| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Integrative Biology and Pharmacology (L.D., G.L.S., D.S.L.-M., G.M.S., P.J.A.D.), University of Texas Houston Health Science Center, Houston, Texas 77030; Department of Pathology (R.B.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030; and Wyeth Research (J.H.P.), Philadelphia, Pennsylvania 19087
Address all correspondence and requests for reprints to: Dr. Peter J. A. Davies, University of Texas Houston Health Science Center, Department of Integrative Biology and Pharmacology, MSB 5.104, 6431 Fannin Street, Houston, Texas 77030. E-mail: peter.j.davies{at}uth.tmc.edu.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Studies in experimental animals have shown that retinoids, particularly all-trans retinoic acid (RA), may play an important role in regulating the effects of estrogens on the endometrium. Studies with vitamin A-deficient rats demonstrated that physiologic levels of RA suppress the endometrial hyperplasia and metaplasia associated with chronic estrogen administration (5). In immature ovariectomized rats, pharmacologic doses of RA suppressed estrogen-induced endometrial stromal cell proliferation (6).
Biologically active retinoids, such as all-trans RA, are generated in tissues by the enzymatic oxidation of vitamin A (all trans-retinol). RA production is regulated by the activity of specific dehydrogenases that catalyze the sequential oxidation of retinol to retinaldehyde and RA. There are three distinct retinaldehyde dehydrogenases (RALDH 1,2 3) that catalyze the oxidation of retinaldehyde into RA (7). RA, generated within a particular cell, may interact with specific nuclear RA receptors (RARs , ß or 
) present within that cell to regulate gene expression (autocrine activity), or the RA may be secreted to regulate gene expression in adjacent cells (paracrine activity). The balance between the autocrine and paracrine activity of RA depends on the level of expression of an intracellular retinoid-binding protein, cytoplasmic retinoic acid-binding protein II (CRABP2), that suppresses the autocrine activity of RA and facilitates its export (8). RA is inactivated by a P450, retinoic acid 4-hydroxylase (RAOH or CYP26A1) (9). The level of expression of CYP26A1 plays a large role in determining whether cells adjacent to RA-producing cells are sensitive or resistant to the paracrine activity of the retinoids.
Some studies have suggested that RA metabolism in the rodent uterus is subject to estrogen regulation. In cultured rat endometrial cells, exogenous estrogen increases the production of RA (10). In mouse uterus, the expression of RALDH1, RALDH2, and CYP26A1 (three critical enzymes of RA metabolism) has been shown to fluctuate during an estrous cycle, suggesting a possible link to cyclic alterations in estrogenization (11). Although there is no direct evidence that estrogen increases RA production in the uterus in vivo, it has been noted that administration of pregnant mare serum gonadotropin (PMSG), a pharmacological preparation that increases estrogen levels, induces RA production in the rat uterus (10).
Given the important links between the effects of estrogens and retinoids on the normal physiology of the endometrium in rodents, there has been obvious interest in the contribution that these regulators might make to the control of uterine function in humans. To address this issue, we examined the effects of estrogen administration to postmenopausal women on the expression of enzymes involved in RA metabolism (RALDH1, RALDH2, RALDH3, and CYP26A1), the RARs and two retinoid-regulated genes, CRABP2 and tissue transglutaminase (tTG). To determine whether physiological levels of endogenous estrogen also regulates RA production and signaling, we measured the expression of these transcripts in the endometria of premenopausal women at different stages of the menstrual cycle. The results we obtained demonstrate that estrogens coordinately regulate the production and signaling of RA in the human endometrium.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The RNA used for the analysis of estrogen-regulated gene expression in postmenopausal women was obtained from a randomly selected subset (n = 30) of a large group of healthy postmenopausal women (n = 210) participating in a clinical trial of estrogen replacement therapy (ERT). These 210 women were randomly divided into three groups receiving one of the following three treatments: 1) placebo; 2) conjugated estrogens (2:1, wt/wt) of estrone and equilin sulfate (EES; 0.625 mg/d, Wyeth Research, Philadelphia, PA); or 3) Premarin (0.625 mg/d; Wyeth Research) for 3 months under conditions approved by the Human Ethics Committee of Escola Paulista de Medicina Universidade Federal de São Paulo, Brazil. Endometrial biopsies were obtained at the end of the 3-month treatment.
Tissues were frozen in liquid nitrogen and stored at -80 C. Frozen tissues were homogenized in TriReagent (Molecular Research Center, Inc., Cincinnati, OH). RNA was precipitated with isopropanol, applied to RNeasy spin columns (QIAGEN, Valencia, CA), eluted, and treated with RNase-free DNase for 30 min at 37 C, followed by heat inactivation at 75 C and storage at -70 C. Aliquots of RNA from the placebo, EES, and Premarin treatment groups (each contains 10 individuals) were used for the studies described here. As shown in Table 1
, the three groups of women were similar in age, time since last menstrual period, serum estradiol, and FSH concentrations before the treatments.
|
Reverse transcription and real-time quantitative PCR
Aliquots (100 ng) of each RNA were reverse transcribed in quadruplicate (including a no reverse transcriptase control) with 300 nM assay-specific reverse primer, 4 mM MgCl2, 500 µM deoxynucleotide triphosphates, and 10 U Muloney murine leukemia virus Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) at 50 C for 30 min, followed by 72 C for 5 min. Forty microliters of PCR mix containing 1 x PCR buffer, 300 nM specific forward and reverse primers, 4 mM MgCl2, Taq DNA polymerase, and 100 nM fluorogenic probe were added to each 10-µl reverse transcription reaction. Amplification was performed by use of the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) at 95 C for 1 min, followed by 40 cycles of a 12-sec step at 95 C and a 1-min step at 60 C. The primer and probe sequences for each assay are listed in Table 2. Synthetic RNA or single-strand DNA amplicon standards were serially diluted in water containing 100 ng/µl yeast tRNA (Invitrogen).
|
Statistical significance of differences between groups was calculated using an unpaired two-sample t test or ANOVA. Correlation between any two transcripts was evaluated by Pearson correlation analysis and confirmed by Spearman’s and Kendal’s tests. A P value less than 0.05 was considered significant.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To evaluate the effects of estrogen on RA metabolism in the human endometrium, we developed real-time quantitative PCR assays for the three major RA biosynthetic enzymes, RALDH 1, 2, and 3, and then used them to measure the levels of transcripts of these enzymes in RNA prepared from endometrial biopsies obtained from placebo- or ERT-treated subjects (Fig. 1, A–C). We used the housekeeping gene cyclophilin mRNA to normalize each sample for loading because its level does not significantly change with either Premarin or EES treatment for three months(data not shown). ERT, with either EES or Premarin, had no effect on the level of expression of RALDH1 but resulted in a marked induction (4-fold, P < 0.01) in the level of RALDH2 expression. The basal level of RALDH3 transcripts in the endometrial biopsies was much lower than either the RALDH1 or RALDH2 transcripts and was decreased significantly by either EES or Premarin treatment (P < 0.01).
|
). The absolute level of CYP26A1 mRNA in postmenopausal endometria was extremely low, only about 0.1% the level of RALDH2 mRNA, and ERT had no effect on its expression. Effects of ERT on the expression of RARs in postmenopausal endometria
RA exerts its functions through the activation of RARs (RAR, ß, and ). We measured the levels of RAR, RARß, and RAR transcripts in the endometria of ERT- and placebo-treated postmenopausal women (Fig. 2). In the placebo-treated subjects, the levels of RAR and RAR transcripts were generally comparable, but RARß mRNA was present at a lower level (about 10% of RAR). ERT with either EES or Premarin increased RAR expression 2-fold (P < 0.05); it had no effect on RAR expression and resulted in a 35–40% decrease in RARß mRNA.
|
CRABP2 and tTG are two genes whose expression highly correlates with the level of RA in vivo (10, 12). To assess the effects of estrogens on retinoid-regulated gene expression in the human endometrium, we measured the level of expression of both biomarkers in endometrial RNA obtained from postmenopausal women receiving placebo or ERT (Fig. 3). ERT significantly increased the expression of both biomarkers, approximately 3-fold.
|
and the level of expression of the two RA-regulated transcripts (CRABP2 and tTG) in the endometrium, we compared the level of expression of these genes in all the individual subjects in the placebo or two modes of ERT groups. As shown in Table 3, the expression of CRABP2 and tTG was highly correlated with both the expression of RALDH2 and RAR but not with that of ER. In the same individual subjects, the levels of RALDH2 and RAR were also highly correlated.
|
To determine the effect of endogenous estrogen on RA metabolism in the human endometrium, we then measured levels of RALDH1, RALDH2, and CYP26A1 transcripts in endometrial tissues obtained from premenopausal women who were at either proliferative or secretory phase of the menstrual cycle at the time of hysterectomy. As illustrated in Fig. 4, both the RALDH1 and RALDH2 transcripts were significantly higher in the estrogen-dominated proliferative phase, compared with the progesterone-dominated secretory phase. In sharp contrast, CYP26A1 mRNA level was about 20 times higher in the secretory phase than in the proliferative phase. When the abundance of these transcripts in the endometria of pre- and postmenopausal women was compared, the levels of RALDH1 and RALDH2 were similar in these two populations, but CYP26A1 mRNA in premenopausal endometria was more than 10 times more abundant than that in the postmenopausal endometria (Figs. 1 and 4).
|
To determine whether physiological levels of endogenous estrogen also regulates RARs in vivo in the normal human endometrium, we measured the levels of expression of RAR, RARß, and RAR in the endometria obtained from premenopausal women (Fig. 5). Among the three RARs, only RAR was differentially expressed during a menstrual cycle, with a significantly higher expression in the proliferative phase (P < 0.05).
|
To determine the effect of physiological levels of endogenous estrogen on retinoid-regulated gene expression, we measured the transcript levels of the RA biomarker genes CRABP2 and tTG in the proliferative and secretory phases of the menstrual cycle in the premenopausal endometria (Fig. 6). The CRABP2 mRNA level was significantly higher in the proliferative than secretory phase, and tTG was much higher in the secretory phase.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The molecular mechanism for the induction of RALDH2 by estrogens is currently unknown. There is a potential estrogen-responsive element in the 5'-flanking DNA of the mouse RALDH2 gene (13), but transfection studies with reporter gene constructs containing this region in a variety of estrogen responsive cell lines failed to show estrogen-dependent regulation (data not shown). Detailed experiments in the rodent models are currently in progress to delineate the molecular mechanism underlying the induction of RALDH2 expression by estrogen.
Although the very limited amounts of endometrial tissues available in these studies precluded the measurements of the levels of RALDH2 protein or RA production, studies in a variety of other biological systems have shown a close correlation between the levels of RALDH2 mRNA and protein and RA production. In developing mouse embryos, RALDH2 expression is associated with the temporal and spatial patterns of both RA synthesis and signaling (14, 15, 16). Targeted disrupting of the RALDH2 gene leads to embryonic death and phenotypes of serious vitamin A deficiency, which can be partially rescued by maternal administration of RA (17). This suggests that RALDH2 is the major enzyme involved in RA synthesis in vivo.
The biological effects of RA are mediated by the activation of nuclear RARs. In breast cancer cell lines, estrogens have been shown to increase RAR expression via the activation of an estrogen-responsive element within the RAR promoter (18). Our results extend this observation to the effects of estrogens in vivo in normal human endometrial tissues. The induction of RAR paralleled the induction of RALDH2 in individual subjects, suggesting that estrogen coordinately up-regulates both RA production and signaling. Although there is evidence that RA regulates RARß expression in cultured cells (19), the regulation of RARß expression in vivo is not well understood. In our study, estrogens induced a small but significant decrease in RARß expression in the postmenopausal endometrium. However, the level of RARß mRNA is much lower (only one tenth of that of RAR or RAR), and the impact of this small change on retinoid signaling is unclear.
In the postmenopausal endometrium, the level of CYP26A1 was extremely low and largely unaffected by estrogens. Mice in which the CYP26A1 gene has been deleted are extremely sensitive to low levels of RA and they die in utero from hypervitaminosis A (20, 21). The very low level of CYP26A1 in the endometrium makes it likely that the uterus is very sensitive to the level of endogenous RA production by RALDH2. If possible, it would be desirable to test this hypothesis by measuring the level of RA in the endometrial biopsies. However, large amounts of tissues are required for the detection of endogenous retinoids (22). Furthermore, the chemical and physical instability of retinoids in biological fluids also precludes these measurements in the clinical specimens that were the subject of this study. As an alternative, we measured the expression of two RA biomarker genes, CRABP2 and tTG, whose promoters contain well-characterized retinoic acid-responsive element (23, 24) and whose expression has been clearly linked to the level of endogenous RA in a number of biological systems including the rodent uterus (10, 12). ERT significantly increased the expression of both biomarkers, and the extent of induction in individual subjects correlated with the induction of both RALDH2 and RAR but not the level of ER. Although these biomarker data are correlative, they support the hypothesis that in human endometrium RA production and signaling is increased by estrogenization. These results are consistent with the observation that uteri of PMSG-treated rats show enhanced RA production (10).
In the premenopausal endometrium, we observed much higher levels of expression of RALDH2, CRABP2, and RAR in the estrogen-dominated proliferative phase of the menstrual cycle, suggesting that estrogen induction of RA production and signaling is not only a pharmacological response but also a physiological aspect of normal endometrial function. Similar changes in the expression of these transcripts have been reported. RALDH2 expression has been shown to fluctuate during a mouse estrous cycle (11); CRABP2 is elevated in the proliferative phase and depressed during the secretory phase of the menstrual cycle in both epithelial and stromal cells (25). However, there is controversy about the changes of RARs throughout the menstrual cycle. Fukunaka et al. (26) recently reported that endometrial RAR expression changes throughout the menstrual cycle, with higher level in the proliferative phase. But the results of Kumarendran et al. (27) suggested that with the possible exception of RARß, RARs (RAR and RAR) and retinoid X receptors do not change through the menstrual cycle. The complex interplay between different levels of estrogens and progesterones at different stages of the menstrual cycle in the premenopausal endometrium likely accounts for the above controversy. In this regard, our other experimental model, ERT in postmenopausal women, clearly has an advantage. ERT with either EES or Premarin significantly and selectively induces RAR expression, showing that estrogens up-regulate RAR in vivo in the human endometrium.
It has been demonstrated that RA strongly inhibits the decidualization of stromal cells in vitro (28). Thus, in the secretory phase, to facilitate the decidualization and subsequent implantation if pregnancy is achieved, the endometrial tissue needs to drastically reduce its RA level. Consistent with this notion, we observed that CYP26A1 expression was markedly up-regulated in the secretory phase in coordination with the decline of RALDH2 and RAR. We also observed in the secretory phase a marked up-regulation of tTG, which has been found necessary for decidualization (29). The up-regulation of CYP26A1 and tTG in the secretory phase suggests that they may be regulated by progesterone. Vermot et al. (11) found that endometrial CYP26A1 expression was strongly induced 24 h after human chorionic gonadotropin treatment, when the progesterone level was high and the endometrium had entered the secretory phase. As for the progesterone regulation of tTG, Fujimoto et al. (29) have observed that tTG mRNA is induced in a dose-dependent manner within 6 h after the addition of progesterone to cultured uterine stromal cells.
The induction of RA production and signaling in response to estrogens is likely to have important effects on hormone-responsive tissues. Retinoids, especially all-trans-RA, suppress estrogen-dependent proliferation of mammary (30, 31) and endometrial (32) cells. Retinoids can also effectively suppress estrogen-stimulated proliferation of a variety of breast cancer cell lines (33) and some endometrial carcinoma cell lines (34). On the other hand, many hormone-dependent malignancies are found to bear defects in the RA pathway. For example, defects in components of the RA pathway are involved in the tumorigenicity of human mammary epithelial cells (35, 36, 37), cervical carcinoma cells (38), and prostatic cells (39). Altered expression of enzymes and RA-binding proteins essential for RA biosynthesis and signaling is an important phenotypic change associated with the development of endometrial cancer (40, 41). Based on our results that estrogen coordinately up-regulates RA production and signaling in the human endometrium, we speculate that endogenous RA may play an important role in opposing the stimulatory effects of estrogen in the normal endometrium and potentially suppressing the development of endometrial hyperplasia and cancer.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received November 22, 2002.
Accepted February 14, 2003.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene in human breast carcinoma cells is mediated via an imperfect half-palindromic estrogen response element and Sp1 motifs. Cancer Res 55:4999–50062, ß2, and 2 in human breast cancer cells. FASEB J 10:1064–1070[Abstract]
and is abnormally down-regulated in cervical carcinoma cells. Cancer Res 57:1460–1467This article has been cited by other articles:
 |
|
 |
|
  M. Zaitseva, B. J. Vollenhoven, and P. A.W. Rogers Retinoids regulate genes involved in retinoic acid synthesis and transport in human myometrial and fibroid smooth muscle cells Hum. Reprod., May 1, 2008; 23(5): 1076 - 1086. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Hanifi-Moghaddam, B. Boers-Sijmons, A. H.A. Klaassens, F. H. van Wijk, W. F. Van Ijcken, P. Van der Spek, H. A.M. Verheul, H. J. Kloosterboer, C. W. Burger, and L. J. Blok Difference in signalling between various hormone therapies in endometrium, myometrium and upper part of the vagina Hum. Reprod., February 1, 2008; 23(2): 298 - 305. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. O. Burney, S. Talbi, A. E. Hamilton, K. C. Vo, M. Nyegaard, C. R. Nezhat, B. A. Lessey, and L. C. Giudice Gene Expression Analysis of Endometrium Reveals Progesterone Resistance and Candidate Susceptibility Genes in Women with Endometriosis Endocrinology, August 1, 2007; 148(8): 3814 - 3826. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. S. McCampbell, R. R. Broaddus, D. S. Loose, and P. J.A. Davies Overexpression of the Insulin-Like Growth Factor I Receptor and Activation of the AKT Pathway in Hyperplastic Endometrium. Clin. Cancer Res., November 1, 2006; 12(21): 6373 - 6378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Strowitzki, A. Germeyer, R. Popovici, and M. von Wolff The human endometrium as a fertility-determining factor Hum. Reprod. Update, September 1, 2006; 12(5): 617 - 630. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Deng, R. R. Broaddus, A. McCampbell, G. L. Shipley, D. S. Loose, G. M. Stancel, J. H. Pickar, and P. J.A. Davies Identification of a Novel Estrogen-Regulated Gene, EIG121, Induced by Hormone Replacement Therapy and Differentially Expressed in Type I and Type II Endometrial Cancer Clin. Cancer Res., December 1, 2005; 11(23): 8258 - 8264. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S Bauersachs, S E Ulbrich, K Gross, S E M Schmidt, H H D Meyer, R Einspanier, H Wenigerkind, M Vermehren, H Blum, F Sinowatz, et al. Gene expression profiling of bovine endometrium during the oestrous cycle: detection of molecular pathways involved in functional changes J. Mol. Endocrinol., June 1, 2005; 34(3): 889 - 908. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. T. Williams, M. Leyk, S. A. Wrighton, P. J. A. Davies, D. S. Loose, G. L. Shipley, and H. W. Strobel Estrogen Regulation of the Cytochrome P450 3A Subfamily in Humans J. Pharmacol. Exp. Ther., November 1, 2004; 311(2): 728 - 735. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-H. Li, B. Kakkad, and D. E. Ong Estrogen Directly Induces Expression of Retinoic Acid Biosynthetic Enzymes, Compartmentalized between the Epithelium and Underlying Stromal Cells in Rat Uterus Endocrinology, October 1, 2004; 145(10): 4756 - 4762. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-L. Gross, M. Adamczak, T. Rabe, N. A. Harbi, J. Krtil, A. Koch, P. Hamar, K. Amann, and E. Ritz Beneficial Effects of Estrogens on Indices of Renal Damage in Uninephrectomized SHRsp Rats J. Am. Soc. Nephrol., February 1, 2004; 15(2): 348 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Zhang, D. Smith, M. Yamamoto, L. Ma, and P. McCaffery The Meninges Is a Source of Retinoic Acid for the Late-Developing Hindbrain J. Neurosci., August 20, 2003; 23(20): 7610 - 7620. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
