Transforming Growth Factors 1 2, and 33 …cgd.aacrjournals.org › cgi › reprint › 5 › 9 ›...

Vol. 5, 919-935, September 1994 Cell Growth & Differentiation 919

Transforming Growth Factors �1 , �2, and �33 MessengerRNA and Protein Expression in Mouse Uterusand Vagina du ring Estrogen-inducedGrowth: A Comparison to OtherEstrogen-regulated Genes

Tsuneo Takahashi,1 Benjamin Eitzman,Nancy 1. Bossert,2 David Walmer,2 Kalvin Sparrow,Kathleen C. Flanders, John McLachlan,3 andKaren Gray Nelson

Laboratory of Reproductive and Developmental Toxicology, NationalInstitute of Environmental Health Sciences, Research Triangle Park, North

Carolina 27709 [T. T., B. E., N. L. B., D. W., K. S., j. M., K. G. N.], andLaboratory of Chemoprevention, National Cancer Institute, NIH, Bethesda,Maryland 20892 [K. C. F.]

Abstrad

Increasing evidence suggests that the differentialregulation of multiple peptide growth fadors by steroidhormones contributes significantly to the pleiotropiceffects elicited in target tissues. We report here anevaluation of the effects of the potent estrogen,diethylstilbestrol, on the expression of the threemammalian transforming growth fador �3 (TGFIJ)isoforms, TGFfi1, TGFfi2, and TGFfJ3, in both theuterus and the vagina of the prepubescent mouse.Immunohistochemical protein detedion, in situhybridization, and Northern RNA analyses demonstrateoverlapping but distind time-dependent and site-specificindudion of all three TGF�3 genes in the reprodudivetrad in response to estrogen. Temporal analysis ofsteady-state levels of the TGFIJ mRNAs in the uterusby Northern blotting clearly demonstrates thatdiethyistilbestrol significantly but transiently up-regulatesTGFf33 mRNA within 30 mm and TGFfi1 and TGFfi2mRNAs by 3 h with decreases to/or below control levelsby 6 h. The vagina also responds to diethylstilbestrolwith similar kinetics of indudion for TGFfi2 and TGFfi3mRNAs as that observed in the uterus; however, TGFfi1mRNA levels increase gradually and peak around 1 6 hafter treatment. Investigation of the steroid specificitydemonstrates predominant estrogen specificity in thecontrol of TGFf3 expression in the immature mousereprodudive trad. In situ hybridization localizes themRNAs for all three TGFI3 isoforms, primarily to theuterine and vaginal epithelium. Unlike the transientnature of TGFI3 mRNA indudion elicited by estrogen,

Received 4/1 5/94; revised 6/1 7/94; accepted 6/27/94.

1 Present address: Department of Obstetrics-Gynecology, Yokohama City

University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236,Japan.

2 Present address: Department of Obstetrics and Gynecology, Duke Univer-sity Medical Center, Durham, NC 27710.3 To whom requests for reprints should be addressed, at Laboratory ofReproductive and Developmental Toxicology, National Institute of Environ-mental Health Sciences, P. 0. Box 1 2233, B-302, Research Triangle Park,

NC 27709.

immunohistochemistry demonstrates that estrogentreatment results in a more prolonged elevation of theproteins for TGFfi1 , TGFf32, and TGF�33 in theepithelium of both tissues. Investigation of specificbinding of ‘251-TGFfi1 by affinity labeling reveals theexistence of the receptor/binding proteins (types I, II,and Ill) in the uterus. Estrogen treatment significantlyreduces binding to each of these components in theuterus, which suggests that estrogen may modulateTGFf� responsiveness at the receptor level. A comparisonof TGFf� mRNA expression to the indudion of otherestrogen-regulated genes, TGFa, insulin-like growth

, c-myc, progesterone receptor andladotransferrin reveals that, in general, the TGF�3transcript levels are regulated in a more transientmanner by estrogen. When the indudion of these genesis correlated to the growth response measured by 5-bromo-2’-deoxy-uridine incorporation into nuclei, withthe exception of ladotransferrin, the mRNA levels forthe TGFj3 isoforms and the other estrogen-regulatedgenes are stimulated many hours prior to the initiationof DNA synthesis. These data associate the TGF�3s alongwith the other genes to a time period at which estrogenis inducing cell cycle transition of the uterine andvaginal epithelium and thus implicating these growthfadors in this process. A particular noteworthy findingfrom our study is that estrogen-mediated vaginal growthinvolves the regulation of a similar array of peptidegrowth fadors and other genes as documented in theuterus. Morphological studies have documented that thelocal readion in the uterus that occurs followingestrogen treatment is comparable to an acuteinflammatory response, similar to that observed withwounding or tissue trauma. Our data demonstrate that anumber of growth fadors associated with wound repair,including the TGFf3s, are stimulated by estrogen in theuterus and vagina prior to the initiation of DNAsynthesis, which provides further evidence that estrogenmediates its mitogenic adion by eliciting a woundhealing-like response.

Introdudion

The uterus and vagina exist in a dynamic balance betweengrowth, differentiation, and apoptosis which is regulated bythe sex steroid hormones, estrogen and progesterone (1-8).Responses to estrogen can be divided temporally into twodistinct phases (9-1 6). 1) Those events occurring directlyafter estrogen treatment (1 5 mm to 4 h) are most like anacute inflammatory response associated with wound heal-ing. For example, vascular permeability is enhanced lead-ing to pronounced edema and increases in the wet weightof the uterus. This is associated with induction of numerous

920 TGFf3 and Estrogen Action in the Genital Tract

cytokines and the invasion of inflammatory cells into theuterus in a time-dependent manner that is reminiscent ofclassical wound healing (17-19). The stimulation of theexpression of protooncogenes, peptide growth factors, andtheir receptors that are associated with cell cycle progres-

sion also occurs (20-29). 2) At later time points (4-24 hafter estrogen treatment), uterine epithelial cells undergosynchronized proliferation, tissue dry weight increases dueto greater cell numbers, and genes associated with DNAsynthesis and differentiation are activated (30-32). In addi-tion to the uterus, the growth and differentiation of anotherrarely studied target tissue, the vagina, is also substantiallyinfluenced by the sex steroids. Little information is availableconcerning the molecular mechanism by which estrogenand progesterone control DNA synthesis and regulate eithersquamous or mucous differentiation of the vagina (33, 34).

It is now well documented that steroid hormones elicitcomplex biological responses in the reproductive tract atthe level of gene transcription mediated by their specificreceptors which coordinate the expression of gene net-works (35-39). In the last decade, efforts to isolate cell- andtissue-specific target genes for estrogens have identified anumber of protooncogenes, enzymes, cytokines, and pep-tide growth factors and their receptors which participate atvarious points within growth pathways (9-32). It is wellestablished that multiple growth factors are required forgrowth of normal cells (36); thus, it should not be surprisingthat the steroid hormones also regulate many of the samegrowth factors and/or their receptors that have been shownto be important in mediating proliferation of cells in cul-ture. The understanding of the role of peptide growthfactors in reproductive tract physiology is still in its in-fancy, and it must be kept in mind that most peptidegrowth factors are multifunctional and can stimulate bothproliferation and differentiation, as well as act as main-tenance or survival factors that block program cell death(36, 40-42). An often neglected aspect of estrogen actionis that growth of the reproductive tract is induced by boththe stimulation of DNA synthesis and the reduction ofapoptosis or cell death (8).

Our laboratory has used the prepubescent (ages 17-19days) female CD-i mouse as an ifi vivo model to examinesex steroid hormone regulation of the expression of peptidegrowth factors that may have potential roles in elicit-ing changes in cell growth and differentiation associatedwith the synchronized physiological responses of the gen-ital tract to the sex steroids. The TGFf3s4 are a family ofubiquitously expressed and highly conserved peptides thatare involved in such complex phenomena as wound heal-ing, embryogenesis, and carcinogenesis (40-55). TGFI3action has been described as a universal biological “switch”which is responsible for transmitting a variety of environ-mental signals to the cell (41 ). The recent demonstrations ofTGF� expression in the uterus following multiple steroidhormone treatment of rats (43) and during the periimplan-tation period of pregnancy in mice (44, 45), as well as in thehuman (46) and rodent (47) ovary, provide evidence that

4 The abbreviations used are: TGF�, transforming growth factor f3; IGF-1,insulin-like growth factor-i ; PR, progesterone receptor; DES, diethylstilbes-trol; kb, kilobase(s); bp, base pair(s); cDNA, complementary DNA; GAPDH,glyceraldehyde 3-phosphate dehydrogenase; DHT, 5a-dihydrotestosterone;DEX, dexamethasone; BrdUrd, 5-bromo-2’-deoxyuridine; SDS, sodium do-decyl sulfate; poly(A)�, polyadenylated; PCR, polymerase chain reaction;SSC, standard saline citrate; BSA, bovine serum albumin; DTT, dithiothreitol.

TGFf3s are involved in reproductive function. In addition,keratinocyte growth and differentiation are modulated by theTGFf3s, which suggest that the TGF�3s play a regulatory role inthe maintenance of squamous epithelia (48-50) and thus raisethe possibility that the TGFf3s may be involved in the mech-anism by which estrogen stimulates vaginal cornification.

We report here the results of a comparative evaluation ofthe effects of estrogen treatment on RNA and protein ex-pression of the three mammalian isoforms, TGFf31 , TGF�2,and TGFI33, in both the uterus and the vagina of the im-mature mouse. Our study demonstrates time-dependentand site-specific induction of all three TGF�3 genes in thereproductive tract in response to estrogen. A comparison ofTGFI3 mRNA expression to other established estrogen-reg-ulated genes (TGFa, IGF-i , c-myc, PR, and lactotransferrin)reveals that the stimulation of TGFI3 mRNA levels is oftenmore transiently regulated. A kinetic study of DNA synthe-sis elicited by DES treatment in the uterine and vaginalepithelium demonstrates the induction of TGF�3s and theother estrogen-regulated genes, except for lactotransferri n,occurs prior to the growth response. Estrogen treatment alsosignificantly decreases the levels of TGF� binding proteinsin the uterus, which suggests that estrogen may modulateTGFI3 responsiveness at the receptor level. Furthermore, weprovide evidence for the first time that estrogen induces an

array of genes in the vagina similar to that documented inthe uterus. As a result of these data, we propose that estro-gen action in reproductive target tissues involves the induc-tion of TGF�3s simultaneously with other potent biologicalregulators, all ofwhich are probably important in mediatingestrogen effects on the growth and differentiation of theuterus and vagina.

Results

Stimulation of Uterine and Vaginal TGF�i1 , TGF�32, andTGF�3 mRNA Levels by Estrogen. The level of mRNAexpression of each TGFI3 isoform (TGFf31 , TGFI32, andTGFj33) was examined in the uterus and vagina of immaturemice at various times following a single dose of the potentsynthetic estrogen, DES. Northern blot analysis (Fig. 1) forTGFf31 (top), TGF�2 (upper middle), and TGF�3 (lowermiddle) revealed that the uterus and vagina of the vehicle-treated control animals contained significant basal mRNAlevels for TGF�31 (2.5 kb) and for TGFf33 (3.5 kb) in com-parison to lower levels of expression found for TGFI32transcripts (4.0 kb and 5 kb). Analysis of DES effects onTGFf31 mRNA levels in the mouse uterus and vagina re-vealed distinct, time-dependent patterns of induction inthese tissues (Fig. 1 , top). In the uterus, treatment with DEStransiently induced TGFj31 mRNA by -‘10 fold within 3 h,which was followed by down-regulation of the transcriptsby 6 h to below control levels. Whereas in the vagina,estrogen stimulation of TGFf31 mRNA (-3 fold) was notapparent until 16 h after treatment. In this blot, there was adecrease in TGF�31 mRNA levels at 1 h in the vagina;however, this was not consistently observed.

Estrogen modulated the expression of TGFf32 transcriptsidentically in the uterus and vagina. Probing the same blotused for the TGFf31 analysis with TGFj32 cDNA revealedthat estrogen, in a coordinate and time-dependent manner,induced the 5.0-kb and 4.0-kb TGFI32 transcripts (-10-fold) in both tissues by 3 h (Fig. 1, upper middle). Like

TGFf31 regulation, this induction was transient, and theTGFI32 mRNAs decreased to or below control levels within24 h after estrogen treatment.

UTERUS

c,J �C’) CO �- C\J

TGF�3 Consecutive hybridization of the blot with a TGFf33-

1 specific probe demonstrated only slight induction of the

VAGINA � major 3.6-kb TGF33 transcript by estrogen at 1 and 3 h in

F- ,� � the uterus and vagina, respectively, followed by a marked

� � � � � c� suppressionwithin24h[Fig.1,lowermiddle(i-24hblot)J.This initial analysis indicated that TGF!33 mRNA expression

, may be regulated by estrogen at time points earlier than 1 h.

. .� * This was confirmed by analyzing time points of 30-mm. increments [Fig. 1, lower middle (30’-180’ blot)], which

demonstrated that the TGFI33 transcripts were significantlyup-regulated in both uterus (-10-fold) and vagina (-5-fold)within 30 mm after estrogen treatment and remained ele-vated for approximately 3 h. Estrogen did not modulate the

expression of TGF�31 or TGFI32 RNA levels at these earlytime points (data not shown). In addition, to ensure thatequivalent RNA quantities were evaluated for each exper-

TGF�32 mental group, the Northern blot was reprobed for the

VAGINA housekeeping enzyme, GAPDH, which is shown in Fig 4. A

�:�-i I .� I noteworthy observation from the Northern analyses was� �- .- �. � � � that all ofthe TGFI3 transcript levels (especially TGF�1 and(� ‘- C’) CO � � � TGF�3) were suppressed below control levels by 24 h after

DES treatment in the uterus and vagina (Fig. 1 ). In fact,S � longer time course studies have demonstrated that this sup-

0 pression of the TGFI3 mRNA levels in the uterus and vaginawas very prolonged, extending to at least 72 h posttreatment

(data not shown). In addition, since DES is a synthetic18s ‘ � estrogen, we also investigated the influence of the natural

estrogen, 1 7f3-estradiol, on TGFI3 mRNA expression in themouse reproductive tract and found that 1 7f3-estradiol elic-ited very similar tissue- and time-dependent effects onTGF�3 mRNA production as that observed for DES (data notshown). Thus, we conclude that DES modulation of uterineand vaginal TGFI3 expression was due to its estrogenic

TGFf33 properties.

Steroid Specificity and Dose-Response Analysis of TGF�

I UTERUS � I VAGINA _� mRNA Induction in the Reprodudive Tract. To determine‘- �- � �- � ‘- �- � whether modulation of the TGF�s in the immature mouse� � � � � (� � � � � � (� genital tract was specific to estrogen in a dose- and a

steroid-dependent manner, the effect of multiple doses of

28s - - DES and of a single dose of other steroids on the steady-stateRNAlevelforeach oftheTGF�3 isoformswasevaluated (Fig.

3.5kb � *S .� �. � � . � . #{149} . . .2). The time point chosen following treatment with the

different doses of DES or with a single dose of other steroids18s . _ to analyze the TGF� mRNAs was based on the results of the

time course study shown in Fig. 1 . Measurement of theTGF�1 RNA levels were done at times shown to be maxi-mal for transcript induction by DES, which was 3 h for theuterus and 1 6 h for the vagina. The 3-h time point following

UTERUS VAGINA treatment was chosen for analyzing TGFI32 and TGFI33

I _ i_ I RNA expression in both tissues. Although 3 h following

� b -0 C - - treatment was not totally optimal for the analysis TGFI33(� � .� � � � � � � � � � expression, TGF/32 and TGF�3 transcripts were signifi-

cantly elevated by DES in comparison to controls at this

3.5kb-� ..... time such that the same set of RNA specimens could beanalyzed for TGFI32 and TGFI33 isoforms simply by reprob-ing the same Northern blot.

Fig. 1. Time-dependentinductionofTGFj3mRNAsintheuterusandvagina Northern analyses showed that DES elicited a dose-de-upon a single treatment of immature female mice with the potent estrogen, pendent induction of mRNA for all of the TGF� isoforms inDES (20 pg/kg). Poly(A)� RNA was isolated from pooled mouse uteri and both the uterus and vagina upon treatment with 1 , 5, or 20vagina at various times after estrogen treatment, which is designated above . . . .

each lane. The control lanes represent RNA isolated from uteri and vagina 3 p�kg of DES (Fig. 2). The most intriguing data obtainedh after treatment of animals with the vehicle, corn oil. The same Northern

blot containing RNA isolated 1 to 24 h following estrogen exposure washybridized consecutively using 32P-labeled probes specific for TGFf31 (top),

TGFf32 (upper middle), and TGFf33 (lower middle). To further analyze theearly induction of TGFf33 mRNA, an additional Northern blot of RNA

isolated from tissue 30 to 1 80 mm after treatment is shown (bottom panel).

Cell Growth & Differentiation 921

Arrows, the approximate sizes of the TGFI3 transcripts in kilobases kb);

positions of the 1 85 and 28S rRNAs are also shown.

UTERUSF-

.-� .�� .c (0

C’) (0 ‘- C’J 0

2.5kb-p-

5.0kb �28s �

4.0kb �

2.5kb-’TGF)�1 --------

GAPD�

Table 1 Steroid-specific and DES dose-dependent induction of epithelialDNA synthesis in the uterus and vagina of 18-day-old female mice 16 hfollowing treatment

5.0 KbTGF)�2

4.0 KbTGF)32

1.4 Kb -�GAPDH

TGF�3

GAPDH �

Fig. 2. Northern RNA analysis demonstrating steroid specificity and dose-

dependent DES effects on TGFf31 )upper), TGF)32 (middle), and TGF)33(lower) expression in the uterus I Ufl and vagina I VA) of immature mice. The

time point at which the TGF� transcripts were analyzed in the uterus was at

3 h following s.c. injection with the various steroids, whereas the vagina wasevaluated for TGF�2 and TGFfI3 expression at 3 h and at 1 6 h for TGF31mRNA induction. P4, progesterone (2 mg/kg); VEX, dexamethasone (1 mpjkg); DHT, 5cr-dihydrotestosterone (1 mgJkg); E,, estriol (20 pgJkg); DES,

diethylstilbestrol I 1, 1 pg/kg; 5, 5 pgjkg; and 20, 20 pg/kg); Control, corn oilvehicle.

922 TGF� and Estrogen Action in the Genital Tract

TGF�1P4 E3 DHT DEX Control 20 DES 5 DES 1 DES

, ui VA UT VA #{149}UT VA UT VA #{149}UT VA #{149}UT VA #{149}UT VA #{149}UT VA’

TGFf�2p4 DHT DEX E3 Control 1 DES 5 DES 20 DES

VA UT VA UT VA UT VA UT VA UT VA UT VA UT VA UT

4 �

‘

DNA synthesis was measured by quantitation of nuclear BrdUrd incorpo-

ration in the epithelium of the uterus and vagina of immature CD-i femalemice 16 h following treatment with the different doses of DES or with the

other steroid hormones. DNA synthesis was calculated as the percentage of

BrdUrd labeled nuclei to total number of nuclei present. The values representthe average and SE from four different animals from each experimentalgroup. (% BrdUrd-labeled nuclei ± SE).

Treatment group Uterus Vagina

Control 1.7 ± 0.6 2.9 ± 1.3

1 �JgJkg DES 38.2 ± 1 1 .5 52.5 ± 5.2

5 pg/kg DES 80.3 ± 5.6 49.1 ± 4.5

20 pg/kg DES 81.0 ± 7.4 42.0 ± 9.8

20 pg/kg Estriol 26.6 ± 5.0 59.4 ± 12.1

2 mg/kg Progesterone 1 .7 ± 0.4 4.4 ± 1 .2

1 mg/kg Dexamethasone 0.8 ± 0.4 0.8 ± 0.7

1 mg/kg 5a-dihydro 4.6 ± 2.5 8.1 ± 5.1testosterone

p4 #{149}DEX E3 , Control #{149}1 DES #{149}5DES

VA UT VA UT VA UT VA UT VA UT VA UT VA UT

� � .

from this dose-response study that may contribute to theunderstanding of the in vivo role(s) was that the dose-dependent induction ofTGFf3s mRNA paralleled the induc-tion of DNA synthesis by estrogen measured at 1 6 h in themouse uterus and to some extent in the vagina (Table 1).Significant transcript elevation of all three TGFf3s wasclearly evident at 5 pg/kg DES, a dose that elicited maximalepithelial DNA synthesis in the uterus (80.3 ± 5.6%) andvagina (49.1 ± 4.5%; Table 1). A more attenuated increase

of the TGFJ3 mRNAs was induced by 1 pg/kg DES in theuterus, a dose where only 38.2 ± 1 1 .5% Of the uterine

epithelial cells undergo DNA synthesis. Like the uterus, theinduction of the TGF�3 mRNAs in the vagina was not assignificant at 1 pg/kg DES as found for 5 and 20 pg/kg DES;however, vaginal DNA synthesis was maximally induced at1 pg/kg DES (52.5 ± 5.2%), which suggested greater sen-sitivity of the vagina to estrogen. This dose-response anal-ysis suggests that, for the uterus, a similar level of receptoroccupancy by estrogen is required to elicit TGFJ3 RNAexpression as that which is needed to stimulate DNAsynthesis.

Fig. 2 also shows steroid specificity for the regulation ofthe TGF�s, which was investigated by comparing the effectsof a single treatment of progesterone, estriol, DHT, or DEXwith that elicited by various doses of DES. Occasionally,treatment with estriol (a weak estrogen for the uterus butmore potent for the vagina) and progesterone stimulatedTGFf3 mRNA expression in the uterus and vagina (Fig. 2) but

always at significantly reduced levels when compared tothe dramatic response elicited by the potent estrogen, DES(Fig. 2). It was not surprising that estriol elicited someincrease in the levels of TGFI3 transcripts, since the dose

used in our study shown in Fig. 2 induced epithelial DNAsynthesis in the uterus (26.6 ± 5.0%) and in the vagina(59.4 ± 1 2.1 0/); whereas, a single treatment with proges-terone did not modulate growth (uterus, 1 .7 ± 0.4%; va-gina, 4.4 ± 1 .2%; Table 1 ). Unlike the female sex steroids,a single dose of either DHT or DEX did not change theexpression of the TGF� mRNAs or affect DNA synthesis inthe reproductive tract of the prepubescent female mice (Fig.2; Table 1 ; data not shown for DHT effects on TGFI33). Themajor conclusion obtained from this steroid specificitystudy is that TGFI3s RNA expression in the uterus and vaginaof immature mice is predominantly regulated by estrogens.

Comparison of TGF� RNA Expression to Other Estro-gen-regulated Genes. The early mRNA induction of thethree TGF� isoforms suggested that the TGFj3s may bemediators of estrogen action. Estrogen also controls theexpression of a number of genes in the uterus which are

Uterus Vagina

ProgesteroneReceptor

7.0Kb� p�1��p ; .:

A. UterineTGFa Expression� ‘-in ..U) �U)0 �W rw ,,ui

(2 ,.,O �oO c,jc,

TGFa4.8kb- 4

Transcript

EthidiumBromide 28S

Staining18S

B. VaginaiTGFa Expression

� ..Cn ..in#{176} �w �w ,�wC.) ,0 ,oO ,‘jO

TGFa4.8kb

Transcript #{149}: � ,

28S

EthidiumBromideStaining

18S

Lactotransferrin

2.4Kb .-� ! � *� .� .‘

GAPDH

1.4Kb��S�I �

Fig. 4. Time-dependent )hr) mRNA induction in the immature mouse

uterus and vagina of two other established estrogen-regulated genes, the PR(7.0-kb transcript) and lactotransferrin (2.4-kb transcript), following a single

treatment with DES (20 pg/kg). Note that both genes exhibit distinct profilesof induction in both the uterus and vagina in response to estrogen. This

Northern blot is the same blot analyzed consecutively for the expression of

the TGFI3 isoforms shown in Fig. 1 . Reprobing of this Northern blot forGAPDH expression (1 .4-kb transcript) demonstrates that the quality and

quantity of RNA evaluated was equivalent for each time point investigated.

H.

lEE.

lime ih�itc�owngestroge�i (2OiiQ�igi

treatmeni -

uterus VaginaI

6 16 24 0 0.5 1 3 6 16 24

lTPI�*4�$IG �

,0 0.5 1 2 3

Utsrus vagina

(mlnuI� following satrogan tr.atmsnt)

0 15’ 3O� 180’ 0 15’ � �

2.4kb-� #{234}�*$ 5’

Fig. 3. Demonstration oftime-dependent regulation of TGFa (I), IGF-i (II),and c-myc (III) transcripts in mouse uterus and vagina following a singe DES(20 pg/kg) treatment of prepubescent mice. I, Northern hybridization clearlydemonstrates that estrogen significantly stimulates TGFa mRNA (4.8-kb

transcript) expression in both the uterus (A) and vagina (8) within 3 h (hr),which remains elevated for up to 24 h posttreatment. Ethidium bromide

staining ofthe 285 and 185 rRNAs is shown to serve as a control to assess thequality and quantity of RNA from each treatment group evaluated. II, estro-

gen regulation of IGF-i mRNA expression (7.0-kb transcript) in mouse uterusand vagina in a time-dependent (hr) manner. Ill, elevated expression of

c-myc RNA in mouse uterus and vagina is found upon Northern hybridiza-tion when analyzed 0 to 1 80 mm ( ‘) following DES exposure. The 0-mm timepoints (parts II and Ill) represent RNA isolated from tissue obtained from

animals immediately after injection of the vehicle, corn oil.


associated with phenotypic changes in growth and differ-entiation elicited as part of the estrogen response (20, 21,25, 29, 32, 54, 55). In order to relate how the expression ofTGF�3 compared to other estrogen-regulated genes, we ex-amined the pattern of expression of mRNAs for IGF-1,TGFct, c-myc, lactotransferrin, and PR in both the uterusand vagina of the immature female mouse (Figs. 3 and 4).

Considerable evidence supports a role for TGFctlepider-mal growth factor and IGF-1 as estrogen-inducible peptidegrowth factors that may be involved in mediating the abilityof estrogen to induce a mitogenic response in target tissuessuch as the uterus and breast (20, 25-29). Our past studieshave documented dose-dependent DES regulation of TGFain the immature female mouse uterus (29). Here we ex-tended these studies to demonstrate for the first time that, inaddition to the uterus (Fig. 3, IA), estrogen also regulated

TGFct mRNA levels in the vagina (Fig. 3, IB). Similar to theinduction of the TGFI3 mRNAs, detectable TGFa transcriptwas clearly evident within 3 h following DES exposure inboth tissues. However, TGFa mRNA induction by DES,unlike the TGFJ3 transcripts, was much more persistent,remaining elevated for up to 24 h posttreatment, the lasttime point investigated.

Examination oflGF-1 expression following DES treatmentshowed significantly heightened gene expression in theuterus within 0.5 h that peaked within 3 h (10-fold) andremained above control levels at 24 h (Fig. 3, II). LikeTGF�3, the extremely early induction of IGF-1 RNA expres-sion by estrogen in the mouse uterus classifies both asimmediate-early genes. However, TGF�3 mRNA was onlytransiently induced, lasting only a few h; whereas IGF-1gene up-regulation by estrogen, similar to TGFa, was moreprolonged. The vagina also responded to DES with theinduction of IGF-1 transcripts by 3 h after exposure; how-ever, the elevation of IGF-1 steady-state RNA levels in thevagina was delayed and was not nearly as dramatic (1 .5-


2-fold) when compared to the response of the uterus. How-

ever, the basal mRNA levels of IGF-1 expression (0 timepoint) in the vagina of control animals was found to begreater (2-fold) than expressed by the uterus.

Since TGFI33 mRNA was found to be regulated withinmm after estrogen treatment, we compared the induction of

the TGFI33 to an “immediate-early” estrogen-regulatedgene, c-myc (Refs. 21 , 36, 37; Fig. 3, III). Northern analysisdemonstrated that estrogen treatment elevated the level of

the 2.4-kb c-myctranscript in both the uterus and vagina ina time- and tissue-specific manner (Fig. 3, Ill). Within 30mm of estrogen treatment, significant induction of c-myc isfound in the uterus (4-fold), which persisted for up to 180

mm. In comparison to the uterus, the vagina exhibited amore transient elevation of c-myc RNA (only at 1 5 mm) anda lower degree of induction (2-fold) in response to estrogen.

The PR is a gene that is now considered to be a classicalgene marker for estrogen action (54, 55). Estrogen responseelements exist within the 5’ region of the PR gene and areimportant in the up-regulation of gene transcription by theligand-activated estrogen receptor. Although biochemicalmeasurements of estrogen induction of PR binding activityin the mouse reproductive tract has been studied (52), thekinetics of induction of PR mRNA by estrogen in the mouse

uterus and vagina has not been analyzed to our knowledge.Probing the same Northern blot used for the evaluation ofthe time-dependent induction of the TGFj3s mRNAs shownin Fig. 1 with mouse PR cDNA revealed estrogen induction

of the major 7.0-kb PR transcript in both uterus (-2-4-fold)

and vagina (-2-1 0-fold; Fig. 4). Although the uterus dis-played considerable basal levels of PR mRNA in the control

animals, by 3 h following estrogen treatment, the expres-sion ofthe 7.0-kb PR transcript was increased and remained

elevated through 16 h. In comparison to the uterus, thevagina exhibited much lower baseline PR mRNA expres-sion in the control animals; however, estrogen treatmentsignificantly up-regulated vaginal progesterone receptortranscript levels within 3 h, which peaked at 16 h before

decreasing at 24 h. As found for TGFa and IGF-1 , the mostnoteworthy difference between the temporal pattern of ex-pression of the PR gene and the TGFI3 mRNAs was thatTGF� transcripts were transiently induced (1-3 h) in con-trastto the prolonged up-regulation (�1 3 h) of PR mRNA inthe uterus and vagina.

Our laboratory has thoroughly documented estrogen reg-ulation of the iron-binding glycoprotein, lactotransferrin, inthe female mouse reproductive tract (32). Although thephysiological role of lactotransferrin is not yet understood,its association with estrogen-induced growth suggests thatthis protein plays a supportive role in the maintenance of

proliferation, possibly in the transport of iron critical formetabolic reactions associated with DNA synthesis. Here inFig. 4, comparison of the pattern of lactotransferrin mRNAinduction to that of the TGF�3s demonstrated that estrogenstimulation of the 2.4-kb lactoferrin transcript in the uterusand vagina occurred much later and was not evident until1 6 to 24 h. In contrast to the early induction of the TGF�3s,IGF-1 , TGFa, and PR genes, lactotransferrin can be consid-ered as a “intermediate” or “late” gene involved in estrogenaction. In addition, to insure that the quality and quantity ofuterine and vaginal RNA was similar for each time pointevaluated in the Northern blot shown in Figs. 1 and 4, theblot was reprobed for the housekeeping enzyme, GAPDH(Fig. 4).

Cell-specific Localization of the RNA and Protein for theTGFIJ Isoforms by in Situ Hybridization and Immunohis-tochemistry. The Northern hybridization results presentedabove clearly demonstrated that estrogen regulated thesteady-state mRNA levels for each TGFI3 isoform in a tem-poral fashion in the reproductive tract of the immaturemouse. In order to better understand the physiological roleof TGFI3 genes in estrogen-mediated growth of the uterusand vagina, it was necessary to identify the cell types withinthese complex tissues that expressed the different TGFI3isoforms. Therefore, cell-specific accumulation of the TGFI3RNA and protein in the uterus and vagina was localized byifl situ RNA hybridization (Fig. 5) and immunohistochemi-cal (Fig. 6) techniques using reagents specific for each of theTGFI3 isoforms. lii situ RNA hybridization was carried outby incubation of tissue sections with gene-specific 35S-labeled antisense and sense riboprobes derived from theprecursor regions of the TGFI3 genes (Fig. 5). Consistentwith our findings of DES regulation of TGFj3 mRNA fromNorthern blot analysis, in situ hybridization also demon-strated that estrogen stimulated the transcripts for eachTGFf3 isoform in the uterus and vagina when compared tothe signals obtained from control tissues. Distinctive epi-thelial cell-specific patterns of RNA expression for the TGFI3isoforms were found in the both the uterus and vagina. InFig. 5, the photomicrographs clearly demonstrate that,within 3 h following DES treatment, the uterine epitheliumexpressed elevated TGF�31 (Fig. 5, A and B), TGF�32 (Fig. 5,C and D), and TGFI33 (Fig. 5, E and F) transcripts, althoughsome degree ofestrogen-induced TGF� expression was alsoevident in the stroma (Fig. 5, A-F). Similarly, in situ analysisof the vagina (Fig. 5, I-N) demonstrated that the transcriptsfor all three TGFf3 isoforms were localized primarily to theepithelium and were stimulated by DES when assayed at 16h for TGF�31 (Fig. 5, I and J) or at 3 h for TGFf32 (Fig. 5, Kand L) and TGFf33 (Fig. 5, M and N). Our in situ studies alsodetected high levels of TGFf3 transcripts in the fat tissue andblood vessels associated with the reproductive tract (datanot shown). Incubation of tissue sections with 35S-labeledsense probes was carried out as controls for nonspecificbinding. These hybridizations always resulted in signifi-cantly less binding than obtained with the correspondingantisense probes. As examples, the nonspecific bindingexhibited by the uterus (Fig. 5, G and H) and vagina (Fig. 5,0 and P) of DES-treated mice upon incubation with theTGF�32 (Fig. 5, C and 0) and TGF�3 (Fig. 5, H and P) senseprobes are shown.

In order to correlate the estrogen-regulated expression ofTGFI3 mRNAs with the presence of the respective proteins,specific polyclonal antibodies to TGF�31 (Fig. 6: uterus, Aand B; vagina, C and H), TGFf32 (Fig. 6: uterus, C and D;

vagina, I and 1), and TGFf33 (Fig. 6: uterus, E and F; vagina,

K and L) proteins were used to examine the effects of DESon TGFI3 protein expression. Unlike the transient inductionof TGFI3 mRNAs, DES treatment was found to increaseimmunodetectable protein for all three TGFj3 isoforms inthe epithelium over a prolonged period oftime ranging from3 through 24 h for TGF�31 and TGF�33 in the uterus and forTGFI32 and TGFI33 in the vagina (3 and 24 h, data notshown). Elevation of TGF�1 protein in the vagina was notapparent until 16 h posttreatment, a point at which TGF�31mRNA levels were also found to be maximally stimulated.The photomicrographs of Fig. 6 clearly show the sustainedprotein induction and the specific localization of the threeTGFI3 isoforms to the epithelium of the uterus (Fig. 6, B, D,

UterusControl Estrogen Control

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Fig. 5. Representative light field (X 1 00) images of tissue sections of mouse uterus and vagina showing in situ hybridization of specific antisense TGFI3 RNAprobes. Probes specific for TGFf31 (A and B), TGF)32 (C and D), and TGFI33 )E and F) were hybridized to uteri obtained 3 h after treatment of immature mice

with estrogen (DES; 20 rig/kg) or the vehicle control. In situ hybridization of the vaginas were performed at 3 h for TGFf32 (K and L) and TGFf33 (M and N) andat 1 6 h for TGF)31 (I and I) following DES treatment (the control vagina shown is from the 3-h time point). The background hybridizations that resulted upon

incubation of the tissues from estrogen-exposed animals with the sense riboprobes for TGFj32 (uterus, C; vagina, 0) and for TGFj33 (uterus, H; vagina. P( are

also shown. To facilitate orientation, the epithelium (e) and the stroma Is) of the uterus (A) and the vagina (I) are labeled with indicated letters.

and F) and vagina (Fig. 6, H, J, and L) at 16 h followingestrogen treatment in comparison to control tissues (Fig. 6:uterus, A, C, and E; vagina, G, I, and K). A comparison ofthe in situ hybridization and immunohistochemical resultsrevealed the colocalization to the epithelium of both pro-tein and RNA for all the TGF� isoforms in the uterus and thevagina at early time points, which indicated concordantestrogen regulation of these genes. However, this relation-ship between RNA and protein expression levels in thereproductive tract was time dependent in that immunode-tectable TGFI3 proteins (Fig. 6) remained elevated in the

epithelium for up to 24 h when TGFI3 RNA levels weresubstantially reduced. Thus, at later time points followingestrogen treatment, there appeared to be a dissociation ofRNA and protein expression for the TGF�3s in the reproduc-tive tract. Also noteworthy was that, by in situ and immu-nohistochemical analyses, considerable levels of expres-

sion ofthe TGF�3s, especially TGFI31 and TGFI33, was foundin the adipose tissue associated with the reproductive tract.In addition, intense staining for TGFI33 protein was foundassociated with the smooth muscle ofthe uterus and vagina,suggesting a role in smooth muscle differentiation. Al-though the TGF�s were expressed in these other cell types,DES treatment did not dramatically influence their RNA orprotein expression (data not shown). In conclusion, theevidence obtained from in situ RNA hybridization and im-munohistochemistry studies revealed epithelial cell-spe-

cific expression patterns for the TGFf3s in the uterus andvagina, which suggest possible roles as autocrine and/orparacrine mediators of estrogen growth.

Relationship of TGFI3 Protein Expression to TGFa and toEstrogen-induced DNA Synthesis. Using immunohisto-chemistry, we also compared the induction of TGFI3 pro-teins to TGFa protein following DES treatment and found

Uterus

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Control Estrogen Control Estrogen

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926 TGFI3 and Estrogen Action in the Genital Tract

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Fig. 6. Photomicrographs (X 50) are shown of inimunohistochemistry using specific antibodies to TGF�1 (uterus, A and B; vagina, C and H), TGF�2 (uterus,

C and D; vagina. I and I), and TGFj33 (uterus, F and F; vagina, K and L) performed on uterine and vaginal sections obtained 1 6 h after treatment of immaturefemale mice with the corn oil vehicle (control) or with estrogen (DES; 20 pgJkg). The epithelium el, stroma (5), blood vessels (by), and muscle (m) of the uterusand vagina are designated with letters. Note the specific accumulation and colocalization of all three TGFf3 isoforms in the epithelium of the uterus and vagina

upon estrogen treatment. Vaginal and uterine smooth muscle also expresses prominent TGFj33 protein expression but in an estrogen-independent manner.

that the induction patterns of the proteins to be strikinglysimilar. The photomicrographs in Fig. 7 show the specificup-regulation of immunodetectable TGFct protein in theuterine and vaginal epithelium within 3 h following estro-gen exposure, which remained elevated at 24 h. WhenTGFa protein was compared to TGFt mRNA levels asshown earlier by Northern analysis (Fig. 3), the TGFamRNA levels reflected the protein changes and exhibitedprolonged concordant induction, in contrast to the transientmRNA induction of the TGFf3s, yet more prolonged expres-sion of the TGFf3 proteins. In situ hybridization also dem-onstrated that TGFt transcripts were coexpressed with theprotein in the epithelium of the uterus and vagina (data notshown). Comparison of the immunohistochemical localiza-tion of the TGF� isoforms (Fig. 6) to the localization of theTGFa protein (Fig. 7) clearly demonstrated that these potentregulatory proteins were coexpressed simultaneously in theepithelium of the uterus and vagina following estrogentreatment. These same cells were shown to be activelyundergoing DNA synthesis (Fig. 8).

Cell cycle analysis has documented that estrogen stimu-lates the epithelium to enter and start to progress through G1by 3 h and to be actively engaged in DNA synthesis andmitosis by 1 6 h following estrogen treatment (1-7, 33). Thephotomicrographs of Fig. 8 (lower) are examples of immu-

nolocalization of BrdUrd-labeled nuclei in the uterus andvagina, which clearly show the synchronized and dramaticDNA synthesis that the epithelium of both tissues is under-

going at 1 6 h in response to a single dose of DES. A detailedtime course study of estrogen induction of epithelial DNAsynthesis as measured by percentage of BrdUrd-labelednuclei in mouse uterus and vagina is also shown in Fig. 8(top). Maximal epithelial DNA synthesis was stimulated byestrogen within 1 6 h in the uterus and by 24 h in the vagina.It is noteworthy that the actively proliferating uterine andvaginal epithelial cells are the same cells that also respondto estrogen by the accumulation of increased protein levelsof TGFa (Fig. 7) and of each TGFf3 isoform (Fig. 6). Onlymodest DNA synthesis occurred in the other cell types inthe uterus and vagina upon estrogen exposure, which wasconsistent with estrogen not dramatically affecting the 1ev-els of protein for TGFa or the TGF�s expressed by thesecells.

In Vivo Evidence of Estrogen Modulation of TGFIJ Bind-ing in the Mouse Uterine Cells. A number of cell surfaceproteins have been identified that bind the TGFf3s, some areinvolved in signal transduction and others are thought toregulate the access of TGFJ3 to cellular targets (56, 57). Togain more insights into the role of the TGFf3s in estrogenaction and to understand the potential mechanism bywhich the TGFf3s elicited physiological effects in the repro-ductive tract, we wanted to obtain information concerningthe type of TGF�3 receptors that existed in the reproductivetract and also to determine whether estrogen treatmentmodulated TGF�3 binding. Affinity labeling ofTGF� bindingproteins was carried out by incubation of uteri removed

UTERUS VAGINA

Fig. 7. Immunodetection of TGF protein shows that es-trogen (DES pg/kg) treatment of immature mice in com-

parison to the vehicle controls (3-h time point) significantlyinduces the expression of TGFa in the epithelium of the

uterus and vagina within 3 h and remains elevated at 24 hposttreatment (hr(.

Control(3 hr)

Estrogen

(3 hr)

Estrogen

(24hr)


from mice treated 1 6 h previously with DES or with the cornoil vehicle with 125l-TGFf31 in the presence (+) or absence(-) of excess unlabeled TGFI31 followed by covalent cross-linking, extraction, and resolution on SDS-polyacrylamidegels (Fig. 9). As documented in other cells, the uterus ofcontrol animals demonstrated three types of binding pro-teins covalently labeled with 125l-TGF�1 with approximatemolecular weights of 55,000 (type I), 75,000 (type II), and �

250,000 (type Ill, j3-glycan). The Mr 75,000 type II and�-glycan proteins appear to be the predominant TGF� bind-ing proteins expressed in the uterus. In addition, a Mr

95,000 binding component was detected. When comparedto the affinity labeling of control uteri, estrogen (DES) treat-ment was found to significantly reduce the abundance of allof the TGF� binding proteins in the uterus. UnlabeledTGFf31 (+) successfully competed with 125I-TGF�31 bindingto the proteins in each group, which demonstrated thespecificity of TGF�31 interaction. Taken together, the detec-tion of specific TGF�3 binding proteins in the uterus dem-onstrated that the receptors necessary to mediate the bio-logical effects ofthe TGFf3s are present and that modulationof the levels of specific TGFj3 binding proteins by estrogensuggest that uterine response to the TGF�s may be anothermechanism by which estrogen controls TGFJ3 action. Inves-tigation of vaginal TGFI3 binding is presently in progress.

DiscussionIt is well documented that estrogen action involves thesequential expression of a number of cellular genes whoseproducts are thought to mediate the physiological response

in target tissues by the initiation of specific genetic programs(20, 38, 39). The study presented here describes the time-

dependent and site-specific effects of estrogen on the in-duction of mRNA and protein of the three TGF� mamma-ian isoforms (�31 , �32, and f33) in the uterus and vagina of

the immature mouse. Our results demonstrate that a single

s.c. administration of the synthetic estrogen DES to imma-ture prepubescent female mice (ages 1 7-1 9 days) sign ifi-

cantly induces mRNA and protein expression of the threemurine isoforms, TGF�31, TGFf32, and TGFI33, in both theuterus and vagina. Immunohistochemical protein detection,in situ hybridization, and Northern RNA analyses demon-strate overlapping time-dependent and cell-specific induc-tion of each isoform of TGFI3 in response to estrogen.Northern hybridization clearly shows that estrogen signifi-

cantly but only transiently up-regulates TGF/33 mRNAwithin 30 mm and TGF�1 and TGFI32 mRNAs by 3 h. The

vaginal tissues also exhibit a similar pattern for estrogen

induction of the TGFI33 and TGFf32 mRNAs as that ob-served in the uterus; however, TGFf31 mRNA levels in thevagina increase gradually and peak around 16 h. Following

the transient mRNA induction of the TGF�s by estrogen,there is prolonged suppression of the transcript levels of allthe TGFI3 isoforms for up to 72 h following estrogen expo-sure when compared to the tissues from the corn oil vehi-cle-treated controls. The significance of this down-regula-tion is not known. Investigation of the steroid specificity ofthe regulation of TGFI3 steady-state mRNA levels made bycomparing the effects of a single treatment of progesterone,estriol, DHT, or DEX with that elicited by various doses of

6 12 15 24

Time atter treatment (hour.)

42

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928 TGF)3 and Estrogen Action in the Genital Tract

A

CONTROL

ESTROGEN

DNA SynthesisMouse Uterus

UTERUS

DNA SynthesisMouse Vagina

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Fig. 8. Time course of estrogeninduction of epithelial DNA syn-

thesis in the immature mouseuterus and vagina by quantitation

of BrdUrd-labeled nuclei. DNAsynthesis was calculated as a per-

centage of labeled nuclei to totalnumber of nuclei present and ispresented as a bar graph (A). Each

bar represents the average and SEfrom four different animals at the

time points evaluated. Statistically

significant differences from controlvalues (P < 0.05) determined by

one-way analysis of variance usingthe least significant difference

method for comparing individualgroups is indicated by an asterisk(C) B, representative photomicro-

graphs (X 100) are shown of theimmunohistochemical detection

of BrdUrd incorporation into the

epithelial nuclei ofthe uterus (left)and vagina (right) of the vehiclecontrol- and estrogen (DES; 20 pg/

kg)-treated mice 1 6 h following in-jection. The results clearly demon-strate that estrogen is inducing

a synchronized wave of DNA syn-

thesis in the uterine and vaginalepithelium, which at this timeof maximal growth, is expressing

the TGFf3 and TGFa proteinssimultaneously.

the potent estrogen DES demonstrates predominant estro-gen specificity in the control of TGF�3 expression in thei mmatu re mouse reproductive tract, although progesteroneand the weak estrogen, estriol, have some effect.

In situ hybridization to identify the cell types responsiblefor the synthesis of the TGFI3 mRNAs in the reproductivetract reveals that the most pronounced stimulation of allthree isoforms occurs in the epithelium of both the uterusand vagina; however, stroma expression is also stimulatedto some extent by estrogen . Fu rthermore, i mmu nostai n i ngwith specific polyclonal antibodies demonstrates that estro-gen stimulates a prolonged and significant elevation of theproteins for TGF�31 , TGFI32, and TGFI33, specifically in theepithelium of both the uterus and vagina, which is the samecell type that demonstrates the most significant elevation ofTGF� transcripts. However, the relationship between TGFf3mRNA and protein levels is discordant in that enhancedexpression of TGF� proteins following estrogen treatmentpersists for a long period of time in comparison to the very

transient expression observed for the TGF� transcripts. Littleor no immunolocalization for the TGF3s is seen in thestroma, even though significant transcript expression is de-tected in these cells, which is indicative of a possible para-crine mode of action. Prominent TGFI33 and some TGFI32protein expression is also associated with the longitudinal

and circular muscle layers of the uterine myometrium andin the smooth muscle cells of blood vessels, suggesting arole of the TGFI3s in muscle function. Neither TGF�2 norTGFI33 expression in the muscle appears to be subject tomodulation by estrogen. Western analysis of partially pun-fied tissue extracts using an antibody which detects bothTGFj31 and TGFI32 has confirmed the presence of Mr1 2,500 TGFI3 monomeric protein in the uterus and vagina(data not shown).

To gain a better understanding of the sequential changesin gene expression for multiple factors occurring in thereproductive tract upon estrogen treatment, we comparedthe kinetics of TGFI3 expression to that of other genes

(-) or (+) excess +unlabeledTGF-Beta

- +

Fig. 9. Affinity labeling of TGFf3 binding proteins I!, M, 55,000 (55k); Ii, M,

75,000 (75k); and III, M, >250,000; denoted by arrows and approx. molec-ular weightsl by incubation of uteri removed from immature mice treated 16

h previously with DES (20 pg/kg) or with the vehicle corn oil (Control) with

125l-TGF�i in the presence (+) or absence (-) of 200-fold molar excess ofunlabeled TGFf31 followed by covalent cross-linking, solubilization, resolu-

tion by 6% SDS-polyacrylamide gel electrophoresis, and autoradiography.


�3OOk -k

Control DESI II

95k- �75k-55k

documented to be susceptible to estrogen control but notnecessarily containing estrogen response elements (20, 21,25, 29, 32, 54, 55). In general, the comparison of theinduction of the TGFf3s to the estrogen-regulated genes,TGFa, IGF-1 , and PR, reveals that the TGF� mRNAs aremore transiently induced, in contrast, to the prolongedup-regulation of the mRNAs for the other genes in both theuterus and vagina. For example, IGF-1 has been proposedto have a “pivotal” position in the control of cell prolifera-tion and is an absolute requirement for entry into the Sphase of the cell cycle for many cell types (36). Murphy etal. (25) have demonstrated estrogen and estrous cycle reg-ulation of IGF1 mRNA in the rat uterus. The study presentedhere, to our knowledge, is the first example of IGF-1 regu-lation by estrogen in both the uterus and the vagina of theimmature mouse. Although estrogen induction of CF-i inthe vagina was not as dramatic as the uterine response, thebasal levels of IGF-1 mRNA expression was considerablyhigher in the vagina than that found in the uterus. Similar toTGFf33, significant induction of IGF-1 mRNA occurs within30 mm in the uterus; however, the expression ofthe TGF�3

transcripts is short lived, whereas elevated IGF-1 RNA 1ev-els are still evident at 1 6 h. Likewise, analysis of TGFa, apotent mitogen in which the gene contains a putative es-trogen response element (58), demonstrates that estrogencoordinately up-regulated TGFa mRNA and protein levels

in both the uterine and vaginal epithelium, starting as early

as 3 h after treatment; but in contrast to TGFI3 mRNAexpression, TGFa mRNA is still elevated at least 24 h.Despite the transient nature ofthe induction ofTGFj3 mRNAby estrogen, analysis of immunodetectable TGFf3 proteinreveals that uterine and vaginal epithelium still containelevated levels of TGFI3 protein up to 24 h after treatment.It should be noted here that, because of the persistentexpression of the TGF� proteins, the TGFf3s are coex-pressed with TGFa in the rapidly dividing uterine and vagi-nal epithelial cells. Published studies suggest that the tran-scniptional and translational regulation ofthe TGFf3s is quitecomplex and exhibits strain variation (48). Similar to ourresults, discordant regulation of TGF�1 mRNA and proteinexpression has been reported during chemical carcinogen-esis ofthe skin, where the TGF�s are proposed to play a rolein the promotion of neoplastic transformation both in vitro

and in vivo (48, 51 , 54). In addition, it has been shown thatcoapplication of purified TGFf31 and TGFx can synergize to

act as a stage 1 tumor promoter in mouse skin. The induc-tion of TGF�3 in collaboration with other growth factors,such as TGFa, during wounding or chronic inflammationelicited by the phorbol ester promoters has been proposedto inadvertently exert a tumor promoter effect my fosteringconditions for uncontrolled growth. It is of interest thatestrogen also elicits a modified wounding response in thereproductive tract, including the induction of TGFt and theTGF�3s, leading one to speculate that unopposed estrogen,which is associated with development of neoplastic dis-eases of the genital tract, may create a promoting environ-ment similar to that shown for the phorbol ester skin tumorpromoters.

The PR is a classical gene marker for estrogen action thatcontains estrogen response elements within the 5’ region(54). The early and prolonged induction of PR mRNA byestrogen in the mouse reproductive tract was found to besimilar to TGFa and IGF-1 expression and thus, distinctfrom the transient regulation of the TGF� transcripts byestrogen.

Another estrogen-inducible gene in the rat uterus is thec-myc protooncogene, but the regulation is probably mdi-rect because the gene does not contain an obvious estrogenresponse element (21, 37, 59). c-Myc is a tightly regulatedand short-lived transcription factor that governs the deci-sion of a cell to either enter the cell cycle or to undergoprogrammed cell death (apoptosis; Refs. 37 and 59) andthus, has been proposed to mediate the action of diversesignaling molecules. Similar to the uterine and vaginal in-duction of TGF�3 by estrogen, c-myc mRNA levels areelevated within 1 5 mm, and the uterine c-myc response ismaintained up to 3 h after estrogen treatment. In contrast,c-myc induction in the vagina is more transient than TGFf33induction and returns to the baseline level by 3 h.

In comparison to the other genes, estrogen does notsignificantly up-regulate gene expression for the iron-bind-ing glycoprotein lactotransfernin until 1 6 h, even though thisgene has been shown to contain an estrogen responseelement (60). The late induction classifies lactotransfernin asan “intermediate” or “late” gene in estrogen action and alsosuggests that other factors may be needed to cooperate with


the estrogen receptor for the transactivation of lactotrans-fennin gene transcription as indicated by the existence ofpeptide hormone response elements in the promoter (60).

Since the TGFf3s control cell proliferation and diffenenti-ation by binding to cell surface receptors (56, 57, 61), wealso investigated the effects of estrogen on TGFf3 binding inthe uterus. Specific binding of 1251-TGFI31 by affinity label-ing documented the existence of the type I (Mr 55,000),

type II (Mr 75,000), and type III/(3-glycan (Mr >250,000)

binding proteins in the intact uterus and that estrogen treat-

ment significantly decreased specific binding to each of thebinding proteins/receptors. Our attempts to perform Scat-chard analysis of TGFI3 binding on isolated uterine mem-branes failed, possibly due to the degradation or masking ofthe receptors during isolation. The mechanism by whichestrogen reduces the number of binding sites for TGF�31 isnot known and may possibly involve multiple pathways(56, 57, 61-67), including a marked down-regulation of thereceptor expression due to increased use on occupancy ofthe receptors by the enhanced production of the ligands.Estrogen may also modulate the number of receptors orfunction due to induction of differentiation or have specificeffects on the biosynthesis of the TGFf3 binding proteins.The relationship between the decrease in TGFf3s bindingsites and the biological effects of the TGF�3s in the genital

tract is not clear. However, in vitro studies demonstrate thatcell-specific variations in the levels of TGFI3 receptors areoften associated with distinct differences in responsivenessto the TGFJ3s (57, 61 -67). For example, in a number of celllines, the loss or decrease in the TGFI3 receptors is associ-ated with the acquisition of a TGF�-resistant phenotype,which has been proposed to lead to a selective growthadvantage. Since the binding proteins are involved in themediation of the physiological response to the TGFf3s, onecan speculate that the decreased levels of receptors de-tected following estrogen treatment may indicate that uten-me cells become less responsive to the TGFf3s during activegrowth. The effects of estrogen on the binding profile of theTGFf3s in the vagina is presently being investigated.

Most often the TGFI3 proteins are usually produced aslatent forms that do not interact with the specific cell surfacereceptors. Even though immunodetectable TGFI3 proteinsare present in the uterine and vaginal epithelium, we do notknow if the TGFf3s are in a latent or active form. A numberof conditions are known to activate the TGFf3s in vitro, suchas exposure to extreme pH conditions, 8 M urea, heat, andproteases (40, 41 , 49, 68-71). However, little is knownabout the mechanisms in vivo that lead to the activation ofthe TGF�s. It has been suggested that, in wound tissue, theacidic microenvironments or high proteolytic activity fromplasmin or urokinase may be sufficient for TGF� activation.Studies of the reproductive tract, breast cancer, and cul-tuned cell lines have demonstrated that estrogen induces anumber of proteases (18, 19). In addition, estrogen induces

a modified inflammatory response in the uterus, which maymodulate the pH and content of proteases contributed bythe influx of the inflammatory cells and the activation ofthrombin (1 0-1 8). Taking these observations into consid-eration, we can speculate that appropriate conditions mayexist in the reproductive tract after estrogen treatment forthe production of activated TGFI3 proteins.

A time course study of estrogen stimulation of epithelialDNA synthesis as measured by the percentage of BrdUrd-labeled nuclei demonstrates that maximal DNA synthesisoccurs at approximately 1 6 h in both the uterus and vagina.

These epithelial cells stimulated to grow by estrogen are thesame cells that accumulate increased levels of TGF� pro-teins. Only modest DNA synthesis occurs in the other celltypes in the uterus and vagina upon estrogen exposure, andestrogen does not dramatically affect the levels of TGF�3proteins in these cells. The kinetics analysis of estrogeninduction of DNA synthesis in the uterus and vagina revealsthat the transient up-regulation of the TGFf3 mRNAs occursprior to the onset of growth; however, immunodetectableTGFf3 proteins persist much longer than the TGF� tran-scripts and remain elevated in the epithelial cells whileundergoing maximal DNA synthesis. This data implicatesthe TGF�s as mediators that may regulate the cell cycle

transition of the uterine and vaginal epithelium in responseto an estrogenic stimulus. Studies have shown that theTGFf3s are bifunctional growth regulators having either p05-itive or negative effects on cell growth, depending on thecell type and the extracellular environment (40, 41 , 72).Most ectodermal-denived epithelial cells, endothelial cells,and lymphoid cells are growth inhibited; whereas cells ofmesenchymal origin are generally stimulated to proliferate.However, the epithelium of the uterus and upper vagina isnot typical in that both are derived from mesoderm, whichmay possibly program the epithelial cells to respond to theTGFf3s in a more mesenchymal-like manner (73).

The molecular mechanism by which estrogen regulatesTGFf3 mRNA expression is not yet known. The promoters ofeach TGFj3 isoform are strikingly different from one an-other, suggesting distinct mechanisms involved in their reg-ulation (40, 41). The TGF�3 promoters do not contain clas-

sical estrogen response elements, which suggests eitherindirect regulation by estrogen or the existence of nonca-nonical estrogen receptor binding sites. Although theTGFf31 promoter contains no TATA box, three AP-1 sitesand a cluster of Spl regions have been shown to be impor-tant in the regulation of TGF31 expression. Evidence sug-gests that the AP-1 sites may mediate the autoinduction ofTGFj31 . Since estrogen is known to up-regulate the expres-sion of c-jun (24) and c-fos (23), which are both compo-nents of AP-1 , it is reasonable to propose that stimulation ofTGFf31 expression by estrogen may be mediated throughactivation of transcription from the AP-1 sites of the TGF�1promoter. Although the TGF�32 and TGFI33 promoters pos-sess classical TATA boxes adjacent to their transcriptionalstart sites, binding sites for other known transcriptionalfactors are rare. However, cAMP response element bindingprotein elements are found in both the TGF�2 and TGFf33promoters and are important in promoter activity. Thesesites may also be important in mediating estrogen effectssince up-regulation of cAMP-dependent protein kinase ac-tivity does occur in the uterus upon estrogen exposure,which would then lead to the phosphorylation and activa-tion of the cAMP response element and subsequent iran-scniptional activation of the TGFI32 and TGF�3 promoters(74). Because at this time we can only speculate, furtherstudies are needed to identify the DNA elements and bind-ing proteins involved in the estrogen regulation of TGFf3

expression.In addition, we demonstrate that the TGFf3s may also be

mediators of estrogen action on the growth and squamousdifferentiation of the vagina. Both in vivo and in vitro stud-ies have provided evidence that the TGFj3s may play rolesas endogenous autoregulators of epidermal growth and dif-ferentiation (48, 49, 50, 75-77). During wound healing,migrating and dividing keratinocytes in the epidermis have

cell Growth & Differentiation 931

been shown to initiate the synthesis of TGFf31 (75-76). Anumber of biological response modifiers, such as the reti-noids and phorbol esters, affect keratinocyte growth anddifferentiation by the modulation of the TGF�s (48, 50).Phorbol ester-mediated skin tumor promotion is associatedwith dramatic changes in the expression of the TGF�3s atboth the RNA and protein levels in the epidermis, which hasbeen correlated to the hyperplastic response of the skin tothe phorbol esters (48). A recent in vivo study of the actionsof retinoic acid on epithelial differentiation found that thevaginal epithelium of vitamin A-deficient mice becamehighly cornified, which was associated with enhanced ex-pression for each TGF�3 isoform (50). This data extends andsupports our results in demonstrating that keratinization ofthe vagina, regardless of the initiating stimulus (estrogen orvitamin A deficiency), is associated with up-regulation ofTGFj3 expression in the epithelium. An important conceptput forward by Roberts and Sporn (41) is that steroids andretinoids mediate their effectsin part by the modulation ofthe expression of the TGFj3s (41 ). Thus, perhaps it shouldnot be surprising that the TGF3s may be involved in the

mechanism of action of estrogen on the epithelium of boththe vagina and the uterus.

Our study of estrogen regulation of the TGF3 isoforms inthe reproductive tract of the immature mouse extends andsupports other investigations showing the regulation of theTGF�s in the female rodent genital tract (43-47). Significantlevels ofthe TGFI3s are expressed in the ovary of human androdents, suggesting roles in the regulation of folliculargrowth, maturation, atresia,and regression (46, 47). Multi-pIe treatments of ovariectomized adult rats with estrogenand/or progesterone has been shown to significantly induceimmunodetectable TGF�3 proteins in the uterus (43).Tamada et al. (44) and Das et al. (45) provide an additionalexample of a link between the ovarian steroid hormonesand the TGFf3s by demonstrating specific expression duringthe periimplantation period of early pregnancy (44, 45). Inthis pregnancy study, steroid hormone-mediated changes inuterine epithelial and stromal cell differentiation is pre-ceded by periods of rapid proliferation associated with theinduction of the TGF�31 and TGFf32, which further demon-strates that the presence of the TGFf3s in the reproductivetract is compatible with growth. These in vivo effects of thesteroid hormones in the uterus resemble in vitro studieswhere cells respond to differentiation-inducing agents byfirst undergoing a few rounds of DNA synthesis prior towithdrawal from the cell cycle and commitment to differ-entiation. Given that steroid hormones not only inducegrowth but often stimulate morphogenesis and differ-entiation, the presence of TGF�3 proteins suggests possibleroles in mediating these physiological changes in the re-productive tract.

Das et al. (45) report that TGFI33 protein expression waslocalized strictly to the myometrium and vascular smoothmuscle of the uterus and did not fluctuate with pregnancy.In concordance with their study, we find significant immu-nodetectable TGF�33 in the smooth muscle cellsof both theuterus and vagina. However, we also demonstrate that asingle estrogen dose significantly induces TGF�33 expres-sion in the epithelium of the uterus and vagina of prepu-bescent mice. The presence of the TGF�33 in the smoothmuscle of the reproductive tract adds to numerous in vivoand in vitro studies that support a role for the TGF3s,particularly TGF(33, in muscle differentiation and function(41, 64, 78).

Our past studies and that of others have clearly docu-mented that TGFa/epidermal growth factor and their recep-tors are essential components of the normal pathway of cellgrowth in the female rodent uterus in response to estrogen(27-29). The work presented here demonstrates that bothTGF�3 and TGFa are coexpressed in the uterine and vaginalepithelium following estrogen treatment of immature mice.Similarly, during the periimplantation period, TGFa expres-sion has been reported to parallel the induction of TGFJ31and TGF32 (79). The coinduction of TGFa and the TGF�3sin either the stroma or epithelium by the sex steroids isassociated with many events involving the initiation ofgrowth and differentiation in these target cells and in manyrespects resembles wound healing. Studies of growth factorexpression in liver regeneration is another example ofwound healing where both TGFa and the TGFj3 isoformsare coinduced within 4 h after partial hepatectomy (68, 80).The authors suggest that the in vivo function of TGF� in theregenerating liverisas growth inhibitorsto regulate hepa-tocyte proliferation and to prevent uncontrolled cellgrowth. This interpretation is based on the documentedgrowth inhibitory effects of the TGF(3 on purified hepato-

cyte cultures in vitro. However, increasing evidence mdi-cates that TGFj3 effects on isolated cell populations in vitromay not always reflect the role of the TGF�s in complexorgan systems in vivo (41 ). It is possible that the function ofthe TGF�s in the wound-healing environment of the regen-erating liver is similar to that proposed for other early genesassociated with hepatocyte growth (TGFa, hepatocytegrowth factor, insulin-like growth factor, ras, and myc); i.e.,to play a role in the initiation and coordination of repair oftissue injury (80).

The results of our work demonstrate that protein for thethree TGFI3 isoforms are up-regulated in the epithelial com-partments of the uterus and vagina undergoing activegrowth and differentiation in response to estrogen. Enoughevidence exists to propose that the TGF�3s may function inthe regulation ofepithelial homeostasis in the rodent genitaltract. The role of the TGFf3s in the reproductive tract is notknown and thus we can only speculate and extrapolatefrom information obtained from other systems. From thesestudies, the following suggestions of TGF� function(s) inreproductive physiology can be put forward: (a) the TGFf3smay actually facilitate growth in vivo, as evident in boneformation, wound healing, tumor promotion, mesenchymalcell proliferation, and fibrosis (40, 41 , 51 , 53, 62, 65, 72,75-77). Some of the growth promoting properties of theTGFf3s may be associated with the recruitment and activa-

tion of cells (fibroblast and macrophages), positive up-reg-ulation offurther TGF� synthesis, and the induction of othergrowth factors (platelet-derived growth factor, tumor necro-sis factor a, interleukin 1); (b) the TGF�s may inhibit pre-mature differentiation of the epithelial cells to allow ade-quate growth to fulfill a specified physiological role beforeterminal differentiation (as seen in the mammary gland andpossibly the epidermis; Refs. 75-77 and 81 ); (c) modulationof the extracellular matrix by the TGFf3s may actually beresponsible for many ofthe physiological effects elicited bythe TGF�3s (40, 41 , 72); (d) the recruitment of inflammatorycells and modulation of their function by the TGF�3s maycontribute to reproductive tract changes (40, 41 , 52); (e) theestablished role of the TGF�3s in tissue remodeling andmorphogenesis associated with epithelial-mesenchymal in-teractions (40, 41 , 42, 70, 75, 81 ). Similar events occurcyclically and during pregnancy in the genital tract under


the control of steroid hormones, suggesting that TGFI3 maybe functioning in these events; (1) the growth inhibitoryeffects of the TGF(3s in many isolated cells in culture (40,41 , 72) suggest that the TGFf3s may prevent uncontrolledgrowth in vivo in the reproductive tract and act as agentsresponsible for restoring equilibrium following estrogen-induced growth; (g) in pregnancy, the TGF�s may be in-strumental in the preparation of the uterus for reception ofthe embryo as well as in the support of the initial stages ofblastocyst development (44, 45); (h) the association of theTGFf3s with many mature, fully differentiated cell types

such as muscle (40, 41 , 46, 64, 72, 81 ) support a role in themaintenance of the differentiation phenotype; and 0) thepotentiation of angiogenesis by the TGFf3s (40, 41 , 72, 78)may be quite important in supporting the growth and in-creased functional demand (nutrition, secretion) of the re-productive tract, especially during pregnancy.

In summary, our detailed study of the time-dependentand cell-specific estrogen induction of TGFf31 , TGF$32, andTGF�3 in the uterus and vagina and the demonstration ofestrogen modulation of uterine TGFf3 binding proteins sup-port the addition of the TGF�s to the list of growth factorsthat can act as coordinators of the synchronized growth andremodeling ofthe reproductive tract that occurs in responseto estrogen. It has become clear that steroid hormone reg-ulation of the reproductive tract is similar to the mechanismof wound repair with the complex involvement of multiplecytokines and cell types. The identification of multifunc-tional regulatory proteins in the reproductive tract signifi-

cantly enhances our ability to interpret estrogen action andprovide potential biomarkers to evaluate disease states,

which in turn may lead to the design of new agonists,antagonists, and neutralizing agents of potential therapeuticuse.

Materials and Methods

Animals. The animals were treated humanely followingInstitute-approved protocols that were based on NIH guide-lines. Female CD-i mice (ages 1 7-1 9 days) received s.c.injections (0.1 ml) of either DES (20 pg/kg), progesterone (2mg/kg), 5a-dihydrotestosterone (1 mg/kg), DEX (20 ig/kg),estriol (20 pg/kg), or corn oil vehicle control and weresacrificed at indicated times (Sigma Chemical Co., St. Louis,MO). Following CO2 asphyxiation, the uteri and vaginaswere collected and either placed in appropriate fixatives orimmediately frozen in liquid nitrogen for RNA purification.

Cloning, Probe Preparation, RNA Isolation, and North-em Blot Analysis. PCR (Perkin-Elmer Corp., Morrisville,NC) using specific primers amplified TGFI3 sequences fromcDNA generated by reverse transcription (GIBCO-BRL,Gaithersburg, MD) of mouse uterine poly(AY� RNA (TGF�31,23-480 bp; TGFf32, 458-836 bp; and TGFI33, 834-1309

bp), which were then filled in with T4 polymerase (2.5units), followed by ligation into the Smal site of pGEM4Z(Promega, Madison, WI). A similar PCR procedure was usedto clone mouse TGFa (322-556 bp), PR (1660-2774 bp),lactotransferrin (774-1 145 bp), IGF-1 (19-253 bp), andGAPDH (313-914 bp). Double-stranded 32P-labeled spe-cific probes for Northern hybridization were generated byPCR in the presence of 1 00 pCi of [32P]dCTP (AmershamCorp., Arlington Heights, IL) using primers (Sp6 and T7)which hybridize to sequences on either side of the cloningsite. Antisense and sense RNA probes for in situ hybridiza-tion were generated from linearized TGFI3 clones using[35S]CTP (500 pCi/reaction) in a standard RNA transcription

reaction containing either SP6 or T7 polymerase (Promega).The 32P- and 35S-labeled probes were purified from unin-

corporated nucleotides using Nuc-Trap columns (Strat-agene Cloning Systems, Inc., La Jolla, CA). Total RNA wasisolated using the standard cesium chloride centrifugationtechnique by homogenization of frozen tissues in a 4 M

guanidmnium thiocyanate solution, followed by oligo(dT)-

cellulose chromatography (Collaborative Biomedical Prod-ucts, Bedford, MA), and was used for the selection ofpoly(A)1 RNA as described previously (82). The p��y(��FRNA was separated by electrophoresis on a 1 .5% formal-dehyde gel and transferred to a nylon membrane (Biotrans;ICN Biochemicals, Costa Mesa, CA). After UV cross-linking,the membranes were prehybridized for 2-4 h at 42#{176}Cin50% formamide, 5X SSC (20X SSC = 3 M sodium chloride-0.3 M sodium citrate, pH 7.0), 5X Denhardt’s solution (1 OOXDenhardt’s = 2% w/v Ficoll, 2% w/v polyvinylpyrrolidone,2% w/v acetylated BSA), 0.05 M sodium phosphate (pH6.8), 0.1 % SDS, 5 mM EDTA, and 50 �jg/ml of both shearedsalmon sperm DNA and yeast RNA. Hybridization wascarried out at 42#{176}Cfor 16-1 8 h in a similar solution with thefollowing differences: 1 X Denhardt’s, 20 mivi sodium phos-phate, 0.2% SDS, and 1 x 1 06 cpm/ml of 32P-labeled DNAprobe. After hybridization, the membranes were washedtwice at room temperature in 2X SSC, 0.1% SDS, followed

by two washes at 55#{176}Cin 0.1 X SSC-0.1 % SDS. The mem-branes were exposed to X-ray film and developed; theintensity of the hybridization signals was quantitated usingan LKB densitometer.

In Situ Hybridization. Animals were perfused withparaformaldehyde to preserve the integrity of the mRNA.Heparin (1000 units/mI) was injected s.c. at 30 mm andProcaine at (10% solution) 5 mm before perfusion (0.1

mI/bOg body weight). Animals were anesthetized with i.p.

50 mg/mI Nembutal (0.1 mI/bOg body weight) and per-fused first with 1% paraformaldehyde and then 4%paraformaldehyde solutions dissolved in phosphate-buff-ered saline. The tissues were excised, fixed for an additional4 h at 4#{176}Cin 4% paraformaldehyde, washed in severalchanges of cold phosphate-buffered saline, and stored in70% ethanol until paraffin embedding. In order to reduceRNase contamination, all solutions were prepared in dieth-ylpyrocarbonate-treated autoclaved water. Tissue sections(7 pm) were placed onto silanated slides (Digene Diagnos-tics, Inc., Beltsville, MD), deparaffinized in xylene andgraded ethanol solutions, and then sequentially pretreatedwith 0.2 N HCI (10 mm, 24#{176}C),1 pg/mI Proteinase K (10mm, 24#{176}C),and 0.1 M triethanolamine with 0.25% acetic

anhydride (5 mm with stirring at 24#{176}C).Sections wererinsed between each step in diethylpyrocarbonate-treatedautoclaved water. Sections were dehydrated through anethanol series, and the prehybridization mixture was pi-peted on the tissue samples [0.3 M NaCI, 20 m� Tris (pH8.0), 1 mM EDTA, 1 X Denhardt’s solution, 1 0% dextransulfate, and 50% formamide), followed by incubation for 2h at 50#{176}C.The sections were then hybridized with 50-i 00p1 of the same solution as above but containing 1 O�-i 0�cpm/pI of the appropriate antisense or sense TGF�3 ribo-probe. 35S-labeled RNA probes were generated from ap-propriately linearized cloned plasmids using the PromegaRNA transcription kit in the presence of at least 500 1iCi of[35S]CTP per reaction (Amersham). The sections were in-cubated overnight at 50#{176}Cin a humid environment. Fol-lowing hybridization, sections were washed as follows: (a)2X SSC and 1 0 mM DYE at 50#{176}Cfor 1 5 mm; (b) 20 pg/mI


RNase A, 10 units/mI RNase T1, 0.5 M NaCI, and 10 mr�iDli at 37#{176}Cfor 15 mm; (c) 2X SSC and 10 mt�i DTT at roomtemperature for 1 5 mm; (d) 2X SSC, 1 0 m&i DIT, and 50%formamide at 50#{176}Cfor 1 5 mm; and (e) twice in 0.1 X SSCand 1 0 mM DII at 65#{176}Cfor 15 mm. Sections were dehy-drated in an ethanol series containing 300 mt’�i ammoniumacetate and dried thoroughly. After dipping in Kodak NTB-2emulsion (diluted 1 :1 with 600 mtvi ammonium acetate),slides were kept in a desiccated environment for 3-6 weeksand developed for 5 mm in Dl 9 developer (1 :1), rinsed 30sec in water, fixed 5 mm in Kodak fixer, and rinsed in water.Sections were stained with hematoxylmn, and coverslipswere applied using an aqueous medium. If not specified,the reagents were obtained from either Sigma, Life Tech-nologies, Inc. (Gaithersburg, MD), or US Biochemical Corp.(Cleveland, OH).

Immunohistochemistry. Tissues designated for TGFI3immunohistochemistry were fixed for 24 h in 1 0% neutralbuffered formalin, transferred to Bouin’s fixative for 4-6 h,rinsed twice in 70% ethanol, and paraffin embedded; 5-pmtissue sections were placed onto gelatin-coated slides. Un-less specified, all incubation were performed at room tem-perature. Sections were deparaffinized, and endogenousperoxidase was blocked by incubating in 0.6% hydrogenperoxide in methanol for 30 mm. Next, the slides were

equilibrated using three 3-mm washes in TBS (0.01 M Tris,pH 7.4-0.85% NaCI) containing 0.1% BSA. Slides werethen incubated in a humidified chamber with the followingseries of solutions: (a) the sections were permeabilized bydigestion with 1 mg/mI bovine testicular hyaluronidase in0.1 M sodium acetate (pH 5.5) and 0.85% NaCI at 37#{176}Cfor30 mm; (b) followed by rinsing as described above; (c) thenonspecific sites were blocked by incubating with TBScontaining 1 % BSA and 5% normal goat serum for 30 mm;

(d) the sections were incubated overnight with primaryantibodies specific for each ofthe TGFf3 isoforms at 4#{176}C(theanti-LC TGF�1 antibody that recognizes the intracellularform was used in this study); (e) sections were washed fourtimes in TBS-0.i % BSA for 3 mm each; ( t) the biotinylatedsecondary antibody (75 p1 normal goat serum and 25 p1biotinylated antibody; Vectastain Elite kit, Vector Laborato-ries, Inc., Burlingame, CA) diluted into 5 ml TBS-i % BSAwas added for 1 h; (g) sections were washed three times for3 mm each in TBS without BSA; (h) the Vectastain ABCreagent was applied for 1 h; (i) the sections were washedthree times for 3 mm each in TBS minus BSA; (j) theperoxidase substrate 0.05% 3,3 ‘-diaminobenzidine dis-solved in 0.05 M Tris (pH 7.4), 0.01 M imidazole, and 0.1 %hydrogen peroxide was added for 1 mm (because of theabundant amount of TGF� protein found in the reproduc-tive tract, we had to use a very short incubation time inorder to distinguish the induction ofthe TGF�3s by estrogen);and (k) following incubation with the substrate was re-moved by rinsing with water, and the sections were stainedwith Mayer’s hematoxylin for 1-2 mm. In addition, to en-sure the specificity of the TGF�3 antibodies, we performedthe appropriate immunohistochemical control experimentsusing normal rabbit lgG (similar concentrations as used forthe primary antibodies) in place of the primary antibodiesand found that these controls did not result in immunos-taming of the uterine and vaginal epithelial cells (data notshown). Documentation of the specificity of the TGFf3 an-tibodies has been adequately shown in other studies (44,45, 50, 83, 84). The immunohistochemistry method used todetect TGFa protein expression was the protocol recom-

mended by the Vectastain Elite antibody detection kit. Thetissues were fixed as described for the in situ hybridizationprocedure, and the specimens were deparaffinized in xy-lene and graded alcohols. The only modification in the Eliteantibody procedure was that the endogenous peroxidasewas blocked in the sections as described above; the incu-bation with the rabbit anti-TGFa antibody (1 :250 dilution;Peninsula Laboratories, Inc., Belmont, CA) was for 2 h; andthe peroxidase substrate concentration and buffer are thesame as described above for the TGFI3 immunodetection. Inaddition, to ensure the specificity of the TGFa antibody, weperformed the appropriate immunohistochemical controlexperiments using normal rabbit serum in place of theprimary antibody or preabsorption of the TGFa antibodywith purified rat TGFa (data not shown).

Analysis of DNA Synthesis by Immunohistochemical Lo-calization of BrdUrd Incorporation. To measure DNA syn-thesis in vivo at various times following treatment with DESor the vehicle alone, the animals received i.p. injections(0.1 ml) 1-2 h prior to necropsy with BrdUrd (Sigma) at aconcentration of 50 mg/kg in phosphate-buffered saline.The reproductive tracts were removed, fixed overnight at4#{176}Cin 4% paraformaldehyde dissolved in phosphate-buff-ered saline, paraffin embedded, and cut into sections of 5-7pm that were placed on silanated slides. A modification ofthe BrdUrd immunolocalization technique was used to de-tect cells in S phase (85). In brief, the sections were depar-affinized, pretreated with 2 N HCI at 37#{176}Cfor 20 mm, rinsedin boric acid-borate buffer (pH 7.6) for 1 mm, digested withtrypsin (0.005 mg/mI 0.05 M Tris-0.1 % CaCI2) at 37#{176}Cfor 3mm, blocked in TBS-1 % BSA at 24#{176}Cfor 1 5 mm, incubatedwith a monoclonal rat anti-BrdUrd antibody (1 :400 dilu-tion, clone BU1/75; Sera-Labs/Accurate Labs, Westbury,NY) for 30 mm at 24#{176}C,washed three times for 1 0 mm eachin TBS-0.i% BSA, incubated for 30 mm at 24#{176}Cwith abiotinylated anti-rat antibody (1 :100 dilution, Sigma),washed three times in TBS-0.i % BSA, and detected by theavidin biotin peroxidase complex method using a Vec-tastain Elite peroxidase kit (Vector Laboratories). Positivenuclei were visualized using the substrate diaminobenzi-dine as described above for the TGFI3 immunodetection,followed by counterstaining with hematoxylmn and mount-ing with Aqua-Mount (Lerner Laboratories, Pittsburgh, PA).

Detedion of TGF� Binding Sites by Affinity Labeling.Affinity labeling was performed using a modification ofpublished procedures (57, 61 , 66, 86). The uteri of mice 16h after treatment with DES (20 pg/kg) or with the vehiclecorn oil were removed and cut cross-wise into 2-mm sec-tions. The tissue from each group was divided into sepa-rated Petri dishes, and then one dish was incubated for 3 hat 4#{176}Cin Hank’s balanced salt solution-i % BSA containing125I-TGFf31 (100 pCi/mI; R&D Systems, Inc., Minneapolis,MN) alone, and the other dish was incubated with the sameconcentration of 125I-TGFf31 in addition to a 100-fold ex-cess of unlabeled TGF31 as a competitor to demonstratespecificity of binding. After incubation, the tissues werewashed two times in binding medium for 5 mm each andthen one time for 5 mm in Hanks’ balanced salt solutionwithout BSA at 4#{176}C.The receptor bound ligand was cross-linked with bis-(sulfosuccinimidyl)-suberate (Pierce Chem-cal Co., Rockford, IL) in 400 p1 of 20 mt�i 2-(4-hydroxym-

ethyl)propanesulfonic acid buffer for 20 mm at 22#{176}Candfollowed with termination by the addition of excess glycmne(28 mM) for 5 mm. The receptors were solubilized by ho-mogenization (Brinkmann polytron, setting 5, 30 s) in 10


mM 2-(4-hydroxymethyl)propanesulfonic acid (pH 7.4), 1%Triton X-1 00, 1 mM EDTA, 1 0 pg/mI leupeptmn, and 1 00 KUaprotinin and centrifugation at 40,000 rpm at 4#{176}Cto re-move insoluble tissue debris. The supernatants were col-lected, and the protein content was assayed using the BCAprotein assay kit (Pierce). 125I-TGFf3-labeled uterine pro-teins (200 pg/sample) and molecular weight protein mark-ers (Sigma) were separated by 6% SDS-polyacrylamide gelelectrophoresis, stained with Coomassie Brilliant BlueR-250 to detect proteins, dried, and exposed to Kodak XARfilm with enhancing screens at -70#{176}C.

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