Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac)

Kusama, Kaoru; Yoshie, Mikihiro; Tamura, Kazuhiro; Kodaka, Yasuhiro; Hirata, Akihiko; Sakurai, ﻿Toshihiro; Bai, Hanako; Imakawa, Kazuhiko; Nishi, Hirotaka; Isaka, Keiichi; Nagai, Takeshi; Nagao, Toshitaka; Tachikawa, Eiichi

doi:10.1016/j.placenta.2012.12.017

Cited by 34 publications

(45 citation statements)

References 31 publications

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“…The membranes were treated with stripping solution (25 mM glycine-HCl (pH 2.0), containing 1% (w/v) SDS) and re-probed with an antibody against b-actin (clone AC-15, 1:5000, Sigma-Aldrich). The relative band intensity was assessed by densitometric analysis of digitalized autographic images using Scion image software (Scion Corp., Fredrick, MD, USA) and normalized to b-actin (Kusama et al 2013). …”

Section: Immunoblottingmentioning

confidence: 99%

“…The amount of active RAP1 was determined using the RAP1 activation assay kit (Millipore) as described previously (Kusama et al 2013). Decidua at implantation site (IS) and uterine endometrium at inter-IS were dissected on day 9 of pregnancy.…”

Section: Rap1 Gtpase Activation Assaymentioning

confidence: 99%

“…After treatment with siRNA for 24 h, the medium was removed and the cells were cultured in fresh medium for 24 h. Concentrations of siRNA sufficient to knockdown the expression of each protein were predetermined (Kusama et al 2013).…”

Section: Treatment With Sirnamentioning

confidence: 99%

“…The sense (S) and antisense (AS) primers were 5 0 -CATGCTTTCT-CACTACATCCAT-3 0 (S) and 5 0 -CTTCAGGAGTAGCTAGG-GAAGA-3 0 (AS) for Prl; 5 0 -ATCCAGCGAGCTGAAGTCAT-3 0 (S) and 5 0 -ATGCCTATACATGCGTGCAA-3 0 (AS) for Prl8a2; and 5 0 -AAAGCTGTGGCGTGATGG-3 0 (S) and 5 0 -TTCAGCTCTGG-GATGACCTT-3 0 (AS) for Gapdh. The fold change in the expression of each gene was calculated using the DDCt method with Gapdh as an internal control (Kusama et al 2013). …”

Section: Rna Extraction and Real-time Rt-pcrmentioning

confidence: 99%

“…For examples, EPAC1/RAP1 pathway promotes the differentiation of cytotrophoblasts into syncytiotrophoblasts (Yoshie et al 2010). Another example is that, during the decidualization of human endometrial stromal cells (ESCs), EPAC/RAP1 signaling enhanced prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) expression induced by ovarian steroids and cAMP analogs, while siRNA-mediated silencing of EPAC1, EPAC2, or RAP1 inhibited the expression of PRL and IGFBP1 and morphological differentiation (Kusama et al 2013). However, we observed expressions and functions of EPAC/RAP1 only in vitro.…”

Section: Introductionmentioning

confidence: 99%

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Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus

Kusama¹,

Yoshie²,

Tamura³

et al. 2014

Reproduction

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The optimal decidualization of endometrial stromal cells (ESCs) following embryo implantation is one of the critical steps to establish pregnancy in rodents and humans. This step is intricately regulated by ovarian hormones. Using in vitro human ESCs model, we previously showed that activation of a cAMP mediator, exchange protein directly activated by cAMP (EPAC), promotes ovarian steroid-or cAMP analog-induced decidualization. However, expressions and functions of EPAC and RAP1 in the uterus during pregnancy have not yet been examined. In this study, we found that the expression of EPAC2 and RAP1 was markedly upregulated in the decidual cells at the implantation sites on days 7 and 9 of pregnancy in rats. Furthermore, both delayed-implantation and artificial decidualization models showed that EPAC2 and RAP1 expression was enhanced in decidual cells. Significant activation of cAMP-responsive element-binding protein (CREB), a central transcriptional factor of cAMP signaling, was observed in decidual cells. These spatiotemporal expressions of protein related EPAC pathway are overlapped by sites with activated cAMP signaling, indicating the association of EPAC signaling with decidualization. Strikingly, further studies in in vitro rat decidualization model showed that the cAMP analog and medroxyprogesterone stimulated the expression of decidual markers, while knockdown of EPAC1/2 and RAP1 attenuated the expressions of these markers. Together, these findings suggest that EPAC and RAP1 are the crucial factors for endometrial decidualization in rat pregnancy.

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Section: Immunoblottingmentioning

confidence: 99%

Section: Rap1 Gtpase Activation Assaymentioning

confidence: 99%

Section: Treatment With Sirnamentioning

confidence: 99%

Section: Rna Extraction and Real-time Rt-pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus

Kusama¹,

Yoshie²,

Tamura³

et al. 2014

Reproduction

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Regulation of human trophoblast cell syncytialization by transcription factors STAT5B and NR4A3

Kusama

Bai

Imakawa

2018

J of Cellular Biochemistry

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In human trophoblast cells, cyclic AMP or its inducer forskolin (FSK) activates two downstream signaling molecules, protein kinase A (PKA) and exchange protein directly activated by cAMP (EPAC), both of which induce syncytialization, cell fusion, and the production of human chorionic gonadotropin (hCG) and progesterone. However, a transcription factor other than GCM1 and molecular mechanisms associated with these events have not been well characterized. To identify novel transcription factors involved in syncytialization of cAMP-stimulated human choriocarcinoma BeWo cells, the microarray analysis was performed with RNAs extracted from PKA- or EPAC-selective cAMP analog-stimulated BeWo cells, from which two up-regulated transcription factors, STAT5 and NR4A3, were found. The knockdown of STAT5B decreased FSK-induced cell fusion and the expression of syncytialization markers, CGB, syncytin1, syncytin2, GCM1, and OVOL1, but NR4A3 knockdown increased FSK-induced cell fusion and the expression of CGB and syncytin2. These findings indicated that cAMP-PKA up-regulated STAT5B, followed by increase in syncytin2 expression through GCM1 and OVOL1, resulting in cell fusion and hCG production, while cAMP-PKA-up-regulated NR4A3 could decrease syncytin2 expression, and suggested that both positive and negative effects of STAT5B and NR4A3, respectively, are required to control the degree of syncytialization in human trophoblast cells.

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Small GTP‐binding protein Rap1 mediates EGF and HB‐EGF signaling and modulates EGF receptor expression in HTR‐8/SVneo extravillous trophoblast cells

Yoshie,

Ohishi,

Ishikawa

et al. 2023

Reprod Medicine & Biology

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PurposeExtravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin‐binding EGF‐like growth factor (HB‐EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP‐binding protein Rap1 in EGF‐ and HB‐EGF‐stimulated EVT invasion.MethodsExpression of Rap1 in the first‐trimester placenta was examined by immunohistochemistry. Effect of EGF or HB‐EGF on Rap1 activation (GTP‐Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR‐8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined.ResultsRap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB‐EGF activated Rap1 and promoted invasion of HTR‐8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF‐ and HB‐EGF‐induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF‐ and HB‐EGF‐induced Rap1 activation and reduced EGFR expression.ConclusionRap1 may function as a mediator of EGF and HB‐EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.

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Regulation of decidualization in human endometrial stromal cells through exchange protein directly activated by cyclic AMP (Epac)

Cited by 34 publications

References 31 publications

Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus

Possible roles of the cAMP-mediators EPAC and RAP1 in decidualization of rat uterus

Regulation of human trophoblast cell syncytialization by transcription factors STAT5B and NR4A3

Small GTP‐binding protein Rap1 mediates EGF and HB‐EGF signaling and modulates EGF receptor expression in HTR‐8/SVneo extravillous trophoblast cells

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