RESUMEN
OBJECTIVE: To determine the role of P and its nuclear receptor PR in the growth of ectopic uterine tissue of mice with or without a disrupted PR gene. DESIGN: Animal study. SETTING: Academic medical center. ANIMAL(S): Female wild-type (WT) and transgenic knockout mice for P receptor (PRKO). INTERVENTION(S): Endometriosis was induced in the following groups of ovariectomized adult mice: [1] untreated WT, [2] estradiol (E(2))-treated WT, [3] P-treated WT, [4] E(2) + P-treated WT, [5] untreated PRKO, [6] E(2)-treated PRKO, and [7] E(2) + P-treated PRKO (n = 5 in each group). MAIN OUTCOME MEASURE(S): The size of ectopic uterine tissue in WT and PRKO mice were compared between the groups subjected to treatments with P or E(2). Tissue proliferating cell nuclear antigen (PCNA) levels were compared among these groups. RESULTS: Treatment with P only significantly decreased the size of WT ectopic uterine tissue. The untreated PRKO ectopic uterine tissue was significantly larger than WT tissue. Estradiol increased the size of ectopic uterine tissues, and this E(2)-dependent growth could be suppressed by P in WT tissues but not in PRKO tissues. Finally, the hormone-dependent changes in ectopic uterine tissue size were accompanied by comparable alterations in PCNA levels. CONCLUSION(S): Intact PR in ectopic uterine tissue is essential to abolish E(2)-dependent or -independent proliferation. We also suggest that ectopic uterine tissue is associated with significantly increased resistance to P action and increased predisposition to E(2)-dependent proliferation in the absence of PR. Overall, these findings support the hypothesis that P resistance in human endometriosis may be due to the absence of sufficient levels of functional PR in this tissue.
Asunto(s)
División Celular/fisiología , Endometriosis/patología , Estradiol/farmacología , Receptores de Progesterona/fisiología , Animales , División Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Endometriosis/genética , Femenino , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptores de Progesterona/deficiencia , Receptores de Progesterona/genéticaRESUMEN
Aromatase p450 (p450arom) is the key enzyme for biosynthesis of estrogen, which is an essential hormone for the establishment and growth of endometriosis. There is no detectable aromatase enzyme activity in normal endometrium; therefore, estrogen is not locally produced in endometrium. Endometriosis tissue, however, contains very high levels of aromatase enzyme, which leads to production of significant quantities of estrogen. Moreover, one of the best-known mediators of inflammation and pain, prostaglandin E (2), strikingly induces aromatase enzyme activity and formation of local estrogen in this tissue. Additionally, estrogen itself stimulates cyclo-oxygenase-2 and therefore increases the formation of prostaglandin E (2) in endometriosis. We were able to target this positive feedback cycle in endometriosis using aromatase inhibitors. In fact, pilot trials showed that aromatase inhibitors could decrease pelvic pain associated with endometriosis.
Asunto(s)
Aromatasa/metabolismo , Endometriosis/enzimología , Endometriosis/tratamiento farmacológico , Inhibidores Enzimáticos/uso terapéutico , Estrógenos/biosíntesis , Femenino , Regulación de la Expresión Génica , Humanos , Células del Estroma/enzimologíaRESUMEN
Binding activity of steroidogenic factor-1 (SF-1) to promoters of the majority of steroidogenic genes in response to gonadotropins is a critical mechanism that regulates steroidogenesis in gonads. Thus, the modulation of SF-1 action may be essential for the differential regulation of formation of sex steroids in the ovary. Aromatase P450 (P450arom) is the rate-limiting enzyme for estrogen formation. In this study, we characterize another nuclear receptor half site in the gonadal aromatase promoter which we show to be important for aromatase regulation. We also show herein that the stimulation of P450arom promoter activity by SF-1 in ovarian granulosa, testicular Sertoli and JEG-3 choriocarcinoma cells is inhibited by two transcription factors, Wilms' tumor suppressor gene (WT1) and dosage sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Given the characterized roles of these transcription factors in gonadal development and function, modulation of SF-1 action by WT1 and DAX-1 may represent an important key mechanism in steroidogenesis.
Asunto(s)
Aromatasa/genética , Proteínas de Unión al ADN/fisiología , Gónadas/metabolismo , Receptores de Ácido Retinoico/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Proteínas WT1/fisiología , Secuencias de Aminoácidos , Animales , Aromatasa/metabolismo , Secuencia de Bases , Línea Celular , Secuencia de Consenso , Receptor Nuclear Huérfano DAX-1 , Regulación hacia Abajo , Femenino , Factores de Transcripción Fushi Tarazu , Regulación de la Expresión Génica , Genes del Tumor de Wilms , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Células de Sertoli/metabolismo , Factor Esteroidogénico 1 , Activación TranscripcionalRESUMEN
The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms' tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E(2) stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.
Asunto(s)
Aromatasa/genética , Proteínas de Unión al ADN/genética , Endometriosis/enzimología , Endometrio/enzimología , Regulación Enzimológica de la Expresión Génica/fisiología , Receptores de Ácido Retinoico/genética , Proteínas Represoras , Células del Estroma/enzimología , Factores de Transcripción/genética , Proteínas WT1/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Biopsia , Receptor Nuclear Huérfano DAX-1 , Endometriosis/patología , Células Epiteliales/enzimología , Células Epiteliales/patología , Femenino , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Factor Esteroidogénico 1RESUMEN
We investigated the regulation of PG production in human endometrial stromal cells (ESC) by IL-1beta. We found that cyclooxygenase-2 (COX-2) mRNA and protein levels and PGE(2) production in ESC were significantly increased by IL-1beta. COX-2 mRNA, protein, and PGE(2) levels in IL-1beta-treated ESC were decreased by a PKA inhibitor, a nuclear factor (NF-kappaB) inhibitor, and an ERK1/2 inhibitor, but not by a p38 MAPK inhibitor or a PKC inhibitor, suggesting the possible involvement of PKA, NF-kappaB, and/or the ERK1/2 signaling pathway(s) in IL-1beta-mediated COX-2 gene induction in ESC. We then transiently transfected deletion mutants of the COX-2 promoter fused to the luciferase reporter gene and variants of -360/+56 bp promoter construct carrying different site-directed mutations of selected cis-acting elements. We determined that a NF-kappaB site (-222/-213 bp), a nuclear factor for IL-6 expression site (NF-IL6, -132/-124 bp), and a cAMP response element (-59/-52 bp) were essential for the baseline COX-2 gene promoter regulation. The addition of IL-1beta, however, did not affect the activity of these COX-2 promoter constructs. To investigate the potential effects of IL-1beta on COX-2 mRNA stability, ESC were treated with actinomycin D, a general transcription inhibitor, in the absence or presence of IL-1beta. We found that 1) IL-1beta significantly increased COX-2 mRNA stability; 2) continuous transcription was not required to sustain the IL-1beta-induced COX-2 mRNA levels; and 3) COX-2 mRNA was highly unstable in the absence of IL-1beta. Additionally, we found that the ERK1/2 signaling pathway was essential for stabilizing COX-2 mRNA. We conclude that levels of COX-2 mRNA, protein, and enzyme activity in ESC are controlled by various signaling pathways, including PKA, ERK1/2, and NF-kappaB. Moreover, posttranscriptional mRNA stability is an important mechanism for IL-1beta-induced elevation of COX-2 expression in ESC.
Asunto(s)
Endometrio/metabolismo , Interleucina-1/farmacología , Isoenzimas/genética , Isoenzimas/metabolismo , Prolina/análogos & derivados , Prostaglandina-Endoperóxido Sintasas/genética , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/metabolismo , Células del Estroma/metabolismo , Butadienos/farmacología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Endometrio/citología , Inhibidores Enzimáticos/farmacología , Espacio Extracelular/metabolismo , Femenino , Humanos , Proteínas de la Membrana , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/fisiología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/fisiología , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Prolina/farmacología , Regiones Promotoras Genéticas/fisiología , Estabilidad del ARN/efectos de los fármacos , Valores de Referencia , Transducción de Señal , Células del Estroma/efectos de los fármacos , Tiocarbamatos/farmacologíaRESUMEN
Aromatase P450 (P450arom) is the key enzyme for the biosynthesis of estrogen that is essential for the growth of human endometriosis, a pathology characterized by endometrium-like tissue on the peritoneal surfaces of abdominal organs manifest by pelvic pain and infertility. Surgically transplanted autologous uterine tissue to ectopic sites on the peritoneum in mice has been used as an animal model to study endometriosis. Using this mouse model, we evaluated the roles of the P450arom gene and aromatase enzyme activity in the growth of endometriosis represented by ectopic uterine tissues in mice. Endometriosis was induced surgically in the following groups of mice: 1) untreated transgenic mice with disrupted P450arom gene (ArKO); 2) ArKO mice treated with systemic estrogen; 3) untreated wild-type (WT) mice; 4) WT mice treated with estrogen; 5) WT mice treated with the aromatase inhibitor, letrozole; and 6) WT mice treated with letrozole and estrogen. Each group contained eight mice; +/+ littermates of ArKO mice were used as WT controls. Treatment with estrogen increased the size of ectopic uterine tissues in ArKO and WT mice significantly. The ectopic uterine lesions in untreated and estrogen-treated ArKO mice were strikingly smaller than those in untreated and estrogen-treated WT controls, respectively. Systemic treatment of WT mice with letrozole significantly decreased the lesion size in a dose-dependent manner. The addition of estrogen to letrozole treatment increased the ectopic lesion size, although these lesions were significantly smaller than those in mice treated with estrogen only. As tissue controls, the effects of these conditions on normally located (eutopic) uterine tissue were evaluated. The effects of disruption of the P450arom gene and treatments with letrozole and estrogen seemed to be more profound on ectopic tissues, suggesting that ectopic tissues might be more sensitive to estrogen for growth. We conclude that both an intact P450arom gene and the presence of aromatase enzyme activity are essential for the growth of ectopic uterine tissue in a mouse model of endometriosis.
Asunto(s)
Aromatasa/fisiología , Endometriosis/fisiopatología , Animales , Aromatasa/genética , Inhibidores de la Aromatasa , División Celular/fisiología , Coristoma/patología , Coristoma/fisiopatología , Endometriosis/patología , Inhibidores Enzimáticos/farmacología , Estrógenos/farmacología , Femenino , Crecimiento/efectos de los fármacos , Letrozol , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados/genética , Nitrilos/farmacología , Triazoles/farmacología , Útero/efectos de los fármacos , Útero/patologíaRESUMEN
We investigated the effects of vascular endothelial growth factor (VEGF) on cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) (PGE(2)) synthesis in human microvascular endothelial cells (HMEC-1). Treatment of HMEC-1 with VEGF resulted in a dose- and time-dependent up-regulation of COX-2 mRNA and protein levels. This up-regulation was accompanied by a 1.6-fold increase in PGE(2) synthesis. Pretreatment of HMEC-1 with a selective COX-2 inhibitor, NS-398, abolished VEGF-induced PGE(2) synthesis, suggesting specific up-regulation of COX-2 activity by VEGF in HMEC-1. Transient transfection assays using deletion mutants of the human COX-2 promoter fused to the luciferase reporter gene indicated critical requirement of a regulatory region spanning -828/-123 bp for VEGF induction of COX-2 promoter activity in HMEC-1. Site-directed mutation analysis demonstrated that a GATA cis-acting element at -685/-680 bp was essential for VEGF- induced COX-2 promoter activity in HMEC-1. These observations are of particular importance given the recent demonstrations of critical requirement of COX-2 isoenzyme for tumor growth and angiogenesis.
Asunto(s)
Factores de Crecimiento Endotelial/farmacología , Endotelio Vascular/metabolismo , Isoenzimas/metabolismo , Linfocinas/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Línea Celular , Ciclooxigenasa 2 , Dinoprostona/biosíntesis , Endotelio Vascular/citología , Humanos , Isoenzimas/genética , Proteínas de la Membrana , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , Prostaglandina-Endoperóxido Sintasas/genética , ARN Mensajero/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Regulación hacia Arriba , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Estrogen is produced in a number of human tissues including the ovary, placenta and extraglandular sites such as adipose tissue, skin and the brain. Aromatase is the key enzyme that regulates estrogen formation in these tissues. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE(2). This results in local production of estrogen, which induces PGE(2) formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE(2) in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of postmenopausal endometriosis using an aromatase inhibitor.
Asunto(s)
Endometriosis/metabolismo , Estrógenos/biosíntesis , Aromatasa/genética , Aromatasa/metabolismo , Inhibidores de la Aromatasa , Endometriosis/tratamiento farmacológico , Endometriosis/etiología , Inhibidores Enzimáticos/uso terapéutico , Estradiol/metabolismo , Femenino , Expresión Génica , Humanos , Inflamación/etiología , Inflamación/metabolismo , Modelos Biológicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
In human endometriotic stromal cells, markedly high levels of aromatase P450 (P450arom) mRNA and promoter II activity are present and can be vigorously stimulated by PGE(2) via a cAMP-dependent pathway to give rise to physiologically significant estrogen biosynthesis. Stromal cells of eutopic endometrium, on the other hand, do not express sufficient levels of P450arom for detectable enzyme activity. Because P450arom is up-regulated in the ovaries of CCAAT/enhancer binding protein (C/EBP) beta knockout mice and activation of the ovarian-type P450arom promoter (II) is responsible for aberrant P450arom expression in endometriosis, we sought here to evaluate the possible roles of C/EBP isoforms in the regulation of P450arom expression in endometriotic vs. eutopic endometrial stromal cells. We previously found that the -517-bp flanking region of promoter II contained the critical cis-acting elements for baseline and cAMP (analog)-induced activity. In this study, we disrupted several potential sequences and found that mutations of a -211/-197-bp cAMP-response element (CRE) and a -317/-304-bp C/EBP binding site abolished both baseline and cAMP-induced promoter II activity. Ectopic expression of C/EBPalpha increased both baseline and cAMP-dependent promoter II activity significantly in endometriotic cells, whereas ectopic expression of C/EBPbeta or C/EBPdelta abolished promoter II activity in both untreated and cAMP-treated endometriotic stromal cells. Comparable changes in promoter II activity were observed using endometrial stromal cells, which showed, however, seemingly diminished levels of baseline and cAMP-induced promoter II activity in comparison with endometriotic cells. EMSA using a probe containing the critical -317/-304-bp C/EBP site upstream of promoter II demonstrated a distinct DNA-protein complex in endometriotic, but not in endometrial stromal cells. This specific complex, however, could not be altered using antibodies against C/EBPalpha, -beta, or -delta. Because CRE is another potential DNA motif that can bind C/EBP isoforms, we next used EMSA using a probe containing the -211/-197-bp CRE and demonstrated that specific DNA-protein complexes contained C/EBPalpha but not C/EBPbeta or C/EBPdelta in endometriotic stromal cells. In contrast, C/EBPbeta and C/EBPdelta but not C/EBPalpha were detected in DNA-protein complexes using nuclear extracts from endometrial stromal cells. Western blotting and immunohistochemistry demonstrated expression of C/EBPalpha, -beta, and -delta in human endometriotic and endometrial stroma and epithelium. Intriguingly, C/EBPbeta was expressed at increased levels in stromal cells of human eutopic endometrium compared with simultaneously biopsied endometriotic tissues. We conclude that both -317/-304 and -211/-197-bp elements in promoter II are critical for the robust cAMP-dependent induction in endometriosis. C/EBPalpha up-regulates, whereas C/EBPbeta and C/EBPdelta inhibit P450arom promoter activity via binding primarily to the -211/-197-bp CRE under in vitro conditions. In vivo down-regulation of C/EBPbeta in endometriotic stromal cells and its up-regulation in endometrial stromal cells may in part account for the induction of P450arom expression in endometriosis and its inhibition in endometrium.