Inhibition Of Store-operated Calcium Entry Research Articles

Effects of Cardiotoxins from Naja oxiana Cobra Venom on Rat Heart Muscle and Aorta: A Comparative Study of Toxin-Induced Contraction Mechanisms.

Cardiotoxins (CaTxs) are a group of snake toxins that affect the cardiovascular system (CVS). Two types (S and P) of CaTxs are known, but the exact differences in the effects of these types on CVS have not been thoroughly studied. We investigated cellular mechanisms of action on CVS for Naja oxiana cobra CaTxs CTX-1 (S-type) and CTX-2 (P-type) focusing on the papillary muscle (PM) contractility and contraction of aortic rings (AR) supplemented by pharmacological analysis. It was found that CTX-1 and CTX-2 exerted dose-dependent effects manifested in PM contracture and AR contraction. CTX-2 impaired functions of PM and AR more strongly than CTX-1. Effects of CaTxs on PM were significantly reduced by nifedipine, an L-type Ca2+ channel blocker, and by KB-R7943, an inhibitor of reverse-mode Na+/Ca2+ exchange. Furthermore, 2-aminoethoxydiphenyl borate, an inhibitor of store-operated calcium entry, partially restored PM contractility damaged by CaTxs. The CaTx influence on AR contracture was significantly reduced by nifedipine and KB-R7943. The involvement of reverse-mode Na+/Ca2+ exchange in the effect of CaTxs on the rat aorta was shown for the first time. The results obtained indicate that CaTx effects on CVS are mainly associated with disturbance of transporting systems responsible for the Ca2+ influx.

P096 Store-Operated Calcium Entry Controls Innate and Adaptive Immune Cell Function in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) represents a major clinical challenge in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in T cells, regulating many of their functional properties. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD. Therefore, we aimed to investigate the effects of SOCE inhibitors (SOCEi) on lymphocytes and epithelial cells isolated from IBD patients. Methods Peripheral blood mononuclear cells (PBMC), lamina propria lymphocytes (LPMCs) and epithelial cells were isolated from IBD patients undergoing colon resection. LPMCs or PBMCs were ex-vivo stimulated with Ionomycin/PMA ± SOCEi and subsequently stained with a panel of 37 immunological markers for mass cytometry acquisition. Ca2+ influx measurements were performed in order to assess the metabolic status of immune (PBMCs and LPMCs) and epithelial cells after SOCE blockade. Finally, murine models of colitis were used in order to investigate the effects of SOCE blockade on the induction of intestinal inflammation in-vivo. Results Data on B, T, NK, and myeloid cells revealed that treatment with SOCEi caused a dose-dependent inhibition of Ca2+ signaling in human gut-resident lymphocytes and PBMCs. The inhibition of SOCE attenuated the production of pathogenic cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by colonic T cells and ILCs, reduced the activation of B cells and the production of IL-6 by myeloid cells, without affecting the viability of primary human epithelial cells. Remarkably, mouse models of transfer colitis, in which SOCE signaling components have been blocked, revealed that SOCE is required for the induction of intestinal inflammation in-vivo. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Simple SummaryDiffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma with a high curability rate. However, 40% of patients will relapse or exhibit refractory disease, and compromised apoptotic pathways is among the prognosis-worsening factors. Therefore, drugging non-apoptotic modalities might be therapeutically promising. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells. Herein, we show that TQ selectively kills DLBCL cells, either cell lines or primary lymphoma cells bearing resistance features to standard treatment. Investigations show that, although TQ induced apoptotic markers, non-apoptotic death was the major mechanism responsible for TQ-induced cellular demise. We demonstrate critical and selective roles of cytosolic calcium and necroptosis in TQ-induced non-apoptotic cell death. Finally, TQ exhibits an improved selectivity profile over conventional chemotherapy. Collectively, this work provides new insights into the mode of action of TQ and points to the therapeutic relevance of non-apoptotic modalities as a fail-safe mechanism for pro-apoptotic DLBCL therapies.Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

P008 Store-operated calcium entry controls immune cell function and activation in Inflammatory Bowel Disease

Abstract Background Inflammatory bowel disease (IBD) comprises ulcerative colitis (UC) and Crohn’s disease (CD), both representing major clinical challenges in need of new treatment modalities to improve patient care. Store-operated calcium entry (SOCE) is the predominant calcium influx pathway in immune cells regulating many of their functional properties. Conditional knockout mice, in which SOCE signaling components are deleted in T cells, have revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, it is currently unknown whether the pharmacologic inhibition of SOCE is a suitable drug target in IBD and it remains elusive, which immune cell subsets are affected by the pharmaceutical blockade of SOCE. Therefore, we here aimed to investigate the effects of the SOCE inhibitor BTP-2 on the function and activation of lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes (LPMC) were isolated from UC and CD patients undergoing colon resection. Cells were ex -vivo stimulated with PMA/Ionomycin in the presence or absence of increasing concentrations of BTP-2, fixed and stored at -80°C until acquisition by mass cytometry. LPMCs were stained using 37 lineage and functional markers targeting B, T, NK or myeloid cells and the resulting flow cytometry standard (FCS) files were analyzed by using cytobank and the R/Bioconductor 9 packages. Additionally, Ca2+ influx measurements were performed in order to assess the impact of SOCE inhibition on the calcium homeostasis of various immune cell subsets. Results Data on B, T, NK, and myeloid cells revealed that treatment with BTP-2 causes a dose-dependent inhibition of calcium influx, demonstrating that BTP-2 is a potent inhibitor of SOCE in human LPMCs and PBMCs. By applying mass cytometry, we investigated how the gradual pharmacologic inhibition of SOCE affects the activation and pro-inflammatory functions of various immune cell subsets obtained from colon specimens of patients with UC and CD, which were characterized by the accumulation of CD4+ effector T cells, IFNγ-producing CD8+ T cells and IL-17-producing innate lymphoid cells (ILCs) compared to non-inflamed specimens. Remarkably, inhibition of SOCE attenuated the production of cytokines including IL-2, IL-4, IL-6, IL-17, TNF-α and IFNγ by T cells and ILCs, reduced the activation of B cells and decreased the production of IFNγ by myeloid cells without affecting the viability of primary human epithelial cells. Conclusion Our data reveal for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis.

Store-Operated Calcium Channels as Drug Target in Gastroesophageal Cancers.

Gastroesophageal cancers, including tumors occurring in esophagus and stomach, usually have poor prognosis and lack effective chemotherapeutic drugs for treatment. The association between dysregulated store-operated calcium entry (SOCE), a key intracellular Ca2+ signaling pathway and gastroesophageal cancers are emerging. This review summarizes the recent advances in understanding the contribution of SOCE-mediated intracellular Ca2+ signaling to gastroesophageal cancers. It assesses the pathophysiological role of each component in SOCE machinery, such as Orais and STIMs in the cancer cell proliferation, migration, and invasion as well as stemness maintenance. Lastly, it discusses efforts towards development of more specific and potent SOCE inhibitors, which may be a new set of chemotherapeutic drugs appearing at the horizon, to provide either targeted therapy or adjuvant treatment to overcome drug resistance for gastroesophageal cancers.

Store‐operated calcium entry mediated high glucose‐induced podocyte injury and mitochondrial impairment

Purpose Diabetic Nephropathy is one of the major complications of diabetes mellitus. Hyperglycemia is a known initiator of diabetes mellitus. Accumulating evidence suggests that podocyte injury is tightly associated with the onset and progression of diabetic nephropathy. However, the mechanisms underlying podocyte injury induced by hyperglycemia or high glucose (HG) is poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in the settings of diabetes is not known. The aim of the present study was to examine if SOCE mediated HG-induced podocyte injury by impairing mitochondrial function. Methods All experiments were carried out in cultured immortalized human podocytes. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and nephrin (a podocyte specific protein). Calcium imaging was used to analyze SOCE. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP). Results HG (25mM) significantly increased podocyte Orai1 protein abundance for time periods ranging from 2 to 12 hours. This HG effect was dose dependent. Consistently, Ca2+ imaging experiments showed that HG treatment (25 mM for 12 hours) significantly enhanced podocyte SOCE. Furthermore, the abundance of nephrin protein decreased in podocytes after exposure to HG, indicating podocyte injury in the presence of ambient HG. However, the HG-induced decrease of nephrin was blunted by BTP2 (an SOCE inhibitor, 4 µM). Moreover, HG treatment (25 mM for 24 hours) decreased MMP, indicating damage of mitochondria by HG. The decrease of MMP was prevented by BTP2, suggesting the contribution of SOCE to the detrimental effect of HG treatment. Conclusion The present study suggests that upregulated SOCE contributes to HG-induced podocyte injury, possibly by impairing mitochondrial function.

Regulation of Neogenin in Glomerular Mesangial Cells

Background Diabetic Nephropathy (DN) is the leading cause of chronic kidney disease. Renal inflammation is known to contribute to the onset and progression of DN. Glomerular mesangial cells (MCs) are the major cell type involved in the pathogenesis of DN. Store operated calcium entry (SOCE) regulates many MC functions. Neogenin is the multifunctional transmembrane receptor that plays an important role in some organ development and homeostasis. Studies also demonstrated that neogenin was associated with inflammatory responses. However, whether neogenin is involved in DN and how it is regulated in MCs is currently not known. The aim of the present study was to determine if and how neogenin protein abundance in MCs was altered by diabetes/high glucose. Methods Human MCs were cultured in normal glucose (NG, 5mM) with/without an activator (thapsigargin-1 μM) or inhibitor (BTP2-5 µM) of SOCE for different time periods. Tunicamycin (10 ug/ml) was used to induce ER stress. High glucose (HG, 25 mM) treatment was used to mimic diabetic environment in vitro. The protein abundance of neogenin was assessed by western blot of the whole cell lysates. Glomerular neogenin expression in the control and eNOS-/- db/db mice at 20 weeks of age was evaluated using immunofluorescence staining in the paraffin embedded kidney sections. Results Thapsigargin significantly reduced the protein abundance of neogenin beginning at 4 h of treatment, which was partially reversed by BTP2. Tunicamycin for 4 h decreased the protein content of neogenin as well. HG treatment for one and two days increased neogenin level. Furthermore, the glomerular neogenin expression in eNOS-/- db/db mice was decreased at 20 weeks of age, possibly indicating the effect of late stage of the disease. Conclusion Neogenin protein content in MCs was reduced by SOCE and ER stress, and neogenin might play a role in the pathogenesis of DN.

Resveratrol sensitizes A549 cells to irradiation damage via suppression of store-operated calcium entry with Orai1 and STIM1 downregulation.

Resveratrol is a natural polyphenol with multiple positive biofunctions and was found to have potential as a radiosensitizer with an intricate molecular mechanism. Store-operated calcium entry (SOCE) is a novel intracellular calcium regulatory pattern that is mainly mediated by iron channels, such as by the stromal interaction molecule (STIM) and calcium release-activated calcium channel protein (Orai) families. SOCE was recently reported to be suppressed via the downregulation of STIM or Orai families for the promotion of tumor cell death induced by resveratrol. In the present study, resveratrol combined with irradiation treatment were found to induce more evident cell damage compared with irradiation treatment alone, as shown with Cell Counting Kit-8 assay and mitochondrial membrane potential detection with rhodamine 123. Additionally, resveratrol combined with irradiation treatment decreased the expression of STIM1 and Orai1, while it had no effects on STIM2, Orai2 and Orai3. Moreover, resveratrol combined with irradiation treatment lead to alleviated thapsigargin-induced SOCE. In addition, overexpression of STIM1 and Orai1 reversed resveratrol-induced SOCE inhibition and reduced death in A549 cells under irradiation. In summary, the present results revealed that resveratrol can significantly enhance the effect of irradiation damage on lung adenocarcinoma A549 cells, and this effect may be mediated by suppression of SOCE with reduced expression of both STIM1 and Orai1.

Presynaptic store-operated Ca2+ entry drives excitatory spontaneous neurotransmission and augments endoplasmic reticulum stress

Store-operated calcium entry (SOCE) is activated by depletion of Ca2+ from the endoplasmic reticulum (ER) and mediated by stromal interaction molecule (STIM) proteins. Here, we show that in rat and mouse hippocampal neurons, acute ER Ca2+ depletion increases presynaptic Ca2+ levels and glutamate release through a pathway dependent on STIM2 and the synaptic Ca2+ sensor synaptotagmin-7 (syt7). In contrast, synaptotagmin-1 (syt1) can suppress SOCE-mediated spontaneous release, and STIM2 is required for the increase in spontaneous release seen during syt1 loss of function. We also demonstrate that chronic ER stress activates the same pathway leading to syt7-dependent potentiation of spontaneous glutamate release. During ER stress, inhibition of SOCE or syt7-driven fusion partially restored basal neurotransmission and decreased expression of pro-apoptotic markers, indicating that these processes participate in the amplification of ER-stress-related damage. Taken together, we propose that presynaptic SOCE links ER stress and augmented spontaneous neurotransmission, which may, in turn, facilitate neurodegeneration.

SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis.

Angiogenesis is a multistep process that controls endothelial cells (ECs) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promote signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated calcium entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor, and Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), in angiogenesis. Here, we show that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as human umbilical vein endothelial cell (HUVEC) tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice, since it reduces vessel length and the number of junctions, while it increases lacunarity. Moreover, we find that SARAF and Orai1 are involved in VEGF-mediated [Ca2+]i increase, and their knockdown using siRNA impairs HUVEC tube formation, proliferation, and migration. Finally, immunostaining and in situ proximity ligation assays indicate that SARAF likely interacts with Orai1 in HUVECs. Therefore, these findings show for the first time a functional interaction between SARAF and Orai1 in ECs and highlight their essential role in different steps of the angiogenesis process.

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry.

3,3'-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca2+ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca2+ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca2+ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca2+ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.

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.

Store-Operated Calcium Entry as a Therapeutic Target in Acute Pancreatitis: Discovery and Development of Drug-Like SOCE Inhibitors.

Store-operated calcium entry (SOCE) is important in the maintenance of calcium homeostasis and alterations in this mechanism are responsible for several pathological conditions, including acute pancreatitis. Since the discovery of SOCE, many inhibitors have been identified and extensively used as chemical probes to better elucidate the role played by this cellular mechanism. Nevertheless, only a few have demonstrated drug-like properties so far. Here, we report a class of biphenyl triazoles among which stands out a lead compound, 34, that is endowed with an inhibitory activity at nanomolar concentrations, suitable pharmacokinetic properties, and in vivo efficacy in a mouse model of acute pancreatitis.

Orai-1 and Orai-2 regulate oral cancer cell migration and colonisation by suppressing Akt/mTOR/NF-κB signalling

Despite remarkable progress in understanding and treating oral cancer (OC), it still remains one of the life-threatening diseases and predominant cancers in the world. Therefore, deciphering the molecular mechanisms of this disease would help us to develop highly efficacious therapies. Multiple lines of evidence suggest that calcium and its dysregulation play significant role in the development of various cancers. As an adaptation of survival mechanism, upon depletion of ER calcium stores, store-operated calcium entry (SOCE) has been induced via SOCE channels (SOCC) in various mammalian cells. SOCC are regulated by Orai-1, Orai-2 and Orai-3 located on plasma membrane and two calcium-sensing ER membrane proteins known as stromal interaction molecules (STIM-1 and STIM-2). Hence, the present study was aimed at analysing the role of Orai-1 and Orai-2 in oral cancer and the underlying mechanism. Our results suggest that both Orai-1 and Orai-2 proteins were overexpressed in oral cancer tissues and cell lines (SAS) compared to normal epithelial tissues and cell lines respectively. In addition, silencing of Orai-1 and Orai-2 via chemical SOCE inhibitors and siRNAs inhibited calcium uptake and suppressed oral cancer cell proliferation, colony formation and migration. Furthermore, silencing of Orai-1 and Orai-2 inhibited Akt/mTOR/NF-κB pathway in oral cancer cells. Interestingly, tobacco carcinogen NNN and synthetic carcinogen 4-NQO, enhanced the expression of Orai-1 and Orai-2 in SAS cells. Therefore, we conclude that Orai-1 and Orai-2 have significant role in oral cancer and can be further explored to develop novel therapies for the treatment of this disease.

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.

Temporal modulation of calcium sensing in hematopoietic stem cells is crucial for proper stem cell expansion and engraftment

Hematopoietic stem cells (HSCs) are known to reside in a bone marrow (BM) niche, which is associated with relatively higher calcium content. HSCs sense and respond to calcium changes. However, how calcium-sensing components modulate HSC function and expansion is largely unknown. We investigated temporal modulation of calcium sensing and Ca2+ homeostasis during ex vivo HSC culture and in vivo. Murine BM-HSCs, human BM, and umbilical cord blood (UCB) mononuclear cells (MNCs) were treated with store-operated calcium entry (SOCE) inhibitors SKF 96365 hydrochloride (abbreviated as SKF) and 2-aminoethoxydiphenyl borate (2-APB). Besides, K+ channel inhibitor TEA chloride (abbreviated as TEA) was used to compare the relationship between calcium-activated potassium channel activities. Seven days of SKF treatment induced mouse and human ex vivo BM-HSC expansion as well as UCB-derived primitive HSC expansion. SKF treatment induced the surface expression of CaSR, CXCR4, and adhesion molecules on human hematopoietic stem and progenitor cells. HSCs expanded with SKF successfully differentiated into blood lineages in recipient animals and demonstrated a higher repopulation capability. Furthermore, modulation of SOCE in the BM-induced HSC content and differentially altered niche-related gene expression profile in vivo. Intriguingly, treatments with SOCE inhibitors SKF and 2-APB boosted the mouse BM mesenchymal stem cell (MSC) and human adipose-derived MSCs proliferation, whereas they did not affect the endothelial cell proliferation. These findings suggest that temporal modulation of calcium sensing is crucial in expansion and maintenance of murine HSCs, human HSCs, and mouse BM-MSCs function.

Store‐Operated Calcium Entry Upregulated IL6 Expression in Glomerular Mesangial Cells through NF‐κB Pathway

BackgroundIL6 is a pleotropic cytokine and functions as a pro‐ as well as anti‐inflammatory mechanism depending on the cell type or the specific pathological environment. It is unclear if a particular signaling pathway confers IL6 with this property. Immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). Locally produced cytokines/chemokines contributes to the early changes and histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. We have previously demonstrated that the Orai‐1 mediated store‐operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine whether and how SOCE in MCs regulated IL6 production by MCs.MethodsOrai1, the channel protein mediating SOCE in MCs was knocked down using the targeted nanoparticle‐siRNA delivery system in wild type C57BLKS/J mice at the age of 16 weeks. Immunohistochemistry was performed on the paraffin embedded kidney sections to examine glomerular IL6 expression. In cultured human MCs, the expression of IL6 was examined in culture media and whole cell lysates using ELISA and Western blot analysis, respectively in the presence of normal glucose (5 mM D‐glucose + 20 mM L‐glucose) with/without an activator (thapsigargin at 1 μM) or inhibitor (GSK‐7975A 10 μM) of SOCE for 15 hrs. To determine the role of NF‐κB in SOCE effects on IL6 production, human MCs were treated with an inhibitor of NF‐κB, helenaline or siRNA against p65 subunit of NF‐κB. Furthermore, the effect of SOCE on the nuclear translocation of p65 was examined by assessing abundance of nuclear p65 protein in response to activation and inhibition of SOCE.ResultsThe IL6 expression level was reduced in the glomeruli of the mice treated with nanoparticle/Orai1 siRNA for 2 weeks compared to that in the control mice. In cultured human MCs, thapsigargin significantly increased IL‐6 level in supernatant media and in whole cell lysates of MCs, and this effect was attenuated by GSK 7975‐A, a selective inhibitor of SOCE. However, tunicamycin, an ER stress inducer did not alter the amount of IL6 as thapsigargin did. Treatment with helenaline, an inhibitor of NF‐κB pathway, or knockdown of p65, a subunit of NF‐κB significantly blunted the thapsigargin‐induced increase in IL6 protein abundance. Moreover, thapsigargin stimulated the nuclear translocation of p65 in human MCs.ConclusionOrai1‐mediated SOCE positively regulates IL6 production by MCs through activation of NF‐κB pathway.Support or Funding InformationResearch Support: NIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate.

Calcium channel Orai1 promotes lymphocyte IL-17 expression and progressive kidney injury.

We hypothesized that the store-operated calcium entry (SOCE) channel, Orai1, participates in the activation of Th17 cells and influences renal injury. In rats, following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T cells and IL-17 expression was restricted to Orai1+ cells. When kidney CD4+ cells of post-acute kidney injury (post-AKI) rats were stimulated with angiotensin II and elevated Na+ (10-7 M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL-17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58483/BTP2 (1 mg/kg) attenuated IL-17+ cell activation, inflammation, and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks after I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis, and impaired renal function. These responses were significantly attenuated by YM58483/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by approximately 10-fold and Th17 cells were elevated by approximately 4-fold in AKI versus non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL-17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression.

Preventive treatment with ginsenoside Rb1 ameliorates monocrotaline-induced pulmonary arterial hypertension in rats and involves store-operated calcium entry inhibition

Context Ginsenoside Rb1, the main active ingredient of ginseng, exhibits ex vivo depression of store-operated calcium entry (SOCE) and related vasoconstriction in pulmonary arteries derived from pulmonary hypertension (PH) rats. However, the in vivo effects of ginsenoside Rb1 on PH remain unclear. Objective This study explored the possibility of using ginsenoside Rb1 as an in vivo preventive medication for type I PH, i.e., pulmonary arterial hypertension (PAH), and potential mechanisms involving SOCE. Materials and methods Male Sprague-Dawley rats (170–180 g) were randomly divided into Control, MCT, and MCT + Rb1 groups (n = 20). Control rats received only saline injection. Rats in the MCT + Rb1 and MCT groups were intraperitoneally administered single doses of 50 mg/kg monocrotaline (MCT) combined with 30 mg/kg/day ginsenoside Rb1 or equivalent volumes of saline for 21 consecutive days. Subsequently, comprehensive parameters related to SOCE, vascular tone, histological changes and hemodynamics were measured. Results Ginsenoside Rb1 reduced MCT-induced STIM1, TRPC1, and TRPC4 expression by 35.00, 31.96, and 32.24%, respectively, at the protein level. SOCE-related calcium entry and pulmonary artery contraction decreased by 162.6 nM and 71.72%. The mean pulmonary artery pressure, right ventricle systolic pressure, and right ventricular mass index decreased by 19.5 mmHg, 21.6 mmHg, and 39.50%. The wall thickness/radius ratios decreased by 14.67 and 17.65%, and the lumen area/total area ratios increased by 18.55 and 15.60% in intrapulmonary vessels with 51–100 and 101–150 μm o.d. Conclusion Ginsenoside Rb1, a promising candidate for PH prevention, inhibited SOCE and related pulmonary vasoconstriction, and relieved MCT-induced PAH in rats.

Inhibiting store-operated calcium entry attenuates white matter secondary degeneration following SCI

Axonal degeneration plays a key role in the pathogenesis of numerous neurological disorders including spinal cord injury. After the irreversible destruction of the white matter elements during the primary (mechanical) injury, spared axons and their supporting glial cells begin to breakdown causing an expansion of the lesion site. Here we mechanistically link external sources of calcium entry through axoplasmic reticulum calcium store depletion that contributes to secondary axonal degeneration through a process called store-operated calcium entry. There is increasing evidence suggesting that store-operated calcium entry impairment is responsible for numerous disorders. Nevertheless, its role following spinal cord injury remains poorly understood. We hypothesize that store-operated calcium entry mediates secondary white matter degeneration after spinal cord injury. We used our previously published model of laser-induced spinal cord injury to focally transect mid cervical dorsal column axons from live 6–8-week-old heterozygous CNPaseGFP/+: Thy1YFP+ double transgenic murine spinal cord preparations (five treated, eight controls) and documented the dynamic changes in axons over time using two-photon excitation microscopy. We report that 1 hour delayed treatment with YM-58483, a potent inhibitor of store-operated calcium entry, significantly decreased intra-axonal calcium accumulation, axonal dieback both proximal and distal to the lesion site, reduced secondary axonal “bystander” damage acutely after injury, and promoted greater oligodendrocyte survival compared to controls. We also targeted store-operated calcium entry following a clinically relevant contusion spinal cord injury model in vivo. Adult, 6–8-week-old Advillin-Cre: Ai9 mice were subjected to a mild 30 kdyn contusion and imaged to observe secondary axonal degeneration in live animals. We found that delayed treatment with YM-58483 increased axonal survival and reduced axonal spheroid formation compared to controls (n = 5 mice per group). These findings suggest that blocking store-operated calcium entry acutely is neuroprotective and introduces a novel target to prevent pathological calcium entry following spinal cord injury using a clinically relevant model.