[CANCER RESEARCH 59, 5737–5744, November 15, 1999]

Human Uterine Leiomyomata Express Higher Levels of Peroxisome Proliferator-activated Receptorg, Retinoid X Receptor a, and all-trans Retinoic AcidThan Myometrium

John C. M. Tsibris,1 Kathy B. Porter,2 Allahyar Jazayeri,2 Georg Tzimas,3 Heinz Nau,4 Hong Huang,5

Khatuna Kuparadze,6 Gregory W. Porter,2 William F. O’Brien, and William N. SpellacyDepartments of Obstetrics and Gynecology [J. C. M. T., K. B. P., A. J., H. H., K. K., G. W. P., W. F. O., W. N. S.] and Biochemistry and Molecular Biology [J. C. M. T.],University of South Florida, Tampa, Florida 33606, and Institute for Clinical Pharmacology and Toxicology, Free University of Berlin, D-14195 Berlin, Germany [G. T., H. N.]

ABSTRACT

Uterine leiomyomata are the main indication for a hysterectomy inthe United States and occur in 25% of women>35 years. Becauseuterine leiomyomata can form when ovariectomized guinea pigs areexposed to estradiol and retinoic acids, we tested whether humanleiomyomata had high levels of retinoic acids and related nuclearreceptors. Compared with normal human myometrium, leiomyomatahad 3- to 5-fold higher levels of peroxisome proliferator-activatedreceptor g (PPARg), retinoid X receptor a proteins, and all-transretinoic acid, but only during the follicular phase of the menstrualcycle. 9-cis Retinoic acid was undetectable in either leiomyomata ormyometrium. PPARg mRNA levels were lower in leiomyomata thanmyometrium, but only during the luteal phase of the cycle. A PPARgagonist, troglitazone, was given to guinea pigs along with estradiol andall-trans retinoic acid and produced the largest leiomyomata seen todate in this model. By contrast, no tumors formed when troglitazonewas given alone or with estradiol or when troglitazone was given withestradiol and 9-cis retinoic acid. New therapies for human leiomyomatamay emerge by combining antagonists for PPARg and retinoid Xreceptor a with selective estrogen receptor modulators.

INTRODUCTION

Uterine leiomyomata, benign tumors of smooth muscle cells, arethe most common tumors in the human female pelvis and the leadingindication for pelvic surgery (1, 2). Leiomyomata can cause anemia,pain, discomfort, menstrual disturbances, and reproductive failure.Therapy with gonadotropin-releasing hormone agonists induces atemporary hypoestrogenic state and reduces leiomyoma size by 50%.This is implemented as a prelude to either myomectomy or hysterec-tomy (3). However, the hypoestrogenism causes significant noncom-pliance because of climacteric-like symptoms, such as hot flashes,vaginal dryness, and decreased libido. Additionally, this therapy hasthe potential for bone loss and adverse cardiovascular changes whenit exceeds 6 months duration (3).

The cause of leiomyomata is unknown, but estrogens produced byleiomyoma aromatase are suspected to promote their growth (4).Rodent models for uterine leiomyomata include the Eker rat (5),carrying a mutation in the tuberoussclerosis-2gene, and two trans-

genic mice (6, 7). In our guinea pig model, exposure solely to E27

silastic implants was associated with leiomyomata forming mainly onthe abdominal wall, whereas exposure to E2 and retinoic acid implants“switched” leiomyoma formation to the uterus (8). This observationprompted us to test human leiomyomata for the expression of RARsand RXRs and for other members of the ligand-activated nuclearreceptor superfamily (9). Encouraging immunoblot data focused ourattention on PPARg and RXRa and their potential relevance to theregulation of leiomyoma growth.

PPARg, one of three mammalian PPAR isoforms (a, b/d, andg),is found in adipose tissues, where it mediates adipocyte differentiation(10), and other insulin-responsive tissues such as skeletal muscle andliver (11–15). RXR is regarded as the master nuclear receptor becauseit forms heterodimers with RAR, PPAR, vitamin D3, or thyroidhormone receptors (9). In the presence of a PPARg ligand, PPARg-RXR heterodimers are formed and bind to peroxisome proliferation-responsive elements; the heterodimers are considered the functionallyactive receptor formsin vivo.

Retinoic acids are the biologically active form of vitamin A; atRAand 9cRA can be isomerized to each other bothin vivo and in vitro.RARs (RARa, RARb, and RARg) bind atRA and 9cRA. The RXRa,RXRb, and RXRg bind 9cRA. RAR and RXR are encoded bydifferent genes, and each subtype (a, b, andg) differs mainly in theirNH2 terminus because of alternate mRNA splicing and use of differ-ent promoters.

Naturally occurring ligands for PPARg are unsaturated fatty acidsand prostaglandins (12, 14, 16, 17). PPARg is found in the smallintestines and colon, and as the receptor for fatty acids, PPARg mayhave a primary role in colon cancer (18). Natural and syntheticPPARg ligands, such as Tro, a thiazolidinedione, are sufficient tostimulate adipocyte differentiation in fibroblast-like preadipocytes(11, 14). The ability of Tro to bind and activate PPARg correlateswith its ability to improve insulin sensitivity in type II diabeticpatients and in animal models of diabetes and obesity (11–14).

Our in vivo guinea pig experiment with Tro (19) demonstrated thatsustained exposure to E2, Tro, and atRA were optimal for uterineleiomyoma growth in this model. On the basis of comparable findingsin human leiomyomata, we hypothesize that new therapeutic modal-ities for human leiomyomata could be based on antagonists forPPARg and RXRa, given singly or in combination with SERMs.

MATERIALS AND METHODS

Human and Animal Tissue Extraction. Tissues were obtained accordingto protocols approved by the Institutional Review Boards for human andanimal research. Uterine samples were obtained within 30 min after excisionin a hysterectomy or myomectomy and were stored at275°C. The humanleiomyomata sampled were.2–3 cm and distant to necrotic, calcified, or

Received 5/21/99; accepted 9/22/99.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Obstetrics andGynecology, University of South Florida, 4 Columbia Drive, Room 524, Tampa, FL33606. Phone: (813) 254-7774; Fax: (813) 254-0940; E-mail: jtsibris@com1.med.usf.edu.

2 Present address: Department of Obstetrics and Gynecology, Texas Tech University,3601 4th Street, Lubbock, TX 79430.

3 Present address: Tzimas-Dimolios, Inc., Edessis 3, Thessaloniki, GR-54625, Greece.4 Present address: Department of Food Toxicology, School of Veterinary Medicine

Hannover, D-30173 Hannover, Germany.5 Present address: Department of Radiology, University of Minnesota Medical School,

Box 292, 420 Delaware Street, S.E., Minneapolis, MN 55455.6 Present address: Zhordania Institute of Human Reproduction, 380009 Tbilisi,

Georgia.

7 The abbreviations used are: E2, 17b-estradiol; PPARg, peroxisome proliferator-activated receptorg; RXR, retinoid X receptor; atRA, all-transretinoic acid; 9cRA, 9-cisRA; 13cRA, 13-cis RA; Tro, troglitazone; SERM, selective estrogen receptor modulator;HPLC, high-performance liquid chromatography; ER, E2 receptor.

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infarcted regions; myometrial samples were free of endometrium. The stage ofthe menstrual cycle was determined by endometrial dating and the patient’shistory.

Frozen human and animal tissues were finely cut and placed (at 150 mg wettissue/ml) in a Tris-HCl (pH 8) buffer containing aprotinin, NP40, NaCl, NaF,phenylmethylsulfonyl fluoride, orthovanadate, and leupeptin (20) and wereextracted overnight at 4°C with gentle tumbling. Recently, in a few homoge-nizations, we included 20mM of the cathepsin-L/calpain inhibitor Z-Leu-Leu-Tyr-fluoromethyl ketone from Enzyme Systems Products (Livermore, CA).After 15 s of Tekmar homogenization and 2 h of tumbling, the homogenateswere centrifuged at 14,0003 g for 30 min. In the supernatant, protein wasmeasured with the BCA kit from Pierce (Rockford, IL) using BSA as astandard.

Guinea Pig Studies.Dunkin-Hartley female guinea pigs (Cavia porcellus)were used in this study and received one or two silastic implants (21) of E2(each contained 50 mg of powder), an implant of atRA from Sigma ChemicalCo. (St. Louis, MO) or 9cRA (a gift from Hoffmann La-Roche, Nutley, NJ),each containing 40 mg of powder, and a small identification chip from AVID(Norco, CA) in the s.c. space of the intrascapular area after a bilateraloophorectomy via laparotomy (or sham operation) in the costovertebral angle.Anesthetics were administered as described before (22). Tro (Rezulin tablets)from Parke-Davis (Ann Arbor, MI) was suspended in an aqueous solution of1% carboxymethylcellulose, 0.81% NaCl, and 0.1% Tween 80 and given dailyp.o. at a dose of 10 mg/kg body weight in 1 ml, followed by 1 ml of fruitpunch. All animals were weighed weekly.

Western Blotting. NuPage Bis-Tris 4–12% gels from Novex (San Diego,CA) and recombinant S-tagged molecular weight markers from Novagen(Madison, WI) were used; 50–100mg protein were loaded per lane. Polyclonalantibodies to RARa-b2-g, RXRa (SC-552, epitope at NH2 terminal), RXRb-gfrom Santa Cruz Biotechnology (Santa Cruz, CA) and Affinity Bioreagents(Golden, CO), monoclonal RXRa antibody 4RX3A2 (a gift from Dr. P.Chambon, IGBMC, Illkirch, France), and an antibody (15) to recombinantPPARg2 (a gift from Dr. B. Spiegelman, Dana-Farber Cancer Institute, Bos-ton, MA) were used. Equal lane loading was confirmed either by ana-desminantibody from Sigma Chemical Co. (St. Louis, MO) or by protein staining ofthe nitrocellulose membrane with BLOT-FastStain from Geno Technology (St.Louis, MO), followed by destaining, blocking, and exposure to the primaryantibody. Secondary antibodies were from Amersham (Arlington Heights, IL)and Novagen, and chemiluminescence kits were from Amersham.

Northern Blots. Total RNA was isolated with the TRI reagent from MRC(Cincinnati, OH), and poly(A)1 RNA from total RNA using the Oligotex kitfrom Qiagen (Valencia, CA). Prehybridization and washings were carried outat room temperature with MRC solution WP-117, and hybridizations wereperformed at 65°C with MRC solution HS-114. cDNA probes (14) for PPARg,RXR (gifts from Dr. B. Spiegelman), anda-desmin (a gift from Dr. Y.Capetanaki, Baylor College of Medicine, Houston, TX) were labeled with therandom-primed DNA labeling kit from Boehringer-Mannheim (Indianapolis,IN) and [a-32P]dCTP (6000 mCi/mol) from NEN (Boston, MA) and werepurified with the DNA Clean-up kit from Promega (Madison, WI).

Analysis of Retinoids by HPLC. All procedures were performed underdim light to prevent photoisomerization of retinoids. Serum samples wereextracted with 2-propanol, as described previously (23). Uterine tissues (200–400 mg) were cut frozen in 2-mm pieces and extracted for 5 min with threevolumes of ice-cold 2-propanol, containing 13mg of butylated hydroxytolu-ene/ml as antioxidant, followed by 10-min sonication at 4°C and shaking for5 min at room temperature. Precipitated cell debris and proteins were removedat 65003g , and the supernatant solutions from either serum or tissues wereextracted on a C2 solid-phase cartridge to recover the retinoids and removeinterfering substances (24). The cartridges were loaded into the HPLC system,and separation of retinoids was achieved on a C18 column with gradient elutionwithin 28 min; this method can resolve 17 retinoids (24, 25). Criteria foridentification of HPLC peaks were the coincidence of retention time and ratioof peak areas at two wavelengths of detection with those of authentic retinoids.For identification of some retinoids, HPLC fractions were collected andrechromatographed, and UV spectra were recorded at 300–370 nm in the flowcell of the UV detector (26). An isocratic method (25, 27) was used forrechromatography of the putative atRA peaks from the uterine samples; thismethod can resolve 13cRA, 9,13-di-cRA, 9cRA, and atRA.

RESULTS AND DISCUSSION

Lessons from the New Guinea Pig Model.Unlike rodents, guineapigs form uterine leiomyomata spontaneously as they age (21). The1930s guinea pig model for leiomyomata was reexamined using E2silastic implants and modern methods (21); uninterrupted administra-tion of E2 caused large smooth muscle cell tumors (leiomyomata),primarily on the abdominal serosa and small leiomyomata on theuterine horns (Fig. 1,a andb). In women, however, leiomyomata arerestricted to the uterus and rarely form in the abdominal cavity(leiomyomatosis peritonealis disseminata), lungs, digestive tract, orarteries. To establish the mechanism of leiomyoma growth and ulti-mately reverse the disease, we exposed (8) ovariectomized guineapigs for 260–300 days to one or two E2 implants plus one atRA(n 5 12) or one implant each of E2 and 9cRA (n 5 5). The E2implants, with or without 9cRA or atRA implants, maintained serumE2 levels in ovariectomized animals at 30–90 pg/ml (gestationallevels in the guinea pig) for up to 400 days (21). Mean serum retinol(6SE, n 5 8) was 270.56 20.0 ng/ml, retinyl palmitate/oleate was22.7 6 4.2, and retinyl stearate was 35.26 6.1 at 260–300 days oftreatment; serum 13cRA was 4.76 0.6 (n5 6), but atRA and 9cRAwere detectable only in six animals at;0.6 ng/ml.

Surprisingly, intramural and serosal uterine, but not abdominal,tumors were formed in 11 of 12 animals on E2 and atRA and 5 of 5animals on E2 and 9cRA (Fig. 1,c andd). The tumors were leiomy-omata, as verified by electron microscopy and desmin immuno-staining (data not shown). In the absence of E2, atRA or 9cRAproduced no leiomyomata. Animals on E2 and either atRA or 9cRAsurvived beyond 400 days, whereas treatment solely with E2 (21) forlonger than 7 months produced extensive abdominal tumors, causingintestinal obstructions that forced the termination of the experiments.

Receptor Levels in Leiomyomata.Nuclear receptor RAR (a, b,and g), RXR (a, b, and g), and PPARg protein levels were deter-mined by immunoblotting of tissue extracts of human leiomyoma andmyometrium and guinea pig leiomyoma and uterine horn cross-sections. PPARg and RXRa expression were higher in human leiomy-omata than myometrium, but only in the follicular phase; higher levelswere noted in guinea pig leiomyomata relative to uterine horn cross-sections (Figs. 2 and 3).

In human tissues, no correlation was found between the leiomyoma:myometrium ratios for PPARg and RXRa levels or between thePPARg leiomyoma:myometrium ratios and body mass index. RARa,RARb, or RARg levels were not consistently different betweenleiomyomata and myometrium (data not shown). The RXRa immu-noblots with an antibody to a RXRa NH2-terminal segment (Fig. 3)suggested that the native RXRa form (Mr ;50,000) was partiallydegraded; the degradation was more pronounced in the human thanguinea pig tissue extracts. TheMr 50,000 andMr 35,000 bands wereincluded in RXRa quantitation (Fig. 3). Monoclonal antibody4RX3A2 to human RXRa gave nearly identical immunoblots as thosein Fig. 3 (data not shown), but it did not react with the guinea pigsamples. Recently, it was reported that cathepsin-L partially degradesRXRa (28, 29). We tested in eight samples whether 20mM Z-LLY-FMK, a new cathepsin-L/calpain inhibitor, added during human tissuehomogenization would prevent RXRa degradation. Indeed, the mainRXRa band was now atMr ;50,000 in both myometrial and leiomy-oma immunoblots (data not shown), suggesting that the partial pro-teolysis of RXRa seen in earlier blots (Fig. 3) occurred during tissuehomogenization by a protease(s) inhibited directly or indirectly byZ-LLY-FMK. Interestingly, Z-LLY-FMK reduced thea-desminbands to two, atMr ;50,000.

In women, the 3- to 5-fold higher expression of PPARg and RXRain leiomyomata depended on the phase of the menstrual cycle (Fig. 4).

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Perhaps, in the follicular phase E2, unopposed by progesterone, is theprimary stimulus for increased PPARg and RXRa expression, leadingto leiomyoma growth. It is possible that progesterone could decreasePPARg or RXRa expression directly, not through decreased E2receptor concentration.

To corroborate the Western blot data, Northern blots and reversephase-PCR of PPARg and RXRa were performed. Fig. 5 shows that

PPARg and RXRa mRNAs were equally expressed in human myo-metrium and leiomyomata throughout the menstrual cycle, except forPPARg mRNA levels that were lower in leiomyomata than myome-trium (P , 0.002) only in the luteal phase. Another Northern blot ofRXRa using total RNA samples from three uteri in the luteal phasealso showed equal mRNA levels between leiomyomata and myome-trium (data not shown). Reverse phase-PCR showed that mRNA for

Fig. 1. Leiomyoma formation in ovariectomizedguinea pigs. Exposure to E2 Silastic implants formedtumors (21) mainly on the abdominal wall (a and b),whereas treatment with E2 and either an atRA or 9cRAimplant formed tumors (8) in the uterine horns (c andd); in c, the scale is in cm.

Fig. 2. Immunoblots with an antibody (14) to recombinantPPARg (A) and a-desmin (B) of human leiomyomata (L),matched myometrium (M), guinea pig uterine horn (U) andleiomyomata in the uterine serosa (SL) or abdomen (AL); uteri1–5 were obtained at the follicular phase, and6–8 were ob-tained at the luteal phase of the menstrual cycle. Guinea pig 1(GP1) was treated with E2 and 9cRA, andGP2 andGP3 withE2 and atRA, respectively;L5 is the leiomyoma from patient 5.Arrows, molecular mass markers (kDa).Bar graph, densito-metric quantitation of immunoblots (means) from human L/Mpairs from the follicular phase (n 5 18;P , 0.001, pairedt test)and the luteal phase (n 5 6) of the cycle;bars,SE.

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total PPARg (segment 146–620; Ref. 30) and RXRa (segment 1318–1430; Ref. 31) were expressed in both myometrium and leiomyomathroughout the cycle (data not shown).

HPLC analysis of retinoids in human tissue extracts (Fig. 6) showed4.5-fold higher atRA levels in leiomyomata than myometrium in thefollicular phase only. By contrast, the presence of atRA in the lutealphase was not confirmed because of interferences that coeluted withatRA; 9cRA was not detectable. Retinol (vitamin A) levels were 25%lower in follicular phase leiomyomata than myometrium (Fig. 6) and;35% lower in luteal phase leiomyomata than myometrium (data notshown). Retinyl ester levels were undetectable in most of thesetissues, or when detected, did not exceed 15 ng/g, a much lowercontent than retinol. Lower retinyl ester levels than retinol are alsofound in guinea pig uterine tissues (data not shown) and in rodentembryos of 11–12 days gestational age (27).

In Vivo Experiment with Troglitazone. Because PPARg expres-sion was higher in human leiomyomata, we tested in guinea pigswhether a PPARg agonist, Tro, could cause leiomyomata. Tro wasgiven daily for 75 days p.o. (10 mg/kg) to seven ovariectomizedguinea pigs primed for 150 days with two E2 (n 5 2) and either oneatRA (n 5 2) or 9cRA (n 5 3) implant; two other animals receivedTro only for 75 days. Only combined exposure to E2, atRA, and Troproduced uterine leiomyomata, 4-fold larger than seen previously in

the guinea pig (Fig. 7e; 4 cm at largest dimension). Tro alone had noeffect on the atrophic uterus of the ovariectomized animals (Fig. 7b)and even prevented abdominal leiomyoma formation in animalstreated with E2 (Fig. 7c). The apparent increase of abdominal fatattributable to Tro alone (Fig. 7a) was not accompanied by weight

Fig. 3. Immunoblots with antibodies to RXRa (A) and a-desmin (B) of humanleiomyomata (L), matched myometrium (M), and guinea pig uterine horn (U), uterine(UL), and abdominal (AL) leiomyomas. Data from three uteri (different from those in Fig.2) at the follicular phase of the cycle are shown; uteri 1 and 3 had three and two separateleiomyomata, respectively.Arrows,molecular mass markers (kDa). Guinea pig 1 (GP1)was treated only with E2 (21), andGP2 was treated with E2 and atRA.Bar graph,densitometric quantitation of immunoblots (means) from human L/M pairs in the follic-ular phase (n5 23; P , 0.0001, pairedt test) and the luteal phase (n5 7) of the cycle.Bars,SE.

Fig. 4. Summary of immunoblot quantitation for PPARg and RXRa, expressed as themean ratio of leiomyoma to myometrium, relative to the phase of the menstrual cycle;Pswere obtained by the pairedt test of densitometry data (those shown in Figs. 2 and 3).Bars,SE.

Fig. 5. Representative Northern blots for PPARg (a, 1.4 kb),a-desmin (bandd, 2.3kb), and RXRa (c, 4.8 kb) in human leiomyomata (L) and matched myometrial (M)samples; 2–4mg of mRNA were loaded per lane. Uteri1 and2 were in the follicular phaseof the cycle, and uteri3 and4 were in the luteal phase of the cycle, respectively. Theseuteri are different from those in Figs. 2 and 3.Bar graph,ratios of L:M values (means)normalized to desmin;numbersinside thecolumnsindicate the total number of uteritested;p, P , 0.002 by the pairedt test, between L and M normalized levels.Bars,SE.

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gain during the 75-day treatment with Tro. Unexpectedly, the com-bination of 9cRA with E2 and Tro produced neither uterine or ab-dominal leiomyomata (Fig. 7f), although 9cRA and E2 produceduterine leiomyomata (Fig. 1,c andd).

Immunoblots for PPARg and RXRa showed (Fig. 8) that Tro given

either alone or with E2 induced measurable PPARg and RXRa levelsboth in the atrophic (Lanes 1aand1a9) and the E2-stimulated uterinehorn (Lanes 5aand5a9); the distribution of these receptors betweenmyometrium and endometrium has not yet been determined. It ap-pears that relatively high expression of PPARg and RXRa in the

Fig. 6. Representative HPLC chromatograms of retinoidsin human leiomyoma and myometrial tissues at the follicularphase of the cycle (a andb). UV absorbance was monitoredat 340 nm. Peaks X (at 10.95 min) and Y (at 13.20 min)coeluted with authentic atRA and retinol, respectively.Inseta, UV spectra of authentic atRA (Amax, 345 nm) and purifiedpeak X (Amax, 344 nm) from follicular phase uterine sam-ples. Inset b,UV spectra of authentic retinol and purifiedpeak Y from follicular phase leiomyoma samples (Amax at328 nm for both). Authentic atRA and purified peak X froma luteal phase leiomyoma displayed dissimilar UV spectra(c). Mean concentrations of atRA were 4.5-fold higher (4.5versus1.0 ng/g wet tissue;n 5 3), and those of retinol were1.3-fold higher (149versus111 ng/g wet tissue) in follicularphase leiomyomata than myometrium (d). Bars,SE.

Fig. 7. Photographs of representative uteri of ovariectomized guinea pigs treated solely with oral Tro (a andb), two E2 implants and Tro (c andd) in addition to one atRA (e) orone 9cRA implant (f). Large accumulation of abdominal fat (longer arrows) occurred in animala; shorter arrows,uterine horns.

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uterine horns (Lanes 1and5) does not support leiomyoma formation.Rather, a combination of additional stimuli, E2 and atRA, is neededfor tumor formation.

Pathways to Leiomyomata.Our hypothesis is that the rise ofPPARg and RXRa proteins in follicular phase leiomyomata reflectsan increase of the transcriptionally active PPARg:RXRa heterodimer,and that the latter is a key to leiomyoma growth. This heterodimer wassuggested as a single-function complex and a molecular target fortreatment of insulin resistance because of beneficial effects seen indiabetic patients treated with the PPARg agonist Tro and in animalmodels treated with RXR agonists and, surprisingly, antagonists ofRXR (32). In leiomyomata, we do not know the inducers of theputative PPARg:RXRa complex or whether estrogens alone stimulatePPARg and RXRa expression and higher atRA levels. Estrogensinduce the formation of a prostaglandin D2 metabolite that activatesPPARg in the duck uropygial gland (33). In the guinea pig model(Fig. 1), E2 stimulation is necessary but not sufficient for uterineleiomyoma growth. Estrogens are considered beneficial to insulinresistance, although they correlate with obesity in women. A modestincrease in relative risk (confidence interval, 1.03–1.59) for uterine

leiomyomata with elevated body mass index was reported recently ina large study of adult women (34).

Additional studies will determine whether “downstream” factors,such as nuclear receptor coactivators, corepressors, and histoneacetyltransferases (35) are differentially expressed and important forleiomyoma growth, in addition to “upstream” inducers of retinolactivation, 15-lipoxygenase and prostaglandin D and J biosynthesisthat may act in leiomyomata. Undoubtedly, vitamin D3 receptorsparticipate in the development of the leiomyoma phenotype, such ascalcification foci, common to human and guinea pig leiomyomata(Fig. 7e; data not shown).

Fig. 9 summarizes our results on the pathways to leiomyomaformation. Three “inducers” are proposed as prerequisites of uter-ine leiomyoma development and growth,i.e., E2 stimulation in theabsence of progesterone, higher atRA levels, and higher PPARg-RXRa levels. Unlike “hits” leading to malignant transformations(36), this mechanism implies that leiomyoma growth is potentiallyreversible by lowering “inducer” levels, as shown in women whobecome hypoestrogenic by gonadotropin-releasing hormone ago-nist therapy (3).

In the presence of Tro, there is an apparent specificity for atRA, interms of tumor growth promotion, because 9cRA seems to preventgrowth. This apparent specificity is reminiscent of the therapeuticefficiency of atRA in cancers involving the bone marrow (acutepromyelocytic leukemia). It is not known if the bone marrow is in thepathway of leiomyoma growth; Tro induces adipogenesis in culturesof bone marrow stromal cells (37).

A direct link at the molecular level between estrogens and theretinoid pathway (RAR, RXR, PPARg) has not been established inleiomyomata. Two reports (38, 39), awaiting confirmation by otherlaboratories, suggested thatin vitro heterodimers form between eitherRARa or RXRa and the ERa andb; no evidence was offered aboutPPARg-ER dimers. One explanation why guinea pigs treated withE2–9cRA-Tro (Fig. 7f) did not develop leiomyomata could be thatincreased 9cRA favors RXRa homodimerization, in effect, removingRXRa from transcriptionally active RXRa-PPARg or other het-erodimers. Perhaps 9cRA alone would be beneficial to patients withleiomyomata. In the model, the ability of 9cRA and E2 to produce

Fig. 8. Immunoblots for PPARg (a, antibody ABR-821 from Affinity Bioreagents) andRXRa (b, antibody SC-553 from Santa Cruz Biotechnology) of representative extracts ofleiomyomata (Leio) and cross-sections of uterine horn (Horn; cut as outlined by the boxin Fig. 7d) from guinea pigs treated solely with Tro (Lane 1), with a combination of Tro,E2, and atRA (Lanes 2and4), a combination of Tro, E2, and 9cRA (Lane 3), or with Troand E2 (Lane 5), as described in the text.a9 andb9, band intensities of PPARg and RXRa,respectively, normalized to equal protein (50mg)/lane.

Fig. 9. Proposed pathways for the development and growth of uterine leiomyomata.a,pathways of leiomyoma induction in ovariectomized guinea pigs.b, proposed inducers ofspontaneous human uterine leiomyomata that are expressed at significantly higher levelsin leiomyoma than myometrium only during the estrogen-dominated follicular phase ofthe menstrual cycle. In guinea pigs (a), E2, atRA, and 9cRA were given via s.c. Silasticimplants, and Tro was given p.o.

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small uterine leiomyomata in the absence of Tro (Figs. 1 and 9) pointsto a pathway that does not require further induction of PPARg. Thefact that Tro in the presence of E2 prevented even abdominal leiomy-oma formation (Figs. 7cand 9) may imply that leiomyoma formationrelies on a narrow heterodimer stoichiometry, which can be disturbedby receptor agonists and antagonists.

In the follicular phase of the menstrual cycle, under the influence ofelevated E2 and atRA, increased levels of the putative ER-RAR andER-RXR heterodimers could form, along with RXRa-PPARg het-erodimers stimulated by endogenous PPARg ligands, also under thecontrol of estrogens (33). Anzanoet al. (40) and Kelleret al. (41)were first to suggest an interaction between estrogen action andRXRs/or 9cRA and/or PPARs, respectively. Nun˘ez et al. (42) alsoreported that RXRb and PPARa are capable of activating estrogen-responsive genes by direct binding to estrogen response elements.

In the luteal phase, E2 levels are lower than those at the preovula-tory stage, and rising progesterone could down-regulate essentialheterodimers, causing arrest of leiomyoma cell growth (increase incell size, hypertrophy) and initiation of mitosis (hyperplasia); moremitotic indices are found in human leiomyomata during the luteal thanfollicular phase (43).

Potential New Therapies for Leiomyomata.If a mechanism ofthree principal “inducers” controls leiomyoma growth (Fig. 9), it isreasonable that new therapies should target these “inducers.” Asynergistic interaction between the antagonists would lower eachantagonist’s effective dose for tumor regression and would mini-mize side effects. For example, the SERM raloxifene (44) orLY383351 (45) given p.o. will undoubtedly cause leiomyomaregression. However, suboptimal SERM doses for leiomyomamonotherapy combined with PPARg antagonists (under develop-ment) or RXRa antagonists and agonists (now in clinical trials fortype II diabetes and breast cancer) may offer an advantage as newcombination therapies for leiomyomata. Moreover, some of theseheterodimers in leiomyomata may have redundant functions, asseen in other systems (46), and it may be necessary to target allthree leiomyoma “inducers” to account for individual differencesin endogenous receptor inducers and isoforms; even among sepa-rate leiomyomata from one uterus, the levels of PPARg and RXRaproteins are not identical. Vaginal application orin situ (laparo-scopic) delivery to the tumors may be preferable than oral admin-istration of the antagonists. At present, there are no tissue-selectiveinhibitors of enzymes,e.g., retinol oxidases, to test the effect oflowering leiomyoma atRA levels.

ACKNOWLEDGMENTS

We thank Drs. Yassemi Capetanaki, Pierre Chambon, Bruce Spiegelman,and Zhidan Wu for reagents, Dr. Santo Nicosia for assistance with leiomyomapathology, and Drs. Ronald Chez and George Wilbanks for reading themanuscript.

REFERENCES

1. Carlson, K. J., Nichols, D. H., and Schiff, I. Indications for hysterectomy. N. Engl.J. Med., 328:856–860, 1993.

2. Lepine, L. A., Hillis, S. D., Marchbanks, P. A., Koonin, L. M., Morrow, B., Kieke,B. A., and Wilcox, L. S. Hysterectomy surveillance: United States, 1980–1993. Mor.Mortal. Wkly Rep. CDC Surveill. Summ.,46: 1–15, 1997.

3. Lemay, A., and Maheux, R. GnRH agonists in the management of uterine leiomyoma.Infert. Reprod. Med. Clin. N. Am.,7: 33–55, 1996.

4. Bulun, S. E., Simpson, E. R., and Word, R. A. Expression ofCYP 19gene and itsproduct aromatase cytochrome P450 in human uterine leiomyoma tissue and cells inculture. J. Clin. Endocrinol. Metab.,7: 736–743, 1994.

5. Everitt, J. I., Wolf, D. C., Howe, S. R., Goldsworthy, T. L., and Walker, C. Rodentmodel of reproductive tract leiomyomata: clinical and pathology features. Am. J.Pathol.,146: 1556–1567, 1995.

6. Romagnolo, B., Molina, T., Leroy, G., Blin, C., Porteux, A., Thomasset, M., Vande-walle, A., Kahn, A., and Perret, T. Estradiol-dependent uterine leiomyomas intransgenic mice. J. Clin. Invest.,98: 777–784, 1996.

7. Webster, M. A., Martin-Soudant, N., Nepveu, A., Cardiff, R. D., and Muller, W. J.The induction of uterine leiomyomas and mammary tumors in transgenic miceexpressing polyomavirus (PyV) large T (LT) antigen is associated with the ability ofPyV LT antigen to form specific complexes with retinoblastoma and CUTL1 familymembers. Oncogene,16: 1963–1972, 1998.

8. Tsibris, J. C. M., Porter, K. B., Nicosia, S. F., Porter, G. W., O’Brien, W. F., andSpellacy, W. N. Retinoic acid involvement in leiomyoma development: an animalmodel. J. Soc. Gynecol. Invest.,3 (Suppl. 2):29A, 1996.

9. Mangelsdorf, D. J., Thummel, C., Beato, M., Herrlich, P., Schutz, G., Umesono, K.,Blumberg, B., Kastner, P., Mark, M., Chambon, P., and Evans, R. M. The nuclearreceptor superfamily: the second decade. Cell,83: 835–839, 1995.

10. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I., and Spiegelman, B. M.mPPARg2: tissue-specific regulator of an adipocyte enhancer. Genes Dev.,8: 1224–1234, 1994.

11. Spiegelman, B. M. PPARg in monocytes: less pain, any gain? Cell,93: 153–155,1998.

12. Willson, T. M., and Wahli, W. Peroxisome proliferator-activated receptor agonists.Curr. Opin. Chem. Biol.,1: 235–241, 1997.

13. Spiegelman, B. M. PPARg. Adipocyte regulator and thiazolidinedione receptor.Diabetes,47: 507–514, 1998.

14. Kliewer, S. A., and Willson, T. M. The nuclear receptor PPARg: bigger than fat. Curr.Opin. Genet. Dev.,8: 576–581, 1998.

15. Mueller, E., Sarraf, P., Tontonoz, P., Evans, R. M., Martin, K. J., Zhang, M., Fletcher,C., Singer, S., and Spiegelman, B. M. Terminal differentiation of human breast cancerthrough PPARg. Mol. Cell, 1: 465–470, 1998.

16. Nagy, L., Tontonoz, P., Alvarez, J. G. A., Chen, H., and Evans, R. M. Oxidized LDLregulates macrophage gene expression through ligand activation of PPARg. Cell, 93:229–240, 1998.

17. Kim, J. B., Wright, H. M., Wright, M., and Spiegelman, B. M. ADD1/SREBP1activates PPARg through the production of endogenous ligand. Proc. Natl. Acad. Sci.USA, 95: 4333–4337, 1998.

18. Sarraf, P., Mueller, E., Jones, D., King, F. J., DeAngelo, D. J., Partridge, J. B.,Holden, S. A., Chen, L. B., Singer, S., Fletcher, C., and Spiegelman, B. M. Differ-entiation and reversal of malignant changes in colon cancer through PPARg. Nat.Med., 4: 1046–1052, 1998.

19. Tsibris, J., Porter, K., Jazayeri, A., Nicosia, S., Porter, G., O’Brien, W., and Spellacy,W. Effect of troglitazone, an inducer of the peroxisome proliferator-activated receptorg (PPARg), on uterine leiomyoma development in the guinea pig model. J. Soc.Gynecol. Invest., 5 (Suppl. 1):180A, 1998.

20. Furukawa, Y., Uenoyama, S., Ohta, M., Tsunoba, A., Griffin, J. D., and Saito, M.Transforming growth factor-b inhibits phosphorylation of the retinoblastoma suscep-tibility gene product in human monocytic leukemia cell line JOSK-1. J. Biol. Chem.,267: 17121–17127, 1992.

21. Porter, K. B., Tsibris, J. C. M., Nicosia, S. V., Murphy, J. M., O’Brien, W. F., Rao,P., and Spellacy, W. N. Estrogen-induced guinea pig model for uterine leiomyomas:do the ovaries protect? Biol. Reprod.,52: 824–832, 1995.

22. Porter, K. B., Tsibris, J. C. M., Porter, G. W., O’Brien, W. F., and Spellacy, W. N.Use of endoscopic and ultrasound techniques in the guinea pig model. Lab. Anim.Sci., 47: 537–539, 1997.

23. Collins, M. D., Eckhoff, C., Chahoud, I., Bochert, G., and Nau, H. 4-Methylpyrazolepartially ameliorated the teratogenicity of retinol and reduced the metabolic formationof all-trans-retinoic acid in the mouse. Arch. Toxicol.,66: 652–659, 1992.

24. Scott, W. J., Jr., Walter, R., Tzimas, G., Sass, J. O., Nau, H., and Collins, M. D.Endogenous status of retinoids and their cytosolic binding proteins in limb buds ofchick vs.mouse embryos. Dev. Biol.,165: 397–409, 1994.

25. Tzimas, G., Sass, J. O., Wittfoht, W., Elmazar, M. M., Ehlers, K., and Nau, H.Identification of 9,13-di-cis-retinoic acid as a major plasma metabolite of 9-cis-retinoic acid and limited transfer of 9-cis-retinoic acid and 9,13-di-cis-retinoic acid tothe mouse and rat embryos. Drug Metab. Dispos.,22: 928–936, 1994.

26. Arnhold, T., Tzimas, G., Wittfoht, W., Plonait, S., and Nau, H. Identification of9-cis-retinoic acid, 9,13-di-cis-retinoic acid, and 14-hydroxy-4,14-retro-retinol inhuman plasma after liver consumption. Life Sci.,59: PL169–PL177, 1996.

27. Tzimas, G., Collins, M. D., Burgin, H., Hummler, H., and Nau, H. Embryotoxic dosesof vitamin A to rabbits result in low plasma but high embryonic concentrations ofall-trans-retinoic acid: risk of vitamin A exposure in humans. J. Nutr.,126: 2159–2171, 1996.

28. Nagaya, T., Murata, Y., Yamaguchi, S., Nomura, Y., Ohmori, S., Fujieda, M.,Katunuma, N., Yen, P. M., Chin, W. W., and Seo, H. Intracellular proteolyticcleavage of 9-cis-retinoic acid receptora by cathepsin L-type protease is a potentialmechanism for modulating thyroid hormone action. J. Biol. Chem.,273: 33166–33173, 1998.

29. Nomura, Y., Nagaya, T., Yamaguchi, S., Katunuma, N., and Seo, H. Cleavage ofRXRa by a lysosomal enzyme, cathepsin L-type protease. Biochem. Biophys. Res.Commun.,254: 388–394, 1999.

30. Auboeuf, D., Rieusset, J., Fajas, L., Vallier, P., Frering, V., Riou, J. P., Staels, B.,Auwerx, J., Laville, M., and Vidal, H. Tissue distribution and quantitation of theexpression of mRNAs of peroxisome proliferator-activated receptors and liver Xreceptor-a in humans. Diabetes,46: 1319–1327, 1997.

31. Gittoes, N. J., McCabe, C. J., Sheppard, M. C., and Franklyn, J. A. Retinoid Xreceptorg mRNA expression is reduced in recurrent non-functioning pituitary ade-nomas. Clin. Endocrinol., 48:425–433, 1998.

32. Mukherjee, R., Davies, P. J. A., Crombie, D. L., Bischoff, E. D., Cesario, R. M., Jow,L., Hamann, L. G., Boehm, M. F., Mondon, C. E., Nadzan, A. M., Paterniti, J. R., Jr.,

5743

PPARg AND RXRa IN UTERINE LEIOMYOMATA

Research. on March 17, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

and Heyman, R. A. Sensitization of diabetic and obese mice to insulin by retinoid Xreceptor agonists. Nature (Lond.),386: 407–410, 1997.

33. Ma, H., Sprechter, H. W., and Kolattukudy, P. E. Estrogen-induced production of aperoxisome proliferator activated receptor (PPAR) ligand in a PPARg-expressingtissue. J. Biol. Chem.,273: 30131–30138, 1998.

34. Marshall, L. M., Spiegelman, D., Manson, J. E., Goldman, M. B., Barbieri, R. L.,Stampfer, M. J., Willett, W. C., and Hunter, D. J. Risk of uterine leiomyomata amongpremenopausal women in relation to body size and cigarette smoking. Epidemiology,9: 511–517, 1998.

35. Dilworth, F. J., Fromental-Ramain, C., Remboutsika, E., Benecke, A., and Chambon,P. Ligand-dependent activation of transcriptionin vitro by retinoic acid receptora/retinoid X receptora heterodimers that mimics transactivation by retinoidsin vivo.Proc. Natl. Acad. Sci. USA,96: 1995–2000, 1999.

36. Knudson, A. G. Hereditary cancer: two hits revisited. J. Cancer Res. Clin. Oncol.,122: 135–140, 1996.

37. Gimble, J. M., Robinson, C. E., Wu, X., Kelly, K. A., Rodriguez, B. R., Kliewer,S. A., Lehmann, J. M., and Morris, D. C. Peroxisome proliferator-activated receptor-gactivation by thiazolidinediones induces adipogenesis in bone marrow stromal cells.Mol. Pharmacol.,50: 1087–1094, 1996.

38. Lee, S-K., Choi, H-S., Song, M-R., Lee, M-O., and Lee J. W. Estrogen receptor, acommon interaction partner for a subset of nuclear receptors. Mol. Endocrinol., 12:1184–1192, 1998.

39. Song, M-R., Lee, S-K., Seo, Y-W., Choi, H-S., Lee, J. W., and Lee, M-O. Differentialmodulation of transcriptional activity of oestrogen receptors by direct protein-proteininteractions with retinoid receptors. Biochem. J.,336: 711–717, 1998.

40. Anzano, M. A., Byers, S. W., Smith, J. M., Peer, C. W., Mullen, L. T., Brown, C. C.,Roberts, A. B., and Sporn, M. B. Prevention of breast cancer in the rat with9-cis-retinoic acid as a single agent and in combination with tamoxifen. Cancer Res.,54: 4614–4617, 1994.

41. Keller, H., Givel, F., Perroud, M., and Wahli, W. Signaling cross-talk betweenperoxisome proliferator-activated receptor/retinoid X receptor and estrogen receptorthrough estrogen response elements. Mol. Endocrinol.,9: 794–804, 1995.

42. Nunez, S. B., Medin, J. A., Braissant, O., Kemp, L., Wahli, W., Ozato, K., and Segars,J. H. Retinoid X receptor and peroxisome proliferator-activated receptor activate anestrogen responsive gene independent of the estrogen receptor. Mol. Cell. Endocri-nol., 127: 27–40, 1997.

43. Kawaguchi, D., Fujii, S., Konishi, I., Nanbu, Y., Nonagaki, H., and Mori, T. Mitoticactivity in uterine leiomyomas during the menstrual cycle. Am. J. Obstet. Gynecol.,160: 637–641, 1989.

44. Porter, K. B., Tsibris, J. C. M., Porter, G. W., Fuchs-Young, R., Nicosia, S. V.,O’Brien, W. F., and Spellacy, W. N. Effects of raloxifene in a guinea pig model forleiomyomata. Am. J. Obstet. Gynecol.,179: 1283–1287, 1998.

45. Porter, K. B., Tsibris, J. C. M., Porter, G., O’Brien, W. F., and Spellacy, W. N. Theeffect of selective estrogen receptor modulator LY353381 (SERM) and Tamoxifen(Tam) on abdominal leiomyoma (L) growth in the guinea pig model, as determinedby ultrasonography. J. Soc. Gynecol. Invest.,6 (Suppl. 1):229A, 1999.

46. Chiba, H., Clifford, J., Metzger, D., and Chambon, P. Specific and redundantfunctions of retinoid X receptor/retinoid acid receptor heterodimers in differentiation,proliferation, and apoptosis of F9 embryonal carcinoma cells. J. Cell Biol.,139:735–747, 1997.

5744

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Research. on March 17, 2020. © 1999 American Association for Cancercancerres.aacrjournals.org Downloaded from

1999;59:5737-5744. Cancer Res   John C. M. Tsibris, Kathy B. Porter, Allahyar Jazayeri, et al.  

Retinoic Acid Than Myometriumtrans, and all-αReceptor , Retinoid XγPeroxisome Proliferator-activated Receptor

Human Uterine Leiomyomata Express Higher Levels of

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