’ii in th(’ I)r(’s(’ii( t’ I it ((i((t’r(’nt

griisx’th (as tiirs. The ( (‘lb (v(’r(’ griiss-n in: .4. insulin: B. insulin .tiiil t( , I ; (

inscilin and PI)GF.BB.

+

+

NeuT

NeuN

Growth +1- EGF

Induction +1- DIP

D

+ + +

- + +

150 Criiss th Fa( tiir Res t’ptiirs ill xi.ininaars EI)itheli,1l (oIls

A

13-cas. -

3dgrowth +/-EGF

4 d confluence +1- EGF4 d induction +1- DIP

C

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16+ + + +

+ + + + - - - - 4d+ 4d+ 3d+ 3d+ 2d+ 2d+ ld+ ld+

- - + + - - + + + + + + + + + +

B

13-cas. -

123

13-cas.- -- --

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16+ + + + + + +

+ + + + + + + +

13-cas.- .-�

- - + + + + - - -

+ + - - + + . - + +

� �-

I 2 3 4 5 6 7 8 9 10CJrowth+/-EGI’ + + + + + + 4. + + +

Conflucricc +1- EGF 5d- Sd- -Id- 4d- 3d- 3d- 2d- 2d- id- Id.

2dDlPinduction + + + + + + + + + +

fig 6. Tr,irisi(’ilt EGF (“.posun’ or .u tiv,ited n(’uT expression is requir(’d to m,lke tIC 1 1 oIls ompetent to respon(I to ac t(igeni( hornlones. \-Vestern

blot arlalssis of il-( ,isein. c’II Iss.it’s were’ pr(’par(’(I, ele trophoresed. and electrobloitod, and the d-c,is(’in )�1. ,as. I l)rot�iil sv,is detec te(I using a specific

alltil)O(I5 tollowed f)s- ‘251-Protein A treatnlent. The Is-sates art’ tronl: (.-\ I H� i i ( oils grown 3 days in niediunl containing insulin and EGF, kept 4 days at( onflueiu e in the s,lnie nl(’(Iiuill ( Lint’s 1 4 1, and induced 4 (I�is’s with DIP (Lanes I and -3( or kept ,ss a ((introl 4 (Ia5’s in insulin al(ine )L.ines I and 2).HC 1 1 ( (‘115 griisvn iii ,i niediuni ( olltaiilirlg insulin ( I ins’s 5 -8 (, kept 4 days at ccintluen( (‘ in the s.snie niediuni, and ili(IIK ed 4 (lays with DIP I Lanes 7 ,aJl(I8 1 or kept ,is ,i ( iiiitriil 4 ((,i5’s in insulin ,sliine I Lint’s and 6 (. (IC 1 1 ((‘115 grosx’il in the ,slls(’nce of EGF I Lines 9- 16 ), kept 4 days ( L,in(’s 9 and 10), 3 daysI Lines I I ,iil(I I 2 (. 2 (lass I Lines I 3 111(11 4 (, or 1 iI,is ( L,iiic’s I ‘ ,uiid I 6 1 at ( onfluen (‘ in the presen e of EGF and indu ed 4 days with DIP. ) B) HC 1 1( (‘(Is grosvn iii ( iiii(Iu(’nc(’ svit(’i ilsulirl I L,ine I (, insulin mci [CF I Lane 2 ), iir illsulin ,uid PI)GF-Bb I L,In(’ I I and indu ed to (Iitf(’rentiat(’ by 4 days of

tre,itnlent 5% rh 1)1 P. (( ( FIC I 1 oIls tr,inst(’(t(’(I ss ith riouj ( Lines I B I or fli’uN ( L,ii�’s 9- 1 6 ),The cells were grossmn to ( onfluonce in medium ( ontaining

insulin .111(1 E(;F or insulin ,ilon(’ ,in(I thc’n 11(1(15t’d 4 ilas-s ss ith i)(P I Lanes 3. 4. 7. 8. 1 1 . 12, 1 5, and I 6 1 or kept ,is a � ontrol 4 (lays in insulin alorle Lanes

I . 2. 5. 6. 9. Ii). I 3. ,-u-id I 4 (. I1) ( ( (C 1 1 ((‘115 griiss n in a iiiediuni ( tititaining insulin ,iiid EGF. wh’n the (‘115 reached coilflcieii y, EGF was removt’cl 5, 4,3 . 2 , sir 1 (1,lV l)(’Ii ire t he 1)1 P md � t ii ill .I 1 ‘\, ( . mu I ), the results ,ire shiisvn for duplk ,sts’ ( ulturos. Ea( h l,irie contains 7 5 pig of protein ext r,u I.

Cell Growth & Differentiation 151

TiLde 4 EfleO of finalcell density and EGF upon #{216}-casein induction inHC11 ((IIS

Final celldensity/cm2

)x 10�)

Level of d-casein bydensitometry analy-

sis (%)

A. Pretreatment (EGF)�Day -3 2.2 ± 0.14 95.0Das--2 1.4±027 88.3Day-i 0.7±0.15 61.3

B. Gross’th Ia tors addedNone 1.3±0.10 0Insulin 1.9 ± 0.12 0

Ins + PDGF�BB 25 ± 0.33 0Ins + EGF 6.5 ± 0.84 100

The oIls were grown in a medium containing Ins for 8 days; 3, 2, or 1

day bef(ire lactogenic hormone induction, EGF was added to the me-dium.

to a defined cell density under different growth condi-tions, and their inducibility was determined (Table 4).HC1 1 cells were grown to near confluency in Ins, andthen EGF was added back to the cultures for 1-3 daysbefore addition of the DIP induction medium. The levels

of /3-casein produced by the cultures were similar tolevels seen in Fig. 6A. They were scored as a percentageof the maximal level achieved by control cells (Table 4).For comparison, the cell density of Ins-, Ins plus PDGF-,or Ins plus EGF-grown cultures and their response to DIPare shown in Table 4A. It can be seen that the final celldensity of cultures treated for 2 days with EGF is less

than that of cultures treated with Ins or Ins plus PDGF,yet the former cells produce high levels of (.�-casein,whereas the latter do not. This makes it unlikely that theEGF effect on cellular competence to respond to lacto-genic hormones is mediated only by a higher cell density.

DiscussionWe have studied the consequences of activation of three

members of the receptor tyrosine kinase family, the FGF,c-erbB-2/neu, and PDGF receptors in HC1 1 mammary

epithelial cells. Effects upon cell growth, upon the ac-quisition of their competence to respond to lactogenichormones, and upon lactogenic hormone-induced /3-casein expression have been analyzed. The results dem-onstrate that the activation of each receptor results in adistinct phenotype (Table 5).

Addition of PDGF and EGF to the medium of restingHC1 1 cells results in a strong stimulation of DNA synthe-sis. It has previously been reported that EGF is a potentmitogen for COMMA-D cells (3, 21), from which HC11cells were derived (18). In addition, EGF has a role inpromoting differentiation of primary mammary epithelialcells growing on collagen gels (27). This is the first reportthat PDGF acts as a mitogen for an in vitro culturedmammary epithelial cell line, and it suggests that PDGFmay also have a role in vivo in mammary gland devel-opment. We are aware of the possibility that PDGFreceptor expression is a result of in vitro culturing of

mammary gland cells. It has been reported that smoothmuscle cells from adult porcine myometrium are inducedto express the receptor following culturing (28), but itwas also demonstrated that, in vivo, the same cells fromyoung animals normally do express PDGF receptors. The

Table 5 Effect of specific receptor activation on HC1 1 (ells

This table summarizes the results presented in this paper and in a

previous publication (20). Three different aspects are measured: (a)Activation of EGF, c-erbB-2/neu, and PDGF receptor stimulates the

growth of HC1 1 cells, as shown by a determination of DNA synthesis

and final cell density. (b) Activation of EGF or c-erbB-2/neu receptor, butnot PDGF receptor, while the cells are growing or confluent, gives

competence to the cells to respond to the subsequent addition oflactogenic hormones. (c) Simultaneous activation of the EGF receptorduring lactogenic hormone induction inhibits #{216}-casein production,whereas activation of the other two receptors does not affect milk protein

synthesis.

Competence toLactogenic hor.

respond to lac.Growth (a) . mone-induced

togenic hor. . .differentiation (c(

mones )b)

EGF Positive Positive Negativec-erbB-2/neu Positive Positive Neutral

PDGF Positive Negative Neutral

in vitro induction of receptor expression merely reflectsthe proliferative state ofthe cells. Another independentlyderived mammary epithelial cell line, 31F, which alsodifferentiates in response to lactogenic hormones, ex-presses PDGF B-type receptors. NOG-8 cells, whichcannot be differentiated, contain no receptors. This sug-gests that the expression of these receptors may not bean artifact of in vitro culturing and that they might play arole in a particular stage of mammary gland development.We are currently investigating PDGF receptor expressionin primary mammary glands isolated from different de-velopmental stages. Many primary human breast tumorsand breast tumor cell lines express PDGF (29-31). Thisligand may have a role in desmoplasia, a stromal cellreaction found in primary breast tumors (32). If normalepithelial cells express receptors, it is conceivable thatPDGF might play a role in their transformation.

Despite the fact that PDGF is as mitogenic as EGF,only HC1 1 cells kept in EGF respond optimally to thesubsequent addition of Iactogenic hormones and synthe-size high levels of /3-casein. Activation of the c-erbB-2/neu receptor also leads to positive effects upon HC1 1cell growth and promotes their competence to differen-tiate. The natural role of the c-erbB-2 receptor in pro-moting cell growth or differentiation has not been ascer-tamed. Studies have been hindered by the lack of apurified ligand for this receptor. Results obtained withchimeric receptors consisting of the ligand-binding do-main of the EGF receptor and the cytoplasmic domainof c-erbB-2 suggest that c-erbB-2 activation can lead toDNA synthesis, depending upon the cellular environ-ment. Following EGF binding, the chimeric receptor gen-erates a mitogenic stimulation in NIH 3T3 fibroblasts (33,34) but not in 32D hemopoietic cells (35). Indirect evi-dence suggests that the c-erbB-2 protein has a functionin the mammary gland. Dati et a!. (36) have shown thatthe level of c-erbB-2 protein varies in the developing ratmammary gland, with the highest amounts detectable infunctionally differentiated tissue. In addition, the fact thatthe gene is amplified and the protein is overexpressed ina high percentage of human breast tumors (37) suggeststhat this receptor may have an important role in thegrowth control of mammary gland cells.

Why does the activation of EGF and c-erbB-2 recep-tors cause HC1 1 cells to become competent to respond

152 Growth Factor Receptors in Manlmiry Epithelial Cells

4 D. Taverna, unpublished observations.

to lactogenic hormones? One possibility is that compe-tent cells produce and attach to extracellular matrix pro-teins. To date, all primary mammary gland cell cultureshave required cocultivation, either with other cell typesor in collagen gels, to achieve milk protein production(27, 38, 39). The COMMA-D line and its subclone HC1 1appear to be exceptions, i.e., they require no cocultiva-tion to respond to lactogenic hormones. Indirect evi-dence suggests that HC1 1 cells treated with EGF synthe-size an extracellular matrix. HC1 1 cells which were grownto, and maintained at, confluency for 2 days in FGF-containing medium were gently removed and replatedon fresh culture dishes or on dishes containing the extra-cellular matrix of HC1 1 cells grown with or without EGF.After 24 h, these cultures were treated with lactogenichormones. The replated competent cells could producea high amount of (1-casein when plated on an extracellularmatrix produced by FGF-grown HC1 1 cells. Moderatelevels of /3-casein were synthesized by competent cellsreplated on a matrix derived from insulin-grown 1-IC1 1cells, whereas very little /3-casein was seen in cells re-plated on plastic (data not shown). This suggests thatextracellular factors, whose production is enhanced byEGF, are important for the production of a high amountof /3-casein. In primary rat mammary gland cultures, thesynthesis of collagen type 4 is stimulated by EGF (40).Interestingly, the in vitro differentiation of baby kidneycells requires cultivation of these cells with EGF and onextracellular matrix.

We have previously reported that the presence of EGFin the lactogenic hormone induction mixture has a neg-ative effect upon [3-casein promoter utilization, and thecells produce only very low levels of (3-casein protein(20). HC1 1 cells expressing an activated c-erbB-2/neureceptor are still responsive to lactogenic hormones. Theaddition of PDGF-BB to the lactogenic hormone mixturehas no effect upon the levels of 13-casein produced (Table5)�4

These results show that an ordered sequence of re-ceptor activation is required to achieve differentiation ofHC1 1 cells. Growing HC1 1 cells are primed for differ-entiation by the activation of either the EGF or the c-erbB-2/neu receptor. Only these competent cells arecapable of responding to the lactogenic hormones pro-lactin and glucocorticoids, and they produce high levelsof /3-casein. If the EGF receptor pathway remains acti-vated while the lactogenic hormones are present, thepositive effect that prolactin and dexamethasone exerton (3-casein expression is overruled by a negative effectthat EGF exerts on (3-casein promoter transcription.

It now becomes possible to analyze the intracellularpathways that are activated by each of these receptortyrosine kinases and distinguish signals that are importantfor growth, competence, or differentiation-specific geneexpression.

Materials and Methods

Reagents and Cell Culture. HC1 1 cells (18), a subcloneof COMMA-iD cells (3), and 31F cells, an epithelialsubclone of the IM-2 mammary cell line (22), both ofwhich were isolated from mammary glands of midpreg-

nant BALB/c mice, were used. They were grown in RPMI1640 medium containing 8% heat-inactivated fetal calfserum, 5 zg/ml bovine insulin, and 10 ng/ml murine EGF(growth medium). In the experiments indicated, humanrecombinant PDGF-BB (5 ng/ml) was substituted for EGF.Two additional cell lines, NOG-8, an epithelial subcloneof nontumorigenic mouse cell line NuMg, originally iso-lated from the mammary gland of NaMru mice (23, 24),and NIH 3T3 fibroblasts, were also used. NOG-8 cellswere grown in DMEM containing 10% heat-inactivatedfetal calf serum, 5 ,�g/ml bovine insulin, and 10 ng/mIEGF. NIH 3T3 cells were grown in DMEM containing 8%synthetic serum (Nu-serum IV; Collaborative Research).HC1 1 cells, transfected with the neuT and neuN expres-sion plasmid, were described previously (20). In somecases, the HC11 cells were grown in F-12-DMFM con-taming insulin (5 �zg/ml), transferrin (10 �zg/ml), fetuin (1mg/mi), EGF (10 ng/ml), or PDGF-BB (5 ng/ml) withoutserum addition. All media contained 2 m�i L-glutamineand 50 �zg/ml gentamicin.

HC1 1 cells were induced for /3-casein protein by main-taming confluent cultures for 2 days in growth mediumfollowed by 4 days of treatment with induction medium[RPMI 1640, 8% FCS, 5 �zg/ml ovine prolactin (Sigma),0.1 �zM dexamethasone (Sigma), and 5 jig/mI insulin].

Recombinant human PDGF-BB, PDGF-AA, ‘251-PDGF-BB (specific activity, 3 �zCi/j.tg), and #{176}5I-PDGF-AA wereprovided by Dr. C. H. Heldin, Uppsala, Sweden (41).Mouse 1251-FGF (specific activity, 68.4 zCi/�zg) was pur-chased from Amersham.

Immunoblot Analysis. Cultures of HC1 1 cells wereinduced with lactogenic hormones and analyzed for /3-

casein synthesis using an immunoblotting analysis. Cellswere washed with PBS and then extracted in a buffercontaining 10 mM 4-(2-hydroxyethyl)-1 -piperazineeth-anesulfonic acid (pH 7.4), 10 m�i NaCI, 1% Triton, and 1mM EDTA. Debris was removed by centrifugation at12,000 x g for 5 mm. Seventy-five jzg of protein extractwere subjected to electrophoresis on 12% SDS-poly-acrylamide gels. Proteins were electroblotted to PVDFmembranes (Millipore) (42). /3-Casein was detected witha rabbit anti-mouse f.3-casein antibody (18) (a gift fromDr. F. Reichmann, Vienna, Austria) followed by 125I

Protein- A treatment.Analysis of DNA Synthesis. BrdUrd incorporation into

HC1 1 cells was used to assay mitogenic activity. Threex iO� cells/6-cm dish were plated. After overnight incu-

bation in growth medium, cells were washed twice withPBS and then incubated in medium containing 1% FCS.DNA synthesis was stimulated by the addition of PDGF-BB (5 ng/mI) or EGF (10 ng/ml) with or without insulinand was monitored 24 h later by a 1.5-h metaboliclabeling with a thymidine analogue, 5-bromo-2’-deox-yuridine (10 �tM). The cells were fixed for 10 mm with70% ethanol, treated 20 s with 0.07 N NaOH, and neu-tralized with 0.1 M Na2B4O7 (pH 8.5). The proliferatingcells were identified using a specific monoclonal anti-BrdUrd antibody (Becton-Dickinson) conjugated withFITC. The nuclei were stained for 5 mm at room temper-ature using propidium iodide (0.2 �zg/ml). The stainedcells were counted using an immunofluorescencemicroscope.

Growth Factor Binding Experiments. PDGF and EGFbinding experiments were performed as follows. HC1 1cells were seeded at 1 x 1O� cells/i .5-cm well and grown

Cell Growth & Differentiation 153

for 1 -2 days to 8O% confluence. To measure binding, themonolayers were washed twice with PBS and incubatedin RPMI 1640 plus 1% FCS for 24 h. Then the mediumwas removed, and the monolayer was washed once withPBS supplemented with 1 mg/mI of BSA. Each well re-ceived 0.25 ml PBS-BSA containing 251-PDGF (AA or BB)or 21-EGF at different concentrations, in the absence orpresence of unlabeled growth factors (PDGF at 500 ng/ml or EGF at 250 or 500 ng/mI). The dishes were incu-bated at 4#{176}Cfor 2 h. The cells were washed five timeswith PBS-BSA and solubilized for 30 mm at room tem-perature with 0.25 ml of a solution containing 1% Triton,10% glycerol, and 20 mr#{176}�i 4-(2-hydroxyethyl)-1-pipera-

zineethanesulfonic acid (pH 7.4). The radioactivity wascounted in a gamma counter. All ofthe assays were madein quadruplicate.

RNA Isolation and Analysis. Poly(A)’� enriched RNAwas selected by chromatography on oligo(dT) cellulose(Boehringer) (43), fractionated on a 1 .2% formaldehyde-

agarose gel, and transferred to a nylon membrane (GeneScreen) in lOx standard saline citrate. The RNA wascross-linked to the membrane by UV light (StratalinkerStratagene) and hybridized with a 32P-oligo-Iabeled nu-cleotide (chain length, 51) corresponding to amino acidpositions 32-48 of the mouse PDGF B-type receptor(26).

Immunoprecipitation and in Vitro Kinase Assay. Thepresence of the mouse PDGF B-type receptor was inves-

tigated using immunoprecipitation followed by an in vitrokinase assay. The cells were lysed in a buffer containing50 mM Tris-HCI (pH 7.5), 5 m�i ethyleneglycol bis(f3-

aminoethyl ether)-N,N,N’,N’-tetraacetic acid, 1% Tritonx-100, 150 mM NaCI, 1 m�i phenylmethylsulfonyl Iluo-ride, 80 �zg/ml aprotinin, 50 �g/ml leupeptin, and 4 �g/ml pepstatin. Cell debris was removed by centrifugationat 12,000 x g for 10 mm. PDGF B-type receptor wasimmunoprecipitated for 2 h at 4#{176}Cusing the rabbitpolyclonal antipeptide antibody PDGFR-B3 (26). As acontrol for the specificity of the antibody, a reaction wasincubated with the peptide used for immunization for 30mm before the addition of cell lysate.

The immunocomplexes were collected using ProteinA-Sepharose (Pharmacia), washed, and incubated withkinase buffer [20 mM Tris (pH 7.5) and 5 m�i MgCI2] and10 �zCi [�y-12P]ATP (carrier-free) for 10 mm at room tem-perature. The beads were washed and boiled for 5 mmin electrophoresis buffer. The proteins were separatedon an 8% SDS-polyacrylamide gel and autoradiographed.

AcknowledgmentsWe would like It) acknowledge the expert technical assistance of U.Stielel. We thank t)r. C. H. Heldin for kindly providing us with reagents

It) study the PDGF receptor and Dr. E. Valverius br introducing us to the

Ligand program. The critical comments of Drs. K. Ballmer and M. Schmitt-Ney are greatly appreciated.

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