Dexamethasone upregulates macrophage PIEZO1 via SGK1, suppressing inflammation and increasing ROS and apoptosis.

The side effects of high-dose dexamethasone in anti-infection include increased ROS production and immune cell apoptosis. Dexamethasone effectively activates serum/glucocorticoid-regulated kinase 1 (SGK1), which upregulates various ion channels by activating store-operated calcium entry (SOCE), leading to Ca2+ oscillations. PIEZO1 plays a crucial role in macrophages' immune activity and function, but whether dexamethasone can regulate PIEZO1 by enhancing SOCE via SGK1 activation remains unclear. The effects of dexamethasone were assessed in a mouse model of sepsis, and primary BMDMs and the RAW264.7 were treated with overexpression plasmids, siRNAs, or specific activators or inhibitors to examine the relationships between SGK1, SOCE, and PIEZO1. The functional and phenotypic changes of mouse and macrophage models were detected. The results indicate that high-dose dexamethasone upregulated SGK1 by activating the macrophage glucocorticoid receptor, which enhanced SOCE and subsequently activated PIEZO1. Activation of PIEZO1 resulted in Ca2+ influx and cytoskeletal remodelling. The increase in intracellular Ca2+ mediated by PIEZO1 further increased the activation of SGK1 and ORAI1/STIM1, leading to intracellular Ca2+ peaks. In the context of inflammation, activation of PIEZO1 suppressed the activation of TLR4/NFκB p65 in macrophages. In RAW264.7 cells, PIEZO1 continuous activation inhibited the change in mitochondrial membrane potential, accelerated ROS accumulation, and induced autophagic damage and cell apoptosis in the late stage. CaMK2α was identified as a downstream mediator of TLR4 and PIEZO1, facilitating high-dose dexamethasone-induced macrophage immunosuppression and apoptosis. PIEZO1 is a new glucocorticoid target to regulate macrophage function and activity. This study provides a theoretical basis for the rational use of dexamethasone.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

In-vitro and in-vivo anti-allergic effects of magnolol on allergic rhinitis via inhibition of ORAI1 and ANO1 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. AIMS OF THE STUDY: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. MATERIALS AND METHODS: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. RESULTS: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 µM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 µM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. CONCLUSION: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR.

Increasing cytosolic Ca2+ levels restore cell proliferation and stem cell potency in aged MSCs.

Aging is an inescapable complex physiological but extendable process, and all cells, including stem cells, are altered over time. Diverse mechanism(s) could modulate stem cell number, their proliferation rate, and promote tissue repair during aging that leads to longevity. However, the factors that could restore aging stem cell potency and would lead to healthy aging are not fully identified. Here we show that maintaining cytosolic Ca2+ levels was essential for modulating stem cells function in aged mesenchymal stem cells (MSCs). Increasing external Ca2+ induced spindle shape stem cell morphology and maintained stem cell surface marker expression in aged bone marrow-derived MSCs. Similarly, stem cell survival and proliferation of aged MSCs was dependent on cytosolic Ca2+ levels. Importantly, Ca2+ entry potentiated cell cycle progression, and stem cell potential was increased in cells incubated with higher external Ca2+. Moreover, blocking Ca2+ entry using SKF 96365, decreased stem cell survival and its proliferation but, treatment with 2-APB did not significantly affected cell proliferation, rather only modulated cell viability. Evaluation of Ca2+ entry channels, showed that TRPC1/Orai1/Orai3 and their regulator STIM1 was essential for MSCs proliferation/viability as gene silencing of Orai1/Orai3/TRPC1/STIM1 significantly inhibited stem cell viability. Finally, MSCs isolated from aged mice that were subjected to higher Ca2+ levels, were able to rescue age-induced loss of MSCs function. Together these results suggest that Ca2+ entry is essential for preventing the loss of aged stem cell function and supplementing Ca2+ not only restored their proliferative potential but, allowed them to develop into younger stem cell lineages that could be critical for regenerative medicine.

The role of STIM1/ORAI1 channel in the analgesic effect of grain-sized moxibustion on inflammatory pain mice model.

The therapeutic effect of grain-sized moxibustion (GS-Moxi) on inflammatory pain has been well recognized clinically, but the mechanism remains unclear. STIM1/ORAI1 is a sensible temperature channel, therefore; this study aimed to investigate the analgesic effect of GS-Moxi and the association with STIM1/ORAI1 expression. CFA-induced inflammatory pain model was established and was treated with GS-Moxi after 3 days of CFA injection. The behavioral test was measured after the GS-Moxi; then, serum was prepared for IL-1ß, IL-6, and TNF-α, and the stimulated skin was used for measuring STIM1 and ORAI1 expression. The results indicated GS-Moxi had an analgesic effect on inflammatory pain and the heat variation was significant for the analgesia. GS-Moxi decreased the expression of IL-1ß, IL-6, and TNF-α. Immunofluorescence and western blot analysis illustrated that heat change was associated with the stimulation of STIM1 and ORAI1. Suggesting that heat variation created by GS-Moxi could be crucial in this therapy and STIM1 and ORAI1 were potential enhancers in regulating analgesia of GS-Moxi.

Vasopressin-stimulated ORAI1 expression and store-operated Ca2+ entry in aortic smooth muscle cells.

Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca2+-channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca2+ entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca2+ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 µM) or SGK1 inhibitor GSK-650394 (1 µM). Transcript levels were measured using q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and store-operated Ca2+ entry from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca2+ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca2+ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca2+ entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: • In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. • VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). • VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. • VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394.

[Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells.

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca2+]i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca2+]i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca2+]i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

Qingyi decoction protects against myocardial injuries induced by severe acute pancreatitis.

BACKGROUND: We studied the protective effects of Qingyi decoction (QYD) (a Traditional Chinese Medicine) against severe acute pancreatitis (SAP)-induced myocardial infarction (MI). AIM: To study the function and mechanism of QYD in the treatment of myocardial injuries induced by SAP. METHODS: Ultrasonic cardiography, hematoxylin and eosin staining, immunohistochemistry, qRT-PCR, western blot, enzyme-linked immunosorbent assays, and apoptosis staining techniques were used to determine the effects of QYD following SAP-induced MI in Sprague-Dawley rats. RESULTS: Our SAP model showed severe myocardial histological abnormalities and marked differences in the symptoms, mortality rate, and ultrasonic cardiography outputs among the different groups compared to the control. The expression of serum cytokines [interleukin (IL)-1ß, IL-6, IL-8, IL-12, amyloid ß, and tumor necrosis factor-α] were significantly higher in the SAP versus QYD treated group (P < 0.05 for all). STIM1 and Orai1 expression in myocardial tissue extracts were significantly decreased post QYD gavage (P < 0.001). There was no significant histological difference between the 2-aminoethyl diphenylborinate inhibitor and QYD groups. The SAP group had a significantly higher apoptosis index score compared to the QYD group (P < 0.001). CONCLUSION: QYD conferred cardio-protection against SAP-induced MI by regulating myocardial-associated protein expression (STIM1 and Orai1).

Flos Magnoliae and its Constituent Linoleic Acid Suppress T Lymphocyte Activation via Store-Operated Calcium Entry.

Intracellular calcium signaling is crucial for type 2 helper T cell and mast cell activation, which is essential for allergic inflammation. It is initiated by antigen-mediated receptor stimulation that triggers store-operated calcium entry (SOCE) via ORAI1 calcium channel. Flos Magnoliae (FM) is widely used to treat allergic diseases such as allergic rhinitis and asthma. Although many studies have reported that FM regulates intracellular calcium signaling, research on the exact type of calcium channel modulated by FM is scarce. Therefore, we hypothesized that the anti-allergic effects of FM might result from ORAI1 inhibition in T cells. We investigated whether a 70% ethanolic extract of FM (FMEtOH) and its constituents inhibit ORAI1 channel activity and subsequent T cell activation. We performed conventional whole-cell patch clamp studies in hSTIM1 and hORAI1-overexpressing HEK293T cells (HEKORAI1). Intracellular calcium concentration was determined using Fura-2 dye and cytokine production measurement in Jurkat T lymphocytes. FMEtOH (0.03 mg/mL) and its fractions, especially hexane fraction (FMHex, 0.01 mg/mL), significantly inhibited SOCE and IL-2 cytokine production in Jurkat T lymphocytes. GC/MS analysis showed linoleic acid (LA) as the major component of FMHex. FMHex at 0.01 mg/mL (equivalent to 10 µM LA) inhibited not only SOCE but also IL-2 production, as well as CD3/CD28 receptor co-stimulation induced calcium signaling in Jurkat T lymphocytes. FMEtOH and LA suppressed CD4+ T lymphocyte activation, at least in part, by inhibiting ISOCE. Thus, ISOCE inhibition may be a potential strategy to inhibit immune responses in inflammation.

[Effects of n-3 polyunsaturated fatty acid on store-operated calcium channels in coronary artery smooth muscle cells derived from diabetic rat].

Objective: To investigate the impact of n-3 polyunsaturated fatty acid (n-3 PUFA) on function and expression of store-operated calcium channels (SOCC) in coronary artery smooth muscle cells (SMC) derived from diabetic rat. Methods: A total of 180 healthy male Sprague-Dawley (SD) rats were randomly divided into normal group (N, n=45), placebo-treated diabetic group (D, n=45), lose dose n-3 PUFA treated diabetic group (DL, n=45) and high dose n-3 PUFAs treated diabetic group (DH, n=45). Streptozotocin-induced diabetic rat animal model was established by two consecutive intraperitoneal injections. After modeling, rats in group DL and DH were treated with 10 mg·kg(-1)·d(-1) and 50 mg·kg(-1)·d(-1) n-3 PUFAs respectively per gavage for eight weeks. After eight weeks, rat coronary artery SMC was isolated by enzyme digestion. Changes of cytosolic calcium concentration in coronary artery SMC were examined by calcium fluorescence imaging technique, coronary artery tension was detected by myograph system, and protein expressions of SOCC on coronary artery SMC were measured by Western blot. Results: SOCC induced ΔF340/F380 of group N, D, DL and DH were 0.425±0.023, 0.838±0.037, 0.342±0.052 and 0.364±0.045 respectively, which was significantly lower in group N, DL, DH than in group D (P<0.05). SOCC induced changes of tensions were 0.94±0.09, 1.95±0.18, 1.35±0.24 and 1.01±0.18 in the group N, D, DL and DH, respectively, which was significantly lower in group N and DH than in group D (P<0.05). Protein expressions of STIM1, Orai1 and TRPC1 were significantly higher in diabetic rat coronary SMC than in group N (P<0.05). STIM1 protein expressions were significantly lower in group DL and DH than in group D, and Orai1 and TRPC1 protein expressions were similar among group. Conclusions: Coronary artery tension, cytosolic calcium concentration and protein expressions of SOCC are higher in diabetic rat coronary artery SMC when compared with normal rats. n-3 PUFA intervention could downregulate the protein expression of SOCC, reduce cytosolic calcium concentration and coronary artery tension, and is protective to the diabetic injury in coronary artery.

Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex.

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

Effects of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil activity in dairy cows with subclinical hypocalcemia1.

Hypocalcemia in dairy cows is often associated with inflammation-related disorders such as metritis and mastitis. The protein encoded by the Ca2+ release-activated calcium modulator 1 (ORAI1) gene is a membrane Ca2+ channel subunit that is activated when Ca2+ stores are depleted. Polymorphonuclear neutrophils (PMNL) have a crucial role in the defense against infection through migration, adhesion, chemotaxis, phagocytosis, and reactive oxygen species (ROS) production in response to pathogens. Whether hypocalcemia affects the activity of PMNL and if ORAI1 is involved remains unknown. To address this, PMNL were isolated at 3 d of calving from dairy cows diagnosed as clinically healthy (n = 20, CONTROL) or with plasma concentration of calcium < 2.0 mmol/L as a criterion for diagnosis of subclinical hypocalcemia (n = 20, HYPOCAL). PMNL isolated from both groups of cows were treated with or without the sarcoendoplasmic Ca2+ ATPase inhibitor thapsigargin, Ca2+ ionophore Ionomycin, and ORAI1 blocker 2APB. The intracellular Ca2+ concentration, ORAI1 abundance, ROS, phagocytosis rate, migration, and adhering capacity of treated PMNL were evaluated. Some of the in vitro assays also included use of small interfering ORAI1 RNA (siORAI1), 100 nM 1,25(OH)2D3, or 100 nM parathyroid hormone (PTH). Intracellular Ca2+ concentration was markedly lower in HYPOCAL. In addition, ORAI1 was detected in PMNL plasma membrane via FACS and was markedly lower in cows with HYPOCAL. Migration, adhesion capacity, and phagocytosis rate of PMNL were lower in response to HYPOCAL. Furthermore, plasma and PMNL concentration of nucleosome assembly protein (NAP2) and pro-platelet basic protein (CXCL7) was markedly lower with HYPOCAL. All these changes were associated with lower ROS production by PMNL. Thapsigargin and ionomycin treatment in vitro increased ORAI1 expression, migration of PMNL, adhering capacity, phagocytosis rate, and ROS production; conversely, those effects were abrogated by siORAI1 and ORAI1 inhibitor 2APB treatment. Also cytosolic Ca2+ concentration and ORAI1 abundance were increased by 1,25(OH)2D3 and PTH supplementation. Overall, the data indicate that failure of PMNL to uptake Ca2+ due to downregulation of ORAI1 during subclinical hypocalcemia is a factor contributing to impaired PMNL function. In addition, plasma PTH or 1,25(OH)2D3 could regulate ORAI1 and also participate in the regulation of PMNL activity.

Resveratrol reduces store-operated Ca2+ entry and enhances the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex

BACKGROUND: Resveratrol was reported to trigger the apoptosis of fibroblast-like synoviocytes In adjuvant arthritis rats but the subcellular mechanism remains unclear. Since ER stress, mitochondrial dysfunction and oxidative stress were involved in the effects of resveratrol with imbalance of calcium bio-transmission, store operated calcium entry (SOCE), a novel intracellular calcium regulatory pathway, may also participate in this process. RESULTS: In the present study, Resveratrol was found to suppress ORAI1 expression of a dose dependent manner while have no evident effects on STIM1 expressive level. Besides, resveratrol had no effects on ATP or TG induced calcium depletion but present partly dose-dependent suppression of SOCE. On the one hand, microinjection of ORAI1 overexpressed vector in sick toe partly counteracted the therapeutic effects of resveratrol on adjuvant arthritis and serum inflammatory cytokine including IL-1, IL-6, IL-8, IL-10 and TNF-α. On the other hand, ORAI1 SiRNA injection provided slight relief to adjuvant arthritis in rats. In addition, ORAI1 overexpression partly diminished the alleviation of hemogram abnormality induced by adjuvant arthritis after resveratrol treatment while ORAI1 knockdown presented mild resveratrol-like effect on hemogram in rats model. CONCLUSION: These results indicated that resveratrol reduced store-operated Ca2+ entry and enhanced the apoptosis of fibroblast-like synoviocytes in adjuvant arthritis rats model via targeting ORAI1-STIM1 complex, providing a theoretical basis for ORAI1 targeted therapy in future treatment with resveratrol on rheumatoid arthritis.

Spirodela polyrhiza and its Chemical Constituent Vitexin Exert Anti-Allergic Effect via ORAI1 Channel Inhibition.

Intracellular calcium signaling cascades are integral to early and late allergic responses involving mast cell degranulation and type 2 helper T cell activation, respectively. Both the responses are accompanied by the movement of calcium through the calcium release-activated calcium (CRAC) channel, encoded by the ORAI1 gene. Spirodela polyrhiza (L.) Schleid (SP) has anti-inflammatory and anti-allergic effects, but its effect on calcium signaling has not been reported. This study investigated whether a 30% ethanolic SP extract (SPEtOH) and its constituents can reduce CRAC currents ([Formula: see text]), and thus inhibit mast cell degranulation and T cell activation. In Jurkat T lymphocytes, we found that 3[Formula: see text]mg/mL SPEtOH inhibited the [Formula: see text] by [Formula: see text]%, whereas one of its constituents vitexin (100[Formula: see text][Formula: see text]M) inhibited the [Formula: see text] by [Formula: see text]%. Furthermore, in the RBL-2H3 mast cell, the [Formula: see text] was inhibited by 3[Formula: see text]mg/mL SPEtOH ([Formula: see text]%) and 100[Formula: see text][Formula: see text]M vitexin ([Formula: see text]%). Investigation of human primary T cell proliferation induced by co-stimulation with antibodies to cluster of differentiation 3 and 28, and of RBL-2H3 mast cell degranulation following IgE-antigen complex stimulation revealed that 100[Formula: see text][Formula: see text]M vitexin inhibited both T-cell proliferation (by [Formula: see text]%) and mast cell degranulation (by [Formula: see text]%). These effects were concentration-dependent, and no cytotoxicity was observed. Our findings suggest that vitexin is a promising candidate compound for the development of therapeutic agents to prevent and treat allergic diseases.

Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca2+ entry.

Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca2+ entry (SOCE) and diminished Ca2+-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Unveiling some FDA-approved drugs as inhibitors of the store-operated Ca2+ entry pathway.

The store-operated calcium entry (SOCE) pathway is an important route for generating cytosolic Ca2+ signals that regulate a diverse array of biological processes. Abnormal SOCE seem to underlie several diseases that notably include allergy, inflammation and cancer. Therefore, any modulator of this pathway is likely to have significant impact in cell biology under both normal and abnormal conditions. In this study, we screened the FDA-approved drug library for agents that share significant similarity in 3D shape and surface electrostatics with few, hitherto best known inhibitors of SOCE. This has led to the identification of five drugs that showed dose-dependent inhibition of SOCE in cell-based assay, probably through interacting with the Orai1 protein which effectively mediates SOCE. Of these drugs, leflunomide and teriflunomide could suppress SOCE significantly at clinically-relevant doses and this provides for an additional mechanism towards the therapeutic utility of these drugs as immunosuppressants. The other three drugs namely lansoprazole, tolvaptan and roflumilast, were less potent in suppressing SOCE but were more selective and thus they may serve as novel scaffolds for future development of new, more efficacious SOCE inhibitors.

Intra-articular lentivirus-mediated gene therapy targeting CRACM1 for the treatment of collagen-induced arthritis.

Abnormal store-operated calcium uptake has been observed in peripheral T lymphocytes of rheumatoid arthritis (RA) patients, and sustained intracellular calcium signalling is known to mediate the functions of many types of immune cells. Thus, it is hypothesized that regulating calcium entry through CRACM1 (the pore-forming subunit of calcium release-activated calcium (CRAC) channels; also known as ORAI1) may be beneficial for the management of RA. Localized CRACM1 knockdown in the joints and draining lymph nodes (DLNs) of mice with collagen-induced arthritis (CIA) was achieved via lentiviral-based delivery of shRNA targeting mouse CRACM1. Consistent with CRACM1 knockdown, calcium influx in synovial cells and the histopathological features of CIA were reduced. These effects were also associated with reduced levels of several notable inflammatory cytokines, such as IL-6, IL-17A, and IFN-γ, in the joints. Additionally, CRACM1-shRNA reduced the number of bone marrow-derived osteoclasts in vitro as well as osteoclasts in CIA joints, which was associated with reduced RANKL levels in the serum and joints. In summary, inhibiting calcium entry by CRACM1 knockdown suppressed arthritis development and may be therapeutically beneficial for RA patients.

Spirodela polyrhiza extract modulates the activation of atopic dermatitis-related ion channels, Orai1 and TRPV3, and inhibits mast cell degranulation.

CONTEXT: Spirodela polyrhiza (L.) Schleid. (Lemnaceae), Spirodelae Herba (SH), has been known to relieve inflammation, urticaria and skin symptoms including pruritus, eczema and rash. OBJECTIVE: The effects of SH extract on two calcium ion channels, Orai1 and TRPV3, and their potential as novel therapeutics for atopic dermatitis (AD) were investigated. The regulatory role of Orai1 on mast cell degranulation was evaluated. MATERIALS AND METHODS: The dried leaves of SH were extracted by 70% methanol. Effects of SH extract (100 µg/mL) in an HEK293T cell line overexpressing human Orai1 or TRPV3 were assessed. Ion channel modulation in transfected HEK293T cells was measured using a conventional whole-cell patch-clamp technique. IgE-antigen complex-stimulated mast cell degranulation was measured by ß-hexosaminidase assay with morphological observation after treatment with 20, 50 and 100 µg/mL SH extract. RESULTS: SH extract (100 µg/mL) significantly inhibited Orai1 activity (63.8 ± 0.97%) in Orai1-STIM1 co-overexpressed HEK293T cells. SH extract significantly increased TRPV3 activity (81.29 ± 0.05% at -100 mV) compared with the positive control 2-APB (100 µM), which induced full activation. SH extract inhibited degranulation in IgE-antigen complex-stimulated RBL-2H3 mast cells by decreasing ß-hexosaminidase activity (3.14 ± 0.03, 2.56 ± 0.12 and 2.29 ± 0.08 mU/mg, respectively). CONCLUSION: Our results suggested that SH extract could treat abnormal skin barrier pathologies in AD through modulation of the activities of the calcium ion channels Orai1 and TRPV3 and inhibition of mast cell degranulation. This is the first report of an herbal effect on the modulation of ion channels associated with skin barrier disruption in AD pathogenesis.

Orai1-Mediated Antimicrobial Secretion from Pancreatic Acini Shapes the Gut Microbiome and Regulates Gut Innate Immunity.

The gut microbiome participates in numerous physiologic functions and communicates intimately with the host immune system. Antimicrobial peptides are critical components of intestinal innate immunity. We report a prominent role for antimicrobials secreted by pancreatic acini in shaping the gut microbiome that is essential for intestinal innate immunity, barrier function, and survival. Deletion of the Ca2+ channel Orai1 in pancreatic acini of adult mice resulted in 60%-70% mortality within 3 weeks. Despite robust activation of the intestinal innate immune response, mice lacking acinar Orai1 exhibited intestinal bacterial outgrowth and dysbiosis, ultimately causing systemic translocation, inflammation, and death. While digestive enzyme supplementation was ineffective, treatments constraining bacterial outgrowth (purified liquid diet, broad-spectrum antibiotics) rescued survival, feeding, and weight gain. Pancreatic levels of cathelicidin-related antimicrobial peptide (CRAMP) were reduced, and supplement of synthetic CRAMP prevented intestinal disease. These findings reveal a critical role for antimicrobial pancreatic secretion in gut innate immunity.