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Endocrinology Vol. 142, No. 7 2985-2995
Copyright © 2001 by The Endocrine Society

ARTICLES

Induction of UO-44 Gene Expression by Tamoxifen in the Rat Uterus and Ovary1

H. Huynh, C. Y. Ng, K. B. Lim, C. K. Ong, C. S. Ong, Evelyne Tran, T. T. Tuyen Nguyen and T. W. Mark G. Chan

Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610

Address all correspondence and requests for reprints to: Hung Huynh, Laboratory of Molecular Endocrinology, Division of Cellular and Molecular Research, National Cancer Centre of Singapore, Singapore 169610. E-mail: cmrhth{at}nccs.com.sg


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A complementary DNA, uterine-ovarian-specific gene 44 (UO-44), has been isolated from tamoxifen-induced rat uterine complementary DNA library using differential display techniques. UO-44 transcripts are found to be abundant in the uterus and ovary. UO-44 gene expression in the uterus is strictly regulated by estrogens, tamoxifen, and GH, whereas the pure antiestrogen ICI 182780 is inhibitory. Treatment of ovariectomized rats and hypophysectomized rats with tamoxifen and GH, respectively, resulted in up-regulation of UO-44 expression in a dose-dependent manner. In situ hybridization revealed that UO-44 gene expression was restricted to the luminal and glandular epithelial cells of the uterus and to granulosa cells of medium-size ovarian follicles. Transfection studies showed that UO-44 was a membrane-associated protein. Because estrogens, tamoxifen, and GH are stimulators of uterine luminal epithelial cell growth in vivo, UO-44 protein may serve as a mediator of the effect of these compounds in inducing epithelial proliferation and differentiation in these tissues.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGENS PROMOTE THE growth, differentiation, and remodeling of the uterus during the estrous cycle and pregnancy (1). Preovulatory ovarian estrogen secretion has been shown to play an important role for uterine cellular proliferation and epithelial differentiation during early stages of pregnancy (2). The biological activity of estrogens is regulated by its interaction with specific high-affinity nuclear estrogen receptors (ERs)-{alpha} and -ß, which function as ligand-inducible transcription factors (3, 4, 5, 6). Estrogens have been shown to modulate the expression of genes involved in the regulation of cell growth and differentiation, including EGF, IGF-I, and their receptors (7, 8, 9, 10). Ultimately, estrogens increase the rate of cell proliferation by recruiting noncycling cells into the cell cycle, and by shortening the overall cell cycle time by reducing the length of the G1 phase (11).

Tamoxifen belongs to the type 1 (nonsteroidal) antiestrogens that exhibit mixed estrogenic/antiestrogenic activity. Tamoxifen and its metabolites form imperfect complexes upon binding to ER and are unable to induce transcription of estrogen target genes in certain cellular context (12). In the mammary gland, tamoxifen acts as an estrogen antagonist and is currently used in the prevention and treatment of breast cancer (13). However, long-term administration of tamoxifen has been reported to be associated with an increased risk of endometrial cancer in postmenopausal women (14), and with some cases of endometrial thickening (15). The molecular mechanisms responsible for tamoxifen-induced endometrial hyperplasia are not well understood. It has been suggested that the above estrogenic effect of tamoxifen on the atrophic postmenopausal endometrium causes hyperplasia that may progress to atypia and cancer in a manner similar to that seen with estrogen-replacement therapy (15). We previously reported that tamoxifen significantly increased uterine weight, whereas ICI 182780 administration suppressed it (16). We also showed that tamoxifen, in addition to inhibiting IGF-I gene expression in the uterus (16), also altered the expression of other genes in the regulation of proliferation (17). To identify additional tamoxifen-inducible genes in the uterus, differential display was used to examine the transcript-expression profile of the ovariectomized uterus, under conditions of tamoxifen supplementation. A tamoxifen-induced complementary DNA (cDNA), UO-44, was isolated from rat uterus cDNA library. By virtue of its activation by GH, estradiol, and tamoxifen tissue- specific expression and localization on the cell membrane, UO-44 protein may be important in normal and neoplastic uterine and ovarian growth. Thus, it may potentially serve as a biomarker for uterine and ovarian cancer and aid in their diagnosis and treatment.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drug administration
Animal experiments were approved by the local Animal Care Committee. Intact, hypophysectomized (Hypox) or ovariectomized (OVX) female Sprague Dawley rats, 50 days old at the beginning of the experiments, were obtained from Charles River Laboratories, Inc., Québec, Canada. Except for ovarian-dependent and time-dependent tamoxifen-induced UO-44 gene expression studies, OVX and Hypox animals were used in these experiments 2 weeks after ovariectomy and hypophysectomy, respectively. To study the effect of GH on UO-44 gene expression, groups of Hypox rats (n = 15) were injected daily with 0.5, 1, 1.5, and 2 µg recombinant human GH (Genentech, Inc., South San Francisco, CA) per gram body weight for 21 days. To study the effect of estradiol on UO-44 gene expression, groups of OVX rats (n = 15) were implanted with 0.5-cm SILASTIC tubes (0.04-inch id; Dow Corning Corp., Midland, MI) containing 17ß-estradiol (Sigma, St. Louis, MO) on the back of their necks. Control rats experienced the same surgical implantation with empty SILASTIC tubes. Based on previous published work (18), the released rate of 17ß-estradiol from SILASTIC implants was documented to be 2.4 µg/cm·day. Tamoxifen (Sigma) was dissolved in castor oil at a concentration of 10 mg/ml. To study the effects of tamoxifen on UO-44 gene expression, groups of OVX rats (n = 15) daily received 1, 2, 3, 4, and 5 mg tamoxifen per kg body weight, via sc injections, for 3 weeks. To study the effects of ovariectomy on UO-44 gene expression in the uterus, groups of rats (n = 15) underwent ovariectomy, and the uteri were collected at 6, 24, 48, 72, 96, 120, and 144 h post ovariectomy. To study the time-dependent tamoxifen-induced UO-44 gene expression, groups of OVX rats (n = 15) were injected with 5 mg tamoxifen, and the uteri were collected at 0, 6, 12, 18, and 24 h post injection. Preformulated ICI 182780 (Astra Zeneca Pharmaceuticals, UK) was supplied at a concentration of 50 mg/ml in castor oil solution. To study the effects of ICI 182780 on UO-44 expression in ovary intact rats, groups of female Sprague Dawley rats (n = 15) received weekly sc injections of castor oil alone or 1, 1.5, or 2 mg of ICI 182780 per kg body weight for 3 weeks. Our pilot study showed that maximal reduction of uterine weight could be achieved at a dose of 1.5 mg ICI per kg body weight per week. At the end of the experiments, animals were killed by carbon dioxide exposure. The uteri or ovaries were excised, trimmed, weighed, and snap-frozen in liquid nitrogen and stored at -70 C for RNA extraction. Parts of the uterus and one ovary were embedded in OCT Jung Tissue Freezing Medium (Leica Instruments GmbH, Germany) for in situ hybridization studies.

Messenger RNA (mRNA) differential display
OVX rats were either untreated (OVX) or treated with 5 mg/kg body weight tamoxifen per day (OVX-TAM) for 14 days. Total RNA was isolated from uteri, using RNAzol premix solution and RNAzol B method (Tel-Test, Friendswood, TX) as previously described (16). Differential display was performed using RNA derived from uteri of OVX and OVX-TAM rats according to the protocol supplied with the RNAmap kit (GenHunter Corp., Nashville, TN). Briefly, 5 µg deoxyribonuclease I-treated total RNA were reverse transcribed with 300 U Moloney murine leukemia virus reverse transcriptase (Amersham Pharmacia Biotech) in the presence of 1 mM T12MG, T12MA, T12MT, or T12MC primer (GenHunter), where M is a mixture containing deoxyguanyl, deoxyadenyl, and deoxycytidyl nucleotides. Two tenths of this reaction was used in the PCR amplification reaction containing 2.5 mM each of deoxynucleotide triphosphates, 10 mCi of [{alpha}-33P]deoxy-ATP (NEN Life Science Products), and two primers: 1 mM T12 oligonucleotide and 2 mM of one of the five arbitrary primers, AP-1 (5'-AGCCAGCGAA-3'); AP-2 (5'-GACCGCTTGT-3'); AP-3 (5'-AGGTGACCGT-3'); AP-4 (5'-GGTACTCCAC-3'); and AP-5 (5'-GTTGCGATCC-3'). These reactions contained 1 U AmpliTag DNA polymerase (Perkin-Elmer Life Sciences). The cycling parameters for PCR were: 94 C for 30 sec, 42 C for 120 sec, 72 C for 45 sec for 45 cycles. After PCR amplification, the PCR-amplified fragments were separated on 6% denaturing polyacrylamide gel. The gel was dried and exposed to XAR film (Eastman Kodak Co., Rochester, NY) with intensifying screens at -70 C, and cDNA representing differentially expressed mRNAs was excised from the dried gels for elution and reamplification as instructed by GenHunter Corporation. Eluted DNA samples were reamplified by PCR using a corresponding pair of primers under the same conditions as described above. Reamplified cDNA fragments were separated in 2.0% low-melting agarose and used as probes in Northern blots to verify their differential expression in uteri. Desired fragments were subcloned into TA vector (Invitrogen, Carlsbad, CA) and subjected to nucleotide sequence.

cDNA library construction
Total RNA was isolated from tamoxifen-treated uteri (pool of 5 rats) using RNAzol premix solution and RNAzol B method (Tel-Test) as described above. Poly A+ RNA was isolated from total RNA using Oligotex mRNA Kits (QIAGEN GmbH, Germany) according to the manufacturer’s protocol. Ten micrograms of poly A+ RNA derived from uteri of OVX-TAM rats were used to construct a unidirectional cDNA library in the vector pcDNA3.0 (Invitrogen Life Technologies) designed for expression in mammalian cells using the CMV promoter. cDNA was primed using the unidirectional NotI "T" primer, giving inserts in the correct orientation for expression. Double-stranded cDNA was size-enriched and transformed into TOP10F cells after ligation into the vector. The 320-bp DNA probe of rat UO-44 cDNA was used to screen this rat uterine cDNA library as described (19). Clones identified by this probe were isolated and sequenced by the Sanger dideoxy chain termination method, and their nucleotide sequences were compared with those deposited in the GenBank and EMBL databases.

In situ hybridization
For mRNA in situ hybridization, recombinant plasmid pcDNA3.0 containing a 500-bp UO-44 fragment (nucleotides 1780–2280 of the UO-44 sequence, GenBank accession number AF022147) was linearized to generate sense and antisense digoxigenin-labeled RNA probes using Dig RNA Labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). Serial 7- to 8-µm OCT-frozen sections from either uterus or ovary were heated for 2 min at 50 C and dried for 30 min. Prehybridization, hybridization, posthybridization, and immunological detection were performed according to the manufacturer’s protocol, and these sections were subsequently counterstained with hematoxylin.

Northern blotting
Total RNA was isolated from indicated tissues of female rats as described (20). Northern blots were performed on total RNA, and blots were hybridized with rat UO-44 or human glyceraldehyde 3-phosphate dehydrogenase (GAPDH (ATCC, Manassas, VA) cDNAs as previously described (20). mRNA levels were determined by densitometric scanning of autoradiographs.

Stably transfected MCF-7 cell lines
The entire coding region of UO-44 cDNA was cloned into the mammalian expression vector pcDNA3.1/His (Invitrogen) to create the UO-44-pcDNA3.1/His expression vector. The UO-44-pcDNA3.1/His sequence was confirmed by sequencing. MCF-7 cells were seeded at a density of 2 x 105 in 100-mm culture dishes in 90% {alpha}-MEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% FCS with Garamycin, 24 h before transfection. Cells were transfected with 5 µg UO-44-pDNA3.1/His DNA or pDNA3.1/His control plasmid DNA and 28 µl Lipofectamine reagent (Life Technologies, Inc.), following recommendations of the manufacturer. Forty-eight hours after transfection, cells were subcultured at 1:10 and replaced with growth medium containing 800 µg/ml G418 (Calbiochem, La Jolla, CA). After 4 weeks, clones were isolated, expanded, and assayed for UO-44 expression by Western blot analysis.

Western blot analysis
To localize the UO-44 protein, controls and UO-transfected MCF-7 cells were grown to 90% confluence. Plasma membrane-enriched subcellular fractions and cytosol were prepared by differential centrifugation as described previously (21). Plasma membrane and cytosolic proteins were subjected to Western blot analysis as described (22). Blots were incubated with mouse anti-6-Histidine antibody (Epitope Tagging) Ab-1 (NeoMarkers, Union City, CA) (1:500 dilution) and horseradish peroxidase-conjugated donkey antimouse secondary antibody (1:7500). Blots were visualized with a chemiluminescent detection system (ECL; Amersham Pharmacia Biotech) and exposed to film for 10–45 sec.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation and sequence analysis of UO-44 cDNA
Using differential display methodology, several differentially displayed bands representing cDNA corresponding to the gene whose expression was up-regulated during tamoxifen treatment were isolated. One of the bands, which was present in the uteri of tamoxifen-treated rats but disappeared in uteri derived from OVX rats, was selected for further characterization (Fig. 1AGo). This 320-bp DNA fragment was used to probe Northern blots of mRNA obtained from uteri of OVX and OVX-tamoxifen-treated rats. A strong signal, corresponding to an approximate molecular weight of 2.2 kb, emerged in RNA isolated from OVX-tamoxifen-treated uteri, whereas no detectable signal was observed in the RNA derived from uteri of OVX rats (Fig. 1BGo). The results suggested that the gene isolated was up-regulated by tamoxifen. This 320-bp DNA fragment was then subcloned into a TA vector and subjected to nucleotide sequence analysis.



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  		Figure 1. Differentially expressed mRNA bands in uterine tissues of control OVX and OVX-tamoxifen-treated rats. Total RNA, isolated from uteri of OVX and OVX-tamoxifen-treated rats, was subjected to differential display. The band representing mRNA that was induced during tamoxifen treatment is marked by an arrowhead. Northern blot analysis of total RNA from uteri of OVX and OVX-tamoxifen-treated rats was used to confirm the presence of a differentially expressed mRNA in the OVX- tamoxifen-treated uteri. Blots were hybridized with a 32P-labeled cDNA fragment that was isolated from the above differential gel (A) or GAPDH cDNA (B).

 
Using this 320-bp DNA probe to screen the cDNA library prepared from tamoxifen-treated OVX rat uterus resulted in the isolation of 14 clones. The longest clone contained a 2.2-kb insert. Comparison of the nucleotide sequence of the cDNA against the nonredundant nucleotide database of GenBank established that this cDNA (GenBank accession no. AF022147) shared 99% with the estrogen-regulated gene 1 (ERG-1) cDNA (23).

The full-length UO-44 cDNA contains 2,282 bp of nucleotide sequence. An initiator ATG codon (position 253) is followed by a single open reading frame of 607 amino acids with a calculated molecular mass of 68,639 Da. The ATG initiation site is contained in the sequence for initiation by eukaryotic ribosomes described by Kozak (24). The open reading frame ends in a TGA terminator codon at position 2074, followed by 208 nucleotides in the 3' untranslated region.

Within the first 265 amino acids of UO-44, 2 regions were identified that bear homology to the CUB motifs (complement subcomponents C1r/Cls, Uegf protein, and bone morphogenetic protein) (25). The first CUB domain began at Cys-32 and the second at Cys-154. UO-44 also contained a zona pellucida domain at amino acids 276–523. The UO-44 amino acid sequence predicted a membrane protein with 2 transmembrane helices. The hydrophobic transmembrane region was 13 amino acids in length and was located between amino acids 5 and 17, whereas the anchor transmembrane region was 19 amino acids in length and is located between amino acids 571 and 589. There was a putative transmembrane domain near the carboxyl terminus. UO-44 terminated in a short 19-amino acid polypeptide presumably positioned within the cytoplasm.

Tissue distribution of UO-44
To determine the expression of rat UO-44 gene in various tissues, total RNA obtained from tissues of mature female rats was analyzed by Northern blotting. Figure 2Go shows that transcription of the UO-44 gene was observed only in uterus and ovary. UO-44 mRNA levels in adipose tissue, mammary gland, liver, kidney, muscle, heart, stomach, small intestine, spleen, brain, pituitary, and muscle were undetectable, suggesting that the UO-44 gene may be expressed at a very low level, or not at all, in these tissues. Because this gene is highly expressed in the uterus and ovary, it has been designated uterine-ovarian-specific gene 44, or UO-44.



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  		Figure 2. Northern blot analysis of UO-44 gene expression in female adult rat tissues. Total RNA derived from various tissues of an 80-day-old female rat was subjected to Northern blot analysis. Blots were hybridized with rat UO-44 (A) and GAPDH (B) cDNAs. Tissues are: Mg, mammary gland; Fa, abdominal fat; Mu, red muscle; Ov, ovary; He, heart; Lu, lung; Li, liver; Sto, stomach; Int, small intestine; Spl, spleen; Pi, pituitary; Br, brain; Ki, kidney; and Ut, uterus.

 
Hormonal regulation of UO-44
To determine whether UO-44 gene expression is steroid-hormone dependent, uteri were collected from rats at different times after ovariectomy. As shown in Fig. 3Go, UO-44 mRNA rapidly disappeared after ovariectomy. Six hours post ovariectomy, UO-44 mRNA levels dropped to 30% of controls, and no UO-44 transcripts were detected at 144 h post ovariectomy (Fig. 3Go). Reexpression of UO-44 gene could be achieved by tamoxifen treatment. Tamoxifen-induced UO-44 gene expression was observed as early as 6 h post tamoxifen injection (Fig. 3Go). Treatment of OVX rats with various doses of tamoxifen resulted in a dose-dependent up-regulation of UO-44 gene expression (Fig. 4Go). ICI 182780 potently attenuated tamoxifen-induced UO-44 gene expression (Fig 4Go). Estradiol was more potent than tamoxifen in inducing UO-44 gene expression. Progesterone was unable to restore UO-44 gene expression (data not shown). In all cases, the changes in expression of UO-44 gene were positively correlated with the changes in uterine weight (Fig. 4CGo).



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  		Figure 3. Ovarian-steroid-dependent UO-44 gene expression. Female rats underwent ovariectomy. The uteri were collected at the indicated times after ovariectomy. Total RNA derived from uteri was subjected to Northern blotting. Blots were hybridized with GAPDH (A and D) and rat UO-44 (B and E) cDNAs. Time-dependent tamoxifen-induced UO-44 gene expression in uteri of OVX-rats is shown in E. Densitometric scanning of the UO-44 band is shown in C and F. Bars with different letters are significantly different from one another, at P < 0.01. Data are expressed as the mean of six replicates ± SEM.

 


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  		Figure 4. Effects of tamoxifen treatment on UO-44 expression in the uteri of OVX rats. OVX rats were treated with the indicated amount of tamoxifen (TAM), 1.2 µg/day estradiol (E2), or 5 mg tamoxifen plus 1 mg ICI 182780 (TAM + ICI) for 3 weeks. Total RNA derived from uteri was subjected to Northern blotting. Blots were hybridized with GAPDH (A) and rat UO-44 (B) cDNAs. Densitometric scanning of the UO-44 band (C) and the effect of each treatment on uterine weight (D) are shown. Uteri of ovary intact (I) serve as a positive control. Bars with different letters are significantly different from one another, at P < 0.01. Data are expressed as the mean of six replicates ± SEM.

 
To further examine the estrogenic effects of tamoxifen on the uterine growth and expression of UO-44 in the uterus, ovary intact rats were treated with various doses of tamoxifen and a pure antiestrogen, ICI 182780. Figure 5Go shows that treatment of rats with tamoxifen for 3 weeks resulted in a significant increase in basal UO-44 expression. At the doses of 2 mg and 5 mg per kg body weight per day, tamoxifen stimulated UO-44 gene expression by 2- and 3-fold, respectively. ICI 182780, on the other hand, was a very potent inhibitor of UO-44 gene expression, completely abolishing UO-44 gene expression in the uterus at the dose of 1 mg per kg body weight per week, as compared with the ovary-intact uterus (Fig. 5BGo).



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  		Figure 5. Effects of tamoxifen and ICI 182780 on UO-44 gene expression and uterine weight of ovary-intact rats. Ovary-intact rats were treated with indicated concentrations of TAM (A) and ICI 182780 (ICI, B) for 3 weeks. Total RNA derived from uteri was subjected to Northern blot analysis. Blots were hybridized with GAPDH and rat UO-44 cDNAs. Densitometric scanning of the UO-44 band and the effect of the treatments on uterine weight are shown. Bars with different letters are significantly different from one another, at P < 0.01. Data are expressed as the mean of six replicates ± SEM.

 
Uterine weight is known to be increased by the influence of GH (16), and this led us to investigate whether UO-44 gene expression in the uterus was under the regulation of GH. Northern blot analysis was performed using uteri and ovaries obtained from Hypox rats treated with vehicle or various doses of recombinant human GH. Compared with uterine weight of ovary intact rats (Fig. 5Go), hypophysectomy caused a significant reduction in uterine weight, which was coincident with the disappearance of UO-44 mRNA (Fig. 6Go). On administration of GH, UO-44 gene expression in the uterus was restored. Hypophysectomy, on the other hand, had very little (or no) effect on UO-44 gene expression in the ovary (Fig. 7Go). However, blockage of estrogenic activity by administration of a pure antiestrogen, ICI 182780, led to a dramatic reduction in UO-44 mRNA in the ovaries of Hypox rats (Fig. 7B).



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  		Figure 6. Effects of GH on UO-44 gene expression in the uterus of Hypox rats. Hypox rats were treated with vehicle or the indicated concentration of human GH per gram body weight for 3 weeks. Total RNA derived from uteri was analyzed by Northern blotting. Blots were hybridized with rat UO-44 or GAPDH cDNAs (A). Densitometric scanning of the UO-44 band (B) and the effect of GH on uterine weight (C) are shown. Bars with different letters are significantly different from one another, at P < 0.01. Data are expressed as the mean of six replicates ± SEM.

 


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  		Figure 7. Effects of tamoxifen, ICI 182780, GH, and estradiol on UO-44 gene expression in the ovaries of Hypox rats. Hypox rats were treated with vehicle (C), 1 µg human GH per gram body weight (GH), 1.0 mg ICI 182780 per kg body weight per week (ICI), 1.2 µg 17ß-E2 per day, and 5 mg tamoxifen per kg body weight per day (TAM). Total RNA derived from ovaries was analyzed by Northern blotting. Blots were hybridized with rat UO-44 (A) or GAPDH cDNAs (B). Hypophysectomy had no effect on UO-44 gene expression in the ovaries

 
Localization of UO-44 in the uterus and ovary
Fig. 5Go shows that UO-44 mRNA was undetectable in uteri derived from OVX rats and was expressed after tamoxifen treatment. To determine whether gene expression in the luminal epithelial cells was induced by tamoxifen, in situ hybridization was performed on sections of uteri derived from control OVX and OVX-tamoxifen-treated rats, using an antisense RNA probe specific for rat UO-44. As expected, no UO-44 signal was detected in uterus of OVX rats (Fig. 8AGo). High levels of UO-44 were detected in the luminal epithelial cells and glandular population of the uteri after tamoxifen treatment (Fig. 8BGo). The effects of tamoxifen on UO-44 expression were abolished by ICI treatment (Fig. 8DGo), suggesting that tamoxifen acts as an estrogen to induce UO-44 expression. No staining was seen in smooth muscle cells. Hybridization with the sense UO-44 RNA probe showed no background staining (Fig. 8CGo).



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  		Figure 8. Detection of UO-44 mRNA in the tamoxifen-treated uteri. In situ hybridization with antisense RNA probe for UO-44 gene expression in the uteri of control OVX (A) and OVX-tamoxifen-treated rats (B). C, Sense control UO-44 probe showing no background staining in uterus of tamoxifen-treated rats. In situ hybridization with antisense RNA probe for UO-44 gene expression in the OVX-tamoxifen-ICI-treated uteri (D). UO-44 mRNA was localized in the luminal secretory epithelial cells and glandular epithelial cells. ICI abolished tamoxifen-induced UO-44 expression in the uteri of OVX rats.

 
In the ovaries, varying amounts of UO-44 mRNA was detected in granulosa cells of a mixed population of follicles (Fig. 9AGo). High levels of UO-44 expression were observed in the granulosa cells of medium-size follicles (Fig. 9AGo). Low-to-moderate UO-44 expression was detected in granulosa cells of small and large follicles (Fig. 9AGo). Furthermore, lack of uniform UO-44 mRNA among granulosa cells within the same follicle was noted (Fig. 9BGo). A control sense UO-44 probe produced no background staining in ovarian tissue (Fig. 9CGo). UO-44 staining signals were almost lost in sections of ovaries from rats treated with ICI 182780 (Fig. 9DGo).



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  		Figure 9. In situ hybridization with antisense RNA probe for UO-44 expression in rat ovaries. Low (A) and high (B) magnification showing the nonuniform UO-44 mRNA distribution in the follicles and granulosa cells. Abundant levels of UO-44 expression were detected in the granulosa cells of medium-size follicles. Moderate levels of UO-44 mRNA were detected in granulosa cells of large and small follicles. C, A sense control UO-44 probe showed no background staining in the ovarian tissue; D, ICI 182780-treated ovary hybridized with antisense UO-44 showed very faint staining signal.

 
Subcellular localization of UO-44 protein
To further demonstrate the subcellular localization of UO-44 protein, human breast cancer MCF-7 cells were transfected with a mammalian expression vector containing full-length UO-44 cDNA (UO-44 pcDNA3.1/His) or control pcDNA3.1/His vector. As shown in Fig. 10Go, 6Go-Histidine antibody recognized a protein of approximately 68–69 kDa in plasma membrane-enriched subcellular fractions of UO-44 transfectants but not in the cytosol. No protein of identical size was detected in mock-transfected cells.



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  		Figure 10. Subcellular localization of UO-44 protein. Human breast cancer MCF-7 cells were transfected with mammalian expression vector containing full-length UO-44 cDNA (UO-44 pcDNA3.1/His) or control pcDNA3.1/His vector, as described under Materials and Methods. Plasma membrane-enriched subcellular fractions and cytosolic proteins were isolated, and Western blot analysis was performed as described under Materials and Methods. Blots were incubated with mouse anti-6-Histidine antibody and horseradish peroxidase-conjugated donkey antimouse secondary antibody. Blots were visualized with a chemiluminescent detection system. Molecular weights of immunoreactive bands are shown. Clones 1 and 2 are mock transfectants, and UO-44–12 and UO-44–15 are UO-44-expressing clones.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have isolated a tamoxifen- and estrogen-induced cDNA, UO-44, using differential display and cDNA library screening. On the basis of homology to other proteins, there is 87% (at the nucleic acid level) and 88% (at the amino acid level) homology between rat UO-44 cDNA and UTCZP (uterine cub motif zona pellucida motif) cDNA (26). In contrast to the mouse UTCZP gene (a cDNA cloned from pregnant mouse uterus, which is expressed only in the uterus during late pregnancy), rat UO-44 is expressed in nonpregnant and pregnant uteri. Chen et al. (23) recently reported a rat ERG-1 cDNA that shares 99% homology with rat UO-44 cDNA. Therefore, the UO-44 and EGR1 cDNAs are identical. Like the UO-44 gene, the ERG-1 gene is strongly induced in rat uterus and oviduct by estradiol treatment, and its expression is restricted to surface epithelium. In addition, our study shows that UO-44 mRNA is also detected in granulosa cells of ovaries.

Our transfection study provides evidence that UO-44 protein is membrane-bound, a feature only suggested by previous work (23) based on the sequence analyses. In addition to the expression of UO-44 in the luminal and glandular epithelial cells of the uterus, as determined by in situ hybridization, UO-44 mRNA is also localized in the granulosa cells of follicles. High levels of UO-44 gene expression are found only in granulosa cells of medium-size follicles but not in small and large follicles within the same ovary. At the moment, the role of UO-44 in the follicle growth and development is unclear, but it is possible that UO-44 protein may be involved in follicular growth and maturation.

The biological function of UO-44 in the uterus and ovaries is yet unclear. A homology search of the UO-44 gene product indicates that it possesses several motifs with a zona pellucida domain in the carboxylterminal region. This domain seems to be involved in sperm-binding function and sperm-egg recognition (27). In the follicle, UO-44 expression is detected in the granulosa cells surrounding the egg. This pattern of expression is similar to the glycoproteins of the extracellular matrix surrounding the oocytes (27). Unlike the UTCZP gene (26), whose expression is temporal and restricted to the gravid uterus, UO-44 transcripts are not only detected in the uterus during pregnancy but also in nonpregnant uteri of mature female rats. The expression of UO-44 in the uteri and ovaries suggests that UO-44 protein may play other roles in these tissues besides events that transpire during pregnancy as proposed by Kasik (26). Because estrogens and tamoxifen, as well as GH, are growth stimulators for uterine luminal epithelial cells in vivo, the increase in UO-44 expression in uteri and ovaries may be associated with hypertrophy of uterine epithelium or cellular proliferation in these tissues. It is possible that UO-44 is an estradiol-induced protein that is involved in cell-cell and cell-matrix interactions during estrogen-induced growth and tumorigenesis, as described for mammalian tumor-necrosis-factor-stimulated gene 6 (28).

The CUB domain is an extracellular domain of approximately 110 residues that is found in functionally diverse proteins (28, 29, 30, 31). At the present time, the biological significance of the two CUB domains in rat UO-44 protein is unknown. It remains to be determined whether UO-44 protein shares similar functions described for bovine acidic seminal fluid protein, which contains a CUB domain and belongs to the spermadhesin family, functioning both as a mitogen and growth factor in vitro and as a stimulator of progesterone secretion in cultured ovarian cells (32). It is also possible that the CUB domains in UO-44 are involved in cell adhesion, as reported for a calcium-independent cell adhesion molecule that functions during the formation of certain neural circuits (33, 34). If this holds true, then UO-44 expression in uterine epithelial cells, in response to increasing levels of circulating estrogens during early pregnancy, may facilitate the attachment of embryos to the uterine wall. UO-44 expression in the granulosa cells surrounding the egg may serve as an adhesion molecule for cell-cell interaction and as a target for egg-sperm recognition, as described for mammalian spermadhesins (35). Experiments are under way to determine the role of UO-44 in cell adhesion.

The high levels of UO-44 transcripts in the uteri and ovaries, but undetectable levels in other tissues examined, suggest that the UO-44 gene may be expressed at a very low level or is silent in these tissues. The rapid reduction in UO-44 gene expression in the uterus after ovariectomy suggests that UO-44 gene expression is strictly dependent on ovarian steroid hormones. Because estradiol, but not progesterone (data not shown), effectively restores UO-44 expression in the uterus, there is speculation that UO-44 expression may be estrogen-dependent. Like estradiol, tamoxifen induces UO-44 gene expression in the uterus of OVX rats; whereas a pure antiestrogen, ICI 182780, is inhibitory, suggesting that tamoxifen may act as an estrogen agonist to induce UO-44 expression in this tissue. It is unlikely that the observed enhancement of UO-44 expression by tamoxifen is a secondary effect of uterine expansion, considering that the induction of UO-44 expression by tamoxifen occurred within 6–24 h and before the changes in uterine weight were noticed (data not shown).

It is interesting to note that, after hypophysectomy, there is a loss of UO-44 in the uterus but not in the ovaries. Because both estrogens and tamoxifen have important actions on the pituitary gland (36, 37), we have considered the possibility that the effects of tamoxifen on the uterus were pituitary-dependent. Because Hypox and Hypox-GH-replaced animals are rendered estrogen-deficient by pituitary ablation (37) and UO-44 expression is estrogen-dependent, estrogen deficiency may down-regulate UO-44 expression in the uterus of Hypox rats. In the ovaries of hypohysectomized rats, local production of estrogens may be sufficient to maintain UO-44 expression in this tissue. It is also possible that GH may directly regulate UO-44 gene expression in the uterus or indirectly enhance estrogen secretion from the ovaries. Taken together, these data indicate that UO-44 gene expression is cell- and tissue-specific and that its expression is regulated not only by estrogens and estrogen agonists but also by GH.

Our study also indicates that the UO-44 gene expression in the ovary is very low during metestrous and diestrous (unpublished data). Furthermore, abundant levels of UO-44 mRNA are detected in the granulosa cells of medium-size follicles. This observation is different from the previous report by Chen et al. (23), where ERG-1 was not detected in the rat ovary using Northern blotting. The disparity between their data and ours may be attributable to: 1) the differences in the stage of the estrous cycle where the ovaries are collected for the analysis; or 2) whether part of the ovary or the whole ovary is used for RNA extraction.

The biochemical mechanism(s) underlying the effects of tamoxifen and estradiol on UO-44 gene expression is not well understood. Binding of tamoxifen and estrogen to the ER stimulates the increased expression of c-fos, c-jun, vascular endothelial growth factor, complement C3 (38, 39, 40), those for some growth factors, and growth factor receptors such as IGF-I, IGF-IR, and EGF-R, resulting in stimulation of DNA synthesis and cell proliferation (30). When a pure antiestrogen, ICI 182780, binds to the ER, the receptor is not available to bind to estrogen or tamoxifen, and the antiestrogen-ER complex fails to effectively stimulate gene expression and DNA synthesis. Whereas expression of c-fos has been considered a key event in estrogen-induced uterine epithelial cell proliferation, estrogen-induced complement C3 expression in the uterus (39, 40) is not related to growth response, even though the dose responses of growth and gene expression do correlate. Tamoxifen has been shown to act as an estrogen agonist to induce c-fos and jun-B expression in the rat uterus (41), whereas constitutive expression of c-Jun in the uterine luminal epithelial cells completely regressed by tamoxifen (42). It has been proposed that overexpression of c-fos and jun-B may contribute to the molecular mechanism underlying the hypertropic effects of tamoxifen on uterine epithelium (42, 43). Because c-fos, c-jun, and jun-B are transcription regulatory factors that can regulate the expression of a gene containing an AP-1 recognition site, it is possible that UO-44 gene expression is regulated by these transcription factors. Experiments are under way to determine whether UO-44 gene promoter contains AP-1 recognition site(s) and its expression is regulated by the products of the above protooncogenes in response to tamoxifen and estrogen treatment.

The common mechanism by which tamoxifen, GH, and estradiol regulate UO-44 gene expression in the rat uterus is not well understood. It has been reported that GH, tamoxifen, and estrogens have similar effects on UO-44 and IGF-I expression in the uterus, whereas ICI 182780 inhibits it (16, 44). This is consistent with studies in experimental animals showing that these compounds produce similar effects on uterine weight. It is possible that the effect of these compounds on UO-44 expression involves IGF-I as a mediator. Experiments are under way to determine this possibility.

Though there is species-to-species variability in the tissue-specific predominance of agonist vs. antagonist actions of tamoxifen at the ER, the results reported here are clinically relevant, because women treated with tamoxifen frequently exhibit uterine hyperplasia (45) and rarely show neoplasia (14, 46, 47). Tamoxifen has been proposed as a treatment for neoplastic conditions of the uterus (48), but results of clinical trials have not been impressive, and there are clinical and laboratory data suggesting that stimulation of endometrial neoplastic growth and leiomyoma growth by tamoxifen is possible (49, 50). It is possible that the effects of tamoxifen on uterine UO-44 gene expression, which we described here, are related to these adverse effects of tamoxifen.

In summary, our data demonstrate that UO-44 gene expression in the uterus is a molecular marker that correlates well with the positive or negative uterotrophic effects of ER antagonists and partial agonists. The characterization of UO-44 protein in uterine and ovarian cells serves to provide new knowledge concerning the roles of UO-44 protein in estradiol- and tamoxifen-induced cellular proliferation and cancer in these tissues.


   Acknowledgments
 
We would like to thank Dr. Kon Oi Lian for critical reading and helpful discussions of the manuscript.


   Footnotes
 
1 This work was supported, in part, by grants from the United States Army Medical Research Materiel Command (DAMD17-97-1-7084) and National Medical Research Council of Singapore (to H.H.). Back

Received January 12, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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