| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Regular Article |
a Medical Sciences, Indiana University School of Medicine, Bloomington, Indiana 47405
b Department of Obstetrics and Gynecology, Indiana University School of Medicine, Indianapolis, Indiana 46202
c Department of Integrative Biology, Pharmacology, and Physiology, University of Texas-Houston, Medical School, Houston, Texas 77030
ABSTRACT
Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17ß or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17ß, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.
estradiol, estradiol receptor, female reproductive tract, hormone action, progesterone, progesterone receptor, steroid hormones, steroid hormone receptors
INTRODUCTION
Estrogens play a central role in controlling multiple reproductive processes, and estrogen receptors (ER) belong to the nuclear receptor superfamily of transcriptional regulatory proteins related by structure and function [1]. On ligand activation, ER forms a dimer that binds to specific DNA sequences, named estrogen-response elements, that are located in the promoters of target genes [2]. Stimulation of the transcription of specific estrogen-responsive genes achieves subsequent physiological responses. Recently, several proteins have been described that associate with ER, and with several other nuclear receptors, in a ligand-dependent manner. These proteins, which enhance ligand-dependent transcriptional activation by ER and other nuclear receptors, have been classified as coactivators, and they include SRC-1 [3] and related proteins such as GRIP1/TIF2/SRC-2 [4–7], RAC3/ACTR/p/CIP/A1B1/SRC-3 [8–12], RIP140/RIP160 [13, 14], ERAP-140/ERAP-160 [15], CBP/p300 [16–21], and L7/ switch protein for antagonist (SPA) [22]. Although coactivator mRNAs are ubiquitously expressed, the levels of expression vary among tissues and organs [4, 23, 24], suggesting that the level of coactivator expression contributes to the variations in tissue hormone responsiveness.
The antiestrogen tamoxifen is the most commonly prescribed treatment for breast cancer [25, 26], and it is the only drug known to prevent breast cancer [27]. An antagonist in the breast, it also acts as an agonist for specific responses in other tissues. In the immature rat uterus, tamoxifen stimulates a dramatic hypertrophy of the luminal epithelium equal to or greater than the effect of estradiol, but it induces only a slight proliferative effect in ovariectomized rat uterus and blocks estradiol-induced hyperplasia [28–30]. In the endometrial stroma, the hyperplastic effect of tamoxifen approaches that induced by estradiol [31]. The uterine stimulatory effect of tamoxifen in humans is associated with an increased incidence of endometrial cancer among women taking the drug to either treat [25, 26] or prevent [27] breast cancer. The remarkable tissue- and response-specific behavior of tamoxifen is mediated by ER, and enhancement of the agonist activity of tamoxifen-occupied ER by coactivators has recently been reported [24, 32, 33], indicating that the relative expression of coactivators and corepressors can modulate the antiestrogen regulation of ER transcriptional activity. In particular, SPA may play a key role [22, 32] in tissue-specific sensitivity to mixed antiestrogens.
Considering these observations, the objectives of the present study were to determine the cell-specific expression pattern of SPA, SRC-1, GRIP1, RAC3, RIP140, and p300 mRNA in the rat uterus and to investigate if the expression of coactivator mRNA levels in vivo is regulated by estradiol or tamoxifen.
MATERIALS AND METHODS
Animals
All animal studies were performed under protocols and procedures approved by the Bloomington Institutional Animal Care and Use Committee and in accord with National Institutes of Health (NIH) standards established by the Guidelines for the Care and Use of Experimental Animals and by the American Veterinary Medical Association. Mature (150–200 g) and immature (~23 days old and 40–45 g) female Sprague-Dawley rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Mature or immature animals were ovariectomized 3 wk or 5 days before an experiment, respectively. Rats were given animal chow and water ad libitum and maintained on a 12L:12D cycle, with lights-on at 0700 h. Animals (n = 4 per group) were injected s.c. in the periscapular area with 40 µg/kg body weight (BW) of estradiol-17ß, 40 mg/kg BW of progesterone (Sigma Chemical Co., St. Louis, MO), and 1 mg/kg BW tamoxifen (citrate salt; Sigma) or sesame oil vehicle (Sigma), and then sacrificed 0, 3, 6, 24, or 72 h later. The dosages of estradiol, progesterone, and tamoxifen were based on our own earlier work [34–36]. Progesterone-treated animals were included, because ovariectomy increases not only ER levels but progesterone receptor expression levels in the uterus as well [37]. Uteri were collected quickly and trimmed of extraneous connective tissues. One uterine horn from each animal was placed immediately in liquid nitrogen and stored at -80°C for isolation of total RNA for Northern blot analysis. The other uterine horn from each rat was placed in 4% paraformaldehyde in PBS overnight at 4°C, washed with ice-cold PBS, and incubated overnight with 30% sucrose in PBS at 4°C. Tissue was then placed in embedding medium (TBS tissue freezing medium; Triangle Biomedical Sciences, Durham, NC) in molds, frozen on the surface of liquid nitrogen, and stored at -80°C for use during in situ hybridization analysis.
Synthesis of cDNA and cRNA Probes
The constructs used in this study were a 2.3-kilobase (kb) SRC-1 fragment in pBluescript (rat SRC-1 in pCDNA; gift from Dr. T.-P. Yao, Dana-Farber Cancer Institute, Boston, MA), rat RAC3 (2.3 kb) in pCMX-F (gift from Dr. D. Chen, University of Massachusetts Medical School, Worcester, MA), mouse RIP140 (7.2 kb) in pBluescript (gift from Dr. M. Parker, Imperial Cancer Research Fund, UK), rat p300 (9.0 kb) in pBluescript (gift from Dr. M. Brown, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA), and the 1-A cDNA (2 kb; gift from Dr. C.R. Lyttle, University of Pennsylvania, Philadelphia, PA). Rat GRIP1 and SPA were both in pBluescript. Full-length rat ER
cDNA (2 kb) in pGEM3Z (pFLER) was kindly supplied by Dr. M. Shupnik (University of Virginia Medical Center, Charlottesville). Rat c-fos cDNA (2.2 kb; gift provided by Dr. T. Curran, St. Jude's Research Hospital, Memphis, TN) was cloned into pBluescript II SK+ as described elsewhere [36]. For Northern blot analysis, cDNAs were digested with appropriate restriction enzymes and labeled with [32P]deoxycytidine 5'-[-32P]-triphosphate, triethylammonium salt using the Rediprime II kit (Amersham Life Science, Arlington Heights, IL). For in situ hybridization, 35S-labeled sense and antisense RNA probes were transcribed using the Promega Riboprobe Gemini Core System II transcription kit (Promega, Madison, WI) according to the manufacturer's protocol. The cDNAs were linearized using appropriate restriction enzymes. To reduce primary transcripts to 200 bases, an alkaline hydrolysis was performed [36, 38, 39], and the probes were purified, air-dried, dissolved in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris [pH, 8.0], 50 mM EDTA [pH, 8.0], 20 mM dithiothreitol, 10 mM Na3PO4, 1x Denhardt's; 10% dextran sulfate, and 0.5 mg/ml yeast tRNA), and stored at -80°C.
RNA Isolation and Northern Blot Analysis
Uteri were pooled according to treatment groups (n = 4 animals per time-point). Total cellular RNA was isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's protocol. As described elsewhere [35], tcRNA (15 µg) was fractionated on denaturing 1% agarose gels, transferred to Duralon-UV membrane (Stratagene, La Jolla, CA) by electrophoresis (2 h at 70 V) in TAE (40 mM Tris acetate [pH 8.0] and 2 mM Na2 EDTA). Membranes were cross-linked by ultraviolet light (Stratagene) and prehybridized at 60°C in Rapid-Hyb buffer (Amersham) for 30 min. Incubation continued for 2.5 h after the addition of 1 x 106 cpm/µl [32P]-cDNA probes. The membranes were washed once at room temperature for 20 min in 2x sodium chloride, sodium citrate buffer (SSC) containing 0.1% SDS, then at 65°C for 15 min in 1x SSC and 0.1% SDS, and finally at 65°C in 0.5% SSC and 1% SDS for 15 min. The membranes were exposed to x-ray film at -80°C for 7–10 days with intensifying screens. The membranes were dehybridized for subsequent use at 65°C for 20 min in 6x SSC and 0.1% SDS containing 50% formamide. Northern blot experiments were repeated on two separate membranes. The optical densities of autoradiograms for coactivator and 1-A bands were measured and quantified using imaging densitometer and Molecular Analyst Software (G5670; Bio-Rad, Hercules, CA). Levels of coactivators were normalized to those of 1-A mRNA, which is a constitutively expressed gene in the rat uterus [40].
In Situ Hybridization
Uteri were cryosectioned (10 µm), and sections were then mounted on slides, fixed in 4% paraformaldehyde in 1x PBS, dehydrated, dried, and stored at -80°C. As described elsewhere [36, 38, 39], sections were refixed in 4% paraformaldehyde in PBS, treated with proteinase K, fixed again in 4% paraformaldehyde, treated with acetic anhydride, rinsed in water, dehydrated, and dried at room temperature. Next, 35S-labeled antisense and sense cRNA probes in hybridization buffer were heat-denatured and then added to tissue sections (15 µl/coverslip, probe concentration of 1 x 104 to 1 x 105 cpm/µl), coverslipped, and incubated at a hybridization temperature of 50–55°C for 16 h. Coverslips were removed, and sections were then washed and treated with RNases to remove unhybridized probe, dried, dipped in Ilford Nuclear Research Emulsion K5 (Polysciences, Warrington, PA), and stored at 4°C for 6–10 days.
RESULTS AND DISCUSSION
In the untreated (i.e., 0-h) rat uterus, constitutive expression of SRC-1, SPA, RAC3, RIP140, GRIP1, p300 and mRNAs was detected in ovariectomized mature and immature animals (Figs. 1 and 2). Rat 1-A was used as an internal control to monitor loading efficiency. SRC-1 was expressed as two high-molecular-weight transcripts of approximately 5.5 and 8.5 kb of approximately equal band intensity, and SPA was expressed as a single mRNA of approximately 0.8 kb. Single, high-molecular-weight mRNAs of approximately 7.5, 7.5, 9, and 9.5 kb were detected for RAC3, RIP140, GRIP1, and p300, respectively. In the mouse, uterine expression of similar-size mRNAs encoding GRIP1/TIF2 and RAC3/p/CIP has been reported [41]. The sizes of the SRC1 and p300 mRNA that we observed in the rat uterus were consistent with those in a recent report concerning expression of coactivator transcripts in a variety of rat tissues, including endocrine and reproductive tract organs [23].
|
To study the effects of estradiol on coactivator mRNAs in vivo, ovariectomized female rats received a single s.c. injection of estradiol and were sacrificed 0, 3, 6, 12, and 24 h after treatment. Expression c-fos mRNA was used as a positive control in these experiments. No constitutive c-fos expression was detected in untreated uteri, and estradiol treatment increased c-fos expression by 3 h after hormone administration (Fig. 1). The constitutive mRNA levels of SPA, SRC-1, GRIP1, RIP140, RAC3, and p300 in the uterus remained unchanged after estradiol treatment of either ovariectomized mature or immature rats (Fig. 1). Based on normalization of the signal to the rat 1-A expression and densitometric evaluation, constitutive expression of these genes appears to be insensitive to estradiol in the rat uterus (data not shown). Constitutive levels of SRC-1, SPA, RAC3, RIP140, GRIP1, and p300 in the uterus were also insensitive to progesterone treatment of ovariectomized immature and mature rats (data not shown). Insensitivity of constitutive coactivator expression to estradiol has been recently reported in human endometrial cancer cells [24] as well as in the rat hypothalamus and pineal gland [23]. However, coactivator expression is estrogen-inducible in human breast cancer cells [24], and estradiol down-regulates coactivator expression in the rat pituitary gland [23]. Interestingly, in that latter study [23], p300 mRNA levels in the pituitary were not altered by estradiol, further suggesting a differential regulation of different coactivators within the same cell type. Transcriptional activation by steroid/thyroid hormone receptors may also involve the displacement of corepressors such as SMRT, which are proteins that associate with unliganded nuclear receptors and repress receptor activity [42–45]. We could detect only very low levels of constitutive SMRT mRNA expression in the uterus by Northern blot analysis, and SMRT expression was unchanged after estradiol administration (unpublished observations). Constitutive SMRT mRNA levels have been reported in endometrial human cancer cell lines [24], and SMRT expression has been increased by estradiol in male rat pituitary, but not in hypothalamus and pineal, glands [23].
We observed a high constitutive level of SPA mRNA expression in the uterus. That SPA is a ribosomal protein might contribute to its high level of expression, but SPA protein has been detected in both the cytoplasm and nucleus of human cell lines [33]. To our knowledge, no known nuclear function of SPA has been reported [22, 33], but SPA has been reported to enhance the agonist activity of tamoxifen-occupied ER [22]. However, SPA has no effect on transcription by ER when liganded by estradiol [22]. Thus, why this antagonistic-specific transcriptional coactivator is so highly expressed in the rat uterus is curious. We investigated if constitutive expression of SPA and other coactivators was sensitive to tamoxifen. Adult and immature ovariectomized female rats received a single s.c. injection of tamoxifen and were killed 0, 3, 6, 12, and 24 h after treatment. Levels of SPA, SRC-1, p300, RIP140, and GRIP1 mRNA in the uterus remained unchanged after tamoxifen treatment of adult rats (Fig. 2). Densitometric evaluation after normalization of the signal to the rat 1-A expression showed no difference in constitutive coactivator expression, suggesting that these genes are insensitive to tamoxifen. This contrasts with the effects of tamoxifen on the level of ER mRNA, which was dramatically reduced by 12–24 h (Fig. 2).
|
The uterus is a complex tissue comprised of several distinct compartments. Our studies using Northern blot analysis of whole uterine homogenates provided information regarding coactivator expression, but they did not provide information on the localization of mRNAs within the uterus. In situ hybridization was used to examine the expression of mRNAs encoding SPA, SRC-1, GRIP1, RIP140, and p300 genes in the uterine compartments. In addition, because distinct differences exist in the proliferative response of the endometrium to estradiol as animals mature, the expression pattern of coactivator mRNAs was studied in both the adult and the immature rat uterus. Only low background levels of sense RNA binding to uterine sections were detected for the coactivator probes (D, H, L, and P in Figs. 3, 4, and 5, C and F). When antisense SPA, SRC-1, GRIP1, RIP140, and p300 probes were used on uterine sections from untreated, ovariectomized mature and immature rats, silver grains were observed in all uterine compartments (columns 1–3 in Figs. 3 and 4 and columns 1 and 2 in Fig. 5). No cell type-specific pattern of coactivator expression was apparent; however, hybridization signals were more intense in the luminal and glandular epithelia compared with the stroma and myometrium, suggesting that specific ER coactivators in the uterine epithelia may contribute to the estrogen sensitivity of this uterine compartment. No sizeable induction of coactivator gene expression was observed after estradiol administration (columns 2 and 3 in Figs. 3 and 4 and column 2 in Fig. 5), which supports our results using Northern blot analysis. The difference between constitutive SPA expression in the epithelia was more distinct than that in the surrounding stroma and myometrium in the adult and juvenile rat uterus (Figs. 3, A–C, and 4, A–C). In situ hybridization studies repeatedly revealed subtle differences in the expression of some uterine coactivators after estradiol treatment. For example, the pattern of SRC-1 expression was more distinct at 6 h after estradiol treatment compared with that at 0 h in the adult rat uterus (Fig. 3, E and F), and GRIP1 expression in the luminal epithelium of the juvenile uterus appeared to be more distinct after estradiol treatment (Fig. 4, I and J). Expression of p300 also appeared to be more distinct in the uterine epithelia after administration of estradiol (Fig. 5, A, B, D, and E). Silver grains corresponding to coactivator mRNAs appeared to be more evenly distributed by 24 h after estradiol treatment compared with earlier time-points or with untreated rats (Figs. 3, D–P, and 4, D–P). At present, we are uncertain if these subtle changes after hormonal treatment represent an induction of uterine epithelial coactivators, perhaps in selected cells, or repression of stromal coactivator mRNA. We will pursue these studies with more sensitive techniques, including separation of various cell types and application of real-time polymerase chain reaction to more accurately monitor coactivator mRNA changes. We believe these studies will be important, because subtle changes in coactivator expression may serve as powerful signals for selective gene expression in various uterine compartments.
|
|
|
ACKNOWLEDGMENTS
The authors wish to thank Betsy Osborne and Douglas Nam for help with figure preparation.
FOOTNOTES
1 Supported by NIH grant CA74748 and the Bert Elwert Research Award to K.P.N. S.M.H. is supported by NIH grant HD-08615. R.M.B. is supported by U.S. Department of Defense grant DAMD 17-98-1-8011. 
2 Correspondence: Kenneth P. Nephew, Medical Sciences, Indiana University, School of Medicine, Jordan Hall, 1001 E. 3rd St., Bloomington, IN 47405-4401. FAX: 812 855 4436; knephew{at}indiana.edu
Accepted: March 8, 2000.
Received: June 4, 1999.
REFERENCES
This article has been cited by other articles:
|
|
 |
|
  T. J. Nakamura, M. T. Sellix, M. Menaker, and G. D. Block Estrogen directly modulates circadian rhythms of PER2 expression in the uterus Am J Physiol Endocrinol Metab, November 1, 2008; 295(5): E1025 - E1031. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mukherjee, S. M. Soyal, R. Fernandez-Valdivia, M. Gehin, P. Chambon, F. J. DeMayo, J. P. Lydon, and B. W. O'Malley Steroid Receptor Coactivator 2 Is Critical for Progesterone-Dependent Uterine Function and Mammary Morphogenesis in the Mouse. Mol. Cell. Biol., September 1, 2006; 26(17): 6571 - 6583. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. C. Lim, L. Li, Z. Desta, Q. Zhao, J. M. Rae, D. A. Flockhart, and T. C. Skaar Endoxifen, a Secondary Metabolite of Tamoxifen, and 4-OH-Tamoxifen Induce Similar Changes in Global Gene Expression Patterns in MCF-7 Breast Cancer Cells J. Pharmacol. Exp. Ther., August 1, 2006; 318(2): 503 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. N Kelly, T J. McKenna, and L. S Young Coregulatory protein-orphan nuclear receptor interactions in the human adrenal cortex J. Endocrinol., July 1, 2005; 186(1): 33 - 42. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Durrer, K. Maerkel, M. Schlumpf, and W. Lichtensteiger Estrogen Target Gene Regulation and Coactivator Expression in Rat Uterus after Developmental Exposure to the Ultraviolet Filter 4-Methylbenzylidene Camphor Endocrinology, May 1, 2005; 146(5): 2130 - 2139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G.-S. Lee, K.-C. Choi, H.-J. Kim, and E.-B. Jeung Effect of Genistein As a Selective Estrogen Receptor Beta Agonist on the Expression of Calbindin-D9k in the Uterus of Immature Rats Toxicol. Sci., December 1, 2004; 82(2): 451 - 457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Smith and B. W. O'Malley Coregulator Function: A Key to Understanding Tissue Specificity of Selective Receptor Modulators Endocr. Rev., February 1, 2004; 25(1): 45 - 71. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. A. Molenda, C. P. Kilts, R. L. Allen, and M. J. Tetel Nuclear Receptor Coactivator Function in Reproductive Physiology and Behavior Biol Reprod, November 1, 2003; 69(5): 1449 - 1457. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Jalabi, M. Cerghet, R. P. Skoff, and M. S. Ghandour Detection of Oligodendrocytes in Tissue Sections Using PCR Synthesis of Digoxigenin-labeled Probes J. Histochem. Cytochem., July 1, 2003; 51(7): 913 - 919. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. J. Tarleton, T. D. Braden, A. A. Wiley, and F. F. Bartol Estrogen-Induced Disruption of Neonatal Porcine Uterine Development Alters Adult Uterine Function Biol Reprod, April 1, 2003; 68(4): 1387 - 1393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Wieser, C. Schneeberger, G. Hudelist, C. Singer, C. Kurz, F. Nagele, C. Gruber, J.C. Huber, and W. Tschugguel Endometrial nuclear receptor co-factors SRC-1 and N-CoR are increased in human endometrium during menstruation Mol. Hum. Reprod., July 1, 2002; 8(7): 644 - 650. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fan, X. Long, J. A. Bailey, C. A. Reed, E. Osborne, E. A. Gize, E. A. Kirk, R. M. Bigsby, and K. P. Nephew The Activating Enzyme of NEDD8 Inhibits Steroid Receptor Function Mol. Endocrinol., February 1, 2002; 16(2): 315 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Leonardsson, M. A. Jacobs, R. White, R. Jeffery, R. Poulsom, S. Milligan, and M. Parker Embryo Transfer Experiments and Ovarian Transplantation Identify the Ovary as the Only Site in Which Nuclear Receptor Interacting Protein 1/RIP140 Action Is Crucial for Female Fertility Endocrinology, February 1, 2002; 143(2): 700 - 707. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
