title: The role of exchange protein directly activated by cyclic AMP 2-mediated calreticulin expression in the decidualization of human endometrial stromal cells.
abstract: Decidualization of human endometrial stromal cells (ESCs) accompanied by the production of prolactin (PRL) and IGF-binding protein (IGFBP) 1 and rounded-cell morphology is indispensable for the establishment and maintenance of pregnancy. Protein kinase A (PKA)-mediated cAMP signaling is known to be crucial for decidualization. We previously reported that activation of a cAMP mediator, called Exchange protein directly activated by cAMP (EPAC) promotes cAMP analog- or ovarian steroid-induced decidualization in cultured human ESCs. In addition, small interfering RNA-mediated knock-down of the EPAC subtypes, EPAC1 or EPAC2, or knock-down of Rap1, a downstream factor of EPAC signaling, blocked functional and morphological decidualization of ESCs. However, factors downstream of EPAC2 other than Rap1 have not been determined. The present study was undertaken to identify additional downstream targets of EPAC2 associated with decidualization. Using proteomic analysis, we identified calreticulin (CRT) as a potential target of EPAC2. Knock-down of CRT expression in cultured ESCs significantly inhibited PKA-selective cAMP analog- or PKA-selective cAMP analog plus EPAC-selective cAMP analog-induced PRL and IGFBP1 expression. Furthermore, CRT knock-down suppressed the ovarian steroid-stimulated PRL and IGFBP1 expression and morphological differentiation, and silencing of EPAC2 or CRT significantly increased senescence-associated β-galactosidase activity with enhanced p21 expression and decreased p53 expression. These results suggest that EPAC2 and CRT are associated with cellular senescence in ESCs. In conclusion, we demonstrate here that EPAC2-mediated CRT expression is essential for the functional and morphological differentiation of ESCs into decidual cells. Furthermore, both EPAC2 and CRT might prevent ESCs from undergoing abnormal cellular senescence during decidualization.
authors.affiliation: Department of Endocrine and Neural Pharmacology (K.K., M.Y., K.T., T.N., E.T.), Tokyo University of Pharmacy and Life Sciences, Tokyo 192-0392, Japan; and Department of Obstetrics and Gynecology (H.N., K.I.), Tokyo Medical University, Tokyo 160-8402, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kusama
authors.forename: Mikihiro
authors.initials: M
authors.lastname: Yoshie
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Tamura
authors.forename: Takahiro
authors.initials: T
authors.lastname: Nakayama
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Nishi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Isaka
authors.forename: Eiichi
authors.initials: E
authors.lastname: Tachikawa
mesh_terms: D037282:Calreticulin / Q000502:physiology*; D002470:Cell Survival; D016922:Cellular Senescence; D002853:Chromatography, Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D003656:Decidua / Q000378:metabolism*; D004717:Endometrium / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000502:physiology*; D006801:Humans; D018970:Insulin-Like Growth Factor Binding Protein 1 / Q000378:metabolism; D040901:Proteomics; D034741:RNA, Small Interfering / Q000378:metabolism; D017154:Stromal Cells / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D037282:Calreticulin; D020662:Guanine Nucleotide Exchange Factors; C032231:IGFBP1 protein, human; D018970:Insulin-Like Growth Factor Binding Protein 1; C442864:RAPGEF4 protein, human; D034741:RNA, Small Interfering; D000242:Cyclic AMP