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
Find the latest version:
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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 55413Abstract
Thebiological activity of
TGF-fl,
an important modulator of cell proliferation and extracellular matrix formation, isgov-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 versusactiveTGF-fl,
norhave eventsassociated 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 latentTGF-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 activeTGF-#1
in tumor xenografts overproducing activeTGF-fil,
without cross-reactivityin tumorsexpressingsimilar levelsof
latent
TGF-fl1.
Wepreviously reportedthatTGF-ft
immunore-activity increasesinmurinemammarygland after whole-body'Co-'y
radiationexposure.Using
differentialantibody staining
wenow showthat radiationexposure
specifically
generatesac-tive
TGF-f1.
While latentTGF-,61
waswidely
distributed inunirradiated tissue, active
TGF-ftl
distributionwasrestricted. ActiveTGF-#1 increased significantly
within1hofirradiation concomitant with decreased latentTGF-ft
immunoreactivity.
Thisrapid shiftinimmunoreactivity providesthefirst evidence foractivation of
TGF-ft
insitu. Thisreciprocalpattern ofex-pression persisted for 3 d andwas
accompanied
by decreasedrecoveryoflatent
TGF-#1
from irradiated tissue.Radiation-in-ducedactivation of
TGF-ft
may haveprofound implications
forunderstanding tissue effects caused by radiation therapy.
(J.
Clin. Invest. 1994.
93:892-899.)
Keywords:ionizing
radiation*transforming
growth factor-,8
* mammarygland
* immunohis-tochemistry* extracellularmatrixIntroduction
TGF-f3
is amajormodulatorofcellularproliferation
and extra-cellular matrix formation. TGF-f 1, the best studied protein ofAddress 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 regulatedTGF-3
iso-forms,is derived from a390-amino acidprecursor cleaved toproducea 112-amino acid carboxy-terminal peptide( 1 ). The
homodimer of this peptide is noncovalently associatedwith a
dimer
of
theprocessedamino-terminal
pro-segment, calledthelatency-associated
peptide(LAP).'
This secreted latentTGF-f3
complex isunable tobindtoTGF-,3
receptorsuntil the biologi-cally active24-kD matureTGF-3
is dissociatedfrom LAP (2,3). Thus, thebiological activityof
TGF-3
is highlycontrolled by its release from the latent complex, which is consideredacritical 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. ElevatedTGF-3
immunoreactivity hasbeen observed in tissues during normalphysiological
(4) and patho-logical (5-7)processes that result in inflammation, tissuere-pair,
orextracellular matrixdeposition.
We recently showed usingimmunofluorescence
thatTGF-O
is elevated inthemu-rinemammarygland after whole-bodyexposure to
'Co-y
radi-ation (8).TGF-j1
immunoreactivitywasrapidly
induced inthe
epithelium
and thepreviously negative adipose
stroma,andwasfollowedbynovel expression of collagentype
III,
a known targetofTGF-3.
However, whetherthealteredTGF-f
immu-nostaining reflects increasedTGF-f
depositionoralteredepi-tope
accessibility,
asmight
occur ifTGF-3
wasdissociated
from thelatentcomplex,could notbe determined with avail-able methods. Whereasaltered
immunoreactivity
isoftenas-sumedtorepresenta
corresponding
change inTGF-f
activity,
the currentmethodology detects
TGF-j
irrespective
of knowl-edge of itsstateofactivation.
Forexample, antibodies
toLAP areexpectedtonotonlyrecognize
latentTGF-j3
butthesamepro-segment after
dissociation
of the latent complex.Likewise,
recognition of
matureTGF-fl1,
theactivepolypeptide
that ispartofthe latentcomplex, may beimpartialto theactivation
state.However, if the
antigenic
site ismasked, forexample
by association with LAP,ordependsonitsconformation,
whichmay changewithactivation, antibody recognition of
TGF-f1
might be wholly dependent upon activation. Furthermore, sinceaccurateimmunolocalization ingeneraldepends
ontar-get
protein preservation
after tissuefixation,
concern about whether tissue processing might activateTGF-f3
has beenraised(9). Thus,
interpretation
ofTGF-f
antibodyreactivity
is confounded by a numberofrestrictions, 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 $2.00regarding 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 demonstratethe 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 latenttoactiveTGF-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 blotanalysis. Approximately 2ng/lane of LAPwasdetected in Western blot assaysusinganantibodyconcentration of 1
gg/ml.
Direct ELISA demonstrated thatthisantibodyisspecific fortheLAPderived from theTGF-#
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 latentTGF-#
1 whereas clone C 19, transfected withanexpressionplasmid foramodifiedTGF-Bl precursor, secretespredominantlyactive
TGF-,l
1.Cellswereculturedtosubconfluenceand 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 inexperimen-talsamples.
Results
Differential
useof antibodies distinguishes latent
and activeTGF-f,.
We compared thespecificityof several antibodiesus-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 inTGF-#
activation between the tu-morclones results fromdirected mutation of two cysteinestoserines in thepre-pro-segment
TGF-#
peptide (11),whichpre-vents disulfide bond formation in LAP resulting in
constituti-vely active
TGF-#
(14). The activation of TGF-f31 secretedfromC 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 ofTGF-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'sfluidalone (15), or 10% neutral buffered formalin alone (6).To potentiate the preservation of latent and active
TGF-fl,
weelected to usecryosections from frozen unfixed tissue for
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 sequenceresidingattheamino 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.
Incomparison, 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 latentTGF-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 shownTGF-#
I immunoreactivity is faint in normal mammary gland but prominent in irradiated mam-maryglandfrom 1 h to7dpostradiation (8). This rapid riseinimmunoreactivitycould resultfrom either increased total pro-tein, i.e., due to newsynthesisand/ordepositionof
TGF-#
1,orFigure1.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.
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 withLC(
1-30). Thedistinct pattern of staining by these two antibodies indicates that while latentTGF-#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 thatLC(
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 latentTGF-#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, activeTGF-ft
(A)wasdetectedprincipallyin theepitheliumand stromalsheath(curvedarrow)aswell as thetissuesepta(filled arrow),whereas latentTGF-#
(C)wasstrongestin theepithelium(curved arrow)andadiposestroma(openarrow).I hafter 5-Gy radiation, active
TGF-#l
wasdramaticallyincreased inepithelium, peri-epithelialstroma, and tissue septa(B).Inaddition,newstainingwasobserved 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.
-
a JAdditional changes in the immunostaining pattern
oc-curred at longer intervals after radiation exposure
(Fig.
3). Anti-LAPimmunoreactivity progressively
decreasedtobarelydetectable 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 extracellularma-trix.Wemeasured therelative abundanceof latent
TGF-,3
pro-tein in medium
conditioned
by tissuefragments
from irra-diated mice using a standard TGF-3 assay based on its inhibition ofDNAsynthesis in mink lung epithelial cells (Fig. 4). Conditioned mediafrom
unirradiated tissue inhibitedDNA 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 timepoints.
Thisactivity
could beneutralizedusing
TGF-f3-specific
antibodies(Fig.
4 B). These data argueagainst
in-creasedTGF-#3I
protein production
as thebasis for elevated LC- 1-30immunoreactivity
andsupportthe observed decrease inLAPimmunoreactivity.Insummary,thisstudyshowsthatradiationexposureleads
to:(a) arapid shift frompredominantlyLAP immunoreactiv-itytoLC( 1-30)
reactivity;
(b)
arapid induction ofLC(
1-30)
immunoreactivity
in theadipose
stroma; (c) parallel lossof
LAP
immunoreactivity;
and(d)
decreasedlatentTGF-13I
in mediaconditioned
by irradiatedmammarygland. These data provide in situevidence
consistent withTGF-13I
activation.Discussion
Thebiologicalactivity of
TGF-f
is governed by dissociation ofmature
TGF-3
from aninactive,
latentTGF-fl
complex in a processthatis criticaltothe roleofTGF-#
invivo. Sofar,
it hasnotbeenpossibletomonitor 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 markersof
thisprocess.Wedescribeamodifiedimmunodetec-tion method basedontheuse
of differential
antibody
detection oftworegions
oflatentTGF-#
that allows thespecific
detection of activeTGF-3
andidentification
ofeventsassociated
with activation. This method was used to evaluate the status ofTGF-3
in tissueafter ionizing radiation
exposureand shows thatirradiation leadstoincreased immunoreactivity using
anti-bodiesspecific
for activeTGF-,B
concomitant with loss oflatentTGF-#
Iimmunoreactivity.
ActiveTGF-,3
was apparentwithin1 hafter radiationexposureand
persisted
for7dpostirradia-tion,
andwasinversely
correlatedwith latentTGF-#lI
detec-tionover asimilar timecourse.Thesetwocomplementary and reciprocal changesarebestexplained by the conversion oftheavailable pooloflatent TGF-,B1 into active
TGF-#31.
Therapidshift 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 eventsmayhave 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 immunoreactivitymayserve 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 activeTGF-3
(16, 17). Thefunctionalinvolvement of
proteasesin latentTGF-3
activationis best substantiated in the studiesoncoculturedendothelial and smooth muscle cells.Inthis culture
system,
proteolytic
conversion ofplasminogen
intoplasmin
results in plasmin-mediated activation of
TGF-3
1(18, 19). Thisplasminogen activator-induced cascademayalso bere-sponsible for the activation of
TGF-#3I
in several othersystemsand
during
woundhealing. Whereas thegenerality
ofthisenzy-matic
cascade inTGF-1 activationremains
tobeestablished,plasmin
may be the responsible agent inradiation-induced
TGF-#3I
activationaswellsince
radiation
elicitsrapid elevation
of
plasminogen
activatorexpression
andactivity
in cultured cells(20),
which is reminiscent of therapid
radiation-inducedTGF-f31
activation
in the murinemammarygland.Thephysiologicalconsequencesof
radiation-induced
TGF-factivation
areapparentfrom therapid
remodeling of
colla-gen types I and IIIobserved in theirradiatedmammary
gland
(8).
Inparticular,
the abundant collagentype IIIexpression
inthe
previously negative
adipose
stroma3 dafter radiation ex-posurecolocalized with therapid
activation ofTGF-#31
(8).
Thetemporal sequence,
unique
expression of
collagen III in theirradiated tissue,
andtheextensively
documentedability
ofTGF-#
to induce secretion ofseveralmajorextracellularma-Figure 3.Immunofluorescentstainingofactive
TGF-,#
using LC(1-30) (A-D) and latentTGF-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.LatentTGF-,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. X20.25 ), our data suggest that
TGF-#lI
activationmaybe theinitialevent.
Clearly, TGF-3 activation by radiation and subsequent
TGF-f3-induced
extracellular matrixremodeling providea new focus for the study of mechanisms oftissueresponse toradia-tion. Since normal tissue tolerance is the mainlimitingfactor
in
radiotherapy
ofmany cancers, TGF-,3 activationmaypro-videanovel
target
formanipulating
thephysiological
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 0Figure4.Mink lungepitheliala
conditioned byirradiatedtissue irradiation.Total
TGF-ft
activit' creasedas afunctionofradiationDNAsynthesis 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-#
(positil100±40cpm(B).
trixproteinsandchemotaxi
suggest that
TGF-fl
activatimatrixremodeling observed (8). Although collagen char
brosis evolveover aprotract(
noreactivity accompanies fil
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