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Progestin-induced heart and neural crest derivatives expressed transcript 2 is associated with fibulin-1 expression in human endometrial stromal cells

      Objective

      To investigate whether heart and neural crest derivatives expressed transcript 2 (HAND2) regulates fibulin-1 (FBLN1) expression during decidualization of human endometrial stromal cells (ESCs).

      Design

      In vitro experiment.

      Setting

      Research laboratory.

      Patient(s)

      Twenty-four patients undergoing hysterectomy for benign reasons.

      Intervention(s)

      ESCs were cultured with various progestins (medroxyprogesterone acetate [MPA], norethisterone, levonorgestrel, dienogest, and P), E2, dexamethasone, and/or 8-bromoadenosine 3′, 5′-cyclic monophosphate (8-Br-cAMP). HAND2 and FBLN1 were silenced by small interfering RNA technology.

      Main Outcome Measure(s)

      HAND2 and FBLN1 expression levels were assessed by real-time polymerase chain reaction and Western blot analysis.

      Result(s)

      MPA or E2 + MPA increased HAND2 mRNA levels in ESCs in a time- and dose-dependent manner, and this stimulatory effect was blocked by RU-486 (P receptor antagonist). HAND2 was increased by E2 + MPA earlier than FBLN1. Simultaneous MPA and 8-Br-cAMP treatment synergistically enhanced HAND2 mRNA levels. P and all the progestins significantly increased HAND2 mRNA levels, whereas E2, 8-Br-cAMP, or dexamethasone alone had no effect. Silencing of HAND2 expression significantly reduced FBLN1 expression, whereas FBLN1 silencing had no effect on HAND2 expression.

      Conclusion(s)

      These results suggest that progestin-induced HAND2 contributes to FBLN1 expression in human ESCs.

      Key Words

      Discuss: You can discuss this article with its authors and with other ASRM members at http://fertstertforum.com/choh-progestin-endometrial-stromal-cells/
      The human uterine endometrium undergoes cyclic proliferation and differentiation controlled by a sequential, carefully timed interplay of the ovarian steroid hormones, E2 and P, during the menstrual cycle. P plays a central role in reproduction, mediating ovulation, embryo implantation, uterine growth, and maintenance of pregnancy (
      • Graham J.D.
      • Clarke C.L.
      Physiological action of progesterone in target tissues.
      ). Problems with implantation and placental development are clinically important. In fact, a postovulatory P level inadequate to prepare this important biological event in the endometrium is associated with infertility and recurrent spontaneous abortion. Cellular responses are predominantly mediated by the progesterone receptor (PR), a member of the superfamily of ligand-inducible transcription factors. Decidualization is a process that occurs in the human endometrium in response to ovarian steroid hormones. Many studies have addressed the molecular mechanisms underlying decidualization in an in vitro human endometrial stromal cell (ESC) model because these cells express functional PR and estrogen receptor (ER) (
      • Carver J.
      • Martin K.
      • Spyropoulou I.
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      An in-vitro model for stromal invasion during implantation of the human blastocyst.
      ,
      • Berkkanoglu M.
      • Guzeloglu-Kayisli O.
      • Kayisli U.A.
      • Selam B.F.
      • Arici A.
      Regulation of Fas ligand expression by vascular endothelial growth factor in endometrial stromal cells in vitro.
      ,
      • Kawano Y.
      • Furukawa Y.
      • Nasu K.
      • Narahara H.
      Thrombin-induced chemokine production in endometrial stromal cells.
      ,
      • Ishikawa T.
      • Harada T.
      • Kubota T.
      • Aso T.
      Testosterone inhibits matrix metalloproteinase-1 production in human endometrial stromal cells in vitro.
      ,
      • Kodama A.
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      • Osuga Y.
      • Harada M.
      • Hasegawa A.
      • Hamasaki K.
      • et al.
      Progesterone decreases bone morphogenetic protein (BMP) 7 expression and BMP7 inhibits decidualization and proliferation in endometrial stromal cells.
      ).
      To investigate the molecular mechanisms underlying decidualization, we attempted to identify the genes induced by progestin in ESCs in a 3-day culture (
      • Okada H.
      • Nakajima T.
      • Yoshimura T.
      • Yasuda K.
      • Kanzaki H.
      Microarray analysis of genes controlled by progesterone in human endometrial stromal cells in vitro.
      ). Microarray analysis showed fibulin-1 (FBLN1) up-regulation after medroxyprogesterone acetate (MPA) treatment of human ESCs. FBLN1 is an extracellular matrix (ECM) scaffolding protein that binds to many ECM proteins, including fibronectin, laminin-1, fibrinogen, nidogen, and the proteoglycans aggrecan and versican (
      • Timpl R.
      • Sasaki T.
      • Kostka G.
      • Chu M.L.
      Fibulins: a versatile family of extracellular matrix proteins.
      ). FBLN1 also plays an essential role in tissue remodeling, affecting cell adhesion, migration, proliferation, and differentiation (
      • de Vega S.
      • Iwamoto T.
      • Yamada Y.
      Fibulins: multiple roles in matrix structures and tissue functions.
      ). ECM remodeling is central to preparing the endometrium for receptivity and is known to be under hormonal influence (
      • Sillem M.
      • Prifti S.
      • Neher M.
      • Runnebaum B.
      Extracellular matrix remodelling in the endometrium and its possible relevance to the pathogenesis of endometriosis.
      ,
      • Aplin J.D.
      • Lacey H.
      • Haigh T.
      • Jones C.J.
      • Chen C.P.
      • Westwood M.
      Growth factor-extracellular matrix synergy in the control of trophoblast invasion.
      ,
      • Bischof P.
      • Campana A.
      Molecular mediators of implantation.
      ). Indeed, FBLN1 transcript levels in the human endometrium are higher during the secretory phase than during the proliferative phase (
      • Haendler B.
      • Yamanouchi H.
      • Lessey B.A.
      • Chwalisz K.
      • Hess-Stumpp H.
      Cycle-dependent endometrial expression and hormonal regulation of the fibulin-1 gene.
      ,
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ). FBLN1 in human endometrial tissues is expressed in the glandular epithelium during the proliferative phase, and this expression is switched to the stroma during the secretory phase (
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ). These results show FBLN1 as an important molecule that mediates P action in human ESC differentiation toward implantation. However, the mechanism underlying induction of FBLN1 expression by MPA remains unclear.
      Recent reports have demonstrated that heart and neural crest derivatives expressed transcript 2 (HAND2) plays a key role in uterine receptivity and is increased in the mouse uterus during decidualization (
      • Li Q.
      • Kannan A.
      • DeMayo F.J.
      • Lydon J.P.
      • Cooke P.S.
      • Yamagishi H.
      • et al.
      The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.
      ,
      • Huyen D.V.
      • Bany B.M.
      Evidence for a conserved function of heart and neural crest derivatives expressed transcript 2 in mouse and human decidualization.
      ). HAND2 is a transcription factor required for the growth and development of the heart, branchial arches, and limb buds (
      • Liu N.
      • Barbosa A.C.
      • Chapman S.L.
      • Bezprozvannaya S.
      • Qi X.
      • Richardson J.A.
      • et al.
      DNA binding-dependent and -independent functions of the Hand2 transcription factor during mouse embryogenesis.
      ). In uterine tissue–specific HAND2 knockout mice, continued induction of paracrine mitogenic mediators in the stroma maintains epithelial proliferation and stimulates E-induced pathways, resulting in impaired implantation (
      • Li Q.
      • Kannan A.
      • DeMayo F.J.
      • Lydon J.P.
      • Cooke P.S.
      • Yamagishi H.
      • et al.
      The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.
      ). These results suggest that HAND2 expression in the stroma is a critical regulator of the uterine stromal-epithelial communication that directs proper steroid regulation conducive to the establishment of pregnancy. Furthermore, HAND2 plays an important role in decidualization (
      • Huyen D.V.
      • Bany B.M.
      Evidence for a conserved function of heart and neural crest derivatives expressed transcript 2 in mouse and human decidualization.
      ). However, the precise molecular and cellular mechanisms that regulate HAND2 expression in the human endometrium are not completely understood.
      We hypothesized that HAND2 regulates FBLN1 expression during decidualization. We aimed to determine whether progestins and/or E2 direct the effects of HAND2 mRNA expression in human primary cultured ESCs. We then silenced HAND2 or FBLN1 with small interfering RNA (siRNA) technology and measured these expression levels by real-time polymerase chain reaction (PCR) and Western blot analysis to investigate their functional roles in the action of female sex steroids on the endometrium.

      Materials and methods

       Tissue Collection and Culture of Cells

      This study was approved by the Institutional Review Board of Kansai Medical University. All human tissues were obtained with informed consent from all patients and in accordance with the Declaration of Helsinki. Human endometrial tissues were obtained from 24 patients in the proliferative phase, aged 41–47 years, with regular menstrual cycles, who underwent hysterectomies for the treatment of myoma uteri without hormone therapy. ESCs were purified by the standard enzyme digestion method as described elsewhere (
      • Okada H.
      • Nie G.
      • Salamonsen L.A.
      Requirement for proprotein convertase 5/6 during decidualization of human endometrial stromal cells in vitro.
      ). ESCs were cultured in DMEM/F-12 medium supplemented with 10% fetal calf serum (FCS) (HyClone), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen) at 37°C under a humidified atmosphere of 5% CO2 in air. The culture medium was replaced 30 minutes after plating to reduce epithelial cell contamination. The percentage of vimentin-positive cells in confluent ESCs was more than 99% by immunohistochemical staining as described elsewhere (
      • Okada H.
      • Nakajima T.
      • Yoshimura T.
      • Yasuda K.
      • Kanzaki H.
      The inhibitory effect of dienogest, a synthetic steroid, on the growth of human endometrial stromal cells in vitro.
      ).

       Steroid Hormones Treatment and Reagents for ESCs

      After passage 0–1 when ESCs were nearly confluent, cells were trypsinized and replated in six-well plates (1 × 106 cells/well) for real-time PCR analyses. To remove the effect of endogenous steroid hormones, cells were cultured until confluent and then the medium was replaced with phenol red-free DMEM/F-12 supplemented with 10% dextran-coated charcoal stripped (DCS)-FCS, 100 IU/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). After 48 hours, ESCs were washed and cultured in DCS-FCS supplemented medium containing E2 (10−8 mol/L; Wako Pure Chemical Co. Ltd.), P (10−7 mol/L), MPA (10−11, 10−9, and 10−7 mol/L), norethisterone (NET; 10−7 mol/L), levonorgestrel (LNG; 10−7 mol/L), RU-486 (PR antagonist; 10−6 mol/L), dexamethasone (DEX; 10−7 mol/L), 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP; 0.5 mmol/L; Sigma-Aldrich Corp.), dienogest (DNG; 10−7 mol/L; Mochida Pharmaceutical Co.), and/or ethanol as vehicle control. The culture medium was changed every 3 days. Each experiment was repeated at least 3 times with different cell preparations.

       RNA Extraction and Real-Time PCR Analysis

      Total RNA was isolated from cultured ESCs using an RNeasy Minikit (Qiagen GmbH) according to the manufacturer's instructions. Quantitative real-time PCR was performed using the SYBR green I nucleic acid Gel Stain (Roche, Diagnostics GmbH) as described elsewhere (
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ). Elongation factor-1α (EF-1α) as an internal control is valid as reference “housekeeping” gene for transcription profiling, which is also used for real-time PCR experiments (
      • Curtis K.M.
      • Gomez L.A.
      • Rios C.
      • Garbayo E.
      • Raval A.P.
      • Perez-Pinzon M.A.
      • et al.
      EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.
      ,
      • Shen Y.
      • Li Y.
      • Ye F.
      • Wang F.
      • Lu W.
      • Xie X.
      Identification of suitable reference genes for measurement of gene expression in human cervical tissues.
      ). Real-time PCR efficiency (E%) for amplification of each gene was calculated using the following formula: E% = [−1 + 10(−1/α)] × 100, where α is the slope of the corresponding amplification plot (
      • Shen Y.
      • Li Y.
      • Ye F.
      • Wang F.
      • Lu W.
      • Xie X.
      Identification of suitable reference genes for measurement of gene expression in human cervical tissues.
      ). Forward (F) and reverse (R) primers used in this study were as follows: HAND2, 5′-AGAGGAAGAAGGAGCTGAACGA-3′ (F) and 5′-CGTCCGGCCTTTGGTTTT-3′ (R); EF-1α, 5′-TCTGGTTGGAATGGTGACAACATGC-3′ (F) and 5′-AGAGCTTCACTCAAAGCTTCATGG-3′ (R); FBLN1, 5′-GGAGCAGTGCTGCCACAG-3′ (F) and 5′-AGCACCTCTTCACAAATGTG-3′ (R); PRL, 5′-ATTCGATAAACGGTATACCCATGGC-3′ (F) and 5′-TTGCTCCTCAATCTCTACAGCTTTG-3′ (R); 18S, 5′-CGGCTACCACATCCAAGGAA-3′ (F) and 5′-GCTGGAATTACCGCGGCT-3′ (R); insulin-like growth factor-binding protein 1 (IGFBP-1), 5′-GCTCTCCATGTCACCAACAT-3′ (F) and 5′-TCTCCTGATGTCTCCTGTGC-3′ (R) (
      • Buzzio O.L.
      • Lu Z.
      • Miller C.D.
      • Unterman T.G.
      • Kim J.J.
      FOXO1A differentially regulates genes of decidualization.
      ); dickkopf-1 (DKK-1), 5′-CATCAGACTGTGCCTCAGGA-3′ (F) and 5′-CCACAGTAACAACGCTGGAA-3′ (R) (
      • Aghajanova L.
      • Velarde M.C.
      • Giudice L.C.
      The progesterone receptor coactivator Hic-5 is involved in the pathophysiology of endometriosis.
      ). PCR of all controls and samples was performed using duplicate reactions, after which a melting curve analysis was performed to monitor PCR product purity. To eliminate the possibility of contamination with genomic DNA during extraction of total RNA, a control reaction with each primer pair was performed at the same time under identical conditions without reverse transcription, and no amplification was detected. Relative expression levels were calculated for each sample after normalization against the housekeeping gene EF-1α, using the ΔΔthreshold cycle (Ct) method for comparing relative fold expression differences (
      • Huusko T.
      • Salonurmi T.
      • Taskinen P.
      • Liinamaa J.
      • Juvonen T.
      • Paakko P.
      • et al.
      Elevated messenger RNA expression and plasma protein levels of osteopontin and matrix metalloproteinase types 2 and 9 in patients with ascending aortic aneurysms.
      ,
      • Livak K.J.
      • Schmittgen T.D.
      Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
      ).

       HAND2 and FBLN1 Silencing with siRNA

      Silencing of HAND2 or FBLN1 expression was performed with two siRNA preparations directed against distinct areas of the gene sequence (Human Stealth Select RNAi; Invitrogen). Two siRNA targeting human HAND2 sequence (catalog no. HSS145155 [HAND2-A] and HSS190355 [HAND2-B]), two siRNA targeting human FBLN1 sequence (catalog no. HSS103558 [FBLN1-A] and HSS176691 [FBLN1-B]), and nonsilencing RNA (catalog no. HSS12935-112) as negative control of the manufacturer's recommendation were purchased from Invitrogen. After optimization of experiment conditions, cells in six-well plates were grown until 50% confluence at which time they were transfected with each siRNA (10 nmol/L) using Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. ESCs were transfected for 2 days and were then changed to fresh medium containing MPA (10−7 mol/L) and E2 (10−8 mol/L). Three days later, the medium was removed and RNA was extracted from cells. Silencing of the HAND2 and FBLN1 gene was verified by real-time PCR and Western blot analysis in this study. Each experiment was repeated at least 3 times with different cell preparations.

       Western Blot Analysis

      For analysis of HAND2 and FBLN1 protein levels, cultured cells were homogenized in lysis buffer containing Mammalian Protein Extraction Reagent (Thermo Fisher Scientific Inc.) and protease inhibitor cocktail. Western blotting was performed as described elsewhere (
      • Tsuzuki T.
      • Okada H.
      • Cho H.
      • Tsuji S.
      • Nishigaki A.
      • Yasuda K.
      • et al.
      Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1alpha and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.
      ). In brief, samples were fractionated on Any kD precast polyacrylamide gel for HAND2 and β-actin and 7.5% precast polyacrylamide gel for FBLN1 (Bio-Rad, Laboratories, Inc.) and transferred to Immun-Blot polyvinylidene difluoride membrane (Bio-Rad). Nonspecific binding sites were blocked with 5% skim milk powder in Tris-buffered saline for 1 hour. Blots were then incubated for 1 hour at room temperature with mouse monoclonal HAND2 antibody (1:250; Abnova), rabbit polyclonal FBLN1 (1:1,000; Santa Cruz Biotechnology, Inc.), or mouse monoclonal β-actin antibody (1:5,000; Sigma-Aldrich) as a primary antibody; and anti-mouse immunoglobulin (Ig) G peroxidase-labeled secondary antibody (1:5,000), anti-rabbit IgG peroxidase-labeled secondary antibody (1:10,000), or anti-mouse IgG peroxidase-labeled secondary antibody (1:10,000; GE Healthcare Life Science) as a secondary antibody, respectively. Immune complexes were visualized by enhanced chemiluminescence plus Western blotting detection reagents (GE Healthcare Life Science). We analyzed the protein levels using ImageJ software and visualized quantitatively. ImageJ is a public domain image-processing program developed by the National Institutes of Health and is freely available at http://rsbweb.nih.gov/ij. Fold increase was calculated by dividing the relative expression of HAND2 and FBLN1 by the relative expression of β-actin.

       Statistical Analysis

      Data are expressed as mean ± SEM. Results were analyzed with a statistical software package, StatView II version 4.0 (Abacus Concepts). Differences in the measured parameters across the different groups were statistically assessed using analysis of variance with repeated measurements, followed by Fisher's protected least significant difference, multiple range test. A level of P<.05 was considered statistically significant.

      Results

       Induction of HAND2 mRNA Levels during MPA-Induced Decidualization in ESCs

      To characterize the effect of sex steroid hormones on mRNA levels of HAND2, FBLN1, and PRL, we analyzed cultured ESCs in the presence of E2 (10−8 mol/L) + MPA (10−7 mol/L) or MPA (10−7 mol/L) for 12 days. We confirmed EF-1α mRNA levels as a reference gene for real-time PCR analysis in cultured ESCs (Supplemental Fig. 1). The amplification efficiencies (E%) of real-time PCR of EF-1α was 100% with high linearity (the correlation coefficient R2 = 0.973). The EF-1α Ct values showed no significant difference among ESCs treated with vehicle, MPA, and E2 + MPA after 12 day (P>.05). Furthermore, EF-1α mRNA levels were calculated after normalization to 18S as another internal control (
      • Okada H.
      • Nie G.
      • Salamonsen L.A.
      Requirement for proprotein convertase 5/6 during decidualization of human endometrial stromal cells in vitro.
      ) and also showed no significant difference in ESCs treated with steroid hormones (P>.05; Supplemental Fig. 1). Real-time PCR analysis showed that E2 + MPA and MPA significantly increased HAND2 mRNA levels after 1 and 6 days, respectively (Fig. 1A). These effects continued to increase until the end of the study on day 12. Similarly, E2 + MPA and MPA significantly increased mRNA levels of FBLN1 and PRL, a typical marker for decidualization (Fig. 1B and C), consistent with previous reports (
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ,
      • Okada H.
      • Okamoto R.
      • Tsuzuki T.
      • Tsuji S.
      • Yasuda K.
      • Kanzaki H.
      Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.
      ). The mRNA levels of FBLN1 and PRL were significantly increased in ESCs treated with E2 + MPA after 3 and 12 days, respectively (Fig. 1B and C). The addition of vehicle control to the culture medium had no significant effect on mRNA levels of HAND2, FBLN1, and PRL.
      Figure thumbnail gr1
      Figure 1Induction of (A) HAND2, (B) FBLN1, and (C) PRL mRNA levels during MPA-induced decidualization in ESCs. ESCs were cultured in the presence of MPA (10−7 mol/L), E2 (10−8 mol/L) + MPA (10−7 mol/L), or vehicle (Control) for the indicated number of hours (h) or days (d). The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Values are significantly different versus control (*P<.05; **P<.01).

       Effect of Steroid Hormones on HAND2 mRNA Levels in ESCs

      To analyze the dose dependence of HAND2 on MPA, ESCs were incubated with various doses of MPA in the presence of E2 for 12 days. MPA significantly increased HAND2 mRNA levels in a dose-dependent manner (MPA doses, 10−11, 10−9, and 10−7 mol/L), resulting in fold increases of 3.3 ± 0.7, 17.8 ± 3.4, and 17.9 ± 2.2, respectively (P<.05; Fig. 2). In contrast, E2 alone did not affect HAND2 mRNA levels. To determine whether PR is necessary for the induction of HAND2 mRNA levels, ESCs were cultured for 12 days with E2 + MPA containing RU-486, an antiprogestin compound with a high affinity for PR. Cotreatment with RU-486 blocked the increase in HAND2 mRNA levels.
      Figure thumbnail gr2
      Figure 2Effects of steroid hormones on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, various doses of MPA (10−11, 10−9, and 10−7 mol/L), E2 (10−8 mol/L), and/or RU-486 (10−6 mol/L) for 12 days. The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of four separate experiments with different cell preparations, and each value represents mean ± SEM; n = 4. Values are significantly different versus control (*P<.05; **P<.01).

       Effect of 8-Br-cAMP and Various Progestins on HAND2 mRNA Levels

      It is well-known that decidualization of ESCs can be stimulated in vitro by 8-Br-cAMP activation of the protein kinase A-dependent pathway, usually within a short time frame of up to 3 days (
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ). ESCs were incubated with 8-Br-cAMP (0.5 mmol/L) and/or steroid hormones for 3 days. As shown in Figure 3A, HAND2 mRNA levels were increased by E2 + MPA (3.2 ± 0.4 fold; P<.01) and were more strongly increased by MPA+8-Br-cAMP (9.8 ± 1.8 fold; P<.01). However, E2, 8-Br-cAMP, and E2 + 8-Br-cAMP did not regulate HAND2 mRNA levels. These results suggest that simultaneous treatment with MPA and 8-Br-cAMP enhanced HAND2 mRNA levels, but treatment with 8-Br-cAMP alone fails to trigger HAND2 expression.
      Figure thumbnail gr3
      Figure 3(A) Effects of 8-Br-cAMP on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, E2 (10−8 mol/L), MPA (10−7 mol/L), and/or 8-Br-cAMP (0.5 mmol/L) for 3 days. (B) Effects of various progestins on HAND2 mRNA levels in ESCs. ESCs were cultured with vehicle, P (10−7 mol/L), MPA (10−7 mol/L), norethisterone (NET; 10−7 mol/L), levonorgestrel (LNG; 10−7 mol/L), dienogest (DNG; 10−7 mol/L), or dexamethasone (DEX; 10−7 mol/L) in the presence of E2 (10−8 mol/L) for 3 days. The mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. Results are combined data of (A) four and (B) seven separate experiments with different cell preparations, and each value represents mean ± SEM; (A) n = 4 and (B) n = 7. Values are significantly different versus control (*P<.05; **P<.01).
      Next we determined whether P, various progestins (MPA, NET, LNG, and DNG), or the glucocorticoid receptor agonist DEX (10−7 mol/L) in the presence of E2 (10−8 mol/L) could affect HAND2 mRNA levels. As shown in Figure 3B, P and all the progestins significantly increased HAND2 mRNA levels (P<.05 vs. control), whereas DEX had no effect on mRNA levels.

       Effects of Silencing with siRNA on mRNA and Protein Levels of MPA-Induced HAND2 or FBLN1

      HAND2 was increased by E2 + MPA earlier than FBLN1. Therefore, we investigated whether HAND2 regulates FBLN1 in cultured ESCs in the presence of E2 + MPA for 3 days, because HAND2 and FBLN1 mRNA levels were significantly increased by these hormones within 3 days. ESCs were pretreated with HAND2 siRNA (HAND2-A or HAND2-B), FBLN1 siRNA (FBLN1-A or FBLN1-B), or nonsilencing RNA (control) for 2 days and then exposed to E2 + MPA for 3 days. As shown in Figure 4A and E, silencing of HAND2 expression by HAND2-A or HAND2-B significantly reduced FBLN1 mRNA and protein levels (P<.05). However, FBLN1 silencing did not have a significant effect on HAND2 mRNA and protein levels (Fig. 4B and F). Real-time PCR and Western blot analysis confirmed that HAND2 and FBLN1 siRNAs effectively suppressed the expression of the respective targets in ESCs after 3 days of E2 + MPA treatment (Fig. 4). Furthermore, silencing of HAND2 and FBLN1 had no effect on the levels of 18S, another internal control (Fig. 4C). As shown in Figure 1C, PRL mRNA levels showed no significant difference in ESCs after 3 days of E2 + MPA treatment. We further investigate the role of HAND2 and FBLN1 in regulating IGFBP-1 and DKK-1, which have been shown to be up-regulated during decidualization (
      • Buzzio O.L.
      • Lu Z.
      • Miller C.D.
      • Unterman T.G.
      • Kim J.J.
      FOXO1A differentially regulates genes of decidualization.
      ,
      • Tulac S.
      • Overgaard M.T.
      • Hamilton A.E.
      • Jumbe N.L.
      • Suchanek E.
      • Giudice L.C.
      Dickkopf-1, an inhibitor of Wnt signaling, is regulated by progesterone in human endometrial stromal cells.
      ). Silencing of HAND2 and FBLN1 had no effect on the levels of IGFBP-1 and DKK-1 (Supplemental Fig. 2).
      Figure thumbnail gr4
      Figure 4Effects of silencing with siRNA on MPA-induced HAND2 or FBLN1 levels. ESCs were pretreated with HAND2 siRNA (HAND2-A or HAND2-B), FBLN1 siRNA (FBLN1-A or FBLN1-B), or nonsilencing RNA (Control) for 2 days and then exposed to E2 (10−8 mol/L) + MPA (10−7 mol/L) for 3 days. The mRNA levels of (A) FBLN1, (B) HAND2, and (C) 18S were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. Values are significantly different versus control (*P<.05; **P<.01). (D) The protein levels of FBLN1, HAND2, and β-actin were analyzed by Western blot analysis. Total cell lysates were obtained from ESCs cultured with the above-described treatment. The protein levels of FBLN1 (E) and HAND2 (F) were quantified using ImageJ. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Columns and vertical bars represent the mean ± SEM for combined data of three separate experiments with different cell preparations; n = 3. Values are significantly different versus control (**P<.01).

      Discussion

      This study showed that MPA, but not E2, is a potent inducer of HAND2 mRNA levels in ESCs. HAND2 mRNA levels are increased in the mouse uterus during the receptive phase and decidualization and in immortalized ESCs after treatment with steroid hormones (
      • Huyen D.V.
      • Bany B.M.
      Evidence for a conserved function of heart and neural crest derivatives expressed transcript 2 in mouse and human decidualization.
      ,
      • Bany B.M.
      • Cross J.C.
      Post-implantation mouse conceptuses produce paracrine signals that regulate the uterine endometrium undergoing decidualization.
      ). However, previous reports have not described whether HAND2 mRNA levels are regulated by progestins and/or E2 in human ESCs. To the best of our knowledge, our study is the first to demonstrate that MPA stimulates HAND2 mRNA levels in a time- and dose-dependent manner, whereas E2 alone has no effect on HAND2 mRNA levels. In addition, mRNA levels of HAND2, FBLN1, and PRL were significantly increased in ESCs stimulated with E2 + MPA after 1, 3, and 12 days of culture, respectively. The variation of HAND2 mRNA levels induced by E2 + MPA may be due to the characteristics of individual human ESCs because we used the primary culture cells obtained from human endometrial tissues.
      We showed that cotreatment with PR antagonist RU-486 abrogated HAND2 mRNA levels, suggesting a pathway by which progestins may induce expression through PR. This is consistent with previous evidence that HAND2 mRNA levels were greatly reduced in the mouse uterus after RU-486 treatment (
      • Li Q.
      • Kannan A.
      • DeMayo F.J.
      • Lydon J.P.
      • Cooke P.S.
      • Yamagishi H.
      • et al.
      The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.
      ). Furthermore, our study describes for the first time that P and clinically relevant progestins (MPA, NET, LNG, and DNG) increase HAND2 mRNA levels in human ESCs. MPA is a 17β-hydroxyprogesterone derivative called pregnane. MPA is a weak glucocorticoid; however, the strong glucocorticoid Dex failed to affect HAND2 mRNA levels. NET and LNG are derivatives of 19-nortestosterone and are known as an estrane and a gonane, respectively. DNG is considered a new progestin that has combined properties of a 19-nortestosterone derivative and a P derivative. Individual synthetic progestins have different steroid receptor selectivity and activity in target tissues (
      • Xu B.
      • Kitawaki J.
      • Koshiba H.
      • Ishihara H.
      • Kiyomizu M.
      • Teramoto M.
      • et al.
      Differential effects of progestogens, by type and regimen, on estrogen-metabolizing enzymes in human breast cancer cells.
      ). However, in our experiments, we were unable to demonstrate different effects of various progestins on HAND2 levels.
      Interestingly, HAND2 mRNA levels were not regulated by the treatment of ESCs with 8-Br-cAMP alone, another known inducer of decidualization, whereas progestins up-regulate HAND2 expression. Progestins induce expression of PRL and IGFBP-1 in ESCs, although only after several days of treatment, by which time the intracellular levels of cAMP are increasing (
      • Brar A.K.
      • Frank G.R.
      • Kessler C.A.
      • Cedars M.I.
      • Handwerger S.
      Progesterone-dependent decidualization of the human endometrium is mediated by cAMP.
      ). Other studies have shown that some genes in human ESCs are uniquely up-regulated by progestin but not by cAMP (
      • Mizuno K.
      • Tanaka T.
      • Umesaki N.
      • Ogita S.
      Establishment and characterization of in vitro decidualization in normal human endometrial stromal cells.
      ,
      • Gellersen B.
      • Brosens J.
      Cyclic AMP and progesterone receptor cross-talk in human endometrium: a decidualizing affair.
      ,
      • Popovici R.M.
      • Kao L.C.
      • Giudice L.C.
      Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology.
      ). This raises an interesting possibility of diversion of the progestin and cAMP regulatory pathways during decidualization of human ESCs. In our results, 8-Br-cAMP in the presence of MPA enhanced HAND2 mRNA levels, suggesting that the regulation of HAND2 is supported through the protein kinase A pathway in the human endometrium.
      Our study showed that simultaneous treatment with MPA and 8-Br-cAMP enhanced HAND2 mRNA levels. The effect of prostaglandin E2 (PGE2) in inducing the decidualization of rodent and human ESCs was attributed to increased intracellular cAMP levels (
      • Kennedy T.G.
      • Ross H.E.
      Temporal- and hormone-dependent changes in uterine sensitization for the decidual cell reaction and decidualization in vitro of rat endometrial stromal cells.
      ,
      • Cong J.
      • Diao H.L.
      • Zhao Y.C.
      • Ni H.
      • Yan Y.Q.
      • Yang Z.M.
      Differential expression and regulation of cylooxygenases, prostaglandin E synthases and prostacyclin synthase in rat uterus during the peri-implantation period.
      ,
      • Pakrasi P.L.
      • Jain A.K.
      Cyclooxygenase-2 derived PGE2 and PGI2 play an important role via EP2 and PPARdelta receptors in early steps of oil induced decidualization in mice.
      ). Therefore, our results are consistent with earlier studies, which state that PGE2 enhances HAND2 levels in an immortalized ESC line undergoing decidualization (
      • Huyen D.V.
      • Bany B.M.
      Evidence for a conserved function of heart and neural crest derivatives expressed transcript 2 in mouse and human decidualization.
      ). Furthermore, several studies have demonstrated that cotreatment with 8-Br-cAMP and progestin maximally increases the levels of PRL and IGFBP-1, suggesting that cAMP enhances expression of P target genes (
      • Buzzio O.L.
      • Lu Z.
      • Miller C.D.
      • Unterman T.G.
      • Kim J.J.
      FOXO1A differentially regulates genes of decidualization.
      ,
      • Brar A.K.
      • Handwerger S.
      • Kessler C.A.
      • Aronow B.J.
      Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization.
      ). However, the mechanisms responsible for cross-talk between the P and cAMP pathways in the decidualization response are unknown.
      We have previously demonstrated that MPA stimulates FBLN1 mRNA levels in a dose-dependent manner and that RU-486 inhibits this stimulatory effect almost completely, but E2 alone does not induce FBLN1 mRNA levels (
      • Nakamoto T.
      • Okada H.
      • Nakajima T.
      • Ikuta A.
      • Yasuda K.
      • Kanzaki H.
      Progesterone induces the fibulin-1 expression in human endometrial stromal cells.
      ). These results suggest that FBLN1 is an important molecule that mediates P action in human ESC differentiation toward implantation. This study demonstrates that the hormonal effects on HAND2 and FBLN1 are similar. HAND2 and FBLN1 mRNA levels were significantly increased in ESCs with E2 + MPA after 1 and 3 days of culture, respectively. Therefore, we investigated the association between HAND2 and FBLN1. Reduced HAND2 levels resulted in a significant decrease in FBLN1 levels, whereas reduced FBLN1 levels did not have a significant effect on HAND2 levels. These results suggest that HAND2 is an essential downstream target of the P pathway required for progestins to induce FBLN1 in ESCs. HAND2 generally binds to the consensus elements termed E-boxes (CANNTG) to regulate expression of the target genes (
      • Vincentz J.W.
      • Barnes R.M.
      • Firulli A.B.
      Hand factors as regulators of cardiac morphogenesis and implications for congenital heart defects.
      ). Indeed, it has been demonstrated that there were several E-boxes in the human FBLN1 gene promoter (
      • Castoldi M.
      • Chu M.L.
      Structural and functional characterization of the human and mouse fibulin-1 gene promoters: role of Sp1 and Sp3.
      ). Therefore, further studies are needed to clarify whether HAND2 directly or indirectly regulates FBLN1 expression.
      HAND2 is a transcription factor and may control the activation and repression of other gene targets important for endometrial function. A recent study has shown that HAND2 suppresses the production of several fibroblast growth factors that act as paracrine mediators of mitogenic effects in the mouse uterus (
      • Li Q.
      • Kannan A.
      • DeMayo F.J.
      • Lydon J.P.
      • Cooke P.S.
      • Yamagishi H.
      • et al.
      The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.
      ). Alkaline phosphatase level, a decidualization marker, decreased in HAND2-silenced mouse endometrial cells; however, PRL family 8, subfamily a, member 2, did not (
      • Huyen D.V.
      • Bany B.M.
      Evidence for a conserved function of heart and neural crest derivatives expressed transcript 2 in mouse and human decidualization.
      ). The study also showed a decrease in IGFBP-1 and forkhead box O1A levels in immortalized ESCs that stably expressed HAND2-targeted small hairpin RNA, but these cells resulted in an increase in PRL mRNA levels. The present study showed that reduced HAND2 levels resulted in a significant decrease in FBLN1 levels but not in IGFBP-1 and DKK-1 levels. We examined the impact of HAND2 suppression on the acute regulation of gene expression by MPA, because siRNA is not retained in prolonged cultures after transfection. It is possible that 3 days of E2 + MPA treatment is not sufficient to observe the significant difference. Further studies are needed to determine the identity and function of downstream target genes of HAND2 and FBLN1 in the endometrium.
      In summary, progestins increased HAND2 mRNA levels in a time- and dose-dependent manner during decidualization of ESCs in vitro and this stimulatory effect was blocked by RU-486. These results may indicate a potential mechanism of female sex steroid activity in the human endometrium and may have various clinical applications. Moreover, progestin-induced HAND2 contributes to the increased FBLN1 levels in human ESCs. This knowledge about FBLN1 and HAND2 in the endometrium may create new insights into our understanding of reproductive processes such as decidualization and the establishment and maintenance of early pregnancy.

      Acknowledgment

      The authors thank Dr. Yutaka Shimizu (Mochida Pharmaceutical Co.) for providing dienogest and technical advice.

      Appendix

      Figure thumbnail fx1
      Supplemental Figure 1Real-time PCR quantification of EF-1α gene expression in ESCs. (A) Validation of the efficiency of amplification of the internal control (EF-1α). Using reverse transcriptase, cDNA was synthesized from 1 μg total RNA isolated from human ESCs. Serial dilutions of cDNA over a 105-fold range were amplified by real-time PCR using gene-specific primers. The most concentrated sample contained cDNA derived from 20 ng total RNA. EF-1α average Ct values were calculated for each cDNA dilution and plotted against log cDNA dilution (n = 3). All data were fit using least squares linear regression analysis. Real-time PCR efficiency (E%) for amplification was calculated as described in the materials and methods. (B) Effects of steroid hormones on EF-1α mRNA levels in ESCs. ESCs were cultured with vehicle (Control), MPA (10−7 mol/L), and E2 (10−8 mol/L) + MPA (10−7 mol/L) for 12 days. The y-axis indicates real-time PCR cycle threshold numbers (Ct values). Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3. (C) The EF-1α mRNA levels were analyzed by real-time PCR analysis and calculated after normalization to 18S mRNA levels. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM; n = 3.
      Figure thumbnail fx2
      Supplemental Figure 2Effects of silencing HAND2 or FBLN1 on IGFBP-1 and DKK1 expression. ESCs were pretreated with HAND2 siRNA (HAND2-A or HAND2-B), FBLN1 siRNA (FBLN1-A or FBLN1-B), or nonsilencing RNA (Control) for 2 days and then exposed to E2 (10−8 mol/L) + MPA (10−7 mol/L) for 3 days. The mRNA levels of (A) IGFBP-1 and (B) DKK-1 were analyzed by real-time PCR analysis and calculated after normalization to EF-1α mRNA levels. The effect of nonsilencing RNA (Control) was assigned a potency of 100%. Results are combined data of three separate experiments with different cell preparations, and each value represents mean ± SEM, n = 3.

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