Expression of transforming growth factor-beta 1 messenger ribonucleic acid and the modulation of deoxyribonucleic acid synthesis by transforming growth factor-beta 1 in human endometrial cells

OBJECTIVE: The purpose of this study was (1) to evaluate the potential sites of transforming growth factor-beta 1 synthesis in human endometrium by analyzing separated endometrial glands and stromal cells for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis of total ribonucleic acid and (2) to investigate the effects of transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelial and stromal cells in culture. STUDY DESIGN: Endometrial glands and stroma from proliferative and secretory endometrium were isolated after collagenase treatment of endometrial tissue minces and were analyzed for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis. We studied the effects of estradiol-17 beta and transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelium and transforming growth factor-beta 1 on stromal cells in culture by evaluating tritiated thymidine incorporation into trichloroacetic acid-precipitable material. RESULTS: Transforming growth factor-beta 1 messenger ribonucleic acid was detected for Northern analysis in separated endometrial stromal cells in levels that were greatest during the secretory phase and in greater levels than in epithelial cells from that same tissue. Transforming growth factor-beta 1 messenger ribonucleic acid in glandular epithelium in culture was not increased to detectable levels by treatment with transforming growth factor-beta 1. Deoxyribonucleic acid synthesis in endometrial glandular epithelium was inhibited by transforming growth factor-beta 1, but transforming growth factor-beta 1 stimulated deoxyribonucleic acid synthesis in endometrial stromal cells in culture. After treatment for 5 days with estradiol-17 beta (10(-8) mol/L), deoxyribonucleic acid synthesis in endometrial glands in culture was decreased by 40%. Transforming growth factor-beta 1 (1 ng/ml) did not alter this effect of estradiol-17 beta on deoxyribonucleic acid synthesis. CONCLUSIONS: Transforming growth factor-beta 1 acts to decrease deoxyribonucleic acid synthesis in epithelial cells and to increase it in stromal cells isolated from human endometrium and maintained in monolayer culture. Transforming growth factor-beta 1, potentially of stromal cell origin, could participate in the regulation of endometrial cell proliferation and differentiation in vivo.     

Regulation of cyclooxygenase-2 and prostaglandin F synthase gene expression by steroid hormones and interferon-tau in bovine endometrial cells

Estradiol (E2) and progesterone are responsible for regulating PG synthesis in the endometrium during the estrous cycle and interferon-tau (IFN-tau) alters PG synthesis during early pregnancy in ruminants. In this study, we examined the effects of these steroid hormones and recombinant bovine IFN-tau (rbIFN-tau) on PG production and on cyclooxygenase-2 (COX-2) and PG F (PGF) synthase (PGFS) gene expression in isolated endometrial cells. E2 decreased both PGF2alpha and PG E2 (PGE2) whereas progesterone increased PGF2alpha secretion in epithelial cells. Steroid hormones had no effect on PG production in stromal cells. rbIFN-tau attenuated both PGF2alpha and PGE2 production in epithelial cells and enhanced their production, and the ratio of PGE2 to PGF2alpha, in stromal cells. Northern blot analysis showed that E2 and rbIFN-tau decreased COX-2 messenger RNA (mRNA) levels in epithelial cells. Conversely, rbIFN-tau increased COX-2 mRNA in stromal cells. Furthermore, rbIFN-tau decreased PGFS mRNA in both cell types and this was associated with the increase in PGE2/PGF2alpha ratio. These results show that the regulation of PG synthesis by steroid hormones is different in endometrial epithelial and stromal cells in vitro. The attenuation of PGF2alpha secretion from epithelial cells and increased PGE2 production in stromal cells by rbIFN-tau are modulated by steroid hormones.     

17Beta-estradiol potentiates the stimulatory effects of progesterone on cadherin-11 expression in cultured human endometrial stromal cells

Cadherin-11 (cad-11) is a novel member of the cadherin gene superfamily of calcium-dependent cell adhesion molecules. To date, the factors capable of regulating this cell adhesion molecule remain poorly characterized. We have recently determined that cad-11 expression in the human endometrium is tightly regulated during the menstrual cycle. The spatiotemporal expression of cad-11 in the stromal cells of the human endometrium during the menstrual cycle suggests that gonadal steroids regulate the expression of this endometrial cell adhesion molecule. In view of these observations, we have examined the ability of progestins, estrogens, and androgens, alone or in combination, to regulate cad-11 expression in isolated human endometrial stromal cells using Northern and Western blot analyses. In these studies, we have determined that progesterone, but not 17beta-estradiol or dihydrotestosterone, is capable of regulating cad-11 messenger RNA and protein expression levels in isolated endometrial stromal cells. In addition, 17beta-estradiol, but not dihydrotestosterone, was capable of potentiating the stimulatory effects of progesterone in a dose-dependent manner. Taken together, these observations suggest that both 17beta-estradiol and progesterone are required for maximal cad-11 expression in human endometrial stromal cells in vitro.     

Expression of telomerase activity in human endometrium is localized to epithelial glandular cells and regulated in a menstrual phase-dependent manner correlated with cell proliferation

Telomerase activity is observed in most malignant tumors and germ cells, whereas normal somatic cells usually do not express it. Human endometrium is composed of glandular and stromal components and exhibits dramatic changes in proliferative activity during the menstrual cycle, which is exquisitely regulated by estrogen function. We previously reported that normal human endometrium expresses telomerase activity. However, it remains unclear which of the above components are the major sources of telomerase activity and how levels of telomerase activity are regulated over the menstrual cycle. Quantitative analysis of telomerase activity revealed that it changes dramatically over the course of the menstrual cycle and is strictly regulated in a menstrual-phase-dependent manner. Maximal activity equivalent to that in endometrial cancer was present in late proliferative phase, and minimal activity in late secretory phase. Postmenopausal endometrium and endometrium treated with anti-estrogen drugs exhibited decreased telomerase activity. Testing isolated epithelial glandular cells and stromal cells, we found that telomerase activity was localized to epithelial glandular cells. In situ RNA hybridization analysis also revealed epithelial-specific expression of human telomerase RNA. In vitro analysis of cultured epithelial cells demonstrated that telomerase activity is correlated with epithelial proliferation but not affected by estrogen treatment. These findings suggest that expression of telomerase activity is specific to epithelial cells and linked to cell proliferative status. The involvement of estrogen in telomerase regulation remains to be elucidated.     

Ovarian steroid-modulated stromelysin-1 expression in human endometrial stromal and decidual cells

This study examined steroid-regulated expression of the metalloproteinase stromelysin-1 in primary human endometrial stromal and decidual cells. Immunoblot analysis using a specific polyclonal antibody against stromelysin-1 revealed that the progestin medroxyprogesterone acetate (MPA) produced a time-dependent reduction in a band at 50,000 mol wt. Although the cells were refractory to estradiol (E2) alone, E2 plus MPA further reduced the intensity of this stromelysin-1 zone. By 6 days of incubation, MPA inhibited levels of secreted stromelysin-1 by one third, and E2 plus MPA inhibited stromelysin-1 levels by two thirds compared with the control values. This differential responsiveness of the stromal cells to the two steroids is reported for several biochemical end points of decidualization. Northern analysis indicated pronounced inhibition of stromelysin-1 messenger ribonucleic acid (mRNA) by E2 plus MPA over a concentration range that simulated circulating progesterone levels of the luteal phase (10(-8) mol/L) through pregnancy (10(-6) mol/L). After suppression of stromelysin-1 expression in the stromal cell monolayers by E2 plus MPA, steroid withdrawal led to a several-fold enhancement of stromelysin-1 mRNA by 4 days and of the stromelysin-1 protein by 7 days. Given its actions in degrading several extracellular matrix components and activating other MMP zymogens, steroid withdrawal-enhanced stromelysin-1 activity could mediate a proteolytic cascade that promotes the rapid tissue destruction and vascular disruption associated with menstruation. Stromelysin-1 expression by cultured decidual cells isolated from first trimester endometrium was also reduced by MPA and synergistically reduced by E2 plus MPA. As activation of the 92-kilodalton gelatinase/type IV collagenase, a crucial mediator of trophoblast invasiveness, is stromelysin-1 dependent, reduced decidual stromelysin-1 production could help to limit trophoblast invasion.     

Biological mechanisms underlying the clinical effects of RU 486: modulation of cultured endometrial stromal cell stromelysin-1 and prolactin expression

During in vitro decidualization of human endometrial stromal cells (HESCs), medroxyprogesterone acetate (MPA) inhibits expression of the potent extracellular matrix (ECM)-degrading protease stromelysin-1 (MMP-3), but enhances PRL expression. Consistent with its priming role in vivo, estradiol (E2) augments these effects. In the current study, immunoblot analysis revealed that coincubation with 10(-6) M RU 486 blocked the inhibition in HESC-secreted MMP-3 levels (50,000 mol wt) evoked by 10(-8) M E2 + 10(-7) M MPA. Although MPA can act as a glucocorticoid, the HESCs were refractory to 10(-7) M dexamethasone added alone or with E2. Because E2 elevates progesterone but not glucocorticoid receptor levels, MPA and RU 486 control MMP-3 expression as a progestin and antiprogestin, respectively. To study RU 486 involvement in steroid withdrawal leading to menstruation, HESCs were decidualized during 10 days incubation with E2 + MPA, and parallel cultures were kept in E2 + MPA or withdrawn to either control or RU 486-containing medium. Compared with E2 + MPA-suppressed HESCs, increases in levels of secreted MMP-3 (2.0-fold), and its 2.1-kilobase messenger RNA (10-fold) were observed in HESCs after 4 days of withdrawal to control medium, with much greater increases seen in RU 486-containing medium (10-fold protein, 100-fold messenger RNA). Previously, we showed that RU 486 up-regulated E2 + MPA-inhibited plasminogen activator expression in the cultured HESCs. Extrapolation of these in vitro observations to endometrial events following RU 486 administration suggests that coordinate enhancement of MMP-3 and plasminogen activator expression promotes proteolysis of the stromal/decidual ECM, which leads to endometrial sloughing. Moreover, destabilization of endometrial microvessels resulting from degradation of their surrounding ECM is consistent with the heavy menstrual bleeding stemming from RU 486 administration. However, in contrast to the marked RU 486-initiated reversal of MMP-3 expression, RU 486 did not significantly reverse E2 + MPA-enhanced PRL secretion by the cultured HESCs. Interestingly, decidual PRL, unlike decidual MMP-3, does not appear to play a role in menstruation. Interleukin-1 beta counteracted E2 + MPA-mediated inhibition of secreted MMP-3 levels, implying that leukocyte/trophoblast-derived cytokines can modulate steroid-regulated MMP-3 expression by stromal/decidual cells during menstruation and pregnancy.     

Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis

The human endometrium undergoes a complex process of vascular and glandular proliferation, differentiation, and regeneration with each menstrual cycle in preparation for implantation. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein that appears to play an important role in both physiological and pathological neovascularization. To investigate whether VEGF may regulate human endometrial angiogenesis, we examined VEGF messenger ribonucleic acid (mRNA) and protein throughout the menstrual cycle and studied the regulation of VEGF by reproductive steroids in isolated human endometrial cells. By ribonuclease protection analysis, VEGF mRNA increased relative to early proliferative phase expression by 1.6-,2.0-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium, respectively. In histological sections, VEGF mRNA and protein were localized focally in glandular epithelial cells and more diffusely in surrounding stroma, with greatest VEGF expression in secretory endometrium. Consistent with these in vivo results, the treatment of isolated human endometrial cells with estradiol (E2), medroxyprogesterone acetate (MPA), or E2 plus MPA significantly increased VEGF mRNA expression over the control value by 3.1-, 2.8-, and 4.7-fold, respectively. The VEGF response to E2 was rapid, with steady state levels of VEGF mRNA reaching 85% maximum 1 h after the addition of steroid. E2 also caused a 46% increase in secreted VEGF protein, and the combination of E2 and MPA caused an 18% increase. VEGF expression in endometriosis, an angiogenesis-dependent, estrogen-sensitive disease was similar to that seen in eutopic endometrium. Peritoneal fluid concentrations of VEGF were significantly higher in women with moderate to severe endometriosis than in women with minimal to mild endometriosis or no disease. VEGF, therefore, may be important in both physiological and pathological angiogenesis of human endometrium, as it is an estrogen-responsive angiogenic factor that varies throughout the menstrual cycle and is elevated in women with endometriosis.     

Transforming growth factor-beta inhibits progesterone-induced enkephalinase expression in human endometrial stromal cells

The specific activity of enkephalinase in endometrial tissue of nonpregnant ovulatory women is correlated in a highly significant, positive manner with the plasma level of progesterone. The specific activity and levels of enkephalinase messenger ribonucleic acid and immunoreactive protein also are increased in human endometrial stromal cells in culture by treatment with a synthetic progestin, medroxyprogesterone acetate (MPA), in a time- and dose-dependent manner. From an analysis of the temporal relationship between the specific activity and half-life of enkephalinase in endometrial tissue and the level of progesterone in plasma, it appeared highly likely that some mechanism, in addition to progesterone withdrawal, was operative to reduce enkephalinase activity in endometrium during the late luteal phase of the ovarian cycle before progesterone levels had declined below those known to be effective for progesterone action. In stromal cells previously (and concurrently) treated with MPA (10(-9) mol/L), the addition of transforming growth factor-beta 1 (TGF beta 1) or TGF beta 2 (1 ng/mL) to the medium caused a decrease in enkephalinase specific activity despite the continued presence of MPA. The half-life of enkephalinase (activity) in stromal cells treated with MPA plus TGF beta 1 was 2.8 days, which is similar to the computed half-life for enkephalinase in endometrial tissue during the mid- to late secretory phase of the endometrial cycle (2.5 days). Simultaneous treatment of endometrial stromal cells with MPA (10(-9) mol/L) and TGF beta 1 (1 ng/ mL) prevented the progestin-induced increase in enkephalinase specific activity and immunoreactive enkephalinase protein. Thus, TGF beta acts to oppose the progesterone-induced increase in enkephalinase expression in endometrial stromal cells, even in the continued presence of MPA.     

Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed-PCR

Angiogenesis is an essential component of endometrial regeneration after menses in preparation for implantation. Vascular endothelial growth factor (VEGF) is a secreted angiogenic peptide with mitogenic activity specific for endothelial and trophoblast cells. VEGF-immunoreactivity was detected in glandular epithelium throughout the menstrual cycle by immunohistochemistry, but, showed cyclic variation in the stroma and the blood vessels. During the early proliferative phase, strong staining was seen in the glandular epithelial cells while staining in the stroma was confined to a subpopulation of stromal cells and endometrial blood vessels appeared negative. In contrast, very intense staining of the endometrial stromal cells was seen in the mid proliferative endometrium possibly due to increased synthesis of VEGF by oestrogen. In the late proliferative endometrium, staining was seen in the endothelial cells and the perivascular stromal cells around the endometrial blood vessels. The greatest degree of immunostaining of stromal cells was observed in the mid to late proliferative endometrium. Throughout the secretory phase no staining was seen around the endometrial blood vessels and staining of endometrial stromal cells was confined to early secretory endometrium. In the late secretory endometrium only the glands were positive to VEGF antibody. The observed increase in the immunostaining of stroma suggests increased production of VEGF from early to mid and late proliferative endometrium which parallels the increase in the oestradiol levels in the proliferative phase of the menstrual cycle. It is proposed that VEGF may serve as a paracrine mediator of the effects of ovarian steroids on endometrial vascular development.     

Localization and sex steroid regulation of androgen receptor gene expression in rhesus monkey uterus

OBJECTIVE: To characterize the cellular sites and hormonal regulation of uterine androgen receptor gene expression in the monkey. METHODS: Ovariectomized rhesus monkeys (five in each group) were treated with placebo (the control group), estradiol (E2), E2 plus progesterone, or E2 plus testosterone by sustained-release pellets administered subcutaneously. After 3 days of treatment, uteri were removed and uterine sections were analyzed by in situ hybridization for androgen receptor messenger RNA (mRNA). RESULTS: Androgen receptor mRNA was detected in endometrial stromal cells and myometrial smooth muscle cells, with lesser expression in endometrial epithelial cells. Both E2 and E2 plus progesterone treatment doubled androgen receptor mRNA levels in stromal cells (P < .01), whereas E2 plus testosterone treatment increased stromal androgen receptor mRNA levels by about five-fold (P < .001) compared with placebo treatment. In the endometrial epithelium, E2 alone did not increase androgen receptor mRNA levels significantly. However, the E2 plus progesterone and E2 plus testosterone treatments increased epithelial androgen receptor mRNA levels by 4.3 and 5 times, respectively (P = .008 and P < .002, respectively). Androgen receptor mRNA was distributed homogeneously in smooth muscle cells across the myometrium. Estradiol treatment alone did not increase myometrial androgen receptor mRNA levels significantly, but the E2 plus progesterone and E2 plus testosterone treatments increased myometrial androgen receptor mRNA levels by 1.8 and 2 times, respectively (P = .001 and P < .001, respectively). CONCLUSION: Androgen receptor gene expression was detected in all uterine cell compartments where it was subject to significant sex steroid regulation. The fact that androgen receptor mRNA levels were consistently up-regulated by a combined E2 plus testosterone treatment while E2 treatment alone had little or no effect shows that a collaborative action of E2 and testosterone enhances androgen receptor expression in the monkey uterus.     

Nuclear envelope acts as a calcium barrier in C6 glioma cells

This study evaluated the expression of the corticosteroid-metabolizing enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) during in vitro decidualization of human endometrial stromal cells. The cultured stromal cells displayed both NADP(+)-dependent (type 1) and NAD(+)-dependent (type 2) 11 beta HSD activities under basal conditions. Although the cells did not respond to estradiol (E2) added alone, catalytic levels of both isoforms were enhanced by medroxyprogesterone acetate (MPA) and further enhanced by E2 plus MPA. Type I messenger RNA (mRNA) was undetected by Northern analysis of total RNA, but was evident as a 1.5-kilobase band in polyadenylated selected RNA from E2- plus MPA-treated cultures. Use of RT-PCR to augment the sensitivity of mRNA detection revealed the presence of type I mRNA as a faint band in the MPA-treated cultures and as an intense band in the E2- plus MPA-treated cultures. Thus, type I mRNA is present as a low abundance message in the cultured stromal cells whose steady state levels parallel progestin-enhanced enzyme activity. As the expression of several progestin-regulated decidualization markers is also augmented by E2, the results of the present study reveal a correlation between enhanced 11 beta HSD expression and the decidualization reaction. Time-course measurements indicated that elevated 11 beta HSD expression is an early event in the decidualization response, which precedes E2- plus MPA-enhanced PRL production by several days. Clear dose-response effects on both type 1 and type 2 11 beta HSD activities were obtained in cells incubated with 10(-8) mol/liter E2 added together with MPA at concentrations that approximated circulating progesterone levels from the luteal phase (10(-9) mol/liter) through pregnancy (10(-7) mol/liter). Corticosteroids are thought to exert toxic and teratogenic effects on the implanting embryo and could influence trophoblast invasion by regulating extracellular matrix turnover. Therefore, the novel finding that decidualization involves marked enhancement of the corticosteroid-metabolizing capacity of stromal cells suggests a mechanism by which decidual cells could affect the health and invasiveness of implanting trophoblastic cells.     

Identification and characterization of cytosolic sulfotransferases in normal human endometrium

Understanding the factors which alter estrogen metabolism and activity in endometrial tissue is important because unopposed estrogen stimulation is an important risk factor in the development of endometrial carcinoma. The cyclic progression of the endometrium through proliferative and secretory phases is normally under the control of the ovarian hormones beta-estradiol (E2) and progesterone. One mechanism by which progesterone inhibits the activity of E2 in secretory endometrium is by elevating the degree of E2 sulfation, thereby reducing its ability to bind to the estrogen receptor and elicit a cellular response. Our laboratories have investigated the cytosolic sulfotransferases (STs) found in biopsies of both proliferative and secretory endometrium obtained from five normal pre-menopausal women who were not taking any drugs or steroids. Two of the human cytosolic STs were detected in human endometrial tissues. The phenol-sulfating form of phenol ST (P-PST) was found at varying levels in cytosol from both proliferative and secretory endometrium in all of the women studied but with no consistent correlation to the phase of the menstrual cycle. In contrast, estrogen ST (EST) was not detected in the proliferative endometrial cytosol of any of the women studied but was consistently found in all of the secretory endometrial cytosols. The presence and levels of these STs was confirmed by ST activity studies, immunoblot analysis and Northern blot analysis. These results indicate that the expression of EST in human endometrial tissues varies with the phase of the menstrual cycle and is most likely regulated by progesterone secreted from the ovaries.     

Effects of sex steroids on silver stained proteins of nucleolar organizer regions (Ag-NOR) in the rabbit uterus

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.     

Cell-cell interactions influence oligosaccharide modifications on mucins and other large glycoproteins

OBJECTIVE: To investigate human endometrial interleukin-6 (IL-6) expression and effects thereon by IL-1 beta. DESIGN: Prospective. SETTING: Academic medical center. PATIENT(S): Endometrial biopsy specimens from normal volunteers (n = 20) at four specific menstrual stages were used for in vivo study. Endometrial specimens for in vitro study were obtained from patients (n = 19) undergoing gynecologic surgery. INTERVENTION(S): Time and dose-response treatment of endometria with IL-1 beta in tissue culture. MAIN OUTCOME MEASURE(S): In vivo IL-6 messenger RNA expression by Northern analysis and in vitro endometrial IL-6 protein production by assay of the conditioned media. RESULT(S): Midsecretory and late secretory phase endometria expressed more IL-6 messenger RNA than late proliferative phase endometria in vivo. Similarly in vitro, in pg/mg endometrium per hour secretory endometria IL-6 protein production, 25.7 +/- 7.1 (mean +/- SEM), exceeded that of proliferative endometria, 4.7 +/- 1.0. With IL-1 beta treatment, secretory endometria IL-6 protein production exceeded that of proliferative endometria. Interleukin-1 beta stimulated endometrial IL-6 protein production in time- and dose-dependent manners. CONCLUSION(S): Human endometrial IL-6 expression varies with the menstrual cycle, occurs more highly in secretory endometria, and in vitro is stimulated by interleukin-1 beta. Human endometrial IL-6 may therefore mediate some actions of IL-1 beta involving the endometrium and trophoblast.