Orai1 Channel Inhibition Preserves Left Ventricular Systolic Function and Normal Ca2+ Handling After Pressure Overload.

BACKGROUND: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. METHODS: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocyte-specific expression of an ion pore-disruptive Orai1R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. RESULTS: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca2+ signaling alterations (increased SOCE, decreased [Ca2+]i transients amplitude and decay rate, lower SR Ca2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. CONCLUSIONS: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

Constitutive calcium entry and cancer: updated views and insights.

Tight control of basal cytosolic Ca2+ concentration is essential for cell survival and to fine-tune Ca2+-dependent cell functions. A way to control this basal cytosolic Ca2+ concentration is to regulate membrane Ca2+ channels including store-operated Ca2+ channels and secondary messenger-operated channels linked to G-protein-coupled or tyrosine kinase receptor activation. Orai, with or without its reticular STIM partner and Transient Receptor Potential (TRP) proteins, were considered to be the main Ca2+ channels involved. It is well accepted that, in response to cell stimulation, opening of these Ca2+ channels contributes to Ca2+ entry and the transient increase in cytosolic Ca2+ concentration involved in intracellular signaling. However, in various experimental conditions, Ca2+ entry and/or Ca2+ currents can be recorded at rest, without application of any experimental stimulation. This led to the proposition that some plasma membrane Ca2+ channels are already open/activated in basal condition, contributing therefore to constitutive Ca2+ entry. This article focuses on direct and indirect observations supporting constitutive activity of channels belonging to the Orai and TRP families and on the mechanisms underlying their basal/constitutive activities.

Molecular Dynamics Simulations of Membrane-Bound STIM1 to Investigate Conformational Changes during STIM1 Activation upon Calcium Release.

Calcium is involved in important intracellular processes, such as intracellular signaling from cell membrane receptors to the nucleus. Typically, calcium levels are kept at less than 100 nM in the nucleus and cytosol, but some calcium is stored in the endoplasmic reticulum (ER) lumen for rapid release to activate intracellular calcium-dependent functions. Stromal interacting molecule 1 (STIM1) plays a critical role in early sensing of changes in the ER's calcium level, especially when there is a sudden release of stored calcium from the ER. Inactive STIM1, which has a bound calcium ion, is activated upon ion release. Following activation of STIM1, there is STIM1-assisted initiation of extracellular calcium entry through channels in the cell membrane. This extracellular calcium entering the cell then amplifies intracellular calcium-dependent actions. At the end of the process, ER levels of stored calcium are reestablished. The main focus of this work was to study the conformational changes accompanying homo- or heterodimerization of STIM1. For this purpose, the ER luminal portion of STIM1 (residues 58-236), which includes the sterile alpha motif (SAM) domain plus the calcium-binding EF-hand domains 1 and 2 attached to the STIM1 transmembrane region (TM), was modeled and embedded in a virtual membrane. Next, molecular dynamics simulations were performed to study the conformational changes that take place during STIM1 activation and subsequent protein-protein interactions. Indeed, the simulations revealed exposure of residues in the EF-hand domains, which may be important for dimerization steps. Altogether, understanding conformational changes in STIM1 can help in drug discovery when targeting this key protein in intracellular calcium functions.

Ofatumumab capacity to deplete B cells from chronic lymphocytic leukaemia is affected by C4 complement exhaustion.

The management of patients with chronic lymphocytic leukaemia (CLL) has improved with the utilisation of ofatumumab as a novel anti-CD20 monoclonal antibody. However, as half of the patients fail to respond to the treatment, the aim of this study was to evaluate circulating CLL cell depletion and clinical response according to the context of complement activation and FcγRIIIA polymorphism in ten CLL patients with relapsed/refractory disease. At the end of the treatment, results indicated that circulating CD5(+) CD19(+) CLL cell depletion was major (<0.01 × 10(9) /L) in 4 of 10 patients, partial (>50% decrease) in 4 of 10 patients and ineffective for the two other patients. No clinical modifications were observed following ofatumumab introduction. Ofatumumab administration leads to a rapid and important exhaustion of complement C4 levels in patients with initial lymphocytosis. C4 exhaustion was accelerated in a non-responder patient, and incomplete in two patients with partial circulating depletion. Moreover, delaying weekly to monthly ofatumumab injections improved CLL cell depletion in two patients. FcγRIIIA 158 polymorphism (FF n = 6 and VF n = 4) was not associated with major and/or partial circulating CLL cell depletion. In conclusion, ofatumumab induces an important C4 exhaustion that needs to be taken into account when treating CLL patients with ofatumumab.

Improved PIEZO1 agonism through 4-benzoic acid modification of Yoda1.

BACKGROUND AND PURPOSE: The protein PIEZO1 forms mechanically activated, calcium-permeable, non-selective cation channels in numerous cell types from several species. Options for pharmacological modulation are limited and so we modified a small-molecule agonist at PIEZO1 channels (Yoda1) to increase the ability to modulate these channels. EXPERIMENTAL APPROACH: Medicinal chemistry generated Yoda1 analogues that were tested in intracellular calcium and patch-clamp assays on cultured cells exogenously expressing human or mouse PIEZO1 or mouse PIEZO2. Physicochemical assays and wire myography assays on veins from mice with genetic disruption of PIEZO1. KEY RESULTS: A Yoda1 analogue (KC159) containing 4-benzoic acid instead of the pyrazine of Yoda1 and its potassium salt (KC289) have equivalent or improved reliability, efficacy and potency, compared with Yoda1 in functional assays. Tested against overexpressed mouse PIEZO1 in calcium assays, the order of potency (as EC50 values, nM) was KC289, 150 > KC159 280 > Yoda1, 600). These compounds were selective for PIEZO1 over other membrane proteins, and the physicochemical properties were more suited to physiological conditions than those of Yoda1. The vasorelaxant effects were consistent with PIEZO1 agonism. In contrast, substitution with 2-benzoic acid failed to generate a modulator. CONCLUSION AND IMPLICATIONS: 4-Benzoic acid modification of Yoda1 improves PIEZO1 agonist activity at PIEZO1 channels. We suggest naming this new modulator Yoda2. It should be a useful tool compound in physiological assays and facilitate efforts to identify a binding site. Such compounds may have therapeutic potential, for example, in diseases linked genetically to PIEZO1 such as lymphatic dysplasia.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Global PIEZO1 Gain-of-Function Mutation Causes Cardiac Hypertrophy and Fibrosis in Mice.

PIEZO1 is a subunit of mechanically-activated, nonselective cation channels. Gain-of-function PIEZO1 mutations are associated with dehydrated hereditary stomatocytosis (DHS), a type of anaemia, due to abnormal red blood cell function. Here, we hypothesised additional effects on the heart. Consistent with this hypothesis, mice engineered to contain the M2241R mutation in PIEZO1 to mimic a DHS mutation had increased cardiac mass and interventricular septum thickness at 8-12 weeks of age, without altered cardiac contractility. Myocyte size was greater and there was increased expression of genes associated with cardiac hypertrophy (Anp, Acta1 and ß-MHC). There was also cardiac fibrosis, increased expression of Col3a1 (a gene associated with fibrosis) and increased responses of isolated cardiac fibroblasts to PIEZO1 agonism. The data suggest detrimental effects of excess PIEZO1 activity on the heart, mediated in part by amplified PIEZO1 function in cardiac fibroblasts.

Endothelial Piezo1 sustains muscle capillary density and contributes to physical activity.

Piezo1 forms mechanically activated nonselective cation channels that contribute to endothelial response to fluid flow. Here we reveal an important role in the control of capillary density. Conditional endothelial cell-specific deletion of Piezo1 in adult mice depressed physical performance. Muscle microvascular endothelial cell apoptosis and capillary rarefaction were evident and sufficient to account for the effect on performance. There was selective upregulation of thrombospondin-2 (TSP2), an inducer of endothelial cell apoptosis, with no effect on TSP1, a related important player in muscle physiology. TSP2 was poorly expressed in muscle endothelial cells but robustly expressed in muscle pericytes, in which nitric oxide (NO) repressed the Tsp2 gene without an effect on Tsp1. In endothelial cells, Piezo1 was required for normal expression of endothelial NO synthase. The data suggest an endothelial cell-pericyte partnership of muscle in which endothelial Piezo1 senses blood flow to sustain capillary density and thereby maintain physical capability.

Shear stress activates ADAM10 sheddase to regulate Notch1 via the Piezo1 force sensor in endothelial cells.

Mechanical force is a determinant of Notch signalling but the mechanism of force detection and its coupling to Notch are unclear. We propose a role for Piezo1 channels, which are mechanically-activated non-selective cation channels. In cultured microvascular endothelial cells, Piezo1 channel activation by either shear stress or a chemical agonist Yoda1 activated a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), a Ca2+-regulated transmembrane sheddase that mediates S2 Notch1 cleavage. Consistent with this observation, we found Piezo1-dependent increase in the abundance of Notch1 intracellular domain (NICD) that depended on ADAM10 and the downstream S3 cleavage enzyme, γ-secretase. Conditional endothelial-specific disruption of Piezo1 in adult mice suppressed the expression of multiple Notch1 target genes in hepatic vasculature, suggesting constitutive functional importance in vivo. The data suggest that Piezo1 is a mechanism conferring force sensitivity on ADAM10 and Notch1 with downstream consequences for sustained activation of Notch1 target genes and potentially other processes.

Cx43 Present at the Leading Edge Membrane Governs Promigratory Effects of Osteoblast-Conditioned Medium on Human Prostate Cancer Cells in the Context of Bone Metastasis.

Among the different interacting molecules implicated in bone metastases, connexin43 (Cx43) may increase sensitivity of prostate cancer (PCa) cells to bone microenvironment, as suggested by our in silico and human tissue samples analyses that revealed increased level of Cx43 expression with PCa progression and a Cx43 specific expression in bone secondary sites. The goal of the present study was to understand how Cx43 influences PCa cells sensitivity and aggressiveness to bone microenvironment. By means of Cx43-overexpressing PCa cell lines, we revealed a Cx43-dependent promigratory effect of osteoblastic conditioned media (ObCM). This effect on directional migration relied on the presence of Cx43 at the plasma membrane and not on gap junctional intercellular communication and hemichannel functions. ObCM stimulation induced Rac1 activation and Cx43 interaction with cortactin in protrusions of migrating PCa cells. Finally, by transfecting two different truncated forms of Cx43 in LNCaP cells, we determined that the carboxy terminal (CT) part of Cx43 is crucial for the responsiveness of PCa cells to ObCM. Our study demonstrates that Cx43 level and its membrane localization modulate the phenotypic response of PCa cells to osteoblastic microenvironment and that its CT domain plays a pivotal role.

STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia.

BACKGROUND: Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. METHODS: An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. RESULTS: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. CONCLUSIONS: These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

CD5 expression promotes IL-10 production through activation of the MAPK/Erk pathway and upregulation of TRPC1 channels in B lymphocytes.

CD5 is constitutively expressed on T cells and a subset of mature normal and leukemic B cells in patients with chronic lymphocytic leukemia (CLL). Important functional properties are associated with CD5 expression in B cells, including signal transducer and activator of transcription 3 activation, IL-10 production and the promotion of B-lymphocyte survival and transformation. However, the pathway(s) by which CD5 influences the biology of B cells and its dependence on B-cell receptor (BCR) co-signaling remain unknown. In this study, we show that CD5 expression activates a number of important signaling pathways, including Erk1/2, leading to IL-10 production through a novel pathway independent of BCR engagement. This pathway is dependent on extracellular calcium (Ca2+) entry facilitated by upregulation of the transient receptor potential channel 1 (TRPC1) protein. We also show that Erk1/2 activation in a subgroup of CLL patients is associated with TRPC1 overexpression. In this subgroup of CLL patients, small inhibitory RNA (siRNA) for CD5 reduces TRPC1 expression. Furthermore, siRNAs for CD5 or for TRPC1 inhibit IL-10 production. These findings provide new insights into the role of CD5 in B-cell biology in health and disease and could pave the way for new treatment strategies for patients with B-CLL.

Calcium Signaling: From Normal B Cell Development to Tolerance Breakdown and Autoimmunity.

Maintenance of self-tolerance of auto-reactive lymphocytes is a fundamental mechanism to prevent the onset of autoimmune diseases. Deciphering the mechanisms involved in the deregulations leading to tolerance disruption and autoimmunity is still a major area of interest to identify new therapeutic targets and options. Ca2+ signaling plays a major role in B cell normal development and is therefore finely tuned by B cell receptor (BCR)-dependent and independent pathways. Developmental changes in the characteristics of BCR-dependent Ca2+ signals as well as the modulation of basal intracellular concentration ([Ca2+]i) contribute strongly to self-tolerance maintaining mechanisms responsible for the physical or functional elimination of autoreactive B cells such as clonal deletion, receptor editing, and anergy. Implication of Ca2+ signals in B tolerance mechanisms mainly occurs through the specific activation of transcriptional programs depending on the amplitude, shape, and duration of Ca2+ signals. A large number of studies reported Ca2+ signaling defects in autoimmune pathology such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and primary SjÓ§gren's syndrome (pSS). However, the precise nature of the molecular events responsible for these deregulations is not fully understood. Moreover, the demonstration of a direct correlation between Ca2+ signaling defects and tolerance disruption is still lacking. The recent identification of proteins involved in B cell Ca2+ signals such as ORAI, stromal interaction molecule and transient receptor potential is opening new horizons for understanding Ca2+ signaling defects observed in autoimmune diseases and for proposing potentially new therapeutic solutions. This review aims to present an overview of the developmental evolution of BCR dependent Ca2+ signaling and to place this signaling pathway in the context of mechanisms involved in tolerance maintenance and breakdown.

Calcium signaling and cell fate: how can Ca2+ signals contribute to wrong decisions for Chronic Lymphocytic Leukemic B lymphocyte outcome?

Ca(2+) signaling is a key regulator of B lymphocyte cell fate and defects in this signaling pathway have been reported in numerous diseases such as Chronic lymphocytic leukemia (CLL). CLL is a B cell clonal disorder characterized by the accumulation of mature monoclonal CD5(+) B cells. Although CLL could be considered to be a proliferative disease, most circulating CLL B cells are arrested in the G0 phase of the cell cycle and present both defects in calcium (Ca(2+)) homeostasis and signaling. The Ca(2+) response to antigen ligation is heterogeneous and related, in part, to defects arising from the incapacity to respond to B cell receptor (BCR) engagement (anergy), to the expression of T cell kinases (e.g. Zap70), and to the presence of negative feedback regulation by phosphatases (e.g. SHP-1). Anergic CD5(+) CLL B cells are characterized by an elevated basal Ca(2+) level, IgM/CD79 downregulation, a constitutive activation of BCR pathway kinases, and an activation of the nuclear factor of activated T cells (NF-AT). Based on the Ca(2+) response, patients are classified into three groups: unresponders, responders with apoptosis, and responders with entry in the cell cycle. Moreover, internal and direct interaction between leukemic BCR-HCDR3 epitopes at the plasma membrane and interaction between Bcl-2 and the IP3-receptor at the endoplasmic reticulum are also suspected to interfere with the intracellular Ca(2+) homeostasis in CLL-B cells. As a whole, the Ca(2+) pathway is emerging to play a key role in malignant CLL-B survival, disease progression, and last but not least, in the therapeutic response.