1. INTRODUCTION

Progesterone drives numerous maternal physiological adaptations on T cells resulting in tolerance rather than immunity, which ensures the acceptance of the semiallogeneic fetus during pregnancy.1 Progesterone is majorly produced by placenta and its concentrations reach high levels there (1–10 μM) and in blood (100–500 nM) during pregnancy.2,3 These higher levels of progesterone maintain an immunotolerogenic microenvironment in placenta, the maternal-fetal interface, and the blood by suppressing human T-cell activation and proliferation.3–7 This immunotolerance seems to be a function of progesterone concentration regardless of the antigen specificity of T cells.6,8 Pregnancy-related remission and subsequent flare-up of autoimmune diseases, including rheumatoid arthritis (RA), psoriasis, and multiple sclerosis (MS), have been found to coincide with changes in progesterone levels during pregnancy and after delivery.9–11 Indeed, progesterone promotes a shift in the cytokine profile and suppresses the proliferation of CD4+ and CD8+ T cells isolated from men and pregnant women.6,8 There is no expression of the classical nuclear progesterone receptor (nPR) in human T cells, and thus the immunotolerance by progesterone is not via nPR.12,13

Progesterone is able to stimulate rapid nongenomic responses that changes various ion fluxes in human resting T cells, such as acidification, an increase in intracellular Ca2+ concentration ([Ca2+]i) and inhibition of Na+/H+-exchange 1 (NHE1). These rapid responses induced by progesterone can all be mimicked by membrane-impermeable progesterone-bovine serum albumin conjugate (progesterone-BSA).6,7 Furthermore, when progesterone is added either at the same time as mitogen phytohemagglutinin (PHA), or 72 hours after PHA, a suppression of PHA-activated T cell proliferation is observed.6 These findings suggest that progesterone targets the plasma membrane sites to exert rapid nongenomic ion flux responses, and this results in a suppression of T cell proliferation.6,7 These various rapid responses induced by progesterone seem to be important for the retention of immunotolerance associated with immunosuppression.6,7,14,15 Until now, it is still unclear which rapid responses by progesterone in T cells are therapeutically relevant to the development of clinical drugs. The drug development of progesterone analogs targeting specific membrane progesterone receptors is difficult because there are a large number of different membrane progesterone receptors that might be involved in the diverse nongenomic responses in T cells and many other cells.

The sustained [Ca2+]i increase due to Ca2+ influx and the activation of protein kinase C (PKC) are two important early mitogenic signals that occur during T-cell activation and proliferation after crosslinking the T-cell receptor (TCR)/CD3 complex with CD3 antibodies or PHA.16–18 The inductions of Ca2+ influx by calcium ionophores and PKC activation by phorbol esters are able to mimic PHA-activated T-cell proliferation.19–21 Blocking PHA-activated Ca2+ influx results in the suppression of T-cell proliferation.15,22,23 In contrast, blocking Ca2+ release from intracellular stores does not affect T-cell proliferation activated by CD3 antibody or PHA.24 When PKC activity is downregulated using phorbol 12-myristate 13-acetate (PMA), the sustained [Ca2+]i increase via Ca2+ influx induced by PHA is enhanced and this can be blocked using an inorganic channel blocker (Ni+).21 Furthermore, PHA-activated sustained Ca2+/Mn2+ influx is rapidly suppressed by progesterone in human T cells.15 In addition, in human resting T cells, conventional PKC (cPKC) inhibitors also enhance the sustained [Ca2+]i increase by progesterone.14 These findings suggest that cPKC activation might participate in the suppression of the sustained [Ca2+]i increase via Ca2+ influx in resting T cells caused by progesterone. One hypothesis is that cPKC activation by progesterone may rapidly suppress Ca2+ influx leading to early inhibition of T-cell proliferation. There are two major cPKC isoforms, cPKCα and cPKCβ, with the latter having two subforms, cPKCβI and cPKCβII, expressed in human T cells.25 cPKCα probably has no effect on Ca2+ influx because it is indispensable to T-cell proliferation.26 Recently, the activation of cPKCβ has been shown to promote antiproliferation in a human T-cell line CCRF-CEM.27 However, the specific roles of cPKCβI and cPKCβII regarding Ca2+ influx and proliferation remain obscure in human resting T cells. Therefore, it has become important to investigate whether either or both cPKCβI and cPKCβII in resting T cells provokes the suppression of the sustained [Ca2+]i increase that occurs via Ca2+ influx and is induced by progesterone.

2. METHODS 2.1. Chemicals and reagents

RPMI 1640 medium (RPMI), N-methyl-D-glucamine (NMDG), progesterone, and PMA were obtained from Sigma-Aldrich. HEPES was bought from BioShop. Charcoal stripped fetal bovine serum (CS-FBS), which has steroid hormones removed, was supplied by Biological Industries. R5020, 02-0, and progesterone-BSA (progesterone 3-O-carboxymethyloxime:BSA) were purchased from PerkinElmer, Axon Medchem, and Steraloids, respectively. Ro318220 was supplied by Enzo Life Sciences. U73122 and U73343 were brought from Calbiochem. A23187, Go6976, and LY333531 were from Cayman Chemicals. CGP53353 was obtained from Tocris Bioscience. Fura-2 acetoxymethyl ester (fura-2 AM) was purchased from Thermo Fisher Scientific. Mouse antibodies against cPKC and cPKCβII were purchased from Santa Cruz Biotechnology. The mouse antibody for β-actin was from Novus Biologicals.

2.2. Preparation of T cells

Peripheral blood was obtained from healthy male volunteers (aged between 20 and 30 years), who gave informed consent, using the experimental procedure approved by the Institutional Review Board of National Yang-Ming University (YM105112E). As previously described, T cells were isolated from the peripheral blood and then incubated in RPMI supplemented with 10% CS-FBS (v/v) at 37°C in the presence of 5% CO2. Assays showed that the T-cell suspension contained nearly 100% CD3-positive cells.20

2.3. Measurement of the Ca2+ influx by Mn2+ quenching fura-2 fluorescence

T cells (1 × 107 cells/ml) were loaded with the fluorescent dye fura-2 AM (5 μM) in RPMI at 37°C for 30 min. Cells (2 × 106 cells) were then resuspended in 2.5 ml of Ca2+-free loading buffer (152 mM NaCl, 1.2 mM MgCl2, 2.2 mM NMDG·Cl, 5 mM KCl, 10 mM HEPES, 10 mM glucose, pH 7.4) before transferred to a cuvette in a dual-wavelength spectrofluorimeter, SPEX model CM1T11I (Spex Industries, Edison, NJ, USA). Using excitation wavelength of 360 nm, the fluorescence emission at 505 nm was continuously measured for 16 minutes at 37°C. As previously described, Mn2+ was used as a Ca2+ surrogate for measuring Ca2+ influx responses by quenching intracellular fura-2 fluorescence; A23187 was added to confirm cell membrane integrity and viability.15,28 Using Spex DM3000 software, every fluorescent signal recording was normalized against its baseline value and represented as F360. The Ca2+ influx index, ∆F360, was obtained by calculating the difference in the normalized fluorescent intensity during the MnCl2 addition.

2.4. Measurement of [Ca2+]i

As described above, fura-2-loaded T cells were resuspended in Ca2+-free loading buffer or Ca2+-containing loading buffer (which replaced the NMDG·Cl with CaCl2) before transferred to a SPEX model CM1T11I. Using excitation wavelengths of 340 nm and 380 nm, the fluorescence emission at 505 nm was continuously measured for 17 min at 37°C. Using Spex DM3000 software, the [Ca2+]i was determined from the ratio of fluorescence intensity according to a previously derived formula.29

2.5. Western blotting analysis

T cells (6 × 106 cells) were homogenized in lysis buffer, and aliquots (40 μg) of protein samples were subjected to 10% SDS-PAGE.7 After protein transfer onto nitrocellulose membranes (Hoefer Scientific Instruments), the membranes were probed with primary antibodies and then with secondary antibodies conjugated to horseradish peroxidase. Protein bands visualized by enhanced chemiluminescence kit (PerkinElmer).

2.6. Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM) from three independent experiments. The t-test was used to compare two groups. One-way ANOVA followed by Fisher’s least significant difference test was used for multiple comparisons. A level of p < 0.05 was set for statistical significance. SPSS Version 24 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis.

3. RESULTS 3.1. Effects of progesterone, its analogs, and progesterone-BSA on Ca2+ influx in resting T cells

Dose-response relationships of progesterone, R5020 and 02-0 (1, 5, 10, 20 μM) and progesterone-BSA (10, 50, 100, 200 nM) on Ca2+ influx were explored in resting T cells. The concentrations of progesterone and analogs >1 μM and progesterone-BSA >10 nM all resulted in significant suppression of Ca2+ influx (Fig. 1). The 50% inhibitory concentration (IC50) values of progesterone, R5020, 02-0, and progesterone-BSA on ∆F360 were 15.3, 14.2, and 9.2 μM and 115.2 nM, respectively. Therefore, 10 μM doses of progesterone and analogs were chosen for the following experiments.

Fig. 1:

Dose effects of progesterone, R5020, 02-0, and progesterone-BSA on the Ca2+ influx in resting T cells. Fura-2-loaded cells were stimulated with (A) progesterone (P4, 1, 5, 10, 20 μM), (B) R5020 (R50, 1, 5, 10, 20 μM), (C) 02-0 (1, 5, 10, 20 μM), (D) progesterone-BSA (P4-BSA, 10, 50, 100, 200 nM) or vehicle control (Con) at the first minute for 6 min, then had 0.5 mM MnCl2 (Mn2+) added for 6 min; this was followed by 250 nM A23187 (filled inverted triangle) for 3 min. The arrows show the times of each addition. Representative traces were obtained from three individual experiments. Data are mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the vehicle control.

3.2. Exploration of the effects of universal inhibitors of cPKC and phosphatidylinositol-phospholipase C on the Ca2+ influx and [Ca2+]i changes induced by progesterone and analogs

A universal cPKC inhibitor, Ro318220,30 was used to investigate the role of cPKC in the Ca2+ influx and [Ca2+]i changes caused by progesterone and analogs in resting T cells. Ro318220 significantly attenuated Ca2+ influx suppression and significantly enhanced [Ca2+]i increase due to progesterone and analogs (Fig. 2).

Fig. 2:

Effects of Ro318220 on the Ca2+ influx and [Ca2+]i changes in resting T cells induced by progesterone and its analogs. Fura-2-loaded cells were pretreated with Ro318220 (Ro, 0, 1 μM) for 30 min. (A) The time course of the Ca2+ influx protocol after stimulation with 10 μM of progesterone (P4), R5020 (R50), 02-0, or vehicle control (Con) was the same as described above. The filled inverted triangle represents A23187. (B) The time course of the [Ca2+]i changes. Cells were stimulated with 10 μM of progesterone, R5020, 02-0, or vehicle control at the second minute for 15 min in Ca2+-containing loading buffer. The representative traces were obtained from three individual experiments. Data are mean ± SEM (n = 3). ***p < 0.001 compared with the vehicle control. #p < 0.05, ##p < 0.01 compared with stimulation by progesterone or its analogs without the presence of Ro318220.

A universal phosphatidylinositol-phospholipase C (PI-PLC) inhibitor, U73122, and its inactive control, U73343,31 were also used to investigate the role of PI-PLC pathway in the Ca2+ influx and [Ca2+]i changes caused by progesterone and analogs. U73122 and U73343 did not affect the Ca2+ influx suppression by progesterone and analogs (Fig. 3A). However, U73122 did significantly decrease the [Ca2+]i increase by progesterone and analogs (Fig. 3B). The results demonstrated that cPKC activation-associated Ca2+ influx suppression by progesterone and analogs was not via the PI-PLC pathway.

Fig. 3:

Effects of U73122 and U73343 on the Ca2+ influx and [Ca2+]i changes in resting T cells after stimulation with progesterone and its analogs. Fura-2-loaded cells were pretreated with 250 nM of U73122 or U73343 for 10 min. (A) The time course of the Ca2+ influx protocol after stimulation with 10 μM of progesterone (P4), R5020 (R50), 02-0 or vehicle control (Con). The filled inverted triangle represents A23187. (B) The time course of the [Ca2+]i changes after stimulation with 10 μM of progesterone, R5020, 02-0 or vehicle control for 15 min was measured in Ca2+-free loading buffer. Representative traces were obtained from three individual experiments. Data are mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the vehicle control. ###p < 0.001 compared with stimulation by progesterone or its analogs in the presence of U73343.

3.3. Determining, using selective inhibitors, the role of the various cPKC isoforms on the Ca2+ influx by progesterone and analogs

To investigate which cPKC isoforms might participate in the Ca2+ influx suppression by progesterone and analogs in resting T cells, a cPKCα/βI-selective inhibitor, Go6976,32 was first used. The Ca2+ influx suppression was not influenced by Go6976 (Fig. 4A).

Fig. 4:

Effects of the various cPKC isoform-selective inhibitors on Ca2+ influx in resting T cells caused by progesterone and its analogs. Fura-2-loaded cells were pretreated with (A) the cPKCα/βI-selective inhibitor Go6976 (Go, 0, 1 μM) or (B) the cPKCβI/βII-selective inhibitor LY333531 (LY, 0, 1 μM) for 30 min. The time course of the Ca2+ influx protocol after stimulation with 10 μM of progesterone (P4), R5020 (R50), 02-0 or vehicle control (Con). The filled inverted triangle represents A23187. Representative traces were obtained from three individual experiments. Data are mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the vehicle control. #p < 0.05, ##p < 0.01 compared with stimulation by progesterone or its analogs without the presence of an inhibitor.

Next, LY333531, a cPKCβI/βII-selective inhibitor,33 was then used. LY333531 significantly attenuated the Ca2+ influx suppression (Fig. 4B). This suggests that the Ca2+ influx suppression did not involve cPKCα and cPKCβI but rather was related to the activation of cPKCβII.

3.4. Comparison the effects of a cPKCβII-specific inhibitor and the status of PKC downregulation on the Ca2+ influx by progesterone and analogs in T cells

To confirm the role of cPKCβII in the Ca2+ influx suppression by progesterone and analogs, a cPKCβII-specific inhibitor, CGP53353,34 was used. CGP53353 did significantly attenuate the Ca2+ influx suppression by progesterone and analogs (Fig. 5A).

Fig. 5:

Effects of cPKCβII and PKC downregulation on the Ca2+ influx in resting T cells caused by progesterone and its analogs. Cells were (A) loaded with fura-2 and then pretreated with the cPKCβII-specific inhibitor CGP53353 (CGP, 0, 10 μM) for 30 min, or (B) loaded with fura-2 after PKC downregulation by PMA (0, 1 μM) for 18 h. The time course of the response to the Ca2+ influx protocol after stimulation with 10 μM of progesterone (P4), R5020 (R50), 02-0 or vehicle control (Con). The filled inverted triangle represents A23187. Representative traces were obtained from three individual experiments. Data are mean ± SEM (n = 3). **p < 0.01, ***p < 0.001 compared with the vehicle control. ##p < 0.01, ###p < 0.001 compared with stimulation by progesterone or its analogs without the presence of CGP53353 or without PKC downregulation by PMA.

Since cPKCβII is one of the PKC isoforms expressed in human T cells and might be affected by PMA-mediated PKC downregulation,21 the effects of PKC downregulation on the Ca2+ influx suppression were observed. After PKC downregulation, the Ca2+ influx suppression by progesterone and analogs was almost abolished (Fig. 5B).

3.5. The status of cPKCβII protein expression after PKC downregulation in T cells

Both the cPKCβII-specific inhibitor, CGP53353, and PKC downregulation are able to attenuate the Ca2+ influx suppression by progesterone and analogs in T cells. Hence, the status of cPKCβII protein expression after PKC downregulation needed to be investigated. After downregulation, the level of cPKCβII protein was significantly reduced to 9.8 ± 4.8% and, furthermore, the level of total cPKC protein was also significantly reduced to 41.3 ± 7.6% (Fig. 6). It should be noted that the reduction in protein level of total cPKC was significantly less than that of cPKCβII (Fig. 6C). Therefore, cPKCβII seems to play a pivotal role in provoking the negative regulation on the Ca2+ influx by progesterone in T cells.

Fig. 6:

Effects of PKC downregulation on the cPKCβII and cPKC expression in resting T cells. After pretreatment with PMA (0, 1 μM) for 18 h, T cell lysates (lane 1-3: three individuals) were collected to determine the protein expression levels of (A) cPKCβII and (B) cPKC by Western blotting analysis. (C) Relative protein levels of cPKCβII and cPKC were normalized against β-actin and are showed as means ± SEM (n = 3). *p < 0.05, **p < 0.01 compared with the control without PMA pretreatment. #p < 0.05 compared with the relative protein levels of cPKCβII and cPKC in the presence of PMA pretreatment.

4. DISCUSSION

Sustained Ca2+ influx is a crucial early mitogenic signal for T-cell activation and proliferation.15,22,23 Progesterone is able to rapidly immunosuppress PHA-activated sustained Ca2+ influx and proliferation in human T cells.15 Therefore, it is important to explore whether this immunosuppressive response on Ca2+ influx by progesterone also existed in human resting T cells. Rapid suppression of Ca2+ influx induced by progesterone and analogs occurs in resting T cells with IC50 values similar to that of PHA-activated T cells.15 Membrane transient receptor potential canonical 3 (TRPC3) channel is known to maintain basal [Ca2+]i level homeostasis in human resting T cells.35 Additionally, TRPC3 inhibitors can blockade Ca2+ influx in PHA-activated T cells.15

Rapid suppression of Ca2+ influx is mimicked by progesterone-BSA in resting T cells. Furthermore, other sex steroids, including 17β-estradiol and testosterone, do not affect Ca2+ influx (unpublished data). This indicates that rapid suppression of Ca2+ influx by progesterone is a unique and specific nongenomic membrane response in resting T cells.

One remaining question is whether cPKC activation is involved in the Ca2+ influx suppression and this then causes the decrease in [Ca2+]i by progesterone and analogs. Ro318220, a universal cPKC inhibitor,30 enhances the [Ca2+]i increase caused by progesterone in resting T cells.14 Ro318220 also attenuates Ca2+ influx suppression and enhances the increase in global [Ca2+]i by progesterone and analogs. This suggests that progesterone might induce activation of cPKC to suppress Ca2+ influx in order to decrease the [Ca2+]i in T cells. In this context, a cPKC activator, thymeleatoxin, can suppress thapsigargin-induced Ca2+ influx in human T cells.36

U7312231 has no effect on the Ca2+ influx suppression in T cells by progesterone and analogs. Thus, cPKC activation-associated Ca2+ influx suppression by progesterone and analogs is not a PI-PLC-mediated downstream phenomenon.

LY333531,33 but not Go6976,32 successfully attenuates Ca2+ influx suppression by progesterone and analogs. This suggests that Ca2+ influx suppression occurs via activation of the cPKCβII rather than cPKCα and cPKCβI. Moreover, the cPKCβII-specific inhibitor CGP5335334 attenuates the suppression of Ca2+ influx by progesterone and analogs, indicating that Ca2+ influx suppression by progesterone occurs via cPKCβII activation in resting T cells. In support of this finding, the cPKCβI/βII-selective inhibitor, LY379196, is known to attenuate the Ca2+ influx suppression in rat retinal arterioles.37

Ca2+ influx suppression is only partially attenuated by the cPKCβII-specific inhibitor, CGP53353, but it is totally abolished after PKC downregulation. In the light of this, cPKCβII protein expression after PKC downregulation in T cells was explored. The level of cPKCβII protein expression was found to be significantly reduced as was total cPKC protein. However, the reduction in total cPKC was significantly less than that of cPKCβII. Thus, the lack of Ca2+ influx suppression by progesterone and analogs is likely to be due to diminished expression of cPKCβII in T cells. These results indirectly support the role of cPKCβII in the suppression of Ca2+ influx by progesterone in human resting T cells. In addition, cPKCβI was found to mediate the suppression of Ca2+ influx in human leukemic Jurkat cells38 and cPKCβII is able to attenuate the suppression of fMet-Leu-Phe-activated Ca2+ influx in human leukemic HL60 cells.39

The present study has revealed a novel rapid nongenomic response in human resting T cells whereby progesterone suppresses Ca2+ influx via the activation of cPKCβII. Furthermore, cPKCβ has been identified to associate with native TRPC3 in a proteomic study.40 A nongenomic pathway has been proposed whereby progesterone changes the physico-chemical properties of plasma membranes in order to indirectly alter the function of various membrane proteins.41 In this context, it is known that progesterone is able to dose-dependently decrease plasma membrane fluidity during meiotic maturation in Rana oocytes42 but also can increase plasma membrane fluidity in human erythrocytes.43 Thus, progesterone might influence membrane fluidity in human T cells in order to alter the functioning of cPKCβII and TRPC3. Thus, investigating the relationship between cPKCβII and TRPC3 in T cells after progesterone stimulation is important.

It is known that the various cPKC isoforms have important roles in the nongenomic responses of various steroid hormones in different cells. The activation of cPKCα by progesterone rapidly suppresses arginine transport via phosphorylation of cationic amino acid transporter-1 in human umbilical vein endothelial cells.44 We have found a novel rapid nongenomic response of cPKCβII, namely activation by progesterone, is able to suppress Ca2+ influx in human resting T cells. However, how progesterone mediates activation of cPKCβII remains unknown. cPKCβII activation by progesterone is independent of the PI-PLC pathway in T cells. This raises an interesting question as to whether progesterone can directly activate cPKCβII in T cells. Studies have shown that endogenous and synthetic steroid hormones are able to directly activate specific cPKC isoforms via plasma membrane sites. Examples included direct activation of cPKCα and cPKCγ by 1,25(OH)2-vitamin D3 of cPKCβ by dexamethasone, and of cPKCα by aldosterone and 17β-estradiol.45–47 Furthermore, the activation of cPKCβ by dexamethasone leads to an inhibition of insulin-induced glucose uptake in rat adipocytes.46

Progesterone has a multitude of different physiological functions that are initiated via nongenomic ion flux responses in various cell types. In human sperm, progesterone stimulates hyperactivation and acrosome reaction via an increase in [Ca2+]i by Ca2+ influx.48 In human T cells, progesterone achieves rapid immunosuppression via suppression of Ca2+ influx and inhibition of NHE1.6,7,14,15 Progesterone promotes a shift in Th1 cytokine towards a more “immunotolerogenic profile”, in peripheral T cells.6,8 These progesterone-driven maternal immunological adaptations promote immunotolerance that ensures a successful pregnancy.

The suppression of Ca2+ influx has been known to cause early T cell immunosuppression.16,18 Our findings suggest that cPKCβII might play a crucial role in the interactions between progesterone and T cell immunotolerance. Progesterone-mediated immunomodulation has been suggested to be involved in the remission of certain Th1-mediated autoimmune diseases during pregnancy including RA and MS.49,50 In addition, reduced expression of the cPKCβII protein has also been observed in psoriatic lesions.51 By way of contrast, systemic lupus erythematosus (SLE) tends to flare during pregnancy.11 Clinically, the expression of cPKCβ protein is remarkable decreased in the T cells of patients with SLE.52 An recent pilot clinical trial applied gestational levels of progesterone in order to mitigate the sometimes fatal cytokine storms that can occur in male patients with COVID-19.53

The activation of cPKCβII is able to mimic the rapid nongenomic response in T cells induced by progesterone. The development of specific cPKCβII modulators should provide a more potent effect than progesterone in terms of immune responses. Specific cPKCβII modulators might be used therapeutically to immunomodulate T cells. This might expand the current treatment arsenal when preventing preterm labor, preventing recurrent pregnancy loss and modulating various autoimmune diseases including RA, psoriasis, MS, and SLE.

ACKNOWLEDGMENTS

This research was supported by grants of Ministry of Education, Aiming for the Top University Plan (105AC-P619) and Cheng Hsin General Hospital and National Yang-Ming University (CY10524, CY10704), Republic of China. The authors are grateful to Dr. Ralph Kirby for his kind assistance in the preparation of the manuscript.

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