steroidal regulation of connective tissue growth factor ...ccn2 (ctgf) in the uterus or the...

Steroidal regulation of connective tissue growthfactor (CCN2; CTGF) synthesis in the mouseuterus

M A E Rageh, E E-D A Moussad, A K Wilson, D R Brigstock

AbstractAims—To determine mechanisms regu-lating the production of connective tissuegrowth factor (CCN2; CTGF) and trans-forming growth factor â1 (TGF-â1) in themouse uterus.Methods—In situ hybridisation and im-munohistochemistry were used to localiseCCN2 (CTGF) and TGF-â1 in uteri fromsexually mature female mice that hadeither been (1) mated with sterile males toinduce pseudopregnancy or (2) ovariect-omised (OVX) and administeredestradiol-17â (E2) or progesterone (P4),either alone or in combination. Uteri col-lected on days 0.5, 1.5, 2.5, 3.5, 4.5, or 5.5of pseudopregnancy or at one, three, six,12, or 24 hours after steroid administra-tion were fixed, sectioned, and incubatedwith specific riboprobes or antibodies topermit detection and localisation ofmRNA or protein for CTGF and TGF-â1.Results—On days 0.5–2.5 of pseudopreg-nancy, CCN2 (CTGF) and TGF-â1 wereprincipally colocalised to uterine epithe-lial cells, with much smaller amounts inthe stroma. On days 3.5–4.5, there was areduction of CCN2 (CTGF) and TGF-â1in the epithelium but an increase instromal and endothelial cells, correspond-ing to a period of extracellular matrixremodelling and neovascularisationwithin the endometrium. In OVX mice,epithelial cells were weakly positive forboth CCN2 (CTGF) and TGF-â1 in theabsence of steroid hormones. EpithelialCTGF mRNA production were stronglybut transiently stimulated in OVX micecells by E2. These eVects were antagonisedby P4, which itself transiently stimulatedepithelial CCN2 (CTGF) production, al-though less robustly than E2. CTGF andTGF-â1 protein amounts were high inepithelial cells throughout steroid treat-ment and were increased in the stroma,where they were relatively long lived.Stromal CCN2 (CTGF) and TGF-â1 werelower after co-administration of E2 and P4

than in response to each hormone indi-vidually. Although ccn2 (ctgf) is a TGF-â1inducible gene in other systems, and bothgrowth factors were often co-localised inuterine tissues in these studies, severaltreatment regimens resulted in highamounts of TGF-â1 protein in stromalcells without the concomitant productionof ccn2 (ctgf) mRNA.

Conclusions—Maternal factors are prin-cipal cues for CCN2 (CTGF) and TGF-â1production in the uterus because (1) theirexpression during pseudopregnancy iscomparable to that seen in pregnancy and(2) they are regulated by ovarian steroids.TGF-â dependent and independentmechanisms of ccn2 (ctgf) gene transcrip-tion exist in the uterus that are variablyregulated by steroid hormones. Collec-tively, the data support a role for CCN2(CTGF) in mediating the eVects of steroidhormones and TGF-â on endometrialfunction.(J Clin Pathol: Mol Pathol 2001;54:338–346)

Keywords: CCN; reproductive tract; oestrogen; proges-terone; connective tissue growth factor; uterus

Connective tissue growth factor (CCN2;CTGF) is a multifunctional mosaic proteinthat is a member of the CCN2 (CTGF)/CCN1(CYR61)/CCN3 (NOV) family.1 CCN2(CTGF) appears to be involved in diverse cel-lular functions such as proliferation, adhesion,migration, apoptosis, and extracellular matrix(ECM) production.1–3 These wide ranging bio-logical activities, together with mRNA andprotein expression studies, have resulted inproposed roles for CCN2 (CTGF) in proc-esses such as embryonic development, tissuediVerentiation, angiogenesis, and wound heal-ing, in addition to various pathologies such asfibrosis, inflammation, and tumour growth.1

CCN2 (CTGF) is produced by and acts on awide variety of cell types including fibroblasts,vascular smooth muscle cells, endothelial cells,epithelial cells, and cells of the nervous system.4

In certain cell types, ccn2 (ctgf) is transcrip-tionally activated by transforming growthfactor â (TGF-â).2 4–11 This mechanism ofinduction has become a central theme inaccounting for certain biological actions thatare shared both by CCN2 (CTGF) andTGF-â, most notably the stimulation of ECMproduction.2 12 Accordingly, CCN2 (CTGF)and TGF-â are frequently coexpressed intissues that are undergoing ECM remodellingor synthesis, such as those that diVerentiateduring development or exhibit pathologicalfibrotic transformation. Although most atten-tion has been focused on the possible role ofCCN2 (CTGF) as a downstream mediator ofthe activity of TGF-â in fibrotic disorders andwound repair, it is nonetheless clear thatCCN2 (CTGF) also has important roles inregulating cellular homeostasis under normalcircumstances. For example, it is present in

J Clin Pathol: Mol Pathol 2001;54:338–346338

Department ofSurgery, The OhioState University, andChildren’s ResearchInstitute, ColumbusOhio 43205, USAM A E Rageh,E E-D A MoussadA K WilsonD R Brigstock

Correspondence to:Dr Brigstock, Children’sResearch Institute, RoomW309, 700 Children’s Drive,Columbus OH 43205, [email protected]

Accepted for publication1 May 2001

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diverse organ systems and tissues duringembryogenesis,13–15 is involved in chrondrogen-esis and endochondral ossification,5 16–20 andoccurs in the uterus and ovary of cycling andpregnant mammals.14 15 21–26

Relatively little is known about the role ofCCN2 (CTGF) in the uterus or the mecha-nisms by which uterine ccn2 (ctgf) genetranscription is regulated. Studies in the pighave revealed that a variety of CCN2 (CTGF)isoforms, ranging from 10 kDa to 38 kDa, arepresent in uterine fluids that exhibit cyclic andpregnancy associated changes in theirconcentrations.24–26 Low mass CCN2 (CTGF)molecules correspond principally to one or twostructural modules located at the C-terminusof the primary translational product and arisethrough limited proteolysis of full length38 kDa CCN2 (CTGF).24 26 A variety ofCCN2 (CTGF) isoforms have also been iden-tified in mouse uterine fluids.14 In the cyclingmouse, CCN2 (CTGF) was localised mainlyin luminal and glandular epithelial cells inwhich the signal is somewhat attenuated at thetime of oestrus.14 CCN2 (CTGF) localisationin pregnant animals mimicked that in cyclinganimals until day 4.5, when the blastocyst wasfirst detected in the uterine lumen. At this time,epithelial expression of CCN2 (CTGF) wassubstantially reduced and followed by highexpression in decidual cells after invasion andimplantation by the embryo.14 Collectively,these data suggest that in the mouse uterus,CCN2 (CTGF) is physiologically important inepithelial cells during the oestrous cycle and indiVerentiating stromal cells during early preg-nancy. Similar observations have been made inwomen, where it was shown that epithelial cellsand decidua expressed relatively high amountsof CCN2 (CTGF) compared with other celltypes.21 Although the pig does not exhibit aclassic decidual cell reaction, epithelial cells area major site of CCN2 (CTGF) synthesis, buthave transiently reduced CTGF during theperi-attachment period, coincident with stro-mal ECM remodelling and neovascularisa-tion.4 In all three species (pig, mouse, human),CTGF expression has been documented inother cell types such as endothelial cells, vascu-lar smooth muscle cells and, to a much lesserextent, stromal cells.4 14 21 These observationssuggest that uterine CCN2 (CTGF) may con-trol a variety of diverse biological processes thatare delicately integrated in time and space bysteroid dependent and independent mecha-nisms. Thus, the purpose of these studies wasto determine whether the expression of CCN2(CTGF) is embryo or steroid dependent and todefine the extent to which CCN2 (CTGF)expression mimics that of TGF-â.

Materials and methodsMATERIALS

Histochoice was obtained from Amresco(Solon, Ohio, USA). Superfrost Plus slides,Crystalmount, and Permount were from FisherScientific (Pittsburgh, Pennsylvania, USA).Phosphate buVered saline (PBS) was obtainedfrom Gibco BRL (Gaithersburg, Maryland,USA). Plasmid pCRII was from Invitrogen

Corp (Carlsbad, California, USA). Normalrabbit IgG was from Vector Laboratories (Bur-lingame, California, USA). Power BlockTM andStrAviGen Multilink kit were purchased fromBioGenex (San Ramon, California, USA).Haematoxylin was obtained from NewcomerSupply (Middleton, Wisconsin, USA). Digoxy-genin RNA labelling kit and sheepantidigoxygenin–alkaline phosphatase werefrom Roche Molecular Diagnostics (Indiana-polis, Indiana, USA). NBT/BCIP chromogenicsubstrates were from Promega Corp (MadisonWisconsin, USA). Other chemicals were fromSigma Chemical Co (St Louis, Missouri, USA)

CCN2 (CTGF) antiserum raised in rabbitsagainst residues 80–93 of mouse CCN2(CTGF) (a peptide region that is not sharedwith other CCN proteins) was produced andaYnity purified as described previously.14 15 27

AYnity purified rabbit anti-TGF-â1 antiserumwas obtained from Santa Cruz Biotechnology(Santa Cruz, California, USA). A cDNAcorresponding to nucleotides −1 to 1046 of thepublished mouse ccn2 (fisp-12) sequence28 wasgenerated from mRNA from TGF-â1 stimu-lated mouse 3T3 cells via the reverse transcrip-tion polymerase chain reaction (RT-PCR)using the primers 5'-gcagggatccatgctcgcctccgtcgca-3' and 5'-ccagcggccgcgaattcttacgccatgtctcc-3'. The same RNA was also used toamplify a 460 bp murine TGF-â1 cDNA usingthe primers 5'-ccggctcctgtccaaactaaggctcgc-3'and 5'-cctctagaccagtgacgtcaaaagacagcc-3'. Theamplified products from each reaction werecloned into pCRII and the resulting pCRII/ccn2 (ctgf) or pCRII/TGF-â1 plasmids werecut with diagnostic restriction enzymes toverify insert directionality. Digoxygenin-UTPlabelled RNA sense and antisense probes weremade using a digoxygenin RNA labelling kit,according to the manufacturer’s instructions.pCRII/ccn2 (ctgf) and pCRII/TGF-â1 werelinearised with EcoRV for SP6 generation ofthe sense probe and with Kpn I for T7 genera-tion of the antisense probe.

ANIMALS

Swiss Webster mice (Harlan Sprague Dawley,Indianapolis, Indiana, USA) were used for allexperiments, which were approved by the insti-tutional animal use and care committee of theChildren’s Research Institute.

PseudopregnancyAdult male mice were vasectomised and keptfor 35 days to clear all viable sperm from thegenital tract. Mating was induced betweenthese sterile males and fertile female mice inthe evening and mating was verified at 8.00 amon the morning of the next day by the appear-ance of the vaginal plug. This time point wasdefined as day 0.5 of pseudopregnancy. Theuteri of pseudopregnant females were har-vested daily at 8.00 am between days 0.5 and5.5 (n = 3/time point), the last time point beingthe probable day of return of oestrus.

Steroid hormone treatmentAdult virgin female mice were ovariectomised(OVX) and kept for two weeks to deplete them

Steroid regulation of uterine CCN2 (CTGF) 339

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of circulating ovarian steroids. On day 15 pos-tovariectomy, animals were given a single sub-cutaneous injection of either estradiol-17â (E2)(250 ng), progesterone (P4) (2 mg), or acombination of both hormones in 200 µlsesame oil (n = 3/treatment group). OVXanimals that received 200 µl sesame oil onlywere used as negative controls. The uteri ofsteroid injected animals or oil injected controlswere collected one, three, six, 12, and 24 hoursafter injection.

TISSUE PROCESSING

Mice were humanely sacrificed at the designatedtimes and the uteri were removed by dissection.Uteri were fixed in Histochoice for 24 hours andkept in 70% ethanol at 4°C until use. ParaYnwax embedded sections (5 µm thick) were cutusing a microtome and placed on SuperfrostPlus slides, which were then heated at 60°C forone to two hours and dewaxed in xylene for fiveminutes. Sections were then hydrated through aseries of graded ethanol washes (95%, 90%,70%, and 50% ethanol) and then placed in dis-tilled water for a few minutes.

IMMUNOHISTOCHEMISTRY FOR CCN2 (CTGF) AND

TGF-â1

Endogenous peroxidase activity was blockedby placing slides in 3% H2O2 for 15 minutes,after which they were rinsed in distilled waterfollowed by PBS. Slides were then incubated inPower Block for up to 10 minutes to blocknon-specific binding sites, and then rinsed withPBS for 10 minutes. Details for CCN2(CTGF) immunohistochemistry were as de-scribed previously,14 15 with some modifica-tions. Sections were incubated for one hour atroom temperature with PBS/2% bovine serumalbumin (BSA) containing either 10 µg/mlanti-CTGF IgG, 2 µg/ml anti-TGF-â1 IgG, orequivalent concentrations of preimmune rabbitIgG (obtained before immunisation with theCCN2 (CTGF) (81–94) peptide) or non-immune rabbit IgG. Slides were rinsed in PBSfor 10 minutes, incubated with a 1/50 dilutionof Multilink biotinylated secondary antibody inPBS/2% BSA for 20 minutes, washed in PBSfor 10 minutes, and then incubated with a 1/50dilution of concentrated strepatividin conju-gated peroxidase for 20 minutes. Slides werewashed for 10 minutes and developed withdiaminobenzidine for three minutes, afterwhich the chromogenic colour reaction wasstopped using distilled water. Slides werecounterstained using haematoxylin, washed indistilled water, dehydrated, and mounted.

IN SITU HYBRIDISATION FOR CCN2 (CTGF) OR

TGF-â1

Prehybridisation was carried out by successiveincubations in water, PBS, PBS/100mM gly-cine, PBS/0.3% Triton X-100, PBS, andTris/EDTA (TE) buVer containing 20 µg/mlproteinase K. All solutions were treated withdiethlpyrocarbonate. Sections were postfixedin PBS/4% paraformaldehyde at 4°C, washedwith PBS, incubated with 0.1M trieth-anolamine buVer (pH 8.0) containing 0.25%

(vol/vol) acetic acid, followed by prehybridisa-tion buVer (4× saline sodium citrate (SSC)containing 50% vol/vol deionised formamide)at 37°C. Slides were then incubated with300 ng/ml digoxygenin labelled sense or anti-sense ccn2 (ctgf) or TGF-â1 RNA probes in300 µl hybridisation buVer (4× SSC containing10% dextran sulphate, 40% deionised forma-mide, 1× Denhardt’s, 10mM dithiothreitol(DTT), 1 mg/ml yeast tRNA, and 1 mg/mldenatured and sheared salmon sperm DNA).Sections were incubated at 42°C overnight in ahumid chamber. Posthybridisation steps were2× SSC for 10 minutes at room temperature,two 15 minute washes with 2× SSC at 37°C ,two 15 minute washes with 1× SSC at 37°C,two 30 minute washes with 0.1× SSC at 37°C,two 10 minute washes with buVer 1 (100mMTris HCl/150mM NaCl, pH 7.5), and a 30minute incubation with blocking solution(buVer 1 containing 0.1% Triton X-100/2%goat serum). Sections were then incubated fortwo hours with PBS/0.1% Triton X-100/1%goat serum containing a 1/750 dilution ofsheep antidigoxygenin–alkaline phosphatase.Sections were washed with buVer 1 and buVer2 (100mM Tris HCl/100mM NaCl/50mMMgCl2, pH 9.5) and developed with NBT/BCIP chromogenic substrate. The reaction wasstopped using 10mM Tris HCl (pH 8.1)containing 1mM EDTA. Slides were washed inwater, counterstained with nuclear fast red,and mounted with crystal mount.

ResultsIn all experiments, verification that theimmunohistochemical signal was specific foreach protein was achieved through the use ofnon-immune controls (data not shown). Simi-larly, sense mRNA in situ hybridisation per-formed on all control and treatment groupswas used to confirm the specificity of staining(data not shown).

PSEUDOPREGNANCY

Both ccn2 (ctgf) and TGF-â1 were transcribedand translated in the uteri of pseudopregnantmice (fig 1). On days 0.5–2.5 of pseudopreg-nancy, strong immunohistochemical staining forCCN2 (CTGF) and TGF-â1 was seen in thecytoplasm of the columnar luminal and glandu-lar cells, whereas moderate to weak staining waspresent in the condensed aggregates of the stro-mal fibroblasts (fig 1). In addition, the smoothmuscle fibres of the myometrium exhibitedmoderate CCN2 (CTGF) protein expression(data not shown). Over the same period, largeamounts of ccn2 (ctgf) and TGF-â1 mRNAwere also colocalised in luminal and glandularepithelial cells, and smaller amounts were foundin the underlying stroma. On days 3.5–4.5, ccn2(ctgf) and TGF-â1 mRNA and protein valueswere decreased in epithelial cells but increasedin stromal fibroblasts. This corresponded tostromal ECM remodelling and massive invasionof blood capillaries, which were also positive forccn2 (ctgf) expression. By the next day (day 5.5;corresponding to the probable return of oe-strus), both ccn2 (ctgf) and TGF-â1 protein andmRNA expression increased in the luminal and

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glandular epithelial cells and was apicallylocalised within the cells. Stromal staining forccn2 (ctgf) or TGF-â1 mRNA was quite low,whereas their respective protein values wererelatively high.

Based on our previously published data,14 thecellular distribution of ccn2 (ctgf) in uterinetissues of pseudopregnant mice was generallycomparable to that found in preimplantationpregnancy. These data suggest that during thepreimplantation period, ccn2 (ctgf) expressionis predominantly regulated by maternal factors.Although embryonic signals may influenceccn2 (ctgf) expression during the peri-implantation or postimplantation period, theseand previously published data suggested thatmaternal factors, such as steroid hormones,drive uterine ccn2 (ctgf) expression in cyclingor early pregnant mice. To investigate this ques-tion, we next evaluated ccn2 (ctgf) and TGF-â1expression in the uteri of OVX mice that hadbeen treated with acute administration of E2 orP4, either alone or in combination.

STEROID HORMONE EFFECTS

Because the steroidal regulation of mRNA andprotein for TGF-â1, TGF-â2, and TGF-â3 inthe mouse uterus has been well establishedfrom previous work,29 30 we focused our investi-gation on the eVects of E2 and P4 on theTGF-â1 protein and performed this in con-junction with a comprehensive analysis of ccn2(ctgf) mRNA and protein.

Epithelial cellsTwo weeks postovariectomy there was a massivereduction in the overall uterine size, a decreasein the height of the luminal and glandularepithelial cells, and invasion of blood capillariesperforating the stroma (fig 2A). This phenotypewas associated with greatly reduced expressionof ccn2 (ctgf) mRNA, especially in epithelialcells, which normally show a very strong ccn2(ctgf) mRNA signal in cycling animals (fig 1;and data not shown14). In control OVX animals,the low basal expression of ccn2 (ctgf) mRNAremained unaltered for up to 24 hours after oiladministration, and was confined to the apicalregion of the cells (fig 2A). However, despitetheir reduced ccn2 (ctgf) mRNA expression, theamount of CCN2 (CTGF) protein detectedimmunohistochemically in these cells was mod-erately high, as was TGF-â1. Both CCN2(CTGF) and TGF-â1 were distributedthroughout the cell and their values were notaltered in response to oil treatment at any of thetime points examined (fig 2A). Thus, whereassteroid deprivation for two weeks after OVXresulted in a significant reduction of ccn2 (ctgf)mRNA expression in epithelial cells, comparedwith cycling or pseudopregnant animals, bothCCN2 (CTGF) and TGF-â1 proteins werequite stable and readily detectable.

Treatment of OVX animals with E2 (fig 2B)resulted in a dramatic but transient increase inthe ccn2 (ctgf) mRNA and protein in uterineepithelial cells, which was detectable within one

Figure 1 Uterine ccn2 (ctgf) and transforming growth factor â (TGF-â) in pseudopregnant mice. Female Swiss Webster mice were mated withvasectomised males and uterine ccn2 (ctgf) and TGF-â mRNA and protein were assessed on the indicated days of pseudopregnancy by in situhybridisation or immmunohistochemistry, respectively.


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hour, had peaked by six hours, and had subsidedby 24 hours (fig 2B). During the inductionphase (one to six hours), ccn2 (ctgf) mRNA waslocalised throughout the epithelial cells, whereasduring the attenuation phase (24 hours), it wasconfined to the apical region of the cells andresembled the localisation seen in control uteri.Greatly increased epithelial CCN2 (CTGF) and

TGF-â1 protein expression was seen at one to24 hours after E2 treatment (fig 2B). P4

treatment of OVX animals resulted in stimula-tion of ccn2 (ctgf) mRNA within one hour inepithelial cells, which was distributed through-out the entire cytoplasm (fig 3A). However, thisinduction was less robust than that stimulatedby E2, and by six to 24 hours of P4 treatment,

Figure 2 Steroidal regulation of uterine ccn2 (ctgf) and transforming growth factor â . Ovariectomised mice were treated with (A) sesame oil control or(B) estradiol-17â for the indicated amounts of time (1-24 hours). The presence of uterine ccn2 (ctgf) and TGF-â was determined by the situ hybridisationor immunohistochemistry.


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ccn2 (ctgf) mRNA expression had declined andwas confined to the apical regions of the cells.However, these changes were not parallelled byccn2 (ctgf) or TGF-â1 proteins, which re-mained at a constant moderate value (compar-able to epithelial cells of control (oil treated)

OVX uteri) over the entire time course (fig 3A).The high amounts of ccn2 (ctgf) mRNAinduced by E2 alone at one to six hours or P4

alone at one hour were absent in animals receiv-ing both hormones at the same time (fig 3B).Nonetheless, at one to six hours, ccn2 (ctgf)

Figure 3 Steroidal regulation of uterine ccn2 (ctgf) and transforming growth factor â (TGF-â). Ovariectomised mice were treated with (A) progesteroneor (B) estradiol-17â and progesterone for the indicated amounts of time (1–24 hours). The presence of uterine ccn2 (ctgf) and TGF-â were determined byin situ hybridisation or immmunohistochemistry.


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mRNA was slightly higher in OVX animalsreceiving E2 and P4 than in OVX animals receiv-ing the oil control. This signal was mainly apicaland its intensity declined to that seen in the oiltreated controls by 24 hours.

STROMAL CELLS

ccn2 (ctgf) mRNA was not detectable in thestromal fibroblasts of OVX animals for one to24 hours after the administration of the oilcontrol. Accordingly, low amounts of CCN2(CTGF) and TGF-â1 proteins were found inthe stroma—lower than that seen in themyometrium (fig 2A). After E2 treatment, therewas a slight increase in ccn2 (ctgf) mRNA in afew scattered stromal cells by six hours aftertreatment, but this increase was far less thanthat seen in the epithelium. Nonetheless,stromal staining for CCN2 (CTGF) andTGF-â1 proteins was higher than in oil treatedcontrols, and appeared to increase modestlywith time. P4 did not appear to stimulate ccn2(ctgf) mRNA production in the stroma,although relatively high amounts of CCN2(CTGF) and TGF-â1 protein were present atall time points (fig 3A). No stromal staining forccn2 (ctgf) mRNA was present in animalsreceiving both E2 and P4, and the CCN2(CTGF) and TGF-â1 protein values weremuch reduced compared with the increasesseen in the stroma in response to each hormoneindividually (fig 3B).

DiscussionThe uterus is an intriguing and uniqueexample of an organ system in which tissueturnover is cyclical and regulated primarily bysteroid hormones. It is now evident that thecyclic changes in uterine cell organisation areattributable, at least in part, to the regulationby E2 and/or P4 of the synthesis of uterinepolypeptide growth factors and their signaltransducing receptors.31 Through the inte-grated action of numerous steroidally regulatedmolecules, including growth factors and recep-tors, endometrial cells undergo proliferation,diVerentiation, ECM remodelling, andregression.32 In addition, during pregnancy,endometrial stromal cells are transformed todecidual cells under the influence of E2 and P4,a process that also involves changes in growthfactor expression. Apart from decidualisation,uterine growth factors are also implicated inother processes during pregnancy, such asembryo–uterine signalling, placental develop-ment, and angiogenesis.32–34 Synchrony be-tween the hormonal status of the uterus and itsreceptiveness for an appropriately developedblastocyst is central to successful implantationin many mammalian species.35–38 This high-lights the pivotal role that steroidally regulatedgrowth factors are likely to play in the embryo–maternal interactions that enable successfulestablishment and maintenance ofpregnancy.32–34 39 Our data establish for the firsttime that maternal sex steroids, including E2

and P4, are among the maternal factors that canregulate the expression of ccn2 (ctgf) in the

uterus, suggesting that CCN2 (CTGF) prob-ably mediates some of the growth and diVeren-tiation processes in uterine tissues that aresteroid dependent. Although steroidal regula-tion of ccn2 (ctgf) has not been reported previ-ously, our data are consistent with an earliersubtractive cDNA library strategy, whichshowed that the CCN2 (CTGF) relatedmolecule CCN1 (CYR61) was induced in ova-riectomised rat uteri after four hours oftreatment with 17-á-ethynyl estradiol.40

Our experiments revealed several featuresregarding the regulation of ccn2 (ctgf) in themouse uterus. Interestingly, the only scenariosin which uterine stromal or connective tissuecells produce large amounts of CCN2 (CTGF)are during the P4 dominated decidualisationphase of pregnancy, after P4 administration, orduring periods of neovascularisation and ECMremodelling during pseudopregnancy.14 In con-trast, one of the most striking features of theseand previous14 data is that epithelial cells incycling, early pregnant, or pseudopregnantanimals are the principal site of uterine CCN2(CTGF) synthesis. These data suggest thatCCN2 (CTGF) may influence uterine func-tion through its action on epithelial cells,directly or by paracrine eVects on the under-lying stroma. CCN2 (CTGF) is produced by avariety of epithelial cell types and regulatesfunctional properties including adhesion,13 dif-ferentiation,41 42 and apoptosis.43 Although di-rect eVects of CCN2 (CTGF) on uterineepithelial cells have yet to be examined, ourresults also support the possibility that CCN2(CTGF) is translocated from the epithelium tothe stroma, as has been suggested previouslyfor TGF-â.44 Evidence for diVusion of CCN2(CTGF) from its site of synthesis is shown bythe presence of large amounts of CCN2(CTGF) protein in both epithelial and stromalcells under conditions in which only theepithelial cells contain appreciable amounts ofccn2 (ctgf) mRNA (for example, on days 0.5–2.5 of pseudopregnancy or in control orhormone treated OVX animals). Thus, CCN2(CTGF) may be one of the mediators ofepithelial–mesenchymal cell interactions thatdrive uterine diVerentiation and support theestablishment and maintenance of pregnancy.

The pseudopregnancy experiments allowedus to investigate which of the pregnancyassociated changes in CCN2 (CTGF) produc-tion are independent of the presence of an acti-vated blastocyst. The data clearly show thatmost of the preimplantation changes in CCN2(CTGF) production that we reported previ-ously14 are embryo independent and, thus,maternally regulated. Similarly, previous stud-ies have shown that the uterine expression ofTGF-â1, TGF-â2, and TGF-â3 appears to beindependent of blastocyst regulation.29 44

Moreover, the pattern of CCN2 (CTGF) pro-duction that we found was comparable to thatof TGF-â1 and was principally epithelial overthe first three days of pseudopregnancy. In viewof previous studies supporting TGF-â depend-ent ccn2 (ctgf) gene induction in various celltypes, including epithelial cells, a functionalrelation between TGF-â and CCN2 (CTGF)


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in the uterine epithelium is plausible. Inaddition, a striking decrease in epithelialCCN2 (CTGF) values was observed on day4.5 in both pregnant14 and pseudopregnant(our present data) mice. This time is thenormal day of implantation and is marked byextensive stromal remodelling and angiogen-esis. Suppression of epithelial CCN2 (CTGF)production may allow for a net degradation ofstromal ECM as an essential prerequisite toECM remodelling and neovascularisation. Inaddition, the presence of CCN2 (CTGF) inthe endothelial cells of the new vessels isconsistent with the proposed role for CCN2(CTGF) in regulating endothelial cell prolif-eration, adhesion, and survival.

The presence of ccn2 (ctgf) mRNA orprotein or TGF-â1 protein was not completelyabolished within the two week period afterovariectomy. Similarly, the mRNA encodingTGF-â1–3 was expressed at high basal valuesin the uterus of immature mice30 and TGF-â2or TGF-â3 mRNA persisted in the uteri ofadult ovariectomised mice.29 These data showthat transcriptional activation of the uterineccn2 (ctgf) and TGF-â genes are, at leastpartly, steroid independent. Nonetheless, ourdata show that both CCN2 (CTGF) andTGF-â1 are individually induced by E2 and P4,although the eVects of each hormone are quitediVerent. In response to a single dose of E2,ccn2 (ctgf) mRNA was induced in epithelialcells within one hour, peaked at six hours, anddeclined by 24 hours. Previous studies of themouse uterus showed that diethylstilbestroltransiently upregulated TGF-â1 and TGF-â2mRNA by three hours and TGF-â3 mRNAwithin 30 minutes, with subsequent decreasesto basal values by six hours.30 In another study,E2 caused a rapid (six hour) but transientinduction of TGF-â2 mRNA, whereas therewas no significant eVect of the hormone onTGF-â3 mRNA in mouse uteri.29 In situhybridisation localised the mRNAs for all threeTGF-â isoforms primarily to the uterineepithelium.29 30 Unlike the transient nature ofTGF-â mRNA induction elicited by E2, theTGF-â proteins persisted in the epithelium fora relatively long time.30 Following E2 stimula-tion, we found that CCN2 (CTGF) andTGF-â1 proteins were long lived in both theepithelium and stroma, and were thus morebroadly distributed than would be predictedfrom their mRNA, which was localised prima-rily to epithelial cells. These data suggest thatepithelial CCN2 (CTGF) or TGF-â act in aparacrine fashion to regulate stromal cell func-tion. Similar conclusions were drawn fromstudies of TGF-â protein localisation duringthe preimplantation period, which showed thatECM of the stroma actively accumulatedTGF-â1 that was synthesised in and secretedfrom the epithelium.44

P4 is an essential hormone in the establish-ment and maintenance of pregnancy, at leastpartly because it is required to sensitise stromalcells to diVerentiate in response to an embry-onic or artificial stimulus and to maintain theirdiVerentiated state. Nonetheless, direct induc-tion of gene expression by P4 is not widely

documented, although some growth factors(such as heparin binding epidermal growthfactor (HB-EGF), insulin-like growth factortype 1 (IGF-1), vascular endothelial growthfactor (VEGF), tumour necrosis factor á(TNF-á), and amphiregulin) and growthfactor receptors or binding proteins (such asthe EGF receptor, IGF binding protein 1, andthe TNF-á receptor type 1) appear to be P4

dependent.32 Although detailed studies of theeVects of P4 on TGF-â are lacking in the litera-ture, neither TGF-â2 nor TGF-â3 werereported to be significantly aVected by P4 treat-ment.29 However, in our studies, a single injec-tion of P4 was eVective in stimulating theexpression of ccn2 (ctgf) and TGF-â1 overcontrol mice, but this response was less robustthan that seen with E2. The P4 responsivenessof the ccn2 (ctgf) and TGF-â genes mayaccount, at least in part, for their highexpression in the P4 dominated decualisationphase following implantation.14 While epithe-lial rather than stromal ccn2 (ctgf) mRNA wasinduced by P4, a similar pattern of regulationhas been reported for other uterine genesincluding amphiregulin, a member of the EGFfamily, which is produced by epithelial cells inresponse to P4.

45 Although co-injection of P4

with E2 did not antagonise the transient E2

stimulated accumulation of TGF-â2 mRNA inan earlier study,29 P4 appeared to antagonise theE2 induced increase in TGF-â1 and ccn2 (ctgf)expression by epithelial cells in our studies.Similar opposing eVects between E2 and P4 ontheir eVector molecules are not uncommonand have been documented previously for sev-eral uterine growth factors including HB-EGF,46 amphiregulin,45 and basic fibroblastgrowth factor.32

The similarity between CCN2 (CTGF) andTGF-â in their patterns of expression andlocalisation are consistent with TGF-â de-pendent mechanisms of ccn2 (ctgf) gene tran-scription, which could be both steroid depend-ent and independent. However, in severalsituations we found high amounts of TGF-âprotein in stromal cells but no detectable ccn2(ctgf) mRNA. These findings may suggest that:(1) TGF-â was present in its inactive latentform, (2) stromal cells were TGF-â responsivebut the ccn2 (ctgf) gene was refractory to theeVect of TGF-â, and/or (3) functional TGF-âreceptors were not present on the stromal cells.However, this last possibility seems unlikelybecause of the previously reported presence ofthe type I and II TGF-â receptors in stromalcells on days 1–3 of pregnancy and of the typeI, II, and III TGF-â receptors in stromal cellson day 4 and the decidua thereafter.47

In conclusion, these and previous data dem-onstrate that maternal factors are principalcues for CCN2 (CTGF) production in theuterus because it is produced in cycling andpseudopregnant animals, its expression duringpseudopregnancy is comparable to that seen inthe preimplantation period, and its expressionis regulated by ovarian steroids. Our studiesfurther suggest that ccn2 (ctgf) gene transcrip-tion in the uterus is not only regulated byfemale sex steroids but also by TGF-â1


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dependent and independent mechanisms.Thus, CCN2 (CTGF) is likely to mediate atleast some of the eVects of steroid hormonesand TGF-â on endometrial function in cyclingand pregnant animals.

This work was supported by USDA grant 9803693.

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