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Heterogeneity in hormone responses and

patterns of collagen synthesis in cloned dermal

fibroblasts.

S R Goldring, … , S M Krane, J H Korn

J Clin Invest.

1990;

85(3)

:798-803.

https://doi.org/10.1172/JCI114506

.

Fibroblasts cultured from normal human dermis are heterogeneous with respect to growth

kinetics, synthetic function, and morphologic features. There are many examples of clonal

heterogeneity in apparently homogeneous connective tissue cell populations, and it has

been suggested that selection of cell populations with particular phenotypic features is the

basis for the development of pathologic connective tissue changes in inflammatory

disorders. In these studies we report characterization of the pattern of matrix biosynthesis

and responses to hormones in cells cloned from normal human dermis. The results indicate

that cloned dermal fibroblasts are heterogeneous with respect to synthesis of collagens as

well as their responses to prostaglandin E2 and parathyroid hormone. Selective expansion

of clonal populations with unique patterns of matrix synthesis and cell surface receptors

could provide the basis for abnormal connective tissue remodeling in certain pathologic

states.

Research Article

(2)

Heterogeneity

in Hormone

Responses and

Patterns

of Collagen Synthesis

in Cloned Dermal

Fibroblasts

Steven R. Goldring, Mary L. Stephenson,Elaine Downie, StephenM. Krane,and Joseph H. Korn

Department ofMedicine, Harvard MedicalSchool, Boston,Massachusetts02115; MedicalServices(Arthritis Unit),NewEngland DeaconessHospital,Boston,Massachusetts02215;Massachusetts GeneralHospital,Boston, Massachusetts02114; and the

Division ofRheumaticDiseases, VeteransAdministrationMedicalCenter, University ofConnecticut

School ofMedicine, Newington, Connecticut 06111

Abstract

Fibroblasts cultured from normal human dermis are heteroge-neous with respect to growth kinetics, synthetic function, and morphologic features. There are many examples of clonal het-erogeneity in apparently homogeneous connective tissue cell populations, and it has been suggested that selection of cell

populationswithparticular phenotypic features is the basis for the development of pathologic connective tissue changes in

inflammatorydisorders. In these studies we report character-ization of the pattern of matrix biosynthesis and responses to hormones in cells cloned from normal human dermis. The re-sultsindicate that cloned dermal fibroblasts are heterogeneous with respect to synthesis of collagens as well as their responses

to prostaglandin E2 and parathyroid hormone. Selective

ex-pansionof clonal populations with unique patterns of matrix

synthesisand cell surface receptors could provide the basis for abnormal connective tissue remodeling in certain pathologic states.(J. Clin. Invest. 1990. 85:798-803.) hormone-collagen *connective tissue cell

Introduction

Examination of the growth kinetics and morphologic features

of fibroblastscultured fromneonatal human skin have dem-onstrated that these cells are genetically and phenotypically diverse(1-7). Examplesofclonalheterogeneity inapparently

homogeneousconnective tissuecell populations have been

re-ported, and it has beensuggested that temporal selection of individual cell populations could be involved in cellular

re-modeling of connective tissues under physiologic conditions

(1-9). Similar mechanisms could also be involved in

patho-logic states in which monoclonal or oligoclonal cell

popula-tionspossessing unfavorableorinappropriate phenotypic

fea-turescould beselected(10-15).

The initialgoalofthesestudieswas todeterminethedegree ofphenotypic diversity in the pattern of matrix biosynthesis

among normal skin fibroblasts. We observed significant dif-ferences in the pattern of collagen

synthesis

in substrains

cloned from normal neonatal foreskin. Thesefindingscanbe

Address correspondence to Dr. Steven R. Goldring, Massachusetts GeneralHospital-East,149 TheNavyYard, 13th Street,Charlestown, MA02129.

Receivedforpublication25 July1989 andinrevisedform 9

No-vember1989.

accounted for in part, by differential regulation ofcollagen

synthesisatthetranscriptionallevel.

The second goal of our studies was to determine whether heterogeneity also exists in responses involving cell surface receptors for hormones known to affect connective tissue re-modeling. We therefore examined the pattern and magnitude

of3'-5'-cAMP responses to PGE2 and PTH. Marked heteroge-neity in the cAMP responses to these hormones was also

ob-served, although there was nocorrelationbetween the rate of collagen synthesis and the magnitude or pattern of hormone responses. Selective expansion of clonal populations in vivo could thus give riseto tissues withunique patterns of matrix

synthesisand altered responses to hormonalregulation.

Methods

Processingoftissue. Samplesofneonatalforeskin obtained from

rou-tine circumcisionweredigested with clostridial collagenase. Substrains

wereisolated fromprimary cultures bylimitingdilution cloning(1-4).

Once confluent, cultures ofthese clones were maintained in basal Eagle'smedium supplemented withFCS andcarried through multiple

passages aftertrypsinization.

Hormone responses. Cellswerepassaged into24-well trays(Costar, Cambridge, MA)at - 5 X 104 cells/well. cAMP responseswere

as-sessedby incubatingcellsfor 20 minat370C inbuffercontaining 1 mM 3-isobutyl-l-methylxanthine (IBMX)' (Aldrich Chemical Co.,

Milwaukee,WI) inthe absence orpresenceof eitherPGE2orPTH. cAMP contentwasdeterminedby radioimmunoassay

(Becton-Dick-inson &Co.,Paramus,NJ)(16,17). PGE2was agiftoftheUpjohn Co.,

Kalamazoo, MI. BovinePTH(- 3,000 U/mg) extracted from TCA precipitates of bovine parathyroid glandsandpurifiedbygelfiltration

and ionexchange chromatographyoncarboxymethyl-cellulosewas a

gift ofDr.Henry T. Keutmann(Massachusetts GeneralHospital, Bos-ton,MA).

Labeling of medium collagens.Cellswereplatedat2X 105cells/

dishin 3.5-cmdishesand 2 d laterincubatedfor 24 h with

L-[5-3H]-proline (30

,tCi/mmol)

in the presence of50jig/ml

beta-aminopro-prionitrile, 50

jg/ml

ascorbicacid,and2 mMglutamineintheabsence of serum. We havepreviouslyfoundthatundertheseconditions

me-diumcollagenaccountsfor>95%of totalcollagensynthesized.

Me-diumproteinswhich includedprocollagensandprocessed procollagen were analyzed bySDS-PAGE with orwithout reduction with 0.5%

#-mercaptoethanol

andfluorographyaspreviouslydescribed(18). La-beled collagens were further characterized by pepsinization at 4°C

followedby SDS-PAGEwithorwithoutdelayedreductionto distin-guishal(I)fromal(III) collagenchains(18, 19).

Analysis of collagen mRNAs. CytoplasmicRNA was extracted from cellsbythe methodof WhiteandBancroft(20).After

denatur-ationin 7.5%formaldehyde,dothybridizationwas

performed

as

de-scribedpreviously usingcDNAprobeslabeledbynick translation(21,

22).ThefollowingcDNAswereused: Hf677,a 1,500-bpcDNA

en-1. Abbreviation usedin this paper: IBMX,

3-isobutyl-l-methylxan-thine.

798 S. R.Goldring,M. L.Stephenson,E.Downie,S.M.Krane, and J. H.Korn

J.Clin.Invest.

© TheAmericanSociety for Clinical Investigation,Inc.

0021-9738/90/03/0798/06 $2.00

(3)

coding partof the al(I)procollagen subunit (23),and Hfl131,a 1,500-bp cDNA encodingpartof the a2(I) procollagen subunit (24) provided byDr.F.Ramirezand Dr. D. J.Prockop. ThecDNAfor the

a1(III)procollagen,pHCIII-I,wasprovided byDr. R.G. Crystal and

Dr. M.Brantly,National Institutes of Health (NIH; 25). Specific

4ctivi-ties ofthelabeledprobeswere - 2.16, 1.77, and 1.45 X108 cpm/yg

DNAforthea1(I), a2(I), anda1(111) procollagen cDNAs, respectively.

For Northern hybridization, total RNA was extracted usingthe guanidine-thiocyanate procedure (21). TotalRNA (5 Ag)was dena-tured bytreatmentwith formaldehyde-formamideand

electropho-resed on0.8%agarosegelscontaining 0.22Mformaldehyde.TheRNA wastransferredtonitrocellulosepaperandhybridizedusing thecDNA

probes describedabove(21). The relativeintensity of the 28Sand18S ribosomal RNA, compared with background as an index of RNA breakdown,wasassessedusingethidium bromide andvisualized under ultravioletlight. Nicktranslation,prehybridization,andhybridization withthe procollagen cDNAs wereperformedasdescribed (21).

Results

We have previously shown that cells cultured from human dermis increase cAMP content in response to PTH orPGE2 (26, 27). To examine the pattern of hormone-induced cAMP responsesin dermal fibroblasts, 24 cell substrains cloned by

limiting dilution from three neonatal foreskin sampleswere

incubated with maximal stimulatory concentrations of PTH orPGE2. The three parent cultures of fibroblasts were

respon-sivetoboth hormones. Allsubstrains preparedfrom the parent cultures from two of thesamples (D.W. and J.R.)increased cAMP contentwhen testedwithPTH orPGE2, althoughthere were marked differences in the magnitude ofthe cAMP re-sponses.Althoughcellsfromthethirdparent culture(L.T.F.)

were responsive to PTH, none ofthe substrains increased

cAMPlevels in response to PTH (Table I). These substrains were, however,responsiveto

PGE2-Fig. 1 shows therelationship between PTH- and PGE-in-duced cAMP responses in clones fromsampleD.W. Although there was a correlation of the PTH and PGE2 responses in

substrains from parent culture D.W.(r= 0.77),there wasno

correlation in the responses to the two hormones in the sub-strains from parentculture J.R. (not shown). Patterns of

re-sponsivenesstoPTHandPGE2remained stableafterpassage; in general,substrainsexhibitingthe greatesthormone-induced increasesin cAMPlevelscontinuedtoshow enhanced sensitiv-itytohormonai stimulation.

TableL ComparisonofPTH-induced cAMP Response inaParentDermalCell Cultureand Two

RepresentativeSubstrains

cAMP

Parent Substrains

L.T.F. C K

pmol/well

Buffer 1.84±0.45 0.55±0.13 0.45±0.06 PTH 7.60±1.82 0.52±0.06 0.57±0.04

PGE2 71.1±1.84 47.7±4.03 6.40±0.93

Cellswereincubated for20 min at 37°C with buffer containing

(1 mM)IBMXwith or without PTH(l0-7M) or±PGE2(2.5 X 10-6 M). Valuesrepresentmean±SEM.

o)

0 U) Q 0 0.

0).

L.=

0

so

U)

C00

2-tO

,

T-0

._0

la I.

0 20 40 60 8 0

PGE2-induced cAMP response (pmol/5X104cells)

Figure1.Comparison of thePTH- andPGE2-inducedcAMP

re-sponses to maximalstimulatory concentrations of hormone in

sub-strains fromparentdermal cultureD.W.Cellsin 24-welltrayswere

incubated 20 minat370 inthe presenceofImM IBMX±PTH(l0-7

M)orPGE2(2.5X 106 M). Pointsrepresent meansof threewells foreach hormone.

We have previously described an inverse relationship

be-tweenendogenous PGE2 production and the magnitude of the PGE2-induced cAMP response in different human connective tissue cell cultures (28-30). As shown in Fig. 2, therewas no

correlation between basal PGE2 levels and the cAMPresponse tothis ligand in the D.W. substrains.

To furtherinvestigate thefunctional heterogeneityof the

dermalcells,substrains from the threeseparate parentcultures were examined for their capacity to synthesize collagen. All substrains produced types I and III collagen (analyzed after pepsinization and resolution of the labeled medium proteins by SDS-PAGE andfluorography),although therewas variabil-ity in the amount and relative abundance of the individual collagentypes.To quantitate the relative capacity of substrains

toproduce individual procollagens, cells were incubated with [3H]proline, and after separation of the pepsin-resistant

me-0

U)-)0)

.=

2-O

0CD

0 -40

0E

-Q1

100

80

60

-40

-20

-O-_

0.0 0.5 1.0 1.5 2.0

PGE (10

8M)

2

Figure2.Comparison of the PGE2-induced cAMPresponseand

basalPGE2 productionin substrainscloned fromonedermal

sam-ple. Cells in 24-welltrayswereincubated 20 minat37°Cinthe

pres-ence orabsence ofamaximalstimulatoryconcentrationofPGE2 (2.5X10-6 M). PGE2 levelsweremeasured after72 h incubationof

substrains(5X 104cells)in culturemedium alone.Values represent

meansofthreewells.

S

0

(4)

B Figure 3. Capacity of

der-

0.-A.< 4000 mal substains to

incorpo-O,. 4000 |-Z@ ok 1 * rate[3H]proline into

._ =L aI(I) procollagen;

com-3000

*

Cf

anparison with basalPGE2

E

3000-

production and

PGE2-in-oE 0~~~ ~ ~ ~ ~ ~~~~~~0m 22000- ducedcAMPresponse.

-w MediumPGE2 was

as-g 2000 * sayed in substrains (5

.2 u

C0~~~~~~~~~~~~~~~~~;-0*

.27Co 00 X

104

cells) incubated 24

0~~~~~~~~

aoe G2 epne

000 * h in culture medium

o . 0 alone.

PGE2

responses

c _ 0 . were' t o-. ,assessed. ,bytest-in-

, .

, .

0 0 1 0 20 3 0 4 0 5 0 cubatingcells(5 x10

0.0 0.2 0.4 0.6 0.8 1.0 cells/well) for 20min

PG18M PGE2-induced cAMP response wt rwtotPE

PGE2(10

M)

(pmol/well)

withorwithoutPGE2

(2.5X 106M). A com-paresPGE2productionto a1(I) procollagensynthesis. Pepsin-resistent labeled medium proteins were separated by SDS-PAGE. The regions of

thegel containingthea1(I)procollagen bands were excised and counted. Counts have been normalized to DNA content in the culture dishes at thetimeof labeling.PGE2productionand a1(I) procollagensynthesis were negatively correlated (r = 0.33). B comparesa1(I)procollagen

syn-thesis withthePGE2-inducedcAMP responses, which were negatively correlated (r = 0.21).

dium proteinsbySDS-PAGE the labeled bands were excised and counted. Fig. 3 shows incorporation of [3H]proline into

a1(I) procollagen in substrains cloned from a single sample of dermis. There was marked heterogeneity in the capacity of the

substrainstosynthesizea1(I)procollagen. With passage of the cells the pattern ofcollagen synthesis among substrains

re-mainedstable. There was no correlation between the amounts

ofa (I)procollagensynthesizedandthe production of PGE2 (r = 0.33) (Fig. 3 A) or the magnitude of the PGE2-induced cAMP response(r=0.21) (Fig. 3 B).

To determine whether the heterogeneity in collagen

syn-thesis amongthe substrains was correlated with steady-state levelsofprocollagen mRNA, cytoplasmic RNA was extracted andhybridizedwith labeled cDNAs for types I and III procol-lagen. Northern blot analysis of total RNA demonstrated that therewas nocross-hybridizationamong the probes (data not

shown). The levelsof procollagen mRNAdeterminedby dot

hybridizationasafunction ofthe secreted labeled procollagen

estimated by [3H]proline incorporation are shown in Fig. 4. Dotblotswerequantitatedbyscanningwith a soft laser densi-tometer and analyzed with an Apple Ile computer using a

Zeineh Videophoresis II electrophoresis reporting program

(Biomed Instruments Inc., Fullerton, CA) as previously

re-ported (21).Therewas acorrelationbetween the steady-state levelsofa1(I)procollagen mRNA and incorporation of

[3H]-proline into al(I)collagen chains(r = 0.68). No correlation wasfound in the pattern of collagen synthesis and hormone

responsiveness.

ThelevelsofaI(I)anda2(I)mRNAswerequantitated in

substrains from one parent culture (L.T.F.) by scanningthe

dotblots after

hybridization

with cDNAprobes. The ratios of

the a1(I) and

a2(I) procollagen

mRNAs inthe substrains ranged from 1.37 to 2.46. As shown in

Fig.

5, the ratios of

a

l(I)/a2(I)

mRNA wereinverselycorrelated with theratioof a

1(III)/a

I(I) plus

a2(I)

(type III/type

Icollagen).

Comparison

ofthe levels oftypes I and III

procollagen

mRNA

(Fig. 6)

revealedasignificant

positive

correlation

(r

=

0.86;

P<

0.001).

Thecorrelationwasbest between the levels ofaI

(III)

and

a2(I)

procollagen (r=0.89;P<

0.001)

compared

with that ofa (III)

andal(I)(r= 0.79;P<0.01).

Discussion

We havepreviously demonstratedthat in responsetoproducts

present inmedium conditioned by PBMC, dermalfibroblast

populations cultured from different individuals exhibit

marked phenotypic diversity in proliferative responses and

synthesis ofPGE2 andcollagenase ( 1-4). This conditioned

me-dium containsmanybiologically active cytokines,suchas IL- 1 ortumor necrosis factor, that could

potentially

influencethe

functions of connective tissue cells. We speculated that the

heterogeneityinproliferative responsestotheseligandscould

provideamechanism for immune-mediated clonal selectionat

sites of inflammation by favoring overgrowth ofclonal cell

populations

with

phenotypic

characteristicsthat promote

dis-easeexpression. Ithas beenproposedthatseveralchronic

in-2000

2 1500

Z~~~~~

0 1000 2000 3000 4000

al(1)

protein(cpm/ggDNA)

Figure 4.Comparisonindermal substrains ofthea1(I)procollagen

mRNAlevelsandthe amountofsecreteda1(I)procollagen

esti-matedby[3H]prolineincorporation. After transfer of cytoplasmic

RNA tonitrocellulose and hybridization with the labeled procollagen

cDNAstheautoradiographswerequantitated by densitometric

scan-ning. Incorporation of[3H]prolineintoal(I)procollagenwas

deter-mined by counting excisedbandsfrom SDS-PAGE gels after

separa-tion oflabeledmediumproteinsasdescribedinFig. 3. Eachpoint

represents the valueforasingle substrain.

(5)

2 1

0'

0 2 4 6

al(III)/Total ca(I)+a2(I)

(Percent)

Figure5.Comparisonin dermal substrains of the ratio ofal(I)/a2(I) procollagenmRNAwith theratio oftypeIII/typeIprocollagen mRNA.After extractioncytoplasmicRNAwastransferredto nitro-cellulose andhybridizedwith32P-labeledaI(I),a2(I),or a

1(III)

pro-collagen probes.RNAwasquantitated by densitometricscanningof theautoradiographs.Eachpointrepresentsthevaluesforasingle

substrain.

flammatory

conditions associated with increased connective tissue

deposition

such as

hepatic cirrhosis,

interstitial

pneu-monitis, nephritis,

and scleroderma (7,

31-33)

could be ex-plained by selectionin vivo of dominantclonesofcells that

proliferateandproduce

relatively

largeamountsofmatrix.

Inthesestudieswefurtherevaluateddermalfibroblast

pop-ulations by measuring

the

production

of extracellularmatrix

macromolecules by substrains derived from parent cultures. Marked differences in the pattern and amounts of

collagen

synthesis

weredemonstrated.

Furthermore,

thedifferencesin the amountsand types of secreted

procollagens

estimated by

[3H]proline

incorporation

correlated with the steady-state

levels of the

corresponding

procollagen mRNAs. Although

these resultsareconsistent with differential

regulation

of

col-lagen synthesis at a

transcriptional

level, they could also be accounted for

by

differences in mRNA

stability.

Direct

mea-Z 100 E

75-0 E050 =-u 25

0*

0 500 1000 1500 2000

Type

I

procollagen

mRNA

(densitometry units)

Figure6.Comparison ofthe levelsoftype III [a

1(III)]

totype I

[al(I)+a2(I)]procollagenmRNAinsubstrains fromparent culture L.T.F.After extraction cytoplasmicRNA was transferred to

nitrocel-luloseandhybridized with32P-labeleda(I), a2(I),oraI(111) procolla-genprobes. RNAwasquantitated by densitometric scanning ofthe

autoradiographs.Eachpointrepresents thevalues (expressed in den-sitometryunits)forasinglesubstrain.

surementsof

transcription

and mRNA

stability

are

required

to

establish the molecular basis for

heterogeneity

in matrix

syn-thetic

capacity

among substrains.

We

previously

noted variation in the ratios of levels of a1

(I)

and

a2(I)

procollagen

mRNAs in cultured human

chon-drocytes

and

synovial

fibroblasts

( 19, 21, 22, 34).

These results

suggested

that theexpressionoftheseprocollagengenes isnot

coordinately regulated.

We

speculated

that the excess

a2(I)

procollagen

mRNAwaseithernottranslatedorthe translated

product

wasnotsecreted

by

the cells. Similar variation in the ratio of the levels of thea

I(I)

and

ac2(I)

procollagen mRNAs

was observed among the dermal substrains in the present re-port, consistent with differential regulation ofthese two

pro-collagen

mRNAs.

Furthermore,

the results would indicate that the normal ratio ofa1

(I)/a2(I)

of 2:1 in parent cultures may reflect the average of constituentpopulationsthat vary

signifi-cantly

from this ratio.

Miskulinetal.

(25)

previously reportedthat the

expression

ofthe type III

collagen

gene is modulated coordinately with that of thetypeI

collagen

genes basedoncomparisonof

pro-collagen mRNA levels in dividingand nondividing cells. In

ourstudieswedidnotobserveacorrelation between

collagen

synthesis

and cell

doubling

times. However,the levels of

type

IIIprocollagen mRNAwerestronglycorrelated with those of type I(r=0.86;P<0.001).This correlationwasbest between

al(III)

and a2(I) (r = 0.89; P < 0.001)compared with that between

alI(III)

and

al(I) (r

=

0.79;

P<0.01).Our resultsare consistent with coordinateregulation of types I and III colla-gen colla-geneexpressioninthese substrain

populations.

Botsteinetal.(7)havereportedthat there is marked

heter-ogeneityin thecapacityof cloned human foreskin fibroblasts

toincorporate [3H]proline into collagens, although thespecific

collagen

types synthesized were not characterized. More

re-cently, Crouchetal. (35)have reported that

morphologically

distinct clones ofahumantumorcellline exhibit

heterogene-ityin theproductionof collagenaswellasfibronectin. Dermal fibroblasts cultured from patients with scleroderma

produce

more types I and III collagen than fibroblasts from normal

subjects (36-38);this enhanced collagen synthesis is accompa-niedbyincreased steady-state levels of types I and III procol-lagenmRNAs. Since the scleroderma phenotype persistsover

many passages in vitro these observations are consistent with the selection of populations of fibroblasts in vivo which are

characterizedby highratesof collagen synthesis. Our

findings

ofheterogeneityin the pattern of matrix synthesis among sub-strains derived from normal dermal fibroblasts providea ratio-nale for apparent clonal selection of fibroblasts in a disease

suchasscleroderma. Of interest in this regard are our earlier observations that short-term exposure of dermal cell cultures to mononuclear cellconditioned mediumresultsin selection of cell populations with enhanced proliferative and

PGE2-syn-thetic responses upon reexposure to this mononuclear cell conditioned medium (1). In analogous studies, Duncan and Berman have shown that transient exposure of dermal fibro-blaststo y-interferon leadsto apersistent decrease incollagen

synthesis (39). Inflammatory cytokines may thus have

long-lasting effects on the phenotype of connective tissue cells. In this report we have also described responses of fibroblast substrains to two hormones that affect connective tissue

re-modeling. Since PGE2 has been shown to modulatecollagen

(6)

there was acorrelation between PGE2 responsiveness and the level of collagen synthesis (22, 34, 40). We had previously shown that theresponsiveness of many different connective tissue cell types (including dermal-, synovial-, and

bone-de-rived cells) to PGE2 is markedly influenced by the levels of endogenous PGE2 (28-30). Treatment of cultured cells with mononuclearcell products such as IL-1/3 or tumor necrosis factor-a, which increased endogenous PGE2synthesis, resulted

in desensitization to PGE2 stimulation. These results were consistent withdownregulation of receptors by high ambient levels of PGE2. In the present studies we did not observe a correlation between endogenous levels ofPGE2 production andPGE2-induced cAMP responses. Basal unstimulated levels ofPGE2were very low (1.6-7.5 ng/ml) in medium from these cells, however, and based on previous studies with skin and

synovial fibroblasts (28, 29) higher levels of PGE2 may be required for receptor downregulation.

We had previously reported that cells cultured from human dermis increase cAMP levels after incubation with PTH(16, 26, 27).Wedemonstrated byphotoaffinity labeling

thatthePTHreceptor in these cells was similar to that in other PTHtargettissues (bone andkidney)(41). Others have

con-firmed our observations concerning PTH responsiveness in dermal cultures, although the kinetics of PTH binding may differ in skin- andbone-derived cells(42-44). We were inter-estedin examining the cells for responsestoPTH, since this hormone may also modulate collagen synthesis (possibly by inducing increasesinintracellularcAMPlevels) (45, 46).Our present resultsindicate that, similar to PGE2, the substrains exhibit markedheterogeneity intheirresponsivenessto PTH. Infibroblast substrainsobtained from one sample, PTHand PGE2 werepositively correlated, butthiswasnot observedin

substrains from anothersample. These results favor

heteroge-neityin cellsurfacereceptorfunctionratherthan ageneralized

up- ordownregulation ofadenylate cyclase activity. We have

not examined thesubstrains forthe capacitytoproducethe recently described PTH-related peptide (47-51). Thisligand

doesincreasecAMP levels inPTH-responsivetissues

includ-ing dermis, and could

potentially

downregulatePTH receptors in a manneranalogoustoendogenous PGE2.

One factor

potentially

contributingtotheheterogeneityin

the patterns of collagen

synthesis

among the substrains

could

be related to differences ingrowth ratesand the effects of in

vitroaging (5, 6)oncellphenotype.This would berelevantto

the datain Table I, in which the parent (L.T.F.) was PTH

responsiveandsubstrainswereunresponsivetothis hormone. The parent cells hadundergonefar fewerdoublingsin

compar-ison

totheclones. However,the

stability

ofsubstrain

pheno-type with respect to collagen synthesis and PGE2 response

found in these studies and in

previous

investigations (1-3)

arguesagainst

aging

or senescence asthecauseof the

pheno-typic diversity.

Inconclusion,ourstudies demonstrate that connective

tis-suecell substrainsclonedfromnormal human dermal tissues areheterogeneousbothintermsof

synthetic products

and in the

activity

of cell surfacereceptorsforhormones that

regulate

connective tissuecellfunction. Selective

expansion

of cell

pop-ulations of connective tissue cells in vivo could

give

riseto

tissues withaberrent patterns of matrix

synthesis.

In

addition,

alterations in responsestohormones that

regulate

these cells

couldcontributetothedisturbance innormalfunctional

activ-ity.

Similarprocesses may also be involved inconnective tissue

remodeling

under

physiologic

conditions.

WethankMicheleAngelo for preparation of the manuscript.

This work wassupported by NIH grantsAM-03564, AR-32343,

and AR-20621,andgrantsfromtheMedicalResearch Service of the VeteransAdministration and the Arthritis Foundation.

This is publicationnumber1056ofthe Robert W. Lovett

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References

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