The non-genomic rapid acidification in peripheral T cells by progesterone depends on intracellular calcium increase and not on Na+/H+-exchange inhibition
Abstract
Progesterone is an endogenous immunomodulator that is able to suppress T cell activation during preg- nancy. An increased intracellular free calcium concentration ([Ca2+]i), acidification, and an inhibition of Na+/H+-exchange 1 (NHE1) are associated with this progesterone rapid non-genomic response that involves plasma membrane sites. Such acidification, when induced by phytohemagglutinin, is calcium dependent in PKC down-regulated T cells. We investigated the relationship between this rapid response involving the [Ca2+]i increase and various membrane progesterone receptors (mPRs). In addition, we explored whether the induction of acidification in T cells by progesterone is a direct result of the [Ca2+]i increase. The results show that the intracellular calcium elevation caused by progesterone is inhibited by SKF96365, U73122, and 2-APB, but not by pertussis toxin or U73343. The elevation is enhanced by the protein tyrosine kinase inhibitor staurosporine and the protein kinase C inhibitors Ro318220 and Go6983. These findings suggest that progesterone does not stimulate the [Ca2+]i increase via the Gi coupled mPRa. Furthermore, progesterone-induced acidification was found to be dependent on Ca2+ entry and blocked by the inorganic channel blocker, Ni2+. However, BAPTA, an intracellular calcium chelator, was found to prevent progesterone-induced acidification but not the inhibition of NHE1. This implies that acid- ification by progesterone is a direct result of the [Ca2+]i increase and does not directly involve NHE1. Taken together, further investigations are needed to explore whether one or more mPRs or PGRMC1 are involved in bringing about the T cell rapid response that results in the [Ca2+]i increase and inhibition of NHE1.
1. Introduction
Steroids acting on their cell surface receptors can initiate non- genomic rapid intracellular signaling and various biological re- sponses. In contrast, steroids acting on intracellular classic nuclear receptors do not usually cause rapid ion fluxes across the cell mem- brane. In our previous study, it was found that only progesterone is able to stimulate a non-genomic rapid response that results in an increase in intracellular free calcium concentration ([Ca2+]i), a de- crease in intracellular pH (pHi), and inhibition of Na+/H+-exchange 1 (NHE1). Other sex steroids, such as estradiol or testosterone, only give rise to a [Ca2+]i increase in T cells [1,2]. A similar transient intracellular Ca2+ increase in the presence of progesterone occurs in human sperm [3,4]. Recently, the rapid non-genomic calcium in- crease in the sperm tail caused by progesterone was found to be activated through a sperm-specific calcium ion (Ca2+) channel called CatSper [5,6].
Three possible receptor candidates for mediating this rapid progesterone response have been proposed; these are membrane progesterone receptors (mPRs); progesterone receptor membrane component 1 (PGRMC1); and nuclear progesterone receptors (nPRs) [7,8]. The mPRs possess seven integral transmembrane domains and mediates signaling through G-protein coupled pathways. These were initially discovered in fish ovaries and have three subtypes,
a, b and c. Previous research has shown that the situation is similar in human T cells [9–12] and, furthermore, that progesterone activates the inhibitory G-protein in fish oocytes, human myocytes and Jurkat T cells via mPRa [11,13,14]. Moreover, mPRs possess an inverted heptahelical membrane topology and belong to a larger class of membrane receptors called PAQRs (progestin and adipoQ receptors) thus may not require heterotrimeric G proteins to respond to progesterone [15,16]. PGRMC1 is a single transmem- brane protein that forms part of a multi-protein complex that binds to progesterone and other steroids, as well as various other pharma- ceutical compounds [17]. PGRMC1 expressed in bacteria is unable to bind progesterone; nonetheless, partially purified PGRMC1-GFP fu- sion protein expressed in mammalian cells does bind progesterone well with high affinity [18]. Unpublished results have indicated that both PGRMC1 mRNA and protein are expressed in human peripheral T cells. One aim of this study was to explore the relationship be- tween the various known progesterone membrane receptors and the non-genomic rapid response in T cells.
Progesterone is a general endogenous immunomodulator, and suppresses T-cell activation [1,2,12,19,20]. However, human peripheral blood lymphocytes and T cells do not express classical nPRs [11] and therefore progesterone-induced immunosuppression can not be mediated via nPRs [21,22]; furthermore, this event does not occur via the glucocorticoid receptor [23]. In these circum- stances, a likely candidate for these non-genomic receptors are one or more of the various mPRs that have seven integral trans- membrane domains or PGRMC1.
One obvious approach to investigating the role of these proteins in the non-genomic rapid responses of T cells is to use mPR siRNAs for knockdown studies. Before such experiments are carried out, the relationship between progesterone membrane receptors and the various non-genomic rapid responses need to be explored. Spe- cifically, there is a need to understand why three rapid responses, a rise in [Ca2+]i, a decrease in pHi and an inhibition of NHE1, are in- duced by two different membrane progesterone receptors, the mPRs and PGRMC1. Related to the above are various other un- known relationships in T cells that occur between these rapid re- sponses due to progesterone need to be investigated. These include whether the inhibition of NHE1 is dependent on either the rise of [Ca2+]i or the decrease of pHi or alternatively whether the decrease in pHi is dependent on either the rise of [Ca2+]i or the inhibition of NHE1.
The rapid increase in [Ca2+]i caused by Ca2+ entry and a prolonged activation of PKC are the two major signals involved in T cell proliferation and differentiation; these event can be induced by treatment with various polyclonal mitogens [24–26]. Blocking either PKC activity [27] or the increase in [Ca2+]i inhibits IL-2 secre- tion and T cell proliferation [28–30]. RU486 is antagonist that af- fects two of the rapid non-genomic responses induced by progesterone, namely the rise in [Ca2+]i and the decrease in pHi, but is synergistic with progesterone with respect to the inhibition of phytohemagglutinin (PHA)-induced T cell proliferation [12]. Our previous research showed that activation of PKC activity seems to increase NHE1 activity with respect to an increase in pHi in T cells in the presence of the mitogens PHA, PMA or LPS [26]. PHA stimu- lates the alkalinization in PKC intact T cells but after down-regula- tion of PKC activity by PMA, acidification due to Ca2+ influx is induced in T cells by PHA [31]. Therefore, in this study, we explore firstly how the various rapid responses, including the rise in [Ca2+]i, are induced via various progesterone membrane receptors and, secondly, whether any two of the rapid responses depend on each other. The approach involves inhibiting the signaling proteins in order to explore the possible pathways in T cells that are involved in the stimulation by progesterone of the [Ca2+]i increase.
2. Experimental
2.1. Materials
BCECF/AM, fura-2/AM, nigericin and valinomycin were pur- chased from Molecular Probes (Eugene, OR, USA). Phytohemagglutinin (PHA), RPMI 1640 medium (RPMI), Hank’s balanced salt solution (HBSS) and fetal calf serum (FCS) were obtained from Gib- co (Grand Island, NY, USA). Progesterone, U73122, U73343, SKF96365, bovine serum albumin (BSA), dimethyl sulfoxide, etha- nol, staurosporine, pertussis toxin, BAPTA/AM, N-methyl-D-gluc- amine (NMDG+), and Ficoll/Hypaque were purchased from Sigma Chemical Co. (St. Louis, MO, USA). 2-aminoethoxydiphenyl borate (2-APB) was purchased from Calbiochem Chemical Co. (San Diego, CA, USA). PHA was dissolved in distilled water. The culture media were supplemented with 10% FCS (v/v). Ro 318220 (Ro) and Gö 6983 (Go) were purchased from Enzo Life Sciences Inc. (Farming- dale, NY, USA). All sera were pretreated with charcoal/dextran to remove small molecules such as steroids and thyroid hormones [1].
2.2. T-cell preparation
Heparinized peripheral blood samples were obtained from healthy male volunteers (age: 20–25 years old) and T cells were isolated from this blood using the Ficoll-Hypaque gradient-density method as previously described [32]. The results showed that the T cell suspension contained almost 100% CD3-positive cells.
2.3. Measurement of [Ca2+]i
T cells (2 × 107 cells/ml) were loaded for 30 min at 37 °C with fura-2/AM (5 lM) in RPMI 1640 containing 10% FCS (v/v), washed free of extracellular fura-2/AM by three washes with RPMI 1640 and resuspended (2 × 107 cells/ml) in RPMI 1640 containing 10% FCS. To determine the [Ca2+]i, the T cells (2 × 106 cells) were then washed twice, resuspended in 2.5 ml of loading buffer [1] and placed in a plastic cuvette at 37 °C in a dual-wavelength spectroflu- orimeter (Spex Industries, model CM1T11I, Edison, NJ, USA). Using excitation wavelengths of 340 and 380 nm, the fluorescence emis- sion at 505 nm was measured and the [Ca2+]i determined from the fura-2 fluorescence-ratio signal using Spex DM3000 software according to the formula derived by Grynkiewicz [33].
2.4. Measurement of the pHi
T cell suspensions (2 × 107 cells/ml) were incubated at 37 °C for 30 min with BCECF/AM (3 lM) in HBSS containing 5 mM glucose and 0.2% BSA; then the cells were washed three times with HBSS and resuspended in RPMI 1640 containing 10% FCS. For pHi mea- surements, 2 × 106 cells were washed twice with HBSS, resus- pended in 2.5 ml of the same solution, transferred to a plastic cuvette at 37 °C, and allowed to stabilize for 15 min before stimula-
tion. Upon excitation at wavelengths of 435 and 500 nm, the BCECF fluorescence emission at 525 nm was measured using a dual-wave- length spectrofluorimeter (model CMIT11I, Spex Industries, Edison, NJ, USA) and the emission ratio calculated. The BCECF fluorescence calibration was carried out as previously described [32]. NHE1 activity was examined by calculating the initial rate of alkaliniza- tion after acute intracellular acidification using the NH4Cl prepulse technique [2]. This method involved suspending cells in NH4Cl for 15 min and then changing the cells into N-methyl-(D)-glucamine (NMDG+) buffer with or without progesterone for 5 min. The treated cells were then immediately placed into a cuvette containing Na+ buffer at a concentration of 145 mM to observe the activity of NHE1.
2.5. Statistical analysis
The pHi, and [Ca2+]i data were analyzed by t test with a significance level set at p < 0.05. All values are quoted as the mean ± SEM.
3. Results
3.1. Characterization of progesterone-induced [Ca2+]i increase in human T cells
3.1.1. The progesterone-induced [Ca2+]i increase is blocked by SKF96365
In order to explore whether the [Ca2+]i increase caused by proges- terone is activated by ligand operated calcium channels in T cells, the T cells were incubated with SKF96365, which acts as a blocker, for 10 min before the addition of progesterone. Starting within 1 min, [Ca2+]i increased by 24.6 ± 1.2 nM (n = 3) from 91.6 ± 0.1 nM in the control experiments without SKF96365 pretreatment. When the T cells were pretreated with SKF96365 (10, 20 lM), the [Ca2+]i only increased by 11.7 ± 0.9 (n = 3, p < 0.05) and 5.6 ± 1.0 (n = 3, p < 0.01), respectively (Fig. 1).
3.1.2. The progesterone-induced [Ca2+]i increase is blocked by inhibitors of phospholipase C and the inositol 1,4,5-trisphosphate receptor but not by an inhibitor of the Gi protein Progesterone is known to activate the inhibitory G-protein in fish oocytes, human myocytes and Jurkat T cells via mPRa. Thus it is a possibility that the [Ca2+]i increase caused by progesterone occurs via mPRa and Gi. Inhibitory G-protein is inhibited by per- tussis toxin, which was used to treat T cells for 30 min before treat- ment with progesterone. [Ca2+]i started increase within 1 min and increased by 26.6 ± 1.0 nM (n = 3) from 90.3 ± 0.7 nM in the control experiments. Interestingly, pretreatment of the T cells with the pertussis toxin (200 ng/ml) did not affect the increase in [Ca2+]i caused by progesterone, which was similar to the control at 26.1 ± 0.7 (n = 3, NS) (Fig. 2).
Another possibility is that the [Ca2+]i increase caused by progesterone is mediated through phospholipase C, phosphatidylinositol- phospholipase C (PI-PLC). To explore this possibility, U-73122,The chemical 2-aminoethoxydiphenyl borate (2-APB) is an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor, which is involved in Ca2+ mobilization and Ca2+ store depletion [34,35].
3.1.3. Progesterone-induced [Ca2+]i increase is enhanced by protein kinase C inhibitors
Staurosporine is an inhibitor of protein kinase C (PKC) and also an inhibitor of protein tyrosine kinases (PTK). Staurosporine was used which is a universal inhibitor, and U-73343 (0, 10 lM), which is the equivalent inactive inhibitor, were used to treat T cells for 10 min and before the stimulation by progesterone. In the control without pretreatment, [Ca2+]i started within 1 min and increased by 30.4 ± 1.0 nM (n = 3) above the resting level of 89.4 ± 0.3 nM. After pretreatment with U73122 (2.5, 5 lM), the [Ca2+]i increase due to progesterone was reduced to 27.0 ± 0.7 (n = 3, NS) and
15.3 ± 1.0 (n = 3, p < 0.05), respectively (Fig. 3a). Equivalent experiments using U73343 (10 lM) gave an increase in [Ca2+]i by progesterone that similar to that of the control at 28.6 ± 1.0 (n = 3, NS) (Fig. 3b).
3.2. The relationship between the [Ca2+]i increase and acidification in progesterone treated T cells
3.2.1. Effect of external calcium on the progesterone-induced [Ca2+]i increase and on acidification
A questions remains as to whether there is a relationship be- tween the [Ca2+]i increase and acidification when T cells are treated with progesterone. An external Ca2+ influx was exploited to deter- mine whether this occurs in T cells. Calcium free buffer containing the calcium chelator EGTA (0.2 mM) was used and the [Ca2+]i changes induced by progesterone monitored. The [Ca2+]i increase to explore whether treatment with this chemical could suppress intracellular Ca2+ mobilization by progesterone. The mitogen, phy- tohemagglutinin (PHA), was used as a control stimulant. T cells were incubated with staurosporine for 3 min before the stimulation with progesterone. The increase in [Ca2+]i, starting within 1 min, in- creased by 35.1 ± 0.7 nM (n = 3) from a resting level 90.8 ± 0.9 nM without staurosporine pretreatment. After pretreatment of the T cells with staurosporine (100, 200 nM), the increase in [Ca2+]i was significantly enhanced to 64.6 ± 1.7 (n = 3, p < 0.05) and 88.1 ± 1.3 (n = 3, p < 0.01), respectively (Fig. 5a). When PHA was used to stim- ulate the cells, the increase in [Ca2+]i, starting within 1 min, in- creased by 152.4 ± 4.0 nM (n = 3) from a resting of 89.4 ± 0.3 nM without staurosporine pretreatment. After pretreatment of the T cells with staurosporine (100 nM), this increase in [Ca2+]i was signif- icantly reduced to 54.4 ± 3.0 nM (n = 3, p < 0.01).
When two other protein kinase C inhibitors, Ro318220 (1 lM) and Go6983 (1 lM) were used to treat T cells, an enhancement was highly suppressed in calcium free buffer (Fig. 6a). However, the removal of extracellular Ca2+ by EGTA did not have a similar ef- fect on intracellular acidification by progesterone (Fig. 6b).
3.2.2. Effect of internal calcium mobilization on the progesterone- induced [Ca2+]i increase and acidification
In order to understand the relationship between internal cal- cium mobilization and acidification by progesterone, the intracel- lular calcium chelator BAPTA was used to monitor the [Ca2+]i and pHi changes caused by progesterone. The progesterone-induced [Ca2+]i increase was abolished when T cells were administered BAPTA/AM for 30 min before the stimulation (Fig. 7a). Under sim- ilar conditions, acidification by progesterone was also found to be abolished by BAPTA (Fig. 7b).
3.2.3. Effects of the inorganic channel blocker Ni2+ on Ca2+ influx and acidification in T cells
In order to further confirm that the acidification by progesterone was induced by Ca2+ influx in T cells, the inorganic channel blocker, Ni2+, was used to block the [Ca2+]i increase. When Ni2+ was added 20 min after progesterone stimulation, it reversed both the proges- terone-induced [Ca2+]i increase (Fig. 8a) and the acidification effect (Fig. 8b).
3.3. The progesterone-induced inhibition of NHE1 activity is independent of intracellular calcium mobilization
When the relationship between internal calcium mobilization and inhibition of Na+/H+-exchange 1 (NHE1) activity by progester- one was explored using the intracellular calcium chelator BAPTA/ AM (50 lM) followed by monitoring [Ca2+]i and NHE1 activity after treatment with progesterone, it was found that the progesterone- induced [Ca2+]i increase was abolished (Fig. 7a). However, the par- allel suppression of NHE1 activity by progesterone was not af- fected by the presence of BAPTA (Fig. 9).
4. Discussion
There are several different routes for Ca2+ influx to stimulate a [Ca2+]i increase in activated T cell. These include the calcium re- lease-activated channels (CRAC), the transient receptor potential (TRP) channels, various inositol-1,4,5-trisphosphate receptors (IP3Rs) and the L-type voltage-gated calcium Ca(v) channels. These channels are regulated either by depletion of internal cal- cium stores or by voltage-gated and Ca2+-activated K+ channel opening; these changes regulate the cell’s membrane potential in such a way that it becomes more negative, which maintains the driving force for Ca2+ influx and keeps T cells in a state of acti- vation [36]. Recently, the expression of multiple TRP isoforms, TRPC1, TRPC3, TRPV1, TRPM2, and TRPM7 have been observed in human T cells and it was found that TRPC3 is highly upregu- lated in activated T cells. Furthermore, down-regulation of TRPC3 reduces not only the proliferation but also resting [Ca2+]i of T cells [37]. Pharmacological blockade of K+ channels is able to cause membrane depolarization; this reduces the driving force for Ca2+ entry through CRAC channels and results in the suppression of T cell activation [38]. The abrogation of store-operated Ca2+ entry stimulated with (0, 10 lM) progesterone. The arrows indicate the time of addition of the individual stimulants. The tracings are from one representative of three experiments.
Several mechanisms have been proposed covering various cell types to explain Ca2+-induced intracellular acidification. For exam- ple, Ca2+-ATPase has been shown to be involved in NMDA-induced intracellular acidification in rat cerebella’s granule cells and the trigeminal ganglion [52,53]. The Ca2+/H+ exchange seems to be [44] and, furthermore, at 10–20 lM is a widely used CRAC inhib- itor that is able to activate or inhibit TRP channels [43,45,46]. Thus, in this study, the blockage of the progesterone-stimulated intracellular calcium increase in T cells due to pretreatment with 2-APB might not attributable to an inhibition of intracellular IP3 receptors (Fig. 4).
U73122 is a universal inhibitor of PLCs and the specific role of PLCc in the [Ca2+]i increase caused by progesterone remains unknown. Both isoforms of PLCc, PLCc1 and PLCc2, are activated by phosphorylation through growth factor receptor tyrosine kinases as well as via non-receptor tyrosine kinases [47]. Treatment in events in pancreatic acinar cells [54], while H+ can be displaced from intracellular buffers by high [Ca2+]i levels in both cardiac tissue and invertebrate neurons [55]. These diverse find- ings confirm the needs for more studies in order to define better the mechanism(s) involved in Ca2+-induced intracellular acidificat- ion by progesterone.
In contrast to the above, the inhibition of NHE1 activity caused by progesterone is not affected by treatment with BAPTA (Fig. 7b, Fig. 9), which implies that the acidification in T cells is dependent on an intracellular calcium increase and is not dependent on NHE1 inhibition by progesterone.
In conclusion, the non-genomic effect on acidification caused by progesterone is dependent on an intracellular calcium increase. Therefore, there are two rapid responses, the [Ca2+]i increase that is associated with the phenomena of acidification as well as the inhibition of NHE1 in T cells by progesterone. Whether these rapid responses are stimulated by membrane progesterone receptors or PGRMC1 in T cells needs to be further explored.