Critical role of TRPC1-mediated Ca²⁺ entry in decidualization of human endometrial stromal cells

Abstract

Decidualization is an ovarian steroid-induced remodeling/differentiation process of uterus essential for embryo implantation and placentation. Here, we investigated the possible involvement of enhanced Ca²⁺ dynamics in the decidualization process in human endometrial stromal cells (hESC) in its connection with a recently emerging nonvoltage-gated Ca²⁺ entry channel superfamily, the transient receptor potential (TRP) protein. Combined application of 17β-estradiol (E₂) (10 nM) and progesterone (P₄) (1 μM) for 7-14 d resulted in morphological changes of hESC characteristic of decidualization (i.e. cell size increase), whereas sole application of E₂ exerted little effects. A 7- to 14-d E₂/P₄ treatment greatly increased the expression level of decidualization markers IGF binding protein-1 (IGFBP-1) and prolactin and also up-regulated the expression of TRPC1, a canonical TRP subfamily member that has been implicated in store-operated Ca²⁺ influx (SOC) in other cell types. In parallel with this up-regulation, SOC activity in hESC, the nuclear translocation of phosphorylated cAMP responsive element binding protein (p-CREB) and the expression of Forkhead box protein 01 were enhanced significantly. Small interfering RNA knockdown of TRPC1 counteracted the E₂/P₄-induced up-regulation of IGFBP-1 and prolactin and enhancement of SOC activity together with the inhibition of hESC size increase, p-CREB nuclear translocation, and FOXO1 up-regulation. Coadministration of SOC inhibitors SK&F96365 or Gd³⁺ with E₂/P₄ also suppressed the up-regulation of IGFBP-1 and hESC size increase. Similar inhibitory effects were observed with extracellularly applied TRPC1 extracellular loop 3-directed antibody, which is known to bind a near-pore domain of TRPC1 channel and block its Ca²⁺ transporting activity. These results strongly suggest that up-regulation of TRPC1 protein and consequent enhancement of SOC-mediated Ca²⁺ influx may serve as a crucial step for the decidualization process of hESC probably via p-CREB-dependent transcriptional activity associated with FOXO1 activation.

Fig. 1.

Enhanced expression of TRPC1 and IGFBP-1 in response to E₂ and P₄. A, mRNA expression pattern of TRPC isoforms in rat brain (left) and those of TRP isoforms and IGFBP-1 in hESC evaluated by conventional RT-PCR immediately after enzymatic dissociation (d 0; right). For the positive control of TRPM and TRPV isoforms, refer to Inoue et al. (56). B–D, Results of quantitative real-time PCR analysis for the mRNA levels of TRPC1 (B), TRPC4 and TRPC6 (C), and IGFBP-1 and PRL (D) after a 7- or 14-d treatment with EtOH (vehicle), E₂ (10 nm), or E₂ (10 nm) plus P₄ (1 μm). Columns and bars indicate the mean ± sem obtained from three to four individual experiments for each condition. To reduce variability among different preparations, data are shown as the relative to those for EtOH at 7 d after normalization to the level of β-actin. E and F, Representative of immunoblot analysis (upper panel) and its summary (lower panel) on the expression of TRPC1 (E) and IGFBP-1 (F) proteins after a 7- or 14-d treatment with EtOH, E₂ only or E₂ plus P₄. Columns and bars indicate the mean ± sem obtained from four individual experiments for each condition. *, P < 0.05 with Tukey's multiple comparison test.

Fig. 2.

Augmentation of SOC by E₂ and P₄. A, Typical Ca²⁺ responses of hESC evoked by store depletion after a 14-d treatment with E₂ alone or E₂ plus P₄. hESC were first perfused with Ca²⁺-free external solution and stimulated by the endoplasmic reticulum Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) (10 μm) to deplete internal stores and then subjected to 1 mm Ca²⁺ to evoke SOC-mediated influx. In contrast, excess K⁺ (75 mm) failed to increase [Ca²⁺]_i, thus suggesting the lack of voltage-dependent Ca²⁺ channels in hESC (data not shown). B and C, Summary of the results such as shown in A for SOC-mediated Ca²⁺ influx (ΔP2; B) and Ca²⁺ release (ΔP1; C) after a 7- or 14-d treatment with EtOH, E₂ only, or E₂ plus P₄. All data points from 8–14 independent experiments are displayed as black crosses, each of which represents the average from at least 100 cells, respectively. Filled circles and vertical bars indicate the mean ± sem calculated from data points. P < 0.01, evaluated by Tukey's multiple comparison test. NS, Statistically not significant.

Fig. 3.

RNA silencing of TRPC1 inhibits the up-regulation of IGFBP-1 by E₂/P₄ treatment. A, Results of real-time PCR analysis for the mRNA levels of IGFBP-1 and PRL after a 14-d treatment with negative control (NCsi) or TRPC1-siRNA (C1si) under continued stimulation with EtOH, E₂ only or E₂ plus P₄. B, Immunoblots of TRPC1 and IGFBP-1 proteins under the same conditions as in A. Representative immunoblots (upper) and relative expression of IGFBP-1 protein to β-actin protein (lower panel). C, Averaged 2D-area of caveolin-1-immunostained hESC (green) after a 14-d treatment with NCsi or C1si, with 4′,6-diamino-2-phenylindole nuclei staining (red). Columns and bars indicate the mean ± sem calculated from 20 cells. D, Typical Ca²⁺ response of hESC after TRPC1 knockdown under E₂/P₄ treatment for 14 d. The procedures employed were the same as in A–C. E, Summary of the effects of TRPC1 knockdown (C1si) on SOC-mediated Ca²⁺ influx (ΔP2) in comparison with negative control (NCsi) under E₂/P₄ stimulation for 7 or 14 d. Columns and bars in A, B, and E indicate the means ± sem obtained from five, four to five, and 9–15 individual experiments, respectively (in E, data are the average from at least 100 cells). * in A–C, P < 0.05 with Tukey's multiple comparison test; ***, P < 0.0005 with unpaired t test. CPA, Cyclopiazonic acid.

Fig. 4.

Suppression of SOC-mediated Ca²⁺ influx attenuates the up-regulation of IGFBP-1 by E₂/P₄ treatment. A and B, Typical Ca²⁺ response to a SOC inhibitor SK&F96365 of hESC from donor 5 (A) and its concentration-dependent inhibitory effects (B) (obtained from 3–11 individual experiments; data are the average from at least 100 cells). The experimental conditions used were the same as in Fig. 2A except that the drug was applied at the dashed bar. C, Representative immunoblots of IGFBP-1 protein after a 7- and/or 14-d E₂/P₄ treatment in the absence and presence of 5 μm SK&F96365 (SK&F) or 1 μm Gd³⁺. Because higher concentrations of SK&F caused progressive death of hESC, its moderate concentration which inhibited SOC-mediated Ca²⁺ influx partially (5 μm) was chosen; 1 μm was enough high for Gd³⁺ to inhibit the Ca²⁺ influx (data not shown). D, Compromised IGFBP-1 protein expression in the presence of SOC inhibitors (either 5 μm SK&F or 1 μm Gd³⁺) under 7- or 14-d E₂/P₄ treatment (obtained from four and five individual experiments, respectively). The relative expression is defined as the ratio of normalized IGFBP-1 protein level by β-actin in the presence (drug) vs. absence (con) of drugs. Data were obtained as a paired set (drug vs. no drug) from the same donor. **, P < 0.01; ***, P < 0.005 with paired t test. E, Averaged 2D-area of hESC after a 14-d E₂/P₄ treatment in the absence and presence of 1 μm Gd³⁺ calculated from 40 cells for each drug. **, P < 0.01 with unpaired t test. CPA, Cyclopiazonic acid.

Fig. 5.

Specific inhibition of TRPC1-associated SOC prevents hESC decidualization. A, Externally applied T1E3-Ab inhibited the Ca²⁺ response of hESC induced by store depletion, as compared with another ineffective antibody (T1-Ab), which specifically recognizes the intracellular domain of TRPC1 protein; 100 cells were used for each condition from donor 6. B, T1E3-Ab inhibited the up-regulation of IGFBP-1 protein by E₂/P₄ treatment (14 d). Representative data. C, Summary of the inhibitory effect of T1E3-Ab on IGFBP-1 expression. Paired data (T1E3-Ab vs. T1-Ab) were obtained for β-actin or IGFBP-1 in hESC under 14-d E₂/P₄ stimulation. Relative expression is defined as the ratio of data with T1E3-Ab vs. T1-Ab for each donor. *, P < 0.05 with paired t test. D, Averaged 2D-area of hESC evaluated from phase-contrast images after a 14-d E₂/P₄ treatment in the presence of T1-Ab or T1E3-Ab. Evaluated from 40 cells for each condition. **, P < 0.01 with unpaired t test. CPA, Cyclopiazonic acid.

Fig. 6.

A 14-d E₂/P₄ treatment up-regulates the nuclear expression level of p-CREB but not of NFAT. A, Representative immunoblots for NFAT extracted from hESC treated with EtOH, E₂, or E₂P₄ for 14 d. To confirm the equal loading and specificity of samples, blots were reprobed for LaminB. B, Histograms show the levels of NFAT proteins relative to a nuclear marker LaminB. Among four isoforms tested, the expression of NFAT3 was most prominent. C, Representative immunoblots for p-CREB (nuclear fractions), CREB (total cell lysate) from EtOH, E₂, or E₂P₄-stimulated hESC. To confirm the equal loading and specificity of samples, blots were reprobed for LaminB or β-actin, respectively. Histograms show the levels of p-CREB and CREB proteins relative to LaminB and β-actin, respectively. D, Nuclear p-CREB expression in hESC treated with TRPC1-siRNA just before E₂/P₄ treatment (14 d). E, Nuclear expression of transcription factors FOXO1, HOXA10, HOXA11, and C/EBP-β in hESC treated with TRPC1-siRNA and E₂/P₄ (14 d). Evaluated by quantitative real-time PCR. Columns and bars in B–E indicate the means ± sem obtained from three to four, four to six, five, and five individual experiments, respectively. * in C–E, P < 0.05 with Tukey's multiple comparison test.