Stromal cells of endometriosis fail to produce paracrine factors that induce epithelial 17beta-hydroxysteroid dehydrogenase type 2 gene and its transcriptional regulator Sp1: a mechanism for defective estradiol metabolism.

OBJECTIVE: In endometrium, stromal progesterone receptors mediate production of paracrine factors, which enhance binding of the transcription factor specific protein-1 to the promoter of the gene encoding the 17beta-hydroxysteroid dehydrogenase type 2 enzyme responsible for converting biologically active estradiol to estrone in epithelium. The objective of this study is to define the cellular defect responsible for the disruption of this stromal-epithelial interaction in endometriosis. STUDY DESIGN: We determined the effects of conditioned media generated from primary human eutopic endometrial stromal cells vs endometriotic stromal cells on Ishikawa malignant endometrial epithelial cells. RESULTS: Conditioned media from progestin-pretreated eutopic endometrial stromal cells but not endometriotic stromal cells significantly stimulated specific protein-1 protein levels, 17beta-hydroxysteroid dehydrogenase type 2 messenger RNA levels and promoter activity, and binding activity of specific protein-1 to the 17beta-hydroxysteroid dehydrogenase type 2 promoter region in Ishikawa cells. CONCLUSION: A stromal cell defect in endometriosis blocks formation of progesterone-dependent production of factors leading to 17beta-hydroxysteroid dehydrogenase type 2 deficiency and defective conversion of estradiol to estrone in epithelium.

Aromatase expression in uterine leiomyomata is regulated primarily by proximal promoters I.3/II.

CONTEXT: Uterine leiomyomata are common tumors that cause irregular uterine bleeding and pregnancy loss and depend on estrogen for growth. Aromatase catalyzes the conversion of androgens to estrogens. Aromatase expression is regulated via alternatively used promoters in the placenta (I.1 and I.2a), fat (I.4, I.3, and II), bone (I.6), and gonads (II). A prostaglandin E(2)/cAMP-dependent pathway regulates coordinately the proximal promoters I.3/II, whereas glucocorticoids and cytokines regulate the distal promoter I.4. Use of each promoter gives rise to a population of aromatase mRNA species with unique 5'-untranslated regions (5'-UTRs). Uterine leiomyoma tissue, but not normal myometrium, overexpresses aromatase leading to estrogen-stimulated cell proliferation. Aromatase inhibitor treatment shrank uterine leiomyomata in a few women. OBJECTIVE AND DESIGN: Promoter I.4 was reported to regulate aromatase expression in uterine leiomyomata from a group of Japanese women. Here, we used two independent techniques to identify the promoters that regulate aromatase expression in uterine leiomyomata (n = 30) from 23 African-American, Hispanic, and white women. RESULTS: Rapid amplification of 5'-cDNA ends of aromatase mRNA species revealed the following distribution of promoter usage in leiomyomata: promoters I.3/II, 61.5%; I.2a, 15.4%; I.6, 15.4%; and I.4, 7.7%. Real-time PCR, which quantifies mRNA species with promoter-specific 5'-UTRs, revealed the following distribution for each 5'-UTR as a fraction of total aromatase mRNA: I.3/II, 69.6%; I.4, 7.3%; and other promoters, 23.1%. CONCLUSIONS: The primary in vivo aromatase promoter in leiomyoma tissues in non-Asian U.S. women is the prostaglandin E(2)/cAMP-responsive I.3/II region. Alternative signals may stimulate aromatase expression that is a common biological phenotype in uterine leiomyomata.

Investigation of the effects of heparin and low molecular weight heparin on E-cadherin and laminin expression in rat pregnancy by immunohistochemistry.

BACKGROUND: Heparin and low molecular weight heparin (LMWH) are used widely to improve the pregnancy outcome in women with thrombophilia, miscarriage, recurrent miscarriage and fetal death. This study was designed to investigate the effects of heparin and LMWHs, enoxaparin and tinzaparin, on E-cadherin and laminin expression in placental and decidual tissues in rat pregnancy. METHODS: Wistar albino female rats (n = 48) were randomly assigned to four study groups (normal saline, heparin, enoxaparin and tinzaparin) in the preconceptional period. Tissue sections of placenta and decidua were immunohistochemically examined for the expression of E-cadherin and laminin. RESULTS: E-cadherin placental staining score of heparin group was significantly lower and E-cadherin decidual staining score of heparin and enoxaparin groups were significantly lower than control group. There were no significant differences in placental and decidual laminin staining scores among the study groups. CONCLUSIONS: Heparin and enoxaparin can reduce E-cadherin expression but not laminin expression in rat pregnancy. They might modulate trophoblast invasion. We suggest that this is the possible underlying mechanism involving in improvement of trophoblast invasion by the use of heparin and LMWH in patients with the history of miscarriage.

SP1 and SP3 mediate progesterone-dependent induction of the 17beta hydroxysteroid dehydrogenase type 2 gene in human endometrium.

The opposing actions of estrogen and progesterone during the menstrual cycle regulate the cyclical and predictable endometrial proliferation and differentiation that is required for implantation. Progesterone indirectly stimulates the expression of 17beta hydroxysteroid dehydrogenase type 2 (HSD17B2), which catalyzes the conversion of biologically potent estradiol to weakly estrogenic estrone in the endometrial epithelium. We previously demonstrated upregulation of the HSD17B2 gene in human endometrial epithelial cells by factors secreted from endometrial stromal cells in response to progesterone. We investigated the underlying mechanism by which these stroma-derived, progesterone-induced paracrine factors stimulate HSD17B2 expression. Here, we show that transcription factors SP1 and SP3 interact with specific motifs in HSD17B2 promoter to upregulate enzyme expression in human endometrial epithelial cell lines. Conditioned medium (CM) from progestin-treated stromal cells increased levels of SP1 and SP3 in endometrial epithelial cells and induced HSD17B2 mRNA expression. Mithramycin A, an inhibitor of SP1-DNA interaction, reduced epithelial HSD17B2 promoter activity in a dose-dependent manner. Serial deletion and site-directed mutants of the HSD17B2 promoter demonstrated that two overlapping SP1 motifs (nt -82/-65) are essential for induction of promoter activity by CM or overexpression of SP1/SP3. CM markedly enhanced, whereas anti-SP1/SP3 antibodies inhibited, binding of nuclear proteins to this region of the HSD17B2 promoter. In vivo, we demonstrated a significant spatiotemporal association between epithelial SP1/SP3 and HSD17B2 levels in human endometrial biopsies. Taken together, these data suggest that HSD17B2 expression in endometrial epithelial cells, and, therefore, estrogen inactivation, is regulated by SP1 and SP3, which are downstream targets of progesterone-dependent paracrine signals originating from endometrial stromal cells.

Comparison of effects of formoterol and BRL 37344 on isolated term-pregnant rat myometrial strips in vitro.

This study was designed to compare the effects of beta-adrenoceptor agonists formoterol and BRL 37344 on spontaneous contractions and the levels of cAMP and cGMP of myometrial strips isolated from timed-pregnant rats. Myometrial strips were obtained from term-pregnant Wistar albino rats (n=12), mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. We evaluated the effect of increasing concentrations of formoterol and BRL 37344 on oxytocin-induced myometrial contractions and on contractions of myometrial smooth muscle pretreated with metoprolol, ICI 118.551 and SR 59230A (beta1, beta2, beta3-adrenoceptor antagonist, respectively, 10(-6) M). Effects of formoterol and BRL 37344 on cAMP and cGMP levels in isolated myometrial strips (n=6) were evaluated by radioimmunoassay kits. Formoterol (10(-12)-10(-8) M) and BRL 37344 (10(-11)-10(-5) M) concentration-dependently decreased the amplitude of oxytocin-induced contractions. E(max) value (100%) of formoterol was increased significantly more than E(max) value (70.6%) of BRL 37344 (P<0.05), with no change in pD(2) value (9.54+/-0.12 and 9.12+/-0.12, respectively). The inhibition of the amplitude of oxytocin-induced contractions by formoterol was antagonized with ICI 118.551 (10(-6) M), but they were not changed by metoprolol (10(-6) M) or SR 59230A (10(-6) M). The inhibition of the amplitude of oxytocin-induced contractions by BRL 37344 were antagonized with SR 59230A (10(-6) M), but they were not changed by metoprolol (10(-6) M) or ICI 118.551 (10(-6) M). Formoterol and BRL 37344 increased cAMP levels. BRL 37344 increased cGMP levels in BRL 37344 group more than control group, but this increase is less significant than cAMP levels (P>0.05). Formoterol and BRL 37344 decreased amplitude of myometrial contractions with similar potency, but efficacy of formoterol was better than BRL 37344.

A novel role of sodium butyrate in the regulation of cancer-associated aromatase promoters I.3 and II by disrupting a transcriptional complex in breast adipose fibroblasts.

The aromatase gene encodes the key enzyme for estrogen formation. Aromatase enzyme inhibitors eliminate total body estrogen production and are highly effective therapeutics for postmenopausal breast cancer. A distal promoter (I.4) regulates low levels of aromatase expression in tumor-free breast adipose tissue. Two proximal promoters (I.3/II) strikingly induce in vivo aromatase expression in breast fibroblasts surrounding malignant cells. Treatment of breast fibroblasts with medium conditioned with malignant breast epithelial cells (MCM) or a surrogate hormonal mixture (dibutyryl (Bt2)cAMP plus phorbol diacetate (PDA)) induces promoters I.3/II. The mechanism of promoter-selective expression, however, is not clear. Here we reported that sodium butyrate profoundly decreased MCM- or Bt2cAMP + PDA-induced promoter I.3/II-specific aromatase mRNA. MCM, Bt2cAMP + PDA, or sodium butyrate regulated aromatase mRNA or activity only via promoters I.3/II but not promoters I.1 or I.4 in breast, ovarian, placental, and hepatic cells. Mechanistically, recruitment of phosphorylated ATF-2 by a CRE (-211/-199, promoter I.3/II) conferred inductions by MCM or Bt2cAMP + PDA. Chromatin immunoprecipitation-PCR and immunoprecipitation-immunoblotting assays indicated that MCM or Bt2cAMP + PDA stabilized a complex composed of phosphorylated ATF-2, C/EBPbeta, and cAMP-response element-binding protein (CREB)-binding protein in the common regulatory region of promoters I.3/II. Overall, histone acetylation patterns of promoters I.3/II did not correlate with sodium butyrate-dependent silencing of promoters I.3/II. Sodium butyrate, however, consistently disrupted the activating complex composed of phosphorylated ATF-2, C/EBPbeta, and CREB-binding protein. This was mediated, in part, by decreased ATF-2 phosphorylation. Together, these findings represent a novel mechanism of sodium butyrate action and provide evidence that aromatase activity can be ablated in a signaling pathway- and cell-specific fashion.

Estrogen regulates expression of tumor necrosis factor receptors in breast adipose fibroblasts.

In breast cancer, a dense layer of undifferentiated fibroblasts is formed around malignant breast epithelial cells and referred to as desmoplastic reaction. These cells provide structural and functional support for tumor growth. Aromatase, the key enzyme in the biosynthesis of estrogen, is overexpressed in these undifferentiated fibroblasts, producing large quantities of estrogen, which in turn influences the growth and progression of malignant epithelial cells. We previously demonstrated that malignant epithelial cells produce large amounts of TNFalpha, which inhibit the differentiation of breast fibroblasts. TNF action is mediated by its two receptors (TNFRs), TNFR1, which mediates inhibition of adipocyte differentiation, and TNFR2, which was linked to the proliferation of thymocytes. We present evidence here that estrogen modulates the synthesis of receptors for TNF in human adipose fibroblasts (HAFs) from breast tissue in a paracrine fashion, which may serve as a mechanism for the inhibition of adipocyte differentiation in breast cancer. Estradiol (E(2)) treatment increased TNFR1 mRNA and protein levels in primary HAFs in a dose- and time-dependent manner, which could be reversed by the estrogen antagonist ICI182,780. Interestingly, higher concentration of E(2) inhibited whereas lower concentrations stimulated TNFR2 mRNA levels in HAFs. To investigate the specific roles of TNFRs in adipocyte differentiation, we incubated breast HAFs with receptor selective muteins of TNF. TNFR1-selective mutein decreased mRNA levels of aP2, a marker for adipogenic differentiation. This antiadipogenic effect was enhanced by cotreatment with E(2). We conclude that high levels of estrogen found in breast tumors promote the antiadipogenic action of TNF on breast adipose fibroblasts by selectively up-regulating TNFR1, which may be a critical mechanism for desmoplastic reaction.

The effect of glyceryl trinitrate on hypertension in women with severe preeclampsia, HELLP syndrome, and eclampsia.

OBJECTIVE: The goal of this study is to evaluate the effect of glyceryl trinitrate (GTN) in the management of hypertension in women with preeclampsia, eclampsia, and HELLP syndrome. STUDY DESIGN: Fifty five women with preeclampsia, eclampsia, and HELLP syndrome administered GTN infusion for the management of hypertension were studied. Demographic, clinical, and perinatal outcome findings were collected for analyses. We recorded initial and maintenance doses of GTN, and duration of its use in prepartum and postpartum periods. We collected systolic and diastolic blood pressures (BPs) at admission and before the administration of GTN infusion. During the GTN infusion, we calculated average diastolic and systolic blood pressures 6 hours apart on the first day, 12 hours apart on the second day, and 24 hours apart on the third day. RESULTS: Of 55 women, 24 with severe preeclampsia, 16 with HELLP syndrome, and 15 with eclampsia were included in this study. In severe preeclampsia group, GTN infusion significantly reduced systolic and diastolic BPs beginning from the second quarter and third quarter, respectively, of first day (p < 0.05). In the HELLP syndrome group, GTN infusion significantly decreased systolic and diastolic blood pressures beginning from the third quarter and second quarter, respectively, of the first day (p < 0.05). In the eclampsia group, GTN infusion significantly reduced systolic and diastolic blood pressures beginning from the third quarter and first quarter, respectively, of the first day (p < 0.05). CONCLUSION: In women with severe preeclampsia, eclampsia, and HELLP syndrome, infusion of GTN can be used as an alternative agent to well-known drugs and causes no significant adverse effect to the mother and fetus.

Reduction of postoperative adhesions by N,O-carboxymethylchitosan and spermine NONOate in rats.

BACKGROUND: Postsurgical adhesions can occur following virtually all types of surgery, resulting in serious clinical complications. Therefore, prevention of adhesions is an important goal of surgical practice. A rat uterine horn model was used to investigate the efficacy of N,O-carboxymethylchitosan (NOCC) and spermine NONOate (SPER/NO) alone and in combination in preventing adhesion formation. METHODS: Sixty Wistar albino rats underwent bilateral uterine horn injury with a unipolar cautery. Study groups were as follows: (i) control group, no adjuvant therapy; and those with adjuvant applied, (ii) normal saline group, 2 ml of normal saline was given; (iii) NOCC group, 2 ml of 2% NOCC gel was given; (iv) SPER/NO group, 2 ml of SPER/NO (0.5 mg/ml) was given, and (v) NOCC plus SPER/NO group, 2 ml of 2% NOCC gel including SPER/NO (0.5 mg/ml) was given. After 14 days, all animals were euthanatized, and a standard adhesion scoring system including extent and severity scores was applied by a blinded examiner. RESULTS: The extent score in NOCC plus SPER/NO group was significantly lower than those of control and normal saline groups (p < 0.05). The extent score in NOCC group was significantly lower than that of normal saline group (p < 0.05). The extent score in NOCC plus SPER/NO group was significantly lower than that of SPER/NO group (p < 0.05). The severity score was significantly lower in NOCC plus SPER/NO and NOCC groups than that of control group (p < 0.05). The severity score was significantly lower in NOCC plus SPER/NO group than that of SPER/NO group (p < 0.05). CONCLUSIONS: Postoperative administration of NOCC gel and SPER/NO alone and especially in combination to the site of peritoneal injury reduces the formation of adhesions in the rat uterine horn model.