hCG activates Epac-Erk1/2 signaling regulating Progesterone Receptor expression and function in human endometrial stromal cells.
How does hCG signal in human endometrial stromal cells (ESCs) and what is its role in regulating ESC function?



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.



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.



This is an in vitro study comprising patients undergoing benign gynecological surgery (n = 46).



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.



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).



N/A.



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.



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.



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.
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Alejandro
A
Tapia-Pizarro
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Sebastián
S
Archiles
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Felipe
F
Argandoña
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Cecilia
C
Valencia
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Keyla
K
Zavaleta
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
M
M
Cecilia Johnson
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Reinaldo
R
González-Ramos
Institute of Maternal and Child Research (IDIMI), Faculty of Medicine, University of Chile, Av. Sta. Rosa 1234, 2do piso, Santiago 8360160, Chile.
Luigi
L
Devoto
D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D000328:Adult; D006063:Chorionic Gonadotropin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D004717:Endometrium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D020662:Guanine Nucleotide Exchange Factors / Q000819:agonists / Q000235:genetics* / Q000378:metabolism; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics* / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics* / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011247:Pregnancy; D061251:Primary Cell Culture; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D017154:Stromal Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014158:Transcription, Genetic
D000262:Adenylyl Cyclases; D006063:Chorionic Gonadotropin; D017868:Cyclic AMP-Dependent Protein Kinases; D020662:Guanine Nucleotide Exchange Factors; D000080905:Homeobox A10 Proteins; D018398:Homeodomain Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011980:Receptors, Progesterone; C116122:RAPGEF3 protein, human; C069634:HOXA10 protein, human; C535150:MAPK1 protein, human
Epac, endometrial stromal cells; Erk1/2; chorionic gonadotropin, hCG, endometrium; progesterone receptor