Regulatory Action of Calcium Ion on Cyclic AMP-Enhanced Expression of Implantation-Related Factors in Human Endometrial Cells

Abstract

Decidualization of human endometrial stroma and gland development is mediated through cyclic AMP (cAMP), but the role of intracellular calcium ion (Ca2+) on cAMP mediated-signaling in human endometrial stroma and glandular epithelia has not been well-characterized. The present study was designed to investigate the role of intracellular Ca2+ on cAMP mediated-decidualization and gland maturation events, which can be identified by the up-regulation of prolactin and IGF-binding protein (IGFBP)1 in human endometrial stromal cells (ESCs), and cyclooxygenase 2 (COX2) and prostaglandin E2 (PGE2) and glandular epithelial EM-1 cells. Increases in decidual prolactin and IGFBP-1 transcript levels, induced by cAMP-elevating agents forskolin or dibutyryl cyclic AMP, were inhibited by Ca2+ influx into ESCs with Ca2+ ionophores (alamethicin, ionomycin) in a dose-dependent manner. Conversely, inhibitors of Ca2+ influx through L-type voltage-dependent Ca2+ channel (VDCC), nifedipine and verapamil, enhanced the decidual gene expression. Furthermore, dantrolene, an inhibitor of Ca2+ release from the intracellular Ca2+ store, up-regulated prolactin and IGFBP-1 expression. Ca2+ ionophores decreased intracellular cAMP concentrations, whereas nifedipine, verapamil or dantrolene increased cAMP concentrations in ESCs. In glandular epithelial cells, similar responses in COX2 expression and PGE2 production were found when intracellular cAMP levels were up-regulated by decreases in Ca2+ concentrations. Thus, a marked decrease in cytosolic Ca2+ levels caused the elevation of cAMP concentrations, resulting in enhanced expression of implantation-related factors including decidual markers. These findings suggest that fluctuation in cytosolic Ca2+ concentrations alters intracellular cAMP levels, which then regulate differentiation of endometrial stromal and glandular epithelial cells.

Fig 1. Ca²⁺ influx inhibits the decidual markers expression in ESCs.

ESCs were treated for 1 h with alamethicin (A-D: 0.25, 0.5, 1 μM) or ionomycin (E-H: 1, 3 μM) and then cultured for 48 h with forskolin (A, B, E, F: 15 μM) or db-cAMP (500 μM). Total RNA was subjected to real-time RT-PCR analysis to determine prolactin and IGFBP1 mRNA levels. GAPDH was used as an internal control. The data from three independent experiments are presented. **p<0.01, *p<0.05 vs. forskolin or db-cAMP alone. Values represent the mean ± SEM.

Fig 2. VDCC blockers promote the decidual markers expression in ESCs.

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 prolactin and IGFBP1 mRNA levels. GAPDH was used as an internal control. The data from three independent experiments are presented. **p<0.01 vs. forskolin or db-cAMP alone. Values represent the mean ± SEM.

Fig 3. Ryanodine receptor (RyR) inhibitor enhances decidual markers expression in ESCs.

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 prolactin and IGFBP1 mRNA levels. GAPDH was used as an internal control. The data from three independent experiments are presented. **p<0.01 vs. forskolin or db-cAMP alone. Values represent the mean ± SEM.

Fig 4. Ca²⁺ modulators alter the concentrations of cAMP in ESCs.

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 vs. forskolin alone-treated group. Values represent the mean ± SEM.

Fig 5. Ca²⁺ modulators affect the cAMP-induced COX2 expression and PGE2 production in epithelial EM1 cells.

EM1 cells 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). A. Total RNA was subjected to real-time RT-PCR analysis to determine COX2 mRNA levels. GAPDH was used as an internal control. The data from three independent experiments are presented. **p<0.01, *p<0.05 vs. forskolin alone. B. The PGE2 levels released into media were determined by EIA. The data from three independent experiments are presented. **p<0.01 vs. forskolin alone. Values represent the mean ± SEM. C. The cAMP levels in the cell lysates were determined by EIA. The data from three independent experiments are presented. **p<0.01, *p<0.05 vs. forskolin alone. Values represent the mean ± SEM.

Fig 6. Ca²⁺ chelator counteracted the effect of Ca²⁺ ionophore in ESCs and EM1 cells.

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 prolactin mRNA levels in ESCs (A) or COX2 mRNA levels in EM1 cells (B). GAPDH was used as an internal control. The data from three independent experiments are presented. **p<0.01, *p<0.05 vs. forskolin alone. ^##p<0.01, ^#p<0.05 vs. treatment without BAPTA (BAPTA(-)). Values represent the mean ± SEM.

Fig 7. Diagram illustrating the potential role of intracellular Ca²⁺ in endometrial cells.

Nifedipine and verapamil inhibit VDCC, whereas dantrolene blocks Ca²⁺ release from ER store which causes decreases in intracellular Ca²⁺ levels. Alamethicin and ionomycin promote Ca²⁺ influx through the cell membrane. Blocking Ca²⁺ causes the alternation of cAMP signaling, resulting in the down-regulation of prolactin and IGFBP1, and COX2 and PGE2 in human endometrial stromal cell and glandular epithelial cell, respectively. Solid arrow: the effect of nifedipine, verapamil, or dantrolene. Open arrow: the effect of alamethicin or ionomycin.