Possible Involvement of miR-98 in the Regulation of PGRMC1 During Decidualization

Human endometrial stromal cells (ESCs) differentiate into decidual cells for embryo implantation during the mid-secretory phase of the menstrual cycle. Decidualization is characterized by enhanced production of insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL) by ESCs and their morphological transformation into polygonal cells. Progesterone (P4) receptor membrane component 1 (PGRMC1) is a member of a P4-binding complex implicated in function in female reproduction. In this study, we explored the mechanisms that regulate PGRMC1 during decidualization of human ESCs. Immunohistochemical analysis of endometrial samples showed that PGRMC1 was expressed in endometrial glandular and luminal epithelial cells and stromal cells throughout the menstrual cycle; however, the protein level in stroma was reduced in the secretory phase. Incubation of ESCs with dibutyryl (db)-cAMP and P4 in vitro, which induces decidualization, decreased the PGRMC1 protein abundance. Further, treatment with a PGRMC1-targeting siRNA or PGRMC1 inhibitor (AG-205) promoted mRNA expression of the db-cAMP/P4- and db-cAMP-induced decidual markers IGFBP1 and PRL. Moreover, the microRNA miR-98, a potential repressor of PGRMC1, was upregulated during decidualization, and transfection of ESCs with a miR-98 mimic decreased the PGRMC1 protein level. These findings suggest that miR-98-mediated downregulation of endometrial PGRMC1 may promote decidualization for the establishment of pregnancy

Possible Involvement of miR-98 in the Regulation of PGRMC1 During Decidualization

Human endometrial stromal cells (ESCs) differentiate into decidual cells for embryo implantation during the mid-secretory phase of the menstrual cycle. Decidualization is characterized by enhanced production of insulin-like growth factor-binding protein 1 (IGFBP1) and prolactin (PRL) by ESCs and their morphological transformation into polygonal cells. Progesterone (P4) receptor membrane component 1 (PGRMC1) is a member of a P4-binding complex implicated in function in female reproduction. In this study, we explored the mechanisms that regulate PGRMC1 during decidualization of human ESCs. Immunohistochemical analysis of endometrial samples showed that PGRMC1 was expressed in endometrial glandular and luminal epithelial cells and stromal cells throughout the menstrual cycle; however, the protein level in stroma was reduced in the secretory phase. Incubation of ESCs with dibutyryl (db)-cAMP and P4 in vitro, which induces decidualization, decreased the PGRMC1 protein abundance. Further, treatment with a PGRMC1-targeting siRNA or PGRMC1 inhibitor (AG-205) promoted mRNA expression of the db-cAMP/P4- and db-cAMP-induced decidual markers IGFBP1 and PRL. Moreover, the microRNA miR-98, a potential repressor of PGRMC1, was upregulated during decidualization, and transfection of ESCs with a miR-98 mimic decreased the PGRMC1 protein level. These findings suggest that miR-98-mediated downregulation of endometrial PGRMC1 may promote decidualization for the establishment of pregnancy

PGRMC1 Regulates Cellular Senescence via Modulating FOXO1 Expression in Decidualizing Endometrial Stromal Cells

The appropriate differentiation of endometrial stromal cells (ESCs) into decidual cells is required for embryo implantation and subsequent placentation into humans. Decidualization is accompanied by the appearance of senescent-like cells. We recently reported the secretory phase-specific downregulation of endometrial progesterone receptor membrane component 1 (PGRMC1) and enhanced decidualization upon PGRMC1 knockdown and inhibition in cultured ESCs. However, it remains unknown whether PGRMC1 is involved in cellular senescence during decidualization. Here, we showed that the small interfering RNA (siRNA)-mediated knockdown of PGRMC1 and the inhibition of PGRMC1 by AG-205 increased the expression of the transcription factor forkhead box protein O1 (FOXO1) and the senescence-associated β-galactosidase activity in cAMP analog- and progesterone-treated ESCs. Furthermore, the knockdown of FOXO1 repressed the decidual senescence induced by siRNA-based PGRMC1 knockdown or AG-205 treatment. Taken together, the decreased PGRMC1 expression in ESCs may accelerate decidualization and cellular senescence via the upregulation of FOXO1 expression for appropriate endometrial remodeling and embryo implantation during the secretory phase

Ca²⁺ modulators alter the concentrations of cAMP in ESCs.

<p>ESCs were treated for 1 h with alamethicin (1 μM), ionomycin (3 μM), nifedipine (20 μM), verapamil (20 μM), or dantrolene (20 μM) and then stimulated for 48 h with forskolin (15 μM). The cAMP levels in the cell lysates were determined by EIA. The amount of cAMP was normalized to the amount of total cellular protein. The data from three independent experiments are presented. **p<0.01 <i>vs</i>. forskolin alone-treated group. Values represent the mean ± SEM.</p

Ryanodine receptor (RyR) inhibitor enhances decidual markers expression in ESCs.

<p>ESCs were treated for 1 h with dantrolene (20 μM) and then stimulated for 48 h with forskolin (A, B: 15 μM) or db-cAMP (C, D: 500 μM). Total RNA was subjected to real-time RT-PCR analysis to determine <i>prolactin</i> and <i>IGFBP1</i> mRNA levels. <i>GAPDH</i> was used as an internal control. The data from three independent experiments are presented. **p<0.01 <i>vs</i>. forskolin or db-cAMP alone. Values represent the mean ± SEM.</p

VDCC blockers promote the decidual markers expression in ESCs.

<p>ESCs were treated for 1 h with nifedipine (20 μM) or verapamil (20 μM) and then stimulated for 48 h with forskolin (A, B: 15 μM) or db-cAMP (C, D: 500 μM). Total RNA was subjected to real-time RT-PCR analysis to determine <i>prolactin</i> and <i>IGFBP1</i> mRNA levels. <i>GAPDH</i> was used as an internal control. The data from three independent experiments are presented. **p<0.01 <i>vs</i>. forskolin or db-cAMP alone. Values represent the mean ± SEM.</p

Ca²⁺ chelator counteracted the effect of Ca²⁺ ionophore in ESCs and EM1 cells.

<p>ESCs or EM1 cells were treated for 1 h with alamethicin (1 μM), nifedipine (20 μM), or dantrolene (20 μM) a in the presence of BAPTA (20 μM) and then stimulated with forskolin (15 μM). Total RNA was subjected to real-time RT-PCR analysis to determine <i>prolactin</i> mRNA levels in ESCs (A) or <i>COX2</i> mRNA levels in EM1 cells (B). <i>GAPDH</i> was used as an internal control. The data from three independent experiments are presented. **p<0.01, *p<0.05 <i>vs</i>. forskolin alone. ^##p<0.01, ^#p<0.05 <i>vs</i>. treatment without BAPTA (BAPTA(-)). Values represent the mean ± SEM.</p