Store-operated calcium entry dysfunction in CRAC channelopathy: Insights from a novel STIM1 mutation.

Store-operated calcium entry (SOCE) plays a crucial role in maintaining cellular calcium homeostasis. This mechanism involves proteins, such as stromal interaction molecule 1 (STIM1) and ORAI1. Mutations in the genes encoding these proteins, especially STIM1, can lead to various diseases, including CRAC channelopathies associated with severe combined immunodeficiency. Herein, we describe a novel homozygous mutation, NM_003156 c.792-3C > G, in STIM1 in a patient with a clinical profile of CRAC channelopathy, including immune system deficiencies and muscle weakness. Functional analyses revealed three distinct spliced forms in the patient cells: wild-type, exon 7 skipping, and intronic retention. Calcium influx analysis revealed impaired SOCE in the patient cells, indicating a loss of STIM1 function. We developed an antisense oligonucleotide treatment that improves STIM1 splicing and highlighted its potential as a therapeutic approach. Our findings provide insights into the complex effects of STIM1 mutations and shed light on the multifaceted clinical presentation of the patient.

Improvement of the rituximab-induced cell death by potentiation of the store-operated calcium entry in mantle cell lymphoma cell lines.

Mantle Cell Lymphoma (MCL) is one of the worst lymphomas with a median overall survival of 3 to 4 years. Even if the use of rituximab was a great step in therapy, patients commonly develop resistance and relapse. New therapies or complement of existing therapies should be developed. Using spectrofluorimetry, we found that the resting cytosolic Ca2+ ion concentration [Ca2+]cyt of MCL patients cells and MCL cell lines was increased. This increase is correlated with a larger store-operated calcium entry (SOCE) amplitude which is responsible for the Ca2+ ions influx. Furthermore, using a SOCE potentiating agent, we demonstrated that in the MCL Rec-1 cell line, the SOCE is already activated in resting conditions. Interestingly, this potentiating agent alone, by disturbing the SOCE, induced the apoptosis of Rec-1 cells with the same efficacy than rituximab. The use of the potentiating agent in addition to rituximab strengthens the rituximab-induced apoptosis of rituximab-sensitive Granta-519 and Rec-1 cells. However, this potentiating agent cannot convert the Jeko-1 rituximab-resistant to a rituximab-sensitive cell line. Our results confirm that the use of compound acting on the Ca2+ homeostasis could be a new target of interest in complement to existing therapies.

Characterization of neutralizing antibodies reacting with the 213-224 amino-acid segment of human galectin-9.

Extra-cellular galectin-9 (gal-9) is an immuno-modulatory protein with predominant immunosuppressive effects. Inappropriate production of gal-9 has been reported in several human malignancies and viral diseases like nasopharyngeal, pancreatic and renal carcinomas, metastatic melanomas and chronic active viral hepatitis. Therefore therapeutic antibodies neutralizing extra-cellular gal-9 are expected to contribute to immune restoration in these pathological conditions. Two novel monoclonal antibodies targeting gal-9 -Gal-Nab 1 and 2-have been produced and characterized in this study. We report a protective effect of Gal-Nab1 and Gal-Nab2 on the apoptotic cell death induced by gal-9 in primary T cells. In addition, they inhibit late phenotypic changes observed in peripheral T cells that survive gal-9-induced apoptosis. Gal-Nab1 and Gal-Nab2 bind nearly identical, overlapping linear epitopes contained in the 213-224 amino-acid segments of gal-9. Nevertheless, they have some distinct functional characteristics suggesting that their three-dimensional epitopes are distinct. These differences are best demonstrated when gal-9 is applied on Jurkat cells where Gal-Nab1 is less efficient than Gal-Nab2 in the prevention of apoptotic cell death. In addition, Gal-Nab1 stimulates non-lethal phosphatidylserine translocation at the plasma membrane and calcium mobilization triggered by gal-9 in these cells. Both Gal-Nab1 and 2 cross-react with murine gal-9. They bind its natural as well as its recombinant form. This cross-species recognition will be an advantage for their assessment in pre-clinical tumor models.

Endoplasmic reticulum stress drives proteinuria-induced kidney lesions via Lipocalin 2.

In chronic kidney disease (CKD), proteinuria results in severe tubulointerstitial lesions, which ultimately lead to end-stage renal disease. Here we identify 4-phenylbutyric acid (PBA), a chemical chaperone already used in humans, as a novel therapeutic strategy capable to counteract the toxic effect of proteinuria. Mechanistically, we show that albumin induces tubular unfolded protein response via cytosolic calcium rise, which leads to tubular apoptosis by Lipocalin 2 (LCN2) modulation through ATF4. Consistent with the key role of LCN2 in CKD progression, Lcn2 gene inactivation decreases ER stress-induced apoptosis, tubulointerstitial lesions and mortality in proteinuric mice. More importantly, the inhibition of this pathway by PBA protects kidneys from morphological and functional degradation in proteinuric mice. These results are relevant to human CKD, as LCN2 is increased in proteinuric patients. In conclusion, our study identifies a therapeutic strategy susceptible to improve the benefit of RAS inhibitors in proteinuria-induced CKD progression.

Potentiation of the store-operated calcium entry (SOCE) induces phytohemagglutinin-activated Jurkat T cell apoptosis.

Store-operated Ca(2+) entry (SOCE) is the main Ca(2+) entry pathway of non-excitable cells. In the past decade, the activation of this entry has been unveiled, with STIM1, a protein of the endoplasmic reticulum able to sense the intraluminal Ca(2+) content, and Orai1, the pore-forming unit of the Ca(2+) release activated Ca(2+) (CRAC) channels. When Ca(2+) ions are released from the endoplasmic reticulum, STIM1 proteins oligomerize and directly interact with Orai1 proteins, allowing the opening of the CRAC channels and a massive Ca(2+) ion influx known as SOCE. As Ca(2+) is involved in various cellular processes, the discovery of new drugs acting on the SOCE should be of interest to control the cell activity. By testing analogs of 2-aminoethyl diphenylborinate (2-APB), a well known, though not so selective effector of the SOCE, we identified methoxy diethylborinate (MDEB), a molecule able to potentiate the SOCE in three leukocyte and two breast cancer cell lines by increasing the Ca(2+) influx amplitude. Unlike 2-APB, MDEB does not affect the Ca(2+) pumps or the Ca(2+) release from the endoplasmic reticulum. MDEB could therefore represent the first member of a new group of molecules, specifically able to potentiate SOCE. Although not toxic for non-activated Jurkat T cells, it could induce the apoptosis of phytohemagglutinin-stimulated cells.

Impact of Exogenous Galectin-9 on Human T Cells: CONTRIBUTION OF THE T CELL RECEPTOR COMPLEX TO ANTIGEN-INDEPENDENT ACTIVATION BUT NOT TO APOPTOSIS INDUCTION.

Galectin-9 (gal-9) is a multifunctional ß-galactoside-binding lectin, frequently released in the extracellular medium, where it acts as a pleiotropic immune modulator. Despite its overall immunosuppressive effects, a recent study has reported bimodal action of gal-9 on human resting blood T cells with apoptosis occurring in the majority of them, followed by a wave of activation and expansion of Th1 cells in the surviving population. Our knowledge of the signaling events triggered by exogenous gal-9 in T cells remains limited. One of these events is cytosolic calcium (Ca(2+)) release reported in some murine and human T cells. The aim of this study was to investigate the contribution of Ca(2+) mobilization to apoptotic and nonapoptotic effects of exogenous gal-9 in human T cells. We found that the T cell receptor (TCR)-CD3 complex and the Lck kinase were required for Ca(2+) mobilization but not for apoptosis induction in Jurkat cells. These data were confirmed in human CD4(+) T cells from peripheral blood as follows: a specific Lck chemical inhibitor abrogated Ca(2+) mobilization but not apoptosis induction. Moreover, Lck activity was also required for the production of Th1-type cytokines, i.e. interleukin-2 and interferon-γ, which resulted from gal-9 stimulation in peripheral CD4(+) T cells. These findings indicate that gal-9 acts on T cells by two distinct pathways as follows: one mimicking antigen-specific activation of the TCR with a mandatory contribution of proximal elements of the TCR complex, especially Lck, and another resulting in apoptosis that is independent of this complex.

Epstein-Barr virus latent membrane protein 1 increases calcium influx through store-operated channels in B lymphoid cells.

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.

The boron-oxygen core of borinate esters is responsible for the store-operated calcium entry potentiation ability.

BACKGROUND: Store-Operated Calcium Entry (SOCE) is the major Ca2+ ion entry pathway in lymphocytes and is responsible of a severe combined immunodeficiency (SCID) when deficient. It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca2+ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca2+-ATPase. RESULTS: A structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE. CONCLUSIONS: This study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators.

Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus latent membrane protein-1.

BACKGROUND: Calcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated. RESULTS: Two Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles. CONCLUSION: The data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.

Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent.

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca(2+)](cyt) enhanced anion channels and impaired H(+)-ATPases.