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Transforming growth factor-beta activation in

irradiated murine mammary gland.

M H Barcellos-Hoff, … , M L Tsang, J A Weatherbee

J Clin Invest.

1994;

93(2)

:892-899.

https://doi.org/10.1172/JCI117045

.

The biological activity of TGF-beta, an important modulator of cell proliferation and

extracellular matrix formation, is governed by dissociation of mature TGF-beta from an

inactive, latent TGF-beta complex in a process that is critical to its role in vivo. So far, it has

not been possible to monitor activation in vivo since conventional immunohistochemical

detection does not accurately discriminate latent versus active TGF-beta, nor have events

associated with activation been defined well enough to serve as in situ markers of this

process. We describe here a modified immunodetection method using differential antibody

staining that allows the specific detection of active versus latent TGF-beta. Under these

conditions, we report that an antibody raised to latency-associated peptide detects latent

beta, and we demonstrate that LC(1-30) antibodies specifically recognize active

TGF-beta 1 in tumor xenografts overproducing active TGF-TGF-beta 1, without cross-reactivity in

tumors expressing similar levels of latent TGF-beta 1. We previously reported that TGF-beta

immunoreactivity increases in murine mammary gland after whole-body 60Co-gamma

radiation exposure. Using differential antibody staining we now show that radiation

exposure specifically generates active TGF-beta 1. While latent TGF-beta 1 was widely

distributed in unirradiated tissue, active beta 1 distribution was restricted. Active

TGF-beta 1 increased significantly within 1 h of irradiation concomitant with decreased latent

TGF-beta immunoreactivity. This rapid shift in immunoreactivity provides the first evidence

for […]

Research Article

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Rapid Publication

Transforming

Growth

Factor-,8

Activation in Irradiated

Murine Mammary

Gland

M. H.Barcellos-Hoff,*R. Derynck,t M. L.-S. Tsang,'and J. A.Weatherbee*

*LawrenceBerkeley Laboratory, Universityof California,Berkeley, California 94720;tDepartmentsof Growth andDevelopment, and Anatomy, Programs in Cell Biology andDevelopmental Biology, UniversityofCalifornia,San Francisco, California94143-0640; and

OR

&DSystems, Minneapolis,Minnesota 55413

Abstract

Thebiological activity of

TGF-fl,

an important modulator of cell proliferation and extracellular matrix formation, is

gov-ernedby dissociation ofmatureTGF-t6 fromaninactive,latent

TGF-f

complex inaprocess thatiscriticaltoitsrole invivo. So far, ithas not beenpossibletomonitor activationinvivo since conventional immunohistochemical detection does not accu-ratelydiscriminatelatent versusactive

TGF-fl,

norhave events

associated with activationbeendefinedwellenoughtoserve as insitu markers of this process. Wedescribehere a modified immunodetection method using differential antibody staining

that allows the

specific

detection of active versus latent

TGF-fl.

Under these conditions, we reportthatan antibody raised to

latency-associated peptidedetects latentTGF-j6,and we demon-strate that

LC(1-30)

antibodies specifically recognize active

TGF-#1

in tumor xenografts overproducing active

TGF-fil,

without cross-reactivityin tumorsexpressingsimilar levelsof

latent

TGF-fl1.

Wepreviously reportedthat

TGF-ft

immunore-activity increasesinmurinemammarygland after whole-body

'Co-'y

radiationexposure.

Using

differential

antibody staining

wenow showthat radiationexposure

specifically

generates

ac-tive

TGF-f1.

While latent

TGF-,61

was

widely

distributed in

unirradiated tissue, active

TGF-ftl

distributionwasrestricted. Active

TGF-#1 increased significantly

within1hofirradiation concomitant with decreased latent

TGF-ft

immunoreactivity.

Thisrapid shiftinimmunoreactivity providesthefirst evidence foractivation of

TGF-ft

insitu. Thisreciprocalpattern of

ex-pression persisted for 3 d andwas

accompanied

by decreased

recoveryoflatent

TGF-#1

from irradiated tissue.

Radiation-in-ducedactivation of

TGF-ft

may have

profound implications

for

understanding tissue effects caused by radiation therapy.

(J.

Clin. Invest. 1994.

93:892-899.)

Keywords:

ionizing

radiation

*transforming

growth factor-,8

* mammary

gland

* immunohis-tochemistry* extracellularmatrix

Introduction

TGF-f3

is amajormodulatorofcellular

proliferation

and extra-cellular matrix formation. TGF-f 1, the best studied protein of

Address correspondenceto Dr. M. H. Barcellos-Hoff, Life Sciences

Division, Building74-159,LawrenceBerkeley Laboratory,University

ofCalifornia, Berkeley,CA 94720.

Receivedfor publication 13July1993 andinrevisedform 10 No-vember 1993.

the three

differentially

expressed and regulated

TGF-3

iso-forms,is derived from a390-amino acidprecursor cleaved to

producea 112-amino acid carboxy-terminal peptide( 1 ). The

homodimer of this peptide is noncovalently associatedwith a

dimer

of

theprocessed

amino-terminal

pro-segment, calledthe

latency-associated

peptide

(LAP).'

This secreted latent

TGF-f3

complex isunable tobindto

TGF-,3

receptorsuntil the biologi-cally active24-kD mature

TGF-3

is dissociatedfrom LAP (2,

3). Thus, thebiological activityof

TGF-3

is highlycontrolled by its release from the latent complex, which is considereda

critical regulatory event for the function of

TGF-3

in vivo.

Although activation has been postulated to occur in a wide

variety of physiologicalandpathologicalsituations in vivo, it has sofarnot beenpossibletoimmunohistochemically

distin-guishlatentandactive

TGF-f3.

Thistechnical limitationhasresulted inaninabilityto

de-fineeventsassociated withorleadingtobiological activation of

TGF-f

in situ. Elevated

TGF-3

immunoreactivity hasbeen observed in tissues during normal

physiological

(4) and patho-logical (5-7)processes that result in inflammation, tissue

re-pair,

orextracellular matrix

deposition.

We recently showed using

immunofluorescence

that

TGF-O

is elevated inthe

mu-rinemammarygland after whole-bodyexposure to

'Co-y

radi-ation (8).

TGF-j1

immunoreactivitywas

rapidly

induced in

the

epithelium

and the

previously negative adipose

stroma,and

wasfollowedbynovel expression of collagentype

III,

a known targetof

TGF-3.

However, whetherthealtered

TGF-f

immu-nostaining reflects increased

TGF-f

depositionoraltered

epi-tope

accessibility,

as

might

occur if

TGF-3

was

dissociated

from thelatentcomplex,could notbe determined with avail-able methods. Whereasaltered

immunoreactivity

isoften

as-sumedtorepresenta

corresponding

change in

TGF-f

activity,

the currentmethodology detects

TGF-j

irrespective

of knowl-edge of itsstateof

activation.

For

example, antibodies

toLAP areexpectedtonotonly

recognize

latent

TGF-j3

butthesame

pro-segment after

dissociation

of the latent complex.

Likewise,

recognition of

mature

TGF-fl1,

theactive

polypeptide

that is

partofthe latentcomplex, may beimpartialto theactivation

state.However, if the

antigenic

site ismasked, for

example

by association with LAP,ordependsonits

conformation,

which

may changewithactivation, antibody recognition of

TGF-f1

might be wholly dependent upon activation. Furthermore, sinceaccurateimmunolocalization ingeneral

depends

on

tar-get

protein preservation

after tissue

fixation,

concern about whether tissue processing might activate

TGF-f3

has been

raised(9). Thus,

interpretation

of

TGF-f

antibody

reactivity

is confounded by a numberof

restrictions, including

questions

1.Abbreviation usedinthis paper: LAP,latency-associated peptide.

892 M.H.Barcellos-HoffR.Derynck,M.L.-S. Tsang,and J. A. Weatherbee

J.Clin.Invest.

©C

The AmericanSocietyfor ClinicalInvestigation,Inc. 0021-9738/94/02/0892/08 $2.00

(3)

regarding antibody specificity in processed tissue, a lack of in-formation on the preservation of endogenous latent and

acti-vate

TGF-f3,

and the inabilityto independently demonstrate

the status of

TGF-f3

in situ.

The specificity of antibody detection usingvarioustissue

fixation methods couldberesolvedbyusingatissue inwhich the activation status of TGF-f is known. Thus, we usedtwo

xenograft tumors constructed to express active and latent

TGF-3 todevelop modified tissue fixation and

immunodetec-tionprocedurespermitting differential antibody staining of ac-tive vs. latent

TGF-#3.

This method was used to evaluate the activation state of TGF-f after irradiation of the murine mam-mary gland in vivo. Immunostaining revealed that radiation inducesarapid shift from predominantly latenttoactive

TGF-3,whichindicatesthatirradiationleads toactivation of

TGF-(31

in situ.

Methods

Antibodies.Polyclonalrabbitantiserum raised against a synthetic pep-tidecorrespondingtotheamino-terminal 30amino acids ofmature

TGF-fl,

designated LC( 1-30) (10),wasprovided by Dr. M. Sporn (Na-tionalInstitutes of Health, Bethesda, MD).

Anti-LAPantibodywasproduced in goatsimmunized with recom-binant human LAP. The recomrecom-binant LAP used as immunogen was purified from medium conditioned byaChinese hamster ovary cell line transfected with an expression plasmid for the human

TGF-f13

pre-pro-protein. LAP was dissociated and purified from mature TGF-(31 byacidificationand reverse phase chromatography. Total IgG was purified by protein Gaffinitychromatography.Both the dimeric LAP anditsmonomers weredetectableusingthisantibodyonWestern blot

analysis. Approximately 2ng/lane of LAPwasdetected in Western blot assaysusinganantibodyconcentration of 1

gg/ml.

Direct ELISA demonstrated thatthisantibodyisspecific fortheLAPderived from the

TGF-#

1 precursor anddoesnotreactwithLAPfrom TGF-(32nor with matureTGF-fl1,TGF-f32,orTGF-133.

Irradiation. Adult(> 12 wkof age) virginfemale BALB/c mice wereirradiated with

'Co--y

radiation usingadose rateof0.35 Gy/min to atotaldose of 5Gy.After 1 h or1, 3,or 7d,animals from each exposure groupwerekilled by cervical dislocation in accordance with animal welfare procedures and the inguinal mammaryglands were removedforimmunohistochemistry.

Tumortissue. Two cell clonesofthe 293 renalsarcomacellline that were transfected with

TGF-#

I expression plasmids were used (11). Clone B9 overproduces latent

TGF-#

1 whereas clone C 19, transfected withanexpressionplasmid foramodifiedTGF-Bl precursor, secretes

predominantlyactive

TGF-,l

1.Cellswereculturedtosubconfluence

and 2X 106 cells wereinjectedinto each flankofadultfemale nude miceasdescribed ( 11).Palpabletumors wereobtained within 3-5wk andwereexcised from mice killed by cervical dislocation in accordance with approved animal welfare guidelines. Tissue was processed as de-scribed below forimmunohistochemistry.

Immunohistochemistry. Tissues were embedded in OCT com-pound(Miles Inc., Elkhart, IN)andfrozen inadryice/ethanolbath. Theblockswerestoredat-70°C untilsectioning.4-Amthick sections were obtained from sectioning at -30 to -35°C. For use with anti-LC (1-30), the sections wereimmediatelyfixedontogelatin-coated

cov-erslipsusing -20°Cmethanol/acetone(1:1),airdried,and storedat -20°C. Anti-LAP antibodieswereusedwith sections fixed for 20 min with2%paraformaldehydeandrinsed threetimes with PBS containing 0.1 Mglycine.The bestfixativewasdetermined bycomparingeach

antibody usingsections fixed in various ways andselectingthe condi-tion under whichreactivitywasstrongest.

Beforeantibodytreatment,thetissuesectionsweretreatedfor 60 min in supernatant fromasolution of 0.5% caseinin

phosphate-buf-fered saline(Na2PO4,0.9%NaCI), pH7.4(blocking buffer).The

sec-tionswerethen incubated with 25Mloftheprimary antibodydilutedto

workingconcentration in blocking buffer and incubated overnightat

40C.Antibody controlswereincubated without primary antibodyor with similar concentrations of normalserumorIgG from thesame

speciesastheprimary antibody.Sectionswerewashed and incubated witha 1:100dilutionof theappropriatefluorescein

isothioycyanate-conjugated secondary antibodyfor 1 h atroomtemperature. After severalwashes,the sectionsweremounted in Vectashield(Vector Labo-ratories, Palo Alto, CA). Sections wereviewed usinga microscope (Olympus Corp. Precision Instr. Div., Lake Success, NY)equipped

with epifluorescence and photographed using Kodak film TriX 400. Identical exposuresweretaken foragiven antigen in eachexperiment

andprinted usingidentical parameters in ordertofacilitate

compari-sons.Eachantibodywasexamined inatleast threeindependent

stain-ing experiments from two independent sets ofirradiated animals.

Antibodycontrolswere negativeincomparisontospecific antibody

staining.

Assayfor TGF-f activity. Mammary glandwasexcisedfrom ani-malsatvarioustimes postirradiation,chopped,andincubatedat370C

for 1 h inDMEM/F12 mediacontaining 2% BSA. 2.5 ul of 0.2 M phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis,MO)

and 5 Ml of aprotonin (- 10 trypsin inhibitor units/ml; Sigma Chemi-calCo.)wereaddedtoeachmilliliter of conditioned mediumsamples,

which were frozen on dry ice and stored at -70'C. Conditioned media were testedfor the presence of latent TGF-(3 using inhibition of mink lung epithelial cell DNA synthesis assayed by

[3HIthymidine

incorpo-ration( 12). Sampleswereheatedat80'C for 10 mintoactivate latent

TGF-fl

( 13 ). 80 Ml ofa 1:1 dilution of each experimental samplewas added 2 h after platingtowells containing 5 x 104cellsin 0.2 ml DMEMcontaining 0.2% serum. 20 h later, the cell cultures were la-beled for 2 h by the addition of 0.5 mCi [3H]thymidine (4mCi/ml;

AmershamCorp., Arlington Heights, IL) and 3H incorporation into DNAwasdetermined by scintillation counting. Neutralizing antibody toTGF-,B (R & D Systems, Minneapolis, MN)wasusedto demon-stratespecificity of

TGF-#

inhibition of DNA synthesis in

experimen-talsamples.

Results

Differential

use

of antibodies distinguishes latent

and active

TGF-f,.

We compared thespecificityof several antibodies

us-ingtwoxenografttumors derived fromtransfected 293 renal

sarcoma cells thatoverexpressTGF-j (11). The clonal tumor celllines B9and C 19 were derived from the same parental cell

lineand secrete similarlevelsof TGF-j 1;however,B9 secretes latent TGF-3 1, whereas C 19 makes predominantly active

TGF-#

1. Thisdifference in

TGF-#

activation between the tu-morclones results fromdirected mutation of two cysteinesto

serines in thepre-pro-segment

TGF-#

peptide (11),which

pre-vents disulfide bond formation in LAP resulting in

constituti-vely active

TGF-#

(14). The activation of TGF-f31 secreted

fromC 19 cells is presumablyaconsequenceof decreased inter-action ofthe mutated LAP withTGF-f31, thus resultingin a

highlevel of dissociation of LAP from

TGF-fl

1. This is in con-trastwith thephysiologicalactivation mechanisms of

TGF-0

1, which are thought to result from proteolytic degradation of LAPandsubsequentrelease of active TGF-fl1.

Previous immunohistochemical detection methods for

TGF-#

have used paraffin-embedded tissue fixed in either Bouin's fluid and neutral buffered formalin(10),Bouin'sfluid

alone (15), or 10% neutral buffered formalin alone (6).To potentiate the preservation of latent and active

TGF-fl,

we

elected to usecryosections from frozen unfixed tissue for

(4)

munodetectionof

TGF-f3.

We then testedvarious fixatives in conjunction withimmunofluorescencedetectionof antibodies raised to distinct regions of the latent TGF-f complex. We chose twoantibodies foruseinthisstudy. LC(1-30) antibodies raised against a30-aminoacid peptide sequenceresidingatthe

amino terminusofmatureTGF-3(10) were usedwith

cryosec-tions fixed with methanol/acetone. Antibodies raisedagainst purifiedrecombinantLAP were usedwith paraformaldehyde-fixed tissue.

Anti-LAPshowedsimilarimmunoreactivity forboth types

oftumorsections (Fig.1,Aand B),indicatingthatthey express

similar levelsofLAPand, byinference, ofmature

TGF-f3.

In

comparison, substantial LC(1-30) immunostaining was ob-servedinCl9-derived tumortissueproducing active TGF-fl1,

whereas negligibleimmunostaining was detected in B9 tumor

sections (Fig. 1, C andD). The lackofimmunostainingin B9 tumordespiteabundantlatentTGF-j3 impliesthat the

embed-dingandstainingproceduredidnotactivateendogenous latent

TGF-f. Furthermore, these data indicate that the

LC(

1-30)

epitope of mature TGF-f Iis masked whenTGF-fl is associated with LAP in the latent complex. Thus, since LC( 1-30) was raised against anepitope of mature

TGF-#

I (10),but did not react with B9 tumor tissue, even though the total level of latent

TGF-f31

wassimilar, LC( 1-30) appears to specifically recog-nize active TGF-p while anti-LAP detects total TGF-p under these conditions.

Active TGF-f increases in mammaryglandafter

irradia-tion. The ability to selectively detect active TGF-,BI by immu-nohistochemistry and to preserve endogenous latent TGF-,Bl allowsexamination ofthe relative distribution of both forms of TGF-f31 in tissues during physiological and pathological

pro-cesses. We used this method to evaluate the distribution of

TGF-fl1

in themurinemammaryglandafterirradiation. We have previously shown

TGF-#

I immunoreactivity is faint in normal mammary gland but prominent in irradiated mam-maryglandfrom 1 h to7dpostradiation (8). This rapid risein

immunoreactivitycould resultfrom either increased total pro-tein, i.e., due to newsynthesisand/ordepositionof

TGF-#

1,or

Figure1.Immunofluorescent localization of anti-LAP (A and B) andLC(1-30) (CandD)immunoreactivityin humanxenografttumors. Tu-morsderived from 293 cloneC19cells (A andC),which overexpress activeTGF-B,andcloneB9 cells(BandD),which overexpress latent TGF-,B,arebothimmunostainedusinganti-LAP but onlyC19 ispositivewith LC( 1-30).Thus,LC( 1-30)reactivityappearstobecontingent

upon activation. It should be noted that theintensityof theimmunofluorescence shouldonlybecomparedforagiven antibodysincedifferences betweendistinct antibodies donotnecessarilyreflect their relative abundance. x20.

(5)

toincreased epitopeaccess, which could occur after local acti-vation of latent TGF-fll. Increased deposition without proteo-lytic activation would presumably result in increased LAP im-munoreactivity since

TGF-#tl

is secreted as a latent complex.

To evaluate these possibilities, we analyzed normal and irradiated mammary gland tissue using the immunofluores-cencedetection procedures outlined above. In the unirradiated mammarygland, anti-LAP was strongly expressed in the epi-thelium and was less abundant in the periepithelial stroma,

while

LC(

1-30) faintlyimmunostainedtheepithelium, perie-pithelial stroma, and tissue septa in normal mammary gland (Fig. 2, A and C). Remarkably, whereas the adipose stroma was stronglypositive for anti-LAP, it was essentially negative with

LC(

1-30). Thedistinct pattern of staining by these two antibodies indicates that while latent

TGF-#3I

isabundant,

ac-tive TGF-3 is restricted and isparticularly limited in the adi-pose stroma.

This pattern of TGF-f immunoreactivity altered signifi-cantly I h after radiationexposure(Fig. 2, B and D). Anti-LAP immunoreactivity was strikingly diminished in the adipose stroma,while LC( 1-30) immunoreactivitywasinduced in the previously negative adipose stroma and wasdramatically in-creased in the epithelium and periepithelial stroma. The

pat-ternof reversal in which

LC(

1-30) immunoreactivitywas in-duced in parallel with loss ofLAP immunoreactivitywas partic-ularly marked in the adipose stroma. Assuming that LAP is degraded ( 16) or otherwise altered after release from the TGF-,B1 latent complex and that the LC(1-30) antigenic site is masked byits association with LAP, these data support the conclusionthat, either directly or indirectly, ionizing radiation exposure leads to TGF-,B1 activation. Further evidence of TGF-,Bl biological activity is that

LC(

1-30) immunostaining in theadipose stroma corresponds with novel collagen type III expression 3 d after irradiation (8).

Figure 2.Immunofluorescent localizationofactive

TGF-,t

usingLC(1-30) (A and B) and latent

TGF-#l1

complexusinganti-LAP(CandD)in normal (A andC) and irradiated (B and D) mammary gland. The nuclei inAarelightlycounterstainedwithpropidiumiodide and thus appear yellow.Inunirradiated mammary gland, active

TGF-ft

(A)wasdetectedprincipallyin theepitheliumand stromalsheath(curvedarrow)aswell as thetissuesepta(filled arrow),whereas latent

TGF-#

(C)wasstrongestin theepithelium(curved arrow)andadiposestroma(openarrow).

I hafter 5-Gy radiation, active

TGF-#l

wasdramaticallyincreased inepithelium, peri-epithelialstroma, and tissue septa(B).Inaddition,new

stainingwasobserved in cells in theadiposestroma(openarrow).Incontrast, latent

TGF.-f

decreased,particularlyin theadiposestroma,

pre-sumablyduetoproteolyticdegradationofLAP(D). Therapidshift inLAP vs.LC( 1-30) immunoreactivity providesthefirst marker ofTGF-flI

activation in situ. X40.

(6)

-

a J

(7)

Additional changes in the immunostaining pattern

oc-curred at longer intervals after radiation exposure

(Fig.

3). Anti-LAP

immunoreactivity progressively

decreasedtobarely

detectable levelsduring the first 3 dafter radiationexposure

and returnedtolevelscomparabletounirradiated tissue onlyat

- 7d.In contrast,LC( 1-30)immunoreactivity remained

ele-vated for7 dpostirradiation.

Thedecreased anti-LAPimmunoreactivity could be dueto

decreased latent

TGF-#3I

ortheresult of masking by associa-tion with otherproteins, suchasthose of the extracellular

ma-trix.Wemeasured therelative abundanceof latent

TGF-,3

pro-tein in medium

conditioned

by tissue

fragments

from irra-diated mice using a standard TGF-3 assay based on its inhibition ofDNAsynthesis in mink lung epithelial cells (Fig. 4). Conditioned media

from

unirradiated tissue inhibited

DNA synthesis more than that from irradiated tissue

frag-ments,whichindicates that therewas nosignificant increase in latent

TGF-3

1 release, and in fact, was somewhat lessatall time

points.

This

activity

could beneutralized

using

TGF-f3-specific

antibodies

(Fig.

4 B). These data argue

against

in-creased

TGF-#3I

protein production

as thebasis for elevated LC- 1-30

immunoreactivity

andsupportthe observed decrease inLAPimmunoreactivity.

Insummary,thisstudyshowsthatradiationexposureleads

to:(a) arapid shift frompredominantlyLAP immunoreactiv-itytoLC( 1-30)

reactivity;

(b)

arapid induction of

LC(

1-30)

immunoreactivity

in the

adipose

stroma; (c) parallel loss

of

LAP

immunoreactivity;

and

(d)

decreasedlatent

TGF-13I

in media

conditioned

by irradiatedmammarygland. These data provide in situ

evidence

consistent with

TGF-13I

activation.

Discussion

Thebiologicalactivity of

TGF-f

is governed by dissociation of

mature

TGF-3

from an

inactive,

latent

TGF-fl

complex in a processthatis criticaltothe roleof

TGF-#

invivo. So

far,

it has

notbeenpossibletomonitor activation invivo, since

conven-tional immunohistochemical detection doesnotaccurately dis-criminate latentvs.

active

TGF-3

norhave eventsassociated with activation been defined well enough to serve asin situ markers

of

thisprocess.Wedescribeamodified

immunodetec-tion method basedontheuse

of differential

antibody

detection oftwo

regions

oflatent

TGF-#

that allows the

specific

detection of active

TGF-3

and

identification

ofevents

associated

with activation. This method was used to evaluate the status of

TGF-3

in tissue

after ionizing radiation

exposureand shows thatirradiation leadsto

increased immunoreactivity using

anti-bodies

specific

for active

TGF-,B

concomitant with loss oflatent

TGF-#

I

immunoreactivity.

Active

TGF-,3

was apparentwithin

1 hafter radiationexposureand

persisted

for7d

postirradia-tion,

andwas

inversely

correlatedwith latent

TGF-#lI

detec-tionover asimilar timecourse.Thesetwocomplementary and reciprocal changesarebestexplained by the conversion ofthe

available pooloflatent TGF-,B1 into active

TGF-#31.

Therapid

shift frompredominantlylatent to activeTGF-,B, togetherwith ourprevious reportthat stromalcollagens rapidlyremodel in theirradiatedmammarygland(8),lead to the conclusion that

radiation exposure induces activation of latent TGF-, I insitu. Radiation-inducedTGF-,B1 activation is the first documented exampleof

TGF-f3

activationinvivo.

Ourstudies indicate thatadiscreteevent,suchas radiation

exposure,leadingtoactivation isaccompanied bytwo

signifi-cantalterations in

TGF-f3

immunoreactivity:increased access toamino-terminal epitopes ofmatureTGF-f31 and decreased stability of,or accessto,LAP. Indeed, whileboth eventsmay

have beenpredictedbased on ourknowledgeof

TGF-f,

activa-tion invitro, therewaslittle prior basis forinterpreting changes in TGF-,3 immunoreactivity in vivo since it is generally

as-sumedthat TGF-,Bantibodiesareinsensitiveto its activation

status.These postirradiation changesare notuniquetothe spe-cificantibodies used herein since increased staining is alsoseen

using CC( 1-30), which was raised to the same peptide se-quence asLC (1-30) (8), and decreased immunoreactivity simi-lartoanti-LAP isseenwithanantibody raisedtorecombinant latentTGF-f complex (Barcellos-Hoff, M. H., and M. L.-S.

Tsang,unpublished data). Thus, the elevated immunoreactiv-ity ofantibodies directed against specific regions ofmature

TGF-f13

in conjunction with loss ofLAP immunoreactivity

mayserve asindicatorsofin situTGF-f activation during

phys-iological andpathologic processes.

Little is as yet known about the in vivo mechanisms of latent

TGF-f3

activation. Proteases arethoughtto beinvolved inselectively degradingLAP,resultinginthereleaseof active

TGF-3

(16, 17). Thefunctional

involvement of

proteasesin latent

TGF-3

activationis best substantiated in the studieson

coculturedendothelial and smooth muscle cells.Inthis culture

system,

proteolytic

conversion of

plasminogen

into

plasmin

results in plasmin-mediated activation of

TGF-3

1(18, 19). Thisplasminogen activator-induced cascademayalso be

re-sponsible for the activation of

TGF-#3I

in several othersystems

and

during

woundhealing. Whereas the

generality

ofthis

enzy-matic

cascade inTGF-1 activation

remains

tobeestablished,

plasmin

may be the responsible agent in

radiation-induced

TGF-#3I

activationaswell

since

radiation

elicits

rapid elevation

of

plasminogen

activator

expression

and

activity

in cultured cells

(20),

which is reminiscent of the

rapid

radiation-induced

TGF-f31

activation

in the murinemammarygland.

Thephysiologicalconsequencesof

radiation-induced

TGF-f

activation

areapparentfrom the

rapid

remodeling of

colla-gen types I and IIIobserved in theirradiatedmammary

gland

(8).

In

particular,

the abundant collagentype III

expression

in

the

previously negative

adipose

stroma3 dafter radiation ex-posurecolocalized with the

rapid

activation of

TGF-#31

(8).

Thetemporal sequence,

unique

expression of

collagen III in the

irradiated tissue,

andthe

extensively

documented

ability

of

TGF-#

to induce secretion ofseveralmajorextracellular

ma-Figure 3.Immunofluorescentstainingofactive

TGF-,#

using LC(1-30) (A-D) and latent

TGF-3

complexusing the anti-LAP antibody (E-H) in mammarygland sectionsatdifferent times after irradiation. (A and E) Unirradiatedtissue;(B andF) Id; (C andG) 3 d; and (D and H)7d afterirradiation. Cross sections ofanepithelialduct(curvedarrow) surrounded byperi-epithelial stroma embedded in adipose stromaareshown. Whereas thereis little activeTGF-,Bin theadiposestromaof the unirradiated tissue (A), it is strongly induced in the adipose stroma of the irradiated tissue (B-D). Increased activeTGF-,B persistedfor7d.Latent

TGF-,3

complex is abundant inunirradiated tissue (E) but radiation exposure(F-H) resulted inaprogressivelossof anti-LAPreactivityin both theepithelium(curvedarrow) andperiepithelial stroma. Anti-LAP immunoreactivitywasbarelydetectable by day 3 but returned to control levels by day 7. X20.

(8)

25 ), our data suggest that

TGF-#lI

activationmaybe theinitial

event.

Clearly, TGF-3 activation by radiation and subsequent

TGF-f3-induced

extracellular matrixremodeling providea new focus for the study of mechanisms oftissueresponse to

radia-tion. Since normal tissue tolerance is the mainlimitingfactor

in

radiotherapy

ofmany cancers, TGF-,3 activationmay

pro-videanovel

target

for

manipulating

the

physiological

conse-quences of radiation

therapy.

Acknowledgments

Wethank Drs. M.Spornand K. Flandersforkindly providing LC(1-30)antibodies and S. Ravani for technical assistance.

3 7 This work was supported bythe National Cancer Institute, Na-tional Institutes of Health(M. H. Barcellos-HoiD;and theOffice of

Days Post-Irradiation Health andEnvironmental Research, Health Effects Research

Divi-sion,of the U.S.DepartmentofEnergy (M.H.Barcellos-Hoff).

B

E 0 CF 0 0.

0L

0 0 C :5 E z~f C, 30000 20000 10000 0

Figure4.Mink lungepitheliala

conditioned byirradiatedtissue irradiation.Total

TGF-ft

activit' creasedas afunctionofradiation

DNAsynthesis comparedwith Resultsshownaremeancpm±S

twoexperiments (A). Thespecif conditionedmediawasdemonst TGF-f3 antibody (B). TGF-13-nc

wereadded (50,g/ml)to medi medium fromunirradiatedmart

diatedmammarygland, 1 d (sa

(sample 5)afterexposure. The

fourdeterminations. [3H]Thym of100 pg/mlof

rTGF-#

(positil

100±40cpm(B).

trixproteinsandchemotaxi

suggest that

TGF-fl

activati

matrixremodeling observed (8). Although collagen char

brosis evolveover aprotract(

noreactivity accompanies fil

References W/ RbigG

W/TGFJ gG

1. Derynck, R.,J.A. Jarrett,E. Y.Chen,D.H. Eaton, J.R. Bell,R. K.

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3.Miyazono,K.,U.Hellman,C.Wernstedt,andC.-H.Heldin. 1988.Latent

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Obberghen-Schil-imarygland(sample 2)orfrom irra- ling,C. Baker, M. E. Kass, L. R. Ellingsworth,A. B.Roberts, and M. B. Sporn.

mple3), 3 d(sample 4),or7 d 1989.Transforming growthfactor-j31: histochemical localizationwithantibodies barsdesignatethe averageoftwoto to distinctepitopes. J. CellBiol. 108:653-660.

idineincorporation in thepresence 1. Arrick, B. A., A. R. Lopez, F.Elfman, R. Ebner, C. H.Damsky,andR.

vecontrol)was775±68cpm(A)and

Derynck.

1992. Altered metabolic and adhesivepropertiesandincreased

tumori-genesisassociated withincreasedexpressionoftransforming growth (1. J. Cell Biol. 118:715-726.

12. Danielpour, D., L. L.Dart, K. C.Flanders, A. B. Roberts,and M. B.

Sporn. 1989. Immunodetection and quantitationof thetwoforms of

transform-ing growthfactor-beta(TGF-#IandTGF-j32). J. Cell.Physiol. 138:79-86.

13.Brown, P.D.,L. M.Wakefield,A.D.Levinson,and M. B.Sporn. 1990.

s offibroblasts

(21, 22),

strongly Physiochem2icalactivation of recombinant latent transforming growth

factor-beta's1,2,and 3.GrowthFactors.3:35-43.

von may initiate the extracellular 14.Brunner, A. M.,H.Marquardt,A.R.Malacko, M. N.Lioubin,and A.F.

within24 hof radiationexposure Purchio.1989. Site-directedmutagenesisofcysteineresidues in theproregionof

iges associated with radiogenic fi- the transforming growth factor

,1

precursor.J.Biol. Chem. 264:13660-13664.

15. Kane,C. J. M., A. M.Knapp, J. N.Mansbridge,andP.C. Hanawalt.

odpenlod(23)andTGF-,( immu- 1990.Transforminggrowthfactor-#l localization innormalandpsoriatic

epider-brosis in irradiated tissue (5,

24,

malkeratinocytesinsitu. J.Cell.Physiol. 144:144-150.

898 M.H.Barcellos-Hoff;R. Derynck, M. L.-S. Tsang, andJ. A. Weatherbee

A

E ._ 0 co A-0 (U L. 0 0 C :0 I--z 5000 4000 3000 2000 1000 0 0

(9)

16. Lyons, R. M., J. Keski-Oja, and H. L. Moses. 1988. Proteolytic activation oflatenttransforming growthfactor-Ofromfibroblast conditioned medium.J. CellBiol. 106:1659-1665.

17.Lyons, R. M., L. E.Gentry,A.F.Purchio, and H. L. Moses. 1990. Mecha-nism ofactivation of latent recombinant transforming growth factor 1 by plas-min. J.Cell Biol. 110:1361-1367.

18.Antonelli-Orlidge, A., K. B. Saunders, S. R. Smith, and P. A. D'Amore. 1989. Anactivatedform oftransforming growth factor#is produced by cocul-turesof endothelial cells and pericytes. Proc.Natl.Acad. Sci. USA. 86:4544-4548.

19.Flaumenhaft, R., M. Abe, P. Mignatti, and D. B. Rifkin. 1992. Basic fibroblast growth factor-induced activation oflatent transforming growth factor , inendothelial cells: regulationof plasminogen activator activity. J. Cell Biol. 118:901-909.

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plasminogen activator byionizing radiation in human malignant melanoma cells. CancerRes. 51:5587-5595.

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22.Ignotz, R. A., and J. Massague.1986. Transforminggrowth factor-B stimu-latestheexpression of fibronectin and collagen and their incorporation into the extracellular matrix. J. Biol. Chem. 261:43374345.

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