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Physiological functions of mitochondrial Na+-Ca2+ exchanger, NCLX, in lymphocytes
Cell Calcium 85 (2020) 102114
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Physiological functions of mitochondrial Na+-Ca2+ exchanger, NCLX, in lymphocytes
T
Ayako Takeuchia,*, Bongju Kimb,c, Satoshi Matsuokaa,c a
Department of Integrative and Systems Physiology, Faculty of Medical Sciences, and Life Science Innovation Center, University of Fukui, Fukui 910-1193, Japan Dental Life Science Research Institute/Clinical Translational Research Center for Dental Science, Seoul National University Dental Hospital, 101 Daehark-ro, Jongno-gu, Seoul, 03080, Republic of Korea c Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan b
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Keywords: Mitochondria NCLX Lymphocyte Ca2+ signaling Chemotaxis Mathematical modeling
Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.
1. Introduction It is well established that Ca2+ signaling in lymphocytes plays crucial roles in regulating cell differentiation and proliferation, gene transcription, motility, and cell death pathway, thereby determining various immune responses depending on the stage and the type of the cells (see reviews [1–3]). Cellular Ca2+ dynamics in lymphocytes has been explained by functional coupling of Ca2+ transporters/channels on plasma membranes and on endoplasmic reticulum (ER), as well as of various Ca2+ binding proteins. In fact, many studies have shown that the defects of the Ca2+ handling proteins in lymphocytes, such as inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) on ER, store-operated Ca2+ channel orai1, ER Ca2+ sensor proteins stromal interaction molecule (stim)1 and stim2, plasma membrane Ca2+ channels Cav1.2-1.4, resulted in severe disturbances in proliferation, differentiation, and cytokine production, which sometimes cause immunodeficiency (see reviews [1,3,4]). Although studies on mitochondrial Ca2+ dynamics had started as early as the 1960’s, and their functional significance have been revealed in both excitable and non-excitable tissues including heart, brain, kidney and liver (see reviews [5,6]), mitochondrial Ca2+ in immune cells did not receive much attention. It is after the molecular identifications of mitochondrial Ca2+ handling proteins in the last decade (putative mitochondrial H+-Ca2+ exchanger Letm1 [7]; mitochondrial Na+-Ca2+ exchanger NCLX [8]; mitochondrial Ca2+
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uniporter MCU [9,10]) that the physiological significance of mitochondrial Ca2+ dynamics in immune cells has begun to be evaluated. This article overviews the recent advances in this field, especially focusing on the roles of mitochondrial Na+-Ca2+ exchanger, NCLX, in B lymphocytes (Fig. 1) [11–14]. 2. NCLX as a cytosolic Na+-dependent mitochondrial Ca2+ efflux system in B lymphocytes Mitochondrial Ca2+ dynamics is balanced by the influx via Ca2+ uniporter and the efflux via Na+-Ca2+ exchanger and H+-Ca2+ exchanger; with the Na+-Ca2+ exchanger accounting for the major component in excitable tissues such as heart and brain, whereas the H+-Ca2+ exchanger being dominant in non-excitable tissues such as liver and kidney (see reviews [5,15]). Regarding lymphocytes, the contribution of the mitochondrial Na+-Ca2+ exchanger had been controversial. Dippenaar and Brand reported that Ca2+ efflux from mitochondria is independent of extra-mitochondrial Na+ in isolated mitochondria from pig mesenteric lymph node [16]. On the other hand, Hoth et al. showed that the depletion of cytosolic Na+ attenuated cytosolic Ca2+ rise triggered by addition of extracellular Ca2+ in human Jurkat T cell line, arguing that mitochondrial Na+-Ca2+ exchanger is functional [17]. Following the identification of NCLX as a mitochondrial Na+-Ca2+ exchanger which is ubiquitously expressed in various
Corresponding author at: Department of Integrative and Systems Physiology, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan. E-mail address: [email protected] (A. Takeuchi).
https://doi.org/10.1016/j.ceca.2019.102114 Received 29 September 2019; Received in revised form 13 November 2019; Accepted 14 November 2019 Available online 16 November 2019 0143-4160/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Possible roles of NCLX in B lymphocytes. NCLX is associated with (a) Ca2+ uptake into ER via SERCA, (b) suppression of spontaneous Ca2+ leak from ER, (c) SOCE activity, and (d) close localization of mitochondria and ER, thereby modulating cytosolic Ca2+ response to BCR stimulation and cell motility/chemotaxis.
tissues including thymus ([8,18], see also reviews [19,20]), we took advantage of molecular technique to clarify the issue using NCLX heterozygous knockout and NCLX knockdown B lymphocyte cell lines, DT40 and A20, respectively. It was demonstrated that Ca2+ efflux from mitochondria in saponin-permeabilized cells is accelerated by cytosolic application of Na+. This Na+-dependent Ca2+ efflux activity was disturbed by silencing NCLX, and the impairment was rescued by overexpressing NCLX, confirming that Na+-Ca2+ exchange system does exist and is mediated by NCLX in B lymphocyte mitochondria [11]. Interestingly, several groups reported that plasma membrane Na+Ca2+ exchanger (NCX) 1–3 expresses and functions in mitochondria of brain and heart [21–23]. These findings raised a possibility that NCX
Fig. 3. Proposed mechanism underlying increased random migration by silencing NCLX in B lymphocytes.
functions similarly in mitochondria of lymphocytes as a Na+-Ca2+ exchanger. However, our preliminary Western blotting experiments showed no positive signal of NCX in mouse spleen mitochondria, though clear signal was confirmed in mitochondria of kidney, brain and Fig. 2. Contribution of NCLX to cytosolic Ca2+ response upon antigen receptor stimulation is larger in B lymphocytes than in T lymphocytes. A. Simulation analysis using a B lymphocyte model predicts that the reduction of mitochondrial Na+-Ca2+ exchanger (NCXmit) abolishes cytosolic Ca2+ response to BCR stimulation. Data are modified from [11]. B. Application of NCLX blocker, CGP-37157, dose dependently inhibited cytosolic Ca2+ response to 10 μg/ml anti-IgM in mouse spleen B lymphocytes. Data are modified from [14]. C. Simulation analysis using a T lymphocyte model, which is obtained by changing scaling factors of Ca2+ handling proteins according to qPCR analysis, predicts that the reduction of NCXmit hardly affects cytosolic Ca2+ response to TCR stimulation. Scaling factors for orai1, IP3R, SERCA, calreticulin, NCXmit, and MCU are reduced to 75, 17, 35, 59, 34, and 73 %, respectively. Note that mRNA expression level of MCU could not be detected by qPCR [14], so expression level of another candidate for mitochondrial Ca2+ uptake, Letm1, was used. D. Application of NCLX blocker, CGP-37157, hardly affected cytosolic Ca2+ response to 10 μg/ml anti-CD3 and anti-CD28 in mouse spleen T lymphocytes. Data are modified from [14].
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Fig. 4. Contribution of NCLX to chemotaxis is larger in mouse spleen B lymphocytes (A, B) than in T lymphocytes (C, D). Data are modified from [14].
refilling activity triggered by applying ATP and Ca2+ to permeabilized cells were disturbed by silencing NCLX. These results are entirely consistent with the model prediction, underlying the pivotal role of mitochondria-ER Ca2+ recycling via NCLX and SERCA for the response to BCR stimulation (Fig. 1). In 2013, we found that NCLX participates in mitochondria-sarcoplasmic reticulum (SR) Ca2+ recycling also in HL-1 cardiomyocytes, thereby modulating automaticity of the cells [24]. It is possible that this coupling via NCLX and SERCA is important for physiological functions of other types of cells. One phenomena, which had not been predicted by the mathematical simulation, is impairment of SOCE activity in the NCLX heterozygous knockout DT40 cells, while mRNA expression levels of stim1 and orai1 are unaltered [11,12]. Similar results were reported by Parnis et al. using NCLX knockdown astrocytes [25], and by Ben-Kasus Nissim et al. using NCLX knockdown HEK293 T cells [26]. The impairment of SOCE activity by NCLX reduction probably further contributes to diminishing the cytosolic Ca2+ increase by BCR stimulation. Contrary to B lymphocytes, pharmacological inhibition of NCLX in mouse spleen T lymphocytes hardly affected the Ca2+ response to TCR stimulation, suggesting minor contribution of NCLX in the cells (Fig. 2D, [14]). What causes the difference between B lymphocytes and T lymphocytes, where the same players are involved in Ca2+ handling? We found by quantitative PCR (qPCR) analyses that the mRNA expression levels of factors determining mitochondria-ER Ca2+ dynamics, such as NCLX, mitochondrial H+-Ca2+ exchanger Letm1, SERCA3, IP3R 1–3, ER Ca2+ binding protein calreticulin, are significantly higher in B lymphocytes than in T lymphocytes [14]. The smaller expression levels of mitochondria-ER Ca2+ crosstalk factors may cause the smaller contribution of NCLX in T lymphocytes. In fact, the smaller cytosolic Ca2+
heart (unpublished observations). Therefore, we consider that contribution of NCX, if any, is negligible in lymphocytes. 3. Role of NCLX in antigen receptor stimulation in B lymphocytes Ca2+ signaling in responses to antigen receptor stimulation is a starting point for subsequent proliferation, differentiation, or apoptosis of lymphocytes. Upon binding of antigen to B cell receptor (BCR) and T cell receptor (TCR) in B lymphocytes and T lymphocytes, respectively, IP3 increases and facilitates Ca2+ release from ER through IP3R, resulting in an increase of cytosolic Ca2+. Then consequent Ca2+ depletion in ER causes translocation of stim1 to the vicinity of plasma membrane, inducing a sustained and oscillatory Ca2+ increase by activation of store-operated Ca2+ entry (SOCE) through orai1 (see reviews [1–3]). Although this scenario had been well investigated, contribution of mitochondrial Ca2+ dynamics were unclear. In order to get insight into it, we constructed a mathematical model of Ca2+ dynamics in B lymphocytes, by formulating biophysical properties of each Ca2+ handling protein on plasma membrane, ER and mitochondria, and by integrating them altogether [11]. Simulation analyses predicted that the NCLX reduction diminishes ER Ca2+ content by decelerating ER Ca2+ uptake via SERCA, resulting in reduced Ca2+ response to BCR stimulation (Fig. 2A, [11]). These hypotheses were thereafter validated experimentally. That is, both the initial increase of cytosolic Ca2+ and the subsequent Ca2+ oscillation after BCR stimulation were diminished in NCLX heterozygous knockout DT40 cells, NCLX knockdown A20 cells, and in native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157 (Fig. 2B, [14]). In addition, ER Ca2+ content evaluated as ionomycin-induced cytosolic Ca2+ increase and ER Ca2+ 3
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Fig. 5. Contribution of NCLX to cell proliferation is larger in mouse spleen B lymphocytes (A, B) than in T lymphocytes (C, D). Mouse spleen B lymphocytes and T lymphocytes were labeled with 10 μM CSFE (ThermoFisher Scientific), and were stimulated with 10 μg/ml anti-IgM (Jackson ImmunoResearch)/50 ng/ml IL-4 (Sigma-Aldrich) and anti-CD3/CD28 beads (ThermoFisher Scientific) for 3 and 4 days, respectively in the absence or presence of CGP37157. Then proliferation was analyzed with FACS Calibur (BD Biosciences). Cells with > M0 were considered as proliferated cells as denoted in the figure. A, C. Representative data for B lymphocytes and T lymphocytes, respectively. B, D. Percentage of proliferated cells (> M0) for B lymphocytes and T lymphocytes, respectively. Data are expressed as mean +/- s.e.m. for N = 3–4 experiments and analyzed for statistical significance using one-way ANOVA followed by Holm-Sidak method. **; p < 0.01 compared with cells in the absence of CGP-37157.
Ca2+ level, possibly by disturbing close localization of mitochondria and ER [11]. This increased level of cytosolic Ca2+ facilitates the actin polymerization and polarized localization of small GTPase Rac1 [14], both of which are well known factors contributing to motility of various cells including T lymphocytes [29–32]. In fact, chelating cytosolic Ca2+ with BAPTA-AM diminished motility of A20 B lymphocytes [14]. The latter phenomenon, the disappearance of directional movement towards CXCL12 by silencing NCLX, is more difficult to explain. Mitochondria distributed relatively uniformly throughout the cells under the control condition, and accumulated in a part of the cells when the cells were treated with CXCL12. This mitochondrial polarization disappeared in NCLX heterozygous knockout DT40 cells and NCLX knockdown A20 cells, though the underlying mechanisms remain unclear [14]. It may be possible that polarized mitochondria contribute to directional movements of the cells by providing ATP to myosin for contraction, as proposed in T lymphocytes [33]. It should be noted that the involvement of NCLX in directional movements towards CXCL12 is applicable to native mouse spleen B lymphocytes, but not to T lymphocytes (Fig. 4). As discussed earlier, the smaller expression levels of mitochondria-ER Ca2+ handling proteins bring the smaller contribution of mitochondria-ER crosstalk via NCLX in T lymphocyte [14].
response to TCR stimulation and the less contribution of NCLX can be reproduced by mathematical model mimicking T lymphocyte, simply by changing the scaling factors of mitochondria-ER Ca2+ crosstalk proteins according to the qPCR analyses (Fig. 2C). 4. Role of NCLX in B lymphocyte migration Migration of lymphocytes is one of the important processes determining immune response. Circulating naive lymphocytes move towards chemoattractants in lymph nodes to be activated and to differentiate into mature cells such as antibody producing plasma cells or CD4+ and CD8+ T cells from naive B lymphocytes and T lymphocytes, respectively [27,28]. There is a growing evidence showing that Ca2+ is one of the key factors regulating lymphocyte migration, because, for instance, chemotaxis-related cellular process such as cytoskeletal remodeling is Ca2+-sensitive, and several Ca2+ handling proteins such as SERCA, stim1/orai1, and IP3R are involved in T lymphocyte migration [29]. However, little information had been available regarding roles of mitochondrial Ca2+ dynamics in migration, especially of B lymphocytes. In 2016, we reported that NCLX is involved in B lymphocyte migration [14]. By cell tracking analysis, we found that silencing NCLX increased the motility of B lymphocyte regardless of manipulation method; heterozygous knockout in DT40 cells and knockdown using siRNA in A20 cells. Pharmacological inhibition of NCLX by CGP-37157 produced similar results. When a chemokine CXCL12 gradient was formed in the chamber, the majority of control cells moved towards it. Strikingly, the directional movements disappeared by silencing NCLX. The former phenomenon, the increased motility by silencing NCLX, is accounted for by disturbed cytosolic Ca2+ signaling (Fig. 3). That is, NCLX reduction increases Ca2+ leak from ER to augment cytosolic
5. Role of NCLX in immune response; implications for future direction From extensive studies investigating the roles of NCLX in lymphocytes [11–14], it has now become clear that NCLX participates in functional coupling of mitochondria and ER [34,35], thereby regulating cellular response to antigen receptor stimulation as well as chemotaxis 4
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of B lymphocytes. Roles of NCLX in immunity is still not clear. One possibility is the involvement in autoimmunity such as systemic lupus erythematosus (SLE). It is well understood that self-reactive antibody producing B cells are eliminated by apoptosis during development and this process is closely related to cytosolic Ca2+ signaling [36–38]. NCLX heterozygous knockout DT40 cells underwent increased apoptosis, as revealed by increased fraction of cells with depolarized mitochondria and increased DNA fragmentation [11]. Since B lymphocytes isolated from patients with SLE showed hyper-response of cytosolic Ca2+ to BCR stimulation [39], it is interesting to test whether NCLX expression is altered in those cells. If the expression alters, NCLX could become a target for SLE treatment, although precaution must be taken against adverse effects such as neurodegeneration and abnormal automaticity of heart [24,40–42]. Another possibility of the role of NCLX is the involvement in proliferation and differentiation of naive B lymphocytes into mature cells. CGP-37157, an NCLX blocker, dose-dependently inhibited proliferation of mouse spleen B lymphocytes stimulated by anti-IgM with IL-4, suggesting the involvements of NCLX in proliferation (Fig. 5). Again, this blockade were not observed in mouse spleen T lymphocytes stimulated by anti-CD3/CD28, implying the minor contribution of NCLX in T lymphocytes (Fig. 5). Recently, Samanta et al. reported that NCLX in forward and reverse mode modulates cytosolic and mitochondrial Ca2+ oscillation triggered by leukotriene C4 receptor and subsequent gene expressions in RBL-1 mast cells, under the condition of mitochondrial depolarization [43]. Although this is an un-physiological condition, it might be possible that NCLX serves as a regulator of gene expressions also in B lymphocytes. Another issue to be considered is a metabolic reprogramming in lymphocytes. It has become clear that lymphocytes in different stages or different types rely on different metabolic state and mitochondrial reactive oxygen species level, which are associated with physiological functions of the cells, such as cytokine production by T cells and antibody production by activated B cells ([44–46], also see review [47]). In 2018, Akkaya et al. extensively studied the relationship between metabolic activity and B cell survival after stimulation by anti-IgM with/ without another stimulus, and involvements of Ca2+ signaling [48]. They discovered that mitochondrial dysfunction after BCR stimulation is mediated by cytosolic Ca2+ increase, especially via SOCE activity, resulting in BCR-activation-induced cell death. Since they also found the defect of mitochondrial Ca2+ handlings in stimulated B cells, it would be interesting to investigate whether NCLX participates in this scenario.
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Declaration of Competing Interest The authors declare no competing interests. Acknowledgements This work was supported by JSPS KAKENHI grant Number 19K22509 (S.M.), Takeda Science Foundation (A.T.), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (A.T.), The Sumitomo Foundation (A.T.) and by Life Science Innovation Center at University of Fukui. References [1] S. Feske, E.Y. Skolnik, M. Prakriya, Ion channels and transporters in lymphocyte function and immunity, Nat. Rev. Immunol. 12 (2012) 532–547. [2] K.S. Friedmann, M. Bozem, M. Hoth, Calcium signal dynamics in T lymphocytes: comparing in vivo and in vitro measurements, Semin. Cell Dev. Biol. 94 (2019) 84–93. [3] M. Trebak, J.P. Kinet, Calcium signalling in T cells, Nat. Rev. Immunol. 19 (2019) 154–169. [4] P.G. Hogan, R.S. Lewis, A. Rao, Molecular basis of calcium signaling in
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Cell Calcium journal homepage: www.elsevier.com/locate/ceca
Physiological functions of mitochondrial Na+-Ca2+ exchanger, NCLX, in lymphocytes
T
Ayako Takeuchia,*, Bongju Kimb,c, Satoshi Matsuokaa,c a
Department of Integrative and Systems Physiology, Faculty of Medical Sciences, and Life Science Innovation Center, University of Fukui, Fukui 910-1193, Japan Dental Life Science Research Institute/Clinical Translational Research Center for Dental Science, Seoul National University Dental Hospital, 101 Daehark-ro, Jongno-gu, Seoul, 03080, Republic of Korea c Center for Innovation in Immunoregulative Technology and Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-konoe, Sakyo-ku, Kyoto 606-8501, Japan b
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A B S T R A C T
Keywords: Mitochondria NCLX Lymphocyte Ca2+ signaling Chemotaxis Mathematical modeling
Roles of mitochondrial Na+-Ca2+ exchanger, NCLX, were studied in B lymphocytes such as heterozygous NCLX knockout DT40 cells, NCLX knockdown A20 cells, and native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157. Cytosolic Ca2+ response to B cell receptor stimulation was impaired in these B lymphocytes, demonstrating importance of mitochondria-ER Ca2+ recycling via NCLX and sarco/endoplasmic reticulum Ca2+-ATPase SERCA, and interaction with store-operated Ca2+ entry. NCLX was also associated with motility and chemotaxis of B lymphocyte. Contrary to B lymphocytes, contribution of NCLX in mouse spleen T lymphocytes was minor.
1. Introduction It is well established that Ca2+ signaling in lymphocytes plays crucial roles in regulating cell differentiation and proliferation, gene transcription, motility, and cell death pathway, thereby determining various immune responses depending on the stage and the type of the cells (see reviews [1–3]). Cellular Ca2+ dynamics in lymphocytes has been explained by functional coupling of Ca2+ transporters/channels on plasma membranes and on endoplasmic reticulum (ER), as well as of various Ca2+ binding proteins. In fact, many studies have shown that the defects of the Ca2+ handling proteins in lymphocytes, such as inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) on ER, store-operated Ca2+ channel orai1, ER Ca2+ sensor proteins stromal interaction molecule (stim)1 and stim2, plasma membrane Ca2+ channels Cav1.2-1.4, resulted in severe disturbances in proliferation, differentiation, and cytokine production, which sometimes cause immunodeficiency (see reviews [1,3,4]). Although studies on mitochondrial Ca2+ dynamics had started as early as the 1960’s, and their functional significance have been revealed in both excitable and non-excitable tissues including heart, brain, kidney and liver (see reviews [5,6]), mitochondrial Ca2+ in immune cells did not receive much attention. It is after the molecular identifications of mitochondrial Ca2+ handling proteins in the last decade (putative mitochondrial H+-Ca2+ exchanger Letm1 [7]; mitochondrial Na+-Ca2+ exchanger NCLX [8]; mitochondrial Ca2+
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uniporter MCU [9,10]) that the physiological significance of mitochondrial Ca2+ dynamics in immune cells has begun to be evaluated. This article overviews the recent advances in this field, especially focusing on the roles of mitochondrial Na+-Ca2+ exchanger, NCLX, in B lymphocytes (Fig. 1) [11–14]. 2. NCLX as a cytosolic Na+-dependent mitochondrial Ca2+ efflux system in B lymphocytes Mitochondrial Ca2+ dynamics is balanced by the influx via Ca2+ uniporter and the efflux via Na+-Ca2+ exchanger and H+-Ca2+ exchanger; with the Na+-Ca2+ exchanger accounting for the major component in excitable tissues such as heart and brain, whereas the H+-Ca2+ exchanger being dominant in non-excitable tissues such as liver and kidney (see reviews [5,15]). Regarding lymphocytes, the contribution of the mitochondrial Na+-Ca2+ exchanger had been controversial. Dippenaar and Brand reported that Ca2+ efflux from mitochondria is independent of extra-mitochondrial Na+ in isolated mitochondria from pig mesenteric lymph node [16]. On the other hand, Hoth et al. showed that the depletion of cytosolic Na+ attenuated cytosolic Ca2+ rise triggered by addition of extracellular Ca2+ in human Jurkat T cell line, arguing that mitochondrial Na+-Ca2+ exchanger is functional [17]. Following the identification of NCLX as a mitochondrial Na+-Ca2+ exchanger which is ubiquitously expressed in various
Corresponding author at: Department of Integrative and Systems Physiology, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan. E-mail address: [email protected] (A. Takeuchi).
https://doi.org/10.1016/j.ceca.2019.102114 Received 29 September 2019; Received in revised form 13 November 2019; Accepted 14 November 2019 Available online 16 November 2019 0143-4160/ © 2019 Elsevier Ltd. All rights reserved.
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Fig. 1. Possible roles of NCLX in B lymphocytes. NCLX is associated with (a) Ca2+ uptake into ER via SERCA, (b) suppression of spontaneous Ca2+ leak from ER, (c) SOCE activity, and (d) close localization of mitochondria and ER, thereby modulating cytosolic Ca2+ response to BCR stimulation and cell motility/chemotaxis.
tissues including thymus ([8,18], see also reviews [19,20]), we took advantage of molecular technique to clarify the issue using NCLX heterozygous knockout and NCLX knockdown B lymphocyte cell lines, DT40 and A20, respectively. It was demonstrated that Ca2+ efflux from mitochondria in saponin-permeabilized cells is accelerated by cytosolic application of Na+. This Na+-dependent Ca2+ efflux activity was disturbed by silencing NCLX, and the impairment was rescued by overexpressing NCLX, confirming that Na+-Ca2+ exchange system does exist and is mediated by NCLX in B lymphocyte mitochondria [11]. Interestingly, several groups reported that plasma membrane Na+Ca2+ exchanger (NCX) 1–3 expresses and functions in mitochondria of brain and heart [21–23]. These findings raised a possibility that NCX
Fig. 3. Proposed mechanism underlying increased random migration by silencing NCLX in B lymphocytes.
functions similarly in mitochondria of lymphocytes as a Na+-Ca2+ exchanger. However, our preliminary Western blotting experiments showed no positive signal of NCX in mouse spleen mitochondria, though clear signal was confirmed in mitochondria of kidney, brain and Fig. 2. Contribution of NCLX to cytosolic Ca2+ response upon antigen receptor stimulation is larger in B lymphocytes than in T lymphocytes. A. Simulation analysis using a B lymphocyte model predicts that the reduction of mitochondrial Na+-Ca2+ exchanger (NCXmit) abolishes cytosolic Ca2+ response to BCR stimulation. Data are modified from [11]. B. Application of NCLX blocker, CGP-37157, dose dependently inhibited cytosolic Ca2+ response to 10 μg/ml anti-IgM in mouse spleen B lymphocytes. Data are modified from [14]. C. Simulation analysis using a T lymphocyte model, which is obtained by changing scaling factors of Ca2+ handling proteins according to qPCR analysis, predicts that the reduction of NCXmit hardly affects cytosolic Ca2+ response to TCR stimulation. Scaling factors for orai1, IP3R, SERCA, calreticulin, NCXmit, and MCU are reduced to 75, 17, 35, 59, 34, and 73 %, respectively. Note that mRNA expression level of MCU could not be detected by qPCR [14], so expression level of another candidate for mitochondrial Ca2+ uptake, Letm1, was used. D. Application of NCLX blocker, CGP-37157, hardly affected cytosolic Ca2+ response to 10 μg/ml anti-CD3 and anti-CD28 in mouse spleen T lymphocytes. Data are modified from [14].
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Fig. 4. Contribution of NCLX to chemotaxis is larger in mouse spleen B lymphocytes (A, B) than in T lymphocytes (C, D). Data are modified from [14].
refilling activity triggered by applying ATP and Ca2+ to permeabilized cells were disturbed by silencing NCLX. These results are entirely consistent with the model prediction, underlying the pivotal role of mitochondria-ER Ca2+ recycling via NCLX and SERCA for the response to BCR stimulation (Fig. 1). In 2013, we found that NCLX participates in mitochondria-sarcoplasmic reticulum (SR) Ca2+ recycling also in HL-1 cardiomyocytes, thereby modulating automaticity of the cells [24]. It is possible that this coupling via NCLX and SERCA is important for physiological functions of other types of cells. One phenomena, which had not been predicted by the mathematical simulation, is impairment of SOCE activity in the NCLX heterozygous knockout DT40 cells, while mRNA expression levels of stim1 and orai1 are unaltered [11,12]. Similar results were reported by Parnis et al. using NCLX knockdown astrocytes [25], and by Ben-Kasus Nissim et al. using NCLX knockdown HEK293 T cells [26]. The impairment of SOCE activity by NCLX reduction probably further contributes to diminishing the cytosolic Ca2+ increase by BCR stimulation. Contrary to B lymphocytes, pharmacological inhibition of NCLX in mouse spleen T lymphocytes hardly affected the Ca2+ response to TCR stimulation, suggesting minor contribution of NCLX in the cells (Fig. 2D, [14]). What causes the difference between B lymphocytes and T lymphocytes, where the same players are involved in Ca2+ handling? We found by quantitative PCR (qPCR) analyses that the mRNA expression levels of factors determining mitochondria-ER Ca2+ dynamics, such as NCLX, mitochondrial H+-Ca2+ exchanger Letm1, SERCA3, IP3R 1–3, ER Ca2+ binding protein calreticulin, are significantly higher in B lymphocytes than in T lymphocytes [14]. The smaller expression levels of mitochondria-ER Ca2+ crosstalk factors may cause the smaller contribution of NCLX in T lymphocytes. In fact, the smaller cytosolic Ca2+
heart (unpublished observations). Therefore, we consider that contribution of NCX, if any, is negligible in lymphocytes. 3. Role of NCLX in antigen receptor stimulation in B lymphocytes Ca2+ signaling in responses to antigen receptor stimulation is a starting point for subsequent proliferation, differentiation, or apoptosis of lymphocytes. Upon binding of antigen to B cell receptor (BCR) and T cell receptor (TCR) in B lymphocytes and T lymphocytes, respectively, IP3 increases and facilitates Ca2+ release from ER through IP3R, resulting in an increase of cytosolic Ca2+. Then consequent Ca2+ depletion in ER causes translocation of stim1 to the vicinity of plasma membrane, inducing a sustained and oscillatory Ca2+ increase by activation of store-operated Ca2+ entry (SOCE) through orai1 (see reviews [1–3]). Although this scenario had been well investigated, contribution of mitochondrial Ca2+ dynamics were unclear. In order to get insight into it, we constructed a mathematical model of Ca2+ dynamics in B lymphocytes, by formulating biophysical properties of each Ca2+ handling protein on plasma membrane, ER and mitochondria, and by integrating them altogether [11]. Simulation analyses predicted that the NCLX reduction diminishes ER Ca2+ content by decelerating ER Ca2+ uptake via SERCA, resulting in reduced Ca2+ response to BCR stimulation (Fig. 2A, [11]). These hypotheses were thereafter validated experimentally. That is, both the initial increase of cytosolic Ca2+ and the subsequent Ca2+ oscillation after BCR stimulation were diminished in NCLX heterozygous knockout DT40 cells, NCLX knockdown A20 cells, and in native mouse spleen B lymphocytes treated with a NCLX blocker, CGP-37157 (Fig. 2B, [14]). In addition, ER Ca2+ content evaluated as ionomycin-induced cytosolic Ca2+ increase and ER Ca2+ 3
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Fig. 5. Contribution of NCLX to cell proliferation is larger in mouse spleen B lymphocytes (A, B) than in T lymphocytes (C, D). Mouse spleen B lymphocytes and T lymphocytes were labeled with 10 μM CSFE (ThermoFisher Scientific), and were stimulated with 10 μg/ml anti-IgM (Jackson ImmunoResearch)/50 ng/ml IL-4 (Sigma-Aldrich) and anti-CD3/CD28 beads (ThermoFisher Scientific) for 3 and 4 days, respectively in the absence or presence of CGP37157. Then proliferation was analyzed with FACS Calibur (BD Biosciences). Cells with > M0 were considered as proliferated cells as denoted in the figure. A, C. Representative data for B lymphocytes and T lymphocytes, respectively. B, D. Percentage of proliferated cells (> M0) for B lymphocytes and T lymphocytes, respectively. Data are expressed as mean +/- s.e.m. for N = 3–4 experiments and analyzed for statistical significance using one-way ANOVA followed by Holm-Sidak method. **; p < 0.01 compared with cells in the absence of CGP-37157.
Ca2+ level, possibly by disturbing close localization of mitochondria and ER [11]. This increased level of cytosolic Ca2+ facilitates the actin polymerization and polarized localization of small GTPase Rac1 [14], both of which are well known factors contributing to motility of various cells including T lymphocytes [29–32]. In fact, chelating cytosolic Ca2+ with BAPTA-AM diminished motility of A20 B lymphocytes [14]. The latter phenomenon, the disappearance of directional movement towards CXCL12 by silencing NCLX, is more difficult to explain. Mitochondria distributed relatively uniformly throughout the cells under the control condition, and accumulated in a part of the cells when the cells were treated with CXCL12. This mitochondrial polarization disappeared in NCLX heterozygous knockout DT40 cells and NCLX knockdown A20 cells, though the underlying mechanisms remain unclear [14]. It may be possible that polarized mitochondria contribute to directional movements of the cells by providing ATP to myosin for contraction, as proposed in T lymphocytes [33]. It should be noted that the involvement of NCLX in directional movements towards CXCL12 is applicable to native mouse spleen B lymphocytes, but not to T lymphocytes (Fig. 4). As discussed earlier, the smaller expression levels of mitochondria-ER Ca2+ handling proteins bring the smaller contribution of mitochondria-ER crosstalk via NCLX in T lymphocyte [14].
response to TCR stimulation and the less contribution of NCLX can be reproduced by mathematical model mimicking T lymphocyte, simply by changing the scaling factors of mitochondria-ER Ca2+ crosstalk proteins according to the qPCR analyses (Fig. 2C). 4. Role of NCLX in B lymphocyte migration Migration of lymphocytes is one of the important processes determining immune response. Circulating naive lymphocytes move towards chemoattractants in lymph nodes to be activated and to differentiate into mature cells such as antibody producing plasma cells or CD4+ and CD8+ T cells from naive B lymphocytes and T lymphocytes, respectively [27,28]. There is a growing evidence showing that Ca2+ is one of the key factors regulating lymphocyte migration, because, for instance, chemotaxis-related cellular process such as cytoskeletal remodeling is Ca2+-sensitive, and several Ca2+ handling proteins such as SERCA, stim1/orai1, and IP3R are involved in T lymphocyte migration [29]. However, little information had been available regarding roles of mitochondrial Ca2+ dynamics in migration, especially of B lymphocytes. In 2016, we reported that NCLX is involved in B lymphocyte migration [14]. By cell tracking analysis, we found that silencing NCLX increased the motility of B lymphocyte regardless of manipulation method; heterozygous knockout in DT40 cells and knockdown using siRNA in A20 cells. Pharmacological inhibition of NCLX by CGP-37157 produced similar results. When a chemokine CXCL12 gradient was formed in the chamber, the majority of control cells moved towards it. Strikingly, the directional movements disappeared by silencing NCLX. The former phenomenon, the increased motility by silencing NCLX, is accounted for by disturbed cytosolic Ca2+ signaling (Fig. 3). That is, NCLX reduction increases Ca2+ leak from ER to augment cytosolic
5. Role of NCLX in immune response; implications for future direction From extensive studies investigating the roles of NCLX in lymphocytes [11–14], it has now become clear that NCLX participates in functional coupling of mitochondria and ER [34,35], thereby regulating cellular response to antigen receptor stimulation as well as chemotaxis 4
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of B lymphocytes. Roles of NCLX in immunity is still not clear. One possibility is the involvement in autoimmunity such as systemic lupus erythematosus (SLE). It is well understood that self-reactive antibody producing B cells are eliminated by apoptosis during development and this process is closely related to cytosolic Ca2+ signaling [36–38]. NCLX heterozygous knockout DT40 cells underwent increased apoptosis, as revealed by increased fraction of cells with depolarized mitochondria and increased DNA fragmentation [11]. Since B lymphocytes isolated from patients with SLE showed hyper-response of cytosolic Ca2+ to BCR stimulation [39], it is interesting to test whether NCLX expression is altered in those cells. If the expression alters, NCLX could become a target for SLE treatment, although precaution must be taken against adverse effects such as neurodegeneration and abnormal automaticity of heart [24,40–42]. Another possibility of the role of NCLX is the involvement in proliferation and differentiation of naive B lymphocytes into mature cells. CGP-37157, an NCLX blocker, dose-dependently inhibited proliferation of mouse spleen B lymphocytes stimulated by anti-IgM with IL-4, suggesting the involvements of NCLX in proliferation (Fig. 5). Again, this blockade were not observed in mouse spleen T lymphocytes stimulated by anti-CD3/CD28, implying the minor contribution of NCLX in T lymphocytes (Fig. 5). Recently, Samanta et al. reported that NCLX in forward and reverse mode modulates cytosolic and mitochondrial Ca2+ oscillation triggered by leukotriene C4 receptor and subsequent gene expressions in RBL-1 mast cells, under the condition of mitochondrial depolarization [43]. Although this is an un-physiological condition, it might be possible that NCLX serves as a regulator of gene expressions also in B lymphocytes. Another issue to be considered is a metabolic reprogramming in lymphocytes. It has become clear that lymphocytes in different stages or different types rely on different metabolic state and mitochondrial reactive oxygen species level, which are associated with physiological functions of the cells, such as cytokine production by T cells and antibody production by activated B cells ([44–46], also see review [47]). In 2018, Akkaya et al. extensively studied the relationship between metabolic activity and B cell survival after stimulation by anti-IgM with/ without another stimulus, and involvements of Ca2+ signaling [48]. They discovered that mitochondrial dysfunction after BCR stimulation is mediated by cytosolic Ca2+ increase, especially via SOCE activity, resulting in BCR-activation-induced cell death. Since they also found the defect of mitochondrial Ca2+ handlings in stimulated B cells, it would be interesting to investigate whether NCLX participates in this scenario.
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Declaration of Competing Interest The authors declare no competing interests. Acknowledgements This work was supported by JSPS KAKENHI grant Number 19K22509 (S.M.), Takeda Science Foundation (A.T.), The Mochida Memorial Foundation for Medical and Pharmaceutical Research (A.T.), The Sumitomo Foundation (A.T.) and by Life Science Innovation Center at University of Fukui. References [1] S. Feske, E.Y. Skolnik, M. Prakriya, Ion channels and transporters in lymphocyte function and immunity, Nat. Rev. Immunol. 12 (2012) 532–547. [2] K.S. Friedmann, M. Bozem, M. Hoth, Calcium signal dynamics in T lymphocytes: comparing in vivo and in vitro measurements, Semin. Cell Dev. Biol. 94 (2019) 84–93. [3] M. Trebak, J.P. Kinet, Calcium signalling in T cells, Nat. Rev. Immunol. 19 (2019) 154–169. [4] P.G. Hogan, R.S. Lewis, A. Rao, Molecular basis of calcium signaling in
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