Synthesis and Characterization of Store-Operated Calcium Entry Inhibitors Active in the Submicromolar Range.

The store-operated calcium entry, better known as SOCE, forms the main Ca2+ influx pathway in non-excitable cells, especially in leukocytes, where it is required for cell activation and the immune response. During the past decades, several inhibitors were developed, but they lack specificity or efficacy. From the non-specific SOCE inhibitor 2-aminoethyl diphenylborinate (2-APB), we synthetized 16 new analogues by replacing/modifying the phenyl groups. Among them, our compound P11 showed the best inhibitory capacity with a Ki ≈ 75 nM. Furthermore, below 1 µM, P11 was devoid of any inhibitory activity on the two other main cellular targets of 2-APB, the IP3 receptors, and the SERCA pumps. Interestingly, Jurkat T cells secrete interleukin-2 under phytohemagglutinin stimulation but undergo cell death and stop IL-2 synthesis when stimulated in the presence of increasing P11 concentrations. Thus, P11 could represent the first member of a new and potent family of immunosuppressors.

[Purinergic receptors and hepatic fibrosis]. / Récepteurs purinergiques et fibrose hépatique.

TITLE: Récepteurs purinergiques et fibrose hépatique. ABSTRACT: Pour la cinquième année, dans le cadre du module d'enseignement « Physiopathologie de la signalisation ¼ proposé par l'université Paris-sud, les étudiants du Master « Biologie Santé ¼ de l'université Paris-Saclay se sont confrontés à l'écriture scientifique. Ils ont sélectionné une quinzaine d'articles scientifiques récents dans le domaine de la signalisation cellulaire présentant des résultats originaux, via des approches expérimentales variées, sur des thèmes allant des relations hôte-pathogène aux innovations thérapeutiques, en passant par la signalisation hépatique et le métabolisme. Après un travail préparatoire réalisé avec l'équipe pédagogique, les étudiants, organisés en binômes, ont ensuite rédigé, guidés par des chercheurs, une Nouvelle soulignant les résultats majeurs et l'originalité de l'article étudié. Ils ont beaucoup apprécié cette initiation à l'écriture d'articles scientifiques et, comme vous pourrez le lire, se sont investis dans ce travail avec enthousiasme ! Trois de ces Nouvelles sont publiées dans ce numéro, les autres le seront dans des prochains numéros.

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.

The P2X4 purinergic receptor regulates hepatic myofibroblast activation during liver fibrogenesis.

BACKGROUND & AIMS: Liver fibrosis is characterized by the accumulation of extracellular matrix produced by hepatic myofibroblasts (hMF), the activation of which is critical to the fibrogenic process. Extracellular ATP, released by dying or stressed cells, and its purinergic receptors, constitute a powerful signaling network after injury. Although the purinergic receptor P2X4 (P2RX4) is highly expressed in the liver, its functions in hMF had never been investigated during liver fibrogenesis. METHODS: In vivo, bile duct ligation was performed and methionine- and choline-deficient diet administered in wild-type and P2x4 knock-out (P2x4-KO) mice. In vitro, hMF were isolated from mouse (wild-type and P2x4-KO) and human liver. P2X4 pharmacological inhibition (in vitro and in vivo) and P2X4 siRNAs (in vitro) were used. Histological, biochemical and cell culture analysis allowed us to study P2X4 expression and its involvement in the regulation of fibrogenic and fibrolytic factors, as well as of hMF activation markers and properties. RESULTS: P2X4 genetic invalidation or pharmacological inhibition protected mice from liver fibrosis and hMF accumulation after bile duct ligation or methionine- and choline-deficient diet. Human and mouse hMFs expressed P2X4, mainly in lysosomes. Invalidation of P2X4 in human and mouse hMFs blunted their activation marker expression and their fibrogenic properties. Finally, we showed that P2X4 regulates calcium entry and lysosomal exocytosis in hMF, impacting on ATP release, profibrogenic secretory profile, and transcription factor activation. CONCLUSION: P2X4 expression and activation is critical for hMF to sustain their activated and fibrogenic phenotype. Therefore, the inactivation of P2X4 may be of therapeutic interest during liver fibrotic diseases. LAY SUMMARY: During chronic injury, the liver often repairs with fibrotic tissue, which impairs liver function, and for which there is currently no treatment. We found that a previously unexplored pathway involving the purinergic receptor P2X4, can modulate fibrotic liver repair. Therefore, this receptor could be of interest in the development of novel therapies for fibrotic liver diseases.

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.

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.

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.

Characterization of novel store-operated calcium entry effectors.

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca(2+) entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1-5µM and inhibition at >30µM. Unfortunately, it is also able to modify the activity of other Ca(2+) transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation. Previously, we have shown that SOCE potentiation by 2-APB depends on the presence of the central boron-oxygen core (BOC) and that the phenyl groups determine the sensitivity of the molecule to inhibit and/or potentiate the SOCE. We hypothesized that by modifying the two phenyl groups of 2-APB, we could identify more efficient and specific analogues. In fact, the addition of methoxyl groups to one phenyl group greatly decreased the potentiation ability without any significant effect on the inhibition. Surprisingly, when the free rotation of the two phenyl groups was blocked by a new hydrocarbon bridge, the BOC was no longer able to potentiate. Furthermore, larger aryl groups than phenyl also impaired the activity of the BOC. Thus, the potentiation site in the Ca(2+) channel is not accessible by the BOC when the lateral groups are too large or unable to freely rotate. However, these molecules are potent inhibitors of store-operated calcium entry with affinities below 1µM, and they can block the activation of the Jurkat T cells. Thus, it is possible to characterize 2-APB analogues with different properties that could be the first step in the discovery of new immunomodulators. This article is part of a special issue entitled "Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

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.

Synthetic partial agonists reveal key steps in IP3 receptor activation.

Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are ubiquitous intracellular Ca2+ channels. IP(3) binding to the IP(3)-binding core (IBC) near the N terminus initiates conformational changes that lead to opening of a pore. The mechanisms underlying this process are unresolved. We synthesized 2-O-modified IP(3) analogs that are partial agonists of IP(3)R. These are similar to IP(3) in their interactions with the IBC, but they are less effective than IP(3) in rearranging the relationship between the IBC and the N-terminal suppressor domain (SD), and they open the channel at slower rates. IP(3)R with a mutation in the SD occupying a position similar to the 2-O substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or the SD therefore block obligatory coupling of the IBC and the SD. Analysis of DeltaG for ligand binding shows that IP(3) is recognized by the IBC and conformational changes then propagate entirely via the SD to the pore.

Counting functional inositol 1,4,5-trisphosphate receptors into the plasma membrane.

Inositol 1,4,5-trisphosphate receptors (IP(3)R) within the endoplasmic reticulum mediate release of Ca(2+) from intracellular stores. Different channels usually mediate Ca(2+) entry across the plasma membrane. In B lymphocytes and a cell line derived from them (DT40 cells), very few functional IP(3)R (approximately 2/cell) are invariably expressed in the plasma membrane, where they mediate about half the Ca(2+) entry evoked by activation of the B-cell receptor. We show that cells reliably count approximately 2 functional IP(3)R into the plasma membrane even when their conductance and ability to bind IP(3) are massively attenuated. We conclude that very small numbers of functional IP(3)R can be reliably counted into a specific membrane compartment in the absence of feedback signals from the active protein.

Ca2+ entry through plasma membrane IP3 receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.

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.

Nephrolithiasis and osteoporosis associated with hypophosphatemia caused by mutations in the type 2a sodium-phosphate cotransporter.

BACKGROUND: Epidemiologic studies suggest that genetic factors confer a predisposition to the formation of renal calcium stones or bone demineralization. Low serum phosphate concentrations due to a decrease in renal phosphate reabsorption have been reported in some patients with these conditions, suggesting that genetic factors leading to a decrease in renal phosphate reabsorption may contribute to them. We hypothesized that mutations in the gene coding for the main renal sodium-phosphate cotransporter (NPT2a) may be present in patients with these disorders. METHODS: We studied 20 patients with urolithiasis or bone demineralization and persistent idiopathic hypophosphatemia associated with a decrease in maximal renal phosphate reabsorption. The coding region of the gene for NPT2a was sequenced in all patients. The functional consequences of the mutations identified were analyzed by expressing the mutated RNA in Xenopus laevis oocytes. RESULTS: Two patients, one with recurrent urolithiasis and one with bone demineralization, were heterozygous for two distinct mutations. One mutation resulted in the substitution of phenylalanine for alanine at position 48, and the other in a substitution of methionine for valine at position 147. Phosphate-induced current and sodium-dependent phosphate uptake were impaired in oocytes expressing the mutant NPT2a. Coinjection of oocytes with wild-type and mutant RNA indicated that the mutant protein had altered function. CONCLUSIONS: Heterozygous mutations in the NPT2a gene may be responsible for hypophosphatemia and urinary phosphate loss in persons with urolithiasis or bone demineralization.