RESUMEN
STUDY QUESTION: How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function? SUMMARY ANSWER: hCG signaling in ESCs activates the extracellular signal-regulated protein kinases 1 and 2 (Erk1/2) pathway through exchange protein activated by cyclic AMP (cAMP) (Epac) and transiently increases progesterone receptor (PR) transcript and protein expression and its transcriptional function. WHAT IS KNOWN ALREADY: hCG is one of the earliest embryo-derived secreted signals in the endometrium, which abundantly expresses LH/hCG receptors. hCG signals through cAMP/protein kinase A (PKA) in gonadal cells, but in endometrial epithelial cells, hCG induces Erk1/2 activation independent of the cAMP/PKA pathway. Few data exist concerning the signal transduction pathways triggered by hCG in ESCs and their role in regulation of ESC function. STUDY DESIGN, SIZE, DURATION: This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46). PARTICIPANTS/MATERIALS, SETTING, METHODS: Endometrial samples were collected from normal cycling women during the mid-secretory phase for ESCs isolation. The study conducted in an academic research laboratory within a tertiary-care hospital. The activation of the Erk1/2 signal transduction pathway elicited by hCG was evaluated in ESC. Signaling pathway inhibitors were used to examine the roles of PKA, PI3K, PKC, adenylyl cyclase and Epac on the hCG-stimulated up-regulation of phospho-Erk1/2 (pErk1/2). Erk1/2 phosphorylation was determined by immunoblot. siRNA targeting Epac was used to investigate the molecular mechanisms. To assess the role of Erk1/2 signaling induced by hCG on ESC function, gene expression regulation was examined by immunofluorescence and real-time quantitative PCR. The role of PR on the regulation of transcript levels was studied using progesterone and the PR antagonist RU486. All experiments were conducted using at least three different cell culture preparations in triplicate. MAIN RESULTS AND THE ROLE OF CHANCE: Addition of hCG to ESCs in vitro induced the phosphorylation of Erk1/2 through cAMP accumulation. Such induction could not be blocked by inhibitors for PKA, PKC and PI3K. Epac inhibition and knockdown with siRNA prevented pErk1/2 induction by hCG. ESCs stimulated with hCG for up to 72 h showed a significant increase in PR mRNA and immunofluorescent label at 48 h only; an effect that was abrogated with the mitogen-activated protein kinase kinase inhibitor UO126. In addition, the hCG-activated Erk1/2 pathway significantly decreased the mRNA levels for secreted frizzled-related protein 4 (SFRP4) at 24 h, whereas it increased those for homeobox A10 (HOXA10) at 48 h (P = 0.041 and P = 0.022 versus control, respectively). Prolactin mRNA levels were not significantly modified. HOXA10 mRNA up-regulation by hCG was not enhanced by co-stimulation with progesterone; however, it was completely abolished in the presence of RU486 (P = 0.036 hCG versus hCG + RU486). LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This is an in vitro study utilizing stromal cell cultures from human endometrial tissues. Furthermore, results obtained should also be confirmed in vivo in the context of the whole human endometrial tissue and hormonal milieu. The in vitro experiments using hCG have been conducted without other hormones/factors that may also modulate the ESCs response to hCG. WIDER IMPLICATIONS OF THE FINDINGS: We have determined that hCG induces the PR through the Erk1/2 pathway in ESCs which may render them more sensitive to progesterone, increasing our understanding about the effects of hCG at the embryo-maternal interface. The activation of such a pathway in the context of the hormonal milieu during the window of implantation might contribute to a successful dialog between the embryo and the uterus, leading to appropriate endometrial function. Defective hCG signaling in the endometrial stromal tissue may lead to an incomplete uterine response, compromising embryo implantation and early pregnancy. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Fund for Scientific and Technological Development, Government of Chile (FONDECYT) grants 11100443 and 1140614 (A.T.-P.). The authors have no conflicts of interest to declare.
Asunto(s)
Gonadotropina Coriónica/farmacología , Factores de Intercambio de Guanina Nucleótido/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Receptores de Progesterona/genética , Células del Estroma/efectos de los fármacos , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Adulto , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endometrio/citología , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Factores de Intercambio de Guanina Nucleótido/agonistas , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas Homeobox A10 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Embarazo , Cultivo Primario de Células , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Progesterona/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Transcripción GenéticaRESUMEN
BACKGROUND: Endometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1. RESULTS: Upstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERa) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified. CONCLUSION: These data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERa and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.