Search Results (77)

STC2 (stanniocalcin 2) controls calcium (Ca²⁺) homeostasis in human platelets and other cell lines. The regulation of intracellular Ca²⁺ homeostasis is crucial for platelet activation; thus, the alteration in intracellular Ca²⁺ concentration or the mechanism involved in its regulation has been proposed to underlie some thrombotic disorders. Our previous studies evidenced that the knockdown of STC2 altered murine platelet activation; furthermore, a reduction in STC2 expression resulted in enhanced Ca²⁺ homeostasis in diabetic patients and, therefore, would contribute to the prothrombotic condition as a hallmark of diabetes mellitus type 2 (DM2). In this study, we examine a possible link between the expression of stanniocalcins (STCs) and different thrombotic events in humans. The expression of STCs was determined by Western blotting (WB); meanwhile, the analysis of protein interaction and phosphorylation was performed by completing a previous immunoprecipitation protocol (IP) of the proteins of interest. Thus, our results from patients with stroke/ictus presented a clear reduction in STC2 expression in their platelets, finding less STC2 content in the youngest thrombotic patients. Furthermore, acetyl-salicylic acid (ASA) administration reversed the decrease in the expression of STC2 in patients who did not suffer additional thrombotic episodes, as evidenced by the longitudinal analysis of up to 10 years of follow-up. Additionally, the increase in STC2 phosphorylation at the serine residues revealed increased activity of STC2 in thrombotic patients. Finally, we suggest that store-operated Ca²⁺ entry (SOCE) is over-activated in patients suffering from stroke/ictus, as revealed by the increase in the STIM1/Orai1 interaction found under resting conditions and, further, because MEG-01 cells transfected with siRNA STC2 to evoke artificial reduction in the STC2 expression presented an increased SOCE with respect to the control cells transfected with siRNA A. Conversely, the expression of the non-capacitative Ca²⁺ channels, Orai3 and TRPC6, was found to be reduced in patients with stroke. Altogether, our data allow us to conclude that STC2 represents a promising marker of stroke/ictus in thrombotic patients. Full article

Benign prostate hyperplasia (BPH) is characterized by prostate enlargement and dynamic alterations contributing to development of lower tract urinary symptoms (LUTS). Prostate hypercontractility has been proposed to contribute to BPH-related LUTS. The aim was to evaluate the effects of inhibiting stromal interaction molecule (STIM)/Orai calcium entry system on adrenergic and neurogenic contractions in prostate (HP) and bladder neck (HB) strips from BPH patients. Effects of STIM/Orai inhibition on adrenergic and neurogenic contractions of HP from organ donors (ODs) without BPH were also evaluated. HP and HB strips were obtained from 20 patients with BPH undergoing radical prostatectomy and from six OD at the time of organ collection for transplantation. Tissues were functionally evaluated for isometric tension recording. STIM-1, Orai1, and Orai3 protein expressions were determined in prostate tissues. STIM-1 was also localized by immunofluorescence in prostate sections. Norepinephrine-induced and neurogenic contractions were significantly reduced by STIM/Orai inhibition with YM-58483 (20 µM) in HP from BPH patients but not in tissues from ODs. STIM/Orai inhibition failed to significantly modify contraction of HB from BPH patients. Protein expression of STIM-1 was significantly elevated in HP from BPH patients. Functional contribution of STIM/Orai system to contractile tone is relevant in prostate when BPH is present, probably related to increased expression of STIM-1. Inhibition of STIM/Orai could have therapeutic implications for the management of BPH patients by alleviating prostatic hypercontraction. Full article

We aimed to determine the influence of donors’ vascular function on renal function in recipients and to evaluate the role of Orai1 calcium channels as a potential marker. A prospective collaborative multicenter study was designed. Blood, aorta (HA), mesenteric arteries (HMAs) and corpus cavernosum (HCC) specimens were obtained from organ donors at the kidney procurement procedure (n = 60). Evolution (up to 2 years) of renal function measured as serum creatinine (SCr) and glomerular filtration rate (GFR) was evaluated in respective recipients (n = 64). Vascular responses were determined in HA, HMA and HCC from donors. Tumor necrosis factor-α, asymmetric dimethyl arginine and Orai1 were measured in plasma. Orai1 protein expression was also evaluated in each donor’s aorta. Endothelium-dependent vasodilation (HMA, HCC) and adrenergic contraction (HA) in donors determined renal function in recipients, 12 months post-transplantation. Donors in the best quartile of vascular function predicted lower SCr and higher GFR in kidney recipients for 12/24 months. Plasma Orai1 in donors was negatively correlated with vascular function and predicted renal function at 3–6 months post-transplantation. Donor Orai1 vascular content was associated with reduced vascular function and with poorer recipient renal function for 1-year post-transplantation. Systemic vascular function of kidney donors determines recipients’ renal function at short/mid-term. Donors’ vascular function and recipients’ renal function are negatively associated with donors’ Orai1 vascular expression, being a potential biomarker of renal outcomes. Full article

From fertilisation to delivery, calcium must be transported into and within the foetoplacental unit for intracellular signalling. This requires very rapid, precisely located Ca²⁺ transfers. In addition, from around the eighth week of gestation, increasing amounts of calcium must be routed directly from maternal blood to the foetus for bone mineralisation through a flow-through system, which does not impact the intracellular Ca²⁺ concentration. These different processes are mediated by numerous membrane-sited Ca²⁺ channels, transporters, and exchangers. Understanding the mechanisms is essential to direct interventions to optimise foetal development and postnatal bone health and to protect the mother and foetus from pre-eclampsia. Ethical issues limit the availability of human foetal tissue for study. Our insight into the processes of placental Ca²⁺ handling is advancing rapidly, enabled by developing genetic, analytical, and computer technology. Because of their diverse sources, the reports of new findings are scattered. This review aims to pull the data together and to highlight areas of uncertainty. Areas needing clarification include trafficking, membrane expression, and recycling of channels and transporters in the placental microvilli; placental metabolism of vitamin D in gestational diabetes and pre-eclampsia; and the vascular effects of increased endothelial Orai expression by pregnancy-specific beta-1-glycoproteins PSG1 and PSG9. Full article

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca²⁺ levels ([Ca²⁺]_i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca²⁺]_i through store-operated Ca²⁺ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca²⁺]_ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca²⁺ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins. Full article

Atopic dermatitis (AD) is a common allergic inflammatory skin condition marked by severe itching, skin lichenification, and chronic inflammation. AD results from a complex immune response, primarily driven by T lymphocytes and environmental triggers, leading to a disrupted epidermal barrier function. Traditional treatments, such as topical corticosteroids, have limitations due to long-term side effects, highlighting the need for safer alternatives. Here, we aimed to show that Agrimonia coreana extract (AC_ext) can be used in treating AD-related dermatologic symptoms. AC_ext could inhibit CRAC (Calcium Release-Activated Calcium) channel activity, reducing Orai1/CRAC currents and decreasing intracellular calcium signaling. This inhibition was further confirmed by the reduced IL-2 levels and T cell proliferation upon AC_ext treatment. In a mouse model of AD, AC_ext significantly ameliorates symptoms, improves histological parameters, and enhances skin barrier function, demonstrating its potential for treating AD. Full article

The ORAI proteins serve as crucial pore-forming subunits of calcium-release-activated calcium (CRAC) channels, pivotal in regulating downstream calcium-related signaling pathways. Dysregulated calcium homeostasis arising from mutations and post-translational modifications in ORAI can lead to immune disorders, myopathy, cardiovascular diseases, and even cancers. Small molecules targeting ORAI present an approach for calcium signaling modulation. Moreover, emerging techniques like optogenetics and optochemistry aim to offer more precise regulation of ORAI. This review focuses on the role of ORAI in cancers, providing a concise overview of their significance in the initiation and progression of cancers. Additionally, it highlights state-of-the-art techniques for ORAI channel modulation, including advanced optical tools, potent pharmacological inhibitors, and antibodies. These novel strategies offer promising avenues for the functional regulation of ORAI in research and may inspire innovative approaches to cancer therapy targeting ORAI. Full article

The incidence and development of cancer are highly dependent on pathological disturbances in calcium homeostasis of the cell. One of the major pathways for calcium entry is store-operated calcium entry (SOCE), which functions in virtually all cell types. Changes in the expression level of the main proteins organizing SOCE are observed during the development of various cancer types, particularly breast cancer (BC). This leads to unique SOCE with characteristics individual for each type of BC and requires particular therapeutic approaches. In this study, we tested the sensitivity of SOCE in various BC cells to selective ORAI channel inhibitors and the less selective compounds Leflunomide and Teriflunomide, approved by the FDA for clinical use. We also analyzed the vulnerability of SOCE to the influence of factors typical of the tumor microenvironment: hypoxia and acidification. We have observed that the SOCE inhibitors Leflunomide and Teriflunomide suppress SOCE in the triple-negative BC cell line MDA-MB-231, but not in the luminal A BC cell line MCF-7. MDA-MB-231 cells also demonstrate higher pH dependence of SOCE compared to MCF-7 cells. In addition, the oxygen scavenger sodium dithionide also affects SOCE, stimulating it in MDA-MB-231 cells but inhibiting in MCF-7 cells. Overall, our data highlight the importance of considering the different sensitivities of various BC cell types to inhibitors and to microenvironmental factors such as hypoxia and acidification when developing targeted drugs. Full article

Many essential biological processes are triggered by the proximity of molecules. Meanwhile, diverse approaches in synthetic biology, such as new biological parts or engineered cells, have opened up avenues to precisely control the proximity of molecules and eventually downstream signaling processes. This also applies to a main Ca²⁺ entry pathway into the cell, the so-called Ca²⁺ release-activated Ca²⁺ (CRAC) channel. CRAC channels are among other channels are essential in the immune response and are activated by receptor–ligand binding at the cell membrane. The latter initiates a signaling cascade within the cell, which finally triggers the coupling of the two key molecular components of the CRAC channel, namely the stromal interaction molecule, STIM, in the ER membrane and the plasma membrane Ca²⁺ ion channel, Orai. Ca²⁺ entry, established via STIM/Orai coupling, is essential for various immune cell functions, including cytokine release, proliferation, and cytotoxicity. In this review, we summarize the tools of synthetic biology that have been used so far to achieve precise control over the CRAC channel pathway and thus over downstream signaling events related to the immune response. Full article

Store-operated calcium entry (SOCE) in cardiomyocytes may be involved in cardiac remodeling, but the underlying mechanisms remain elusive. We hypothesized that SOCE may increase nuclear calcium, which alters gene expression via calcium/calmodulin-dependent enzyme signaling, and elucidated the underlying cellular mechanisms. An experimental protocol was established in isolated adult rat cardiomyocytes to elicit SOCE by re-addition of calcium following complete depletion of sarcoplasmic reticulum (SR) calcium and to quantify SOCE in relation to the electrically stimulated calcium transient (CaT) measured in the same cell before SR depletion. Using confocal imaging, calcium changes were recorded simultaneously in the cytosol and in the nucleus of the cell. In ventricular myocytes, SOCE was observed in the cytosol and nucleus amounting to ≈15% and ≈25% of the respective CaT. There was a linear correlation between the SOCE-mediated calcium increase in the cytosol and nucleus. Inhibitors of TRPC or Orai channels reduced SOCE by ≈33–67%, whereas detubulation did not. In atrial myocytes, SOCE with similar characteristics was observed in the cytosol and nucleus. However, the SOCE amplitudes in atrial myocytes were ≈two-fold larger than in ventricular myocytes, and this was associated with ≈1.4- to 3.6-fold larger expression of putative SOCE proteins (TRPC1, 3, 6, and STIM1) in atrial tissue. The results indicated that SOCE in atrial and ventricular myocytes is able to cause robust calcium increases in the nucleus and that both TRPC and Orai channels may contribute to SOCE in adult cardiomyocytes. Full article

Emerging evidence indicates that intracellular calcium (Ca²⁺) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca²⁺ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation. Full article

Vascular territories display heterogeneous sensitivity to the impacts of aging. The relevance of the STIM/Orai system to vascular function depends on the vascular bed. We aimed to evaluate the contribution of the STIM/Orai system to aging-related vascular dysfunction in rat coronary circulation. Vascular function was evaluated according to myography in coronary arteries from young (three-month-old) and older (twenty-month-old) rats. The effects of aging and STIM/Orai inhibition on the contraction and relaxation of the coronary arteries and on the protein expression of STIM-1, Orai1, and Orai3 in these vessels were determined. Aging-related hypercontractility to serotonin and endothelin-1 in arteries from male rats was reversed by STIM/Orai inhibition with YM-58483 or by specifically blocking the Orai1 channel with Synta66. The inhibitory effects of Synta66 on coronary vasoconstriction were also observed in older female rats. YM-58483 relaxed serotonin- but not KCl-contracted arteries from males. STIM/Orai inhibition improved defective endothelial vasodilations in aged arteries, even in the presence of NO synthase and cyclooxygenase inhibitors, but not in KCl-contracted segments. YM-58483 significantly enhanced relaxations to calcium-activated potassium channel stimulation in aged vessels. Increased protein expression of Orai1 and Orai3 was detected in arterial homogenates and sections from older rats. Upregulation of the Orai channel contributes to aging-related coronary dysfunction, revealing a potential target in reducing CVD risk. Full article

A major route for the influx of calcium ions into neurons uses the STIM-Orai1 voltage-independent channel. Once cytosolic calcium ([Ca²⁺]i) elevates, it activates mitochondrial and endoplasmic calcium stores to affect downstream molecular pathways. In the present study, we employed a novel drug, carbonyl cyanide chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, to explore the role of mitochondria in cultured neuronal morphology. CCCP caused a sustained elevation of [Ca²⁺]i and, quite surprisingly, a massive increase in the density of dendritic filopodia and spines in the affected neurons. This morphological change can be prevented in cultures exposed to a calcium-free medium, Orai1 antagonist 2APB, or cells transfected with a mutant Orai1 plasmid. It is suggested that CCCP activates mitochondria through the influx of calcium to cause rapid growth of dendritic processes. Full article

Zinc (Zn²⁺) is released by platelets during a hemostatic response to injury. Extracellular zinc ([Zn²⁺]_o) initiates platelet activation following influx into the platelet cytosol. However, the mechanisms that permit Zn²⁺ influx are unknown. Fluctuations in intracellular zinc ([Zn²⁺]_i) were measured in fluozin-3-loaded platelets using fluorometry and flow cytometry. Platelet activation was assessed using light transmission aggregometry. The detection of phosphoproteins was performed by Western blotting. [Zn²⁺]_o influx and subsequent platelet activation were abrogated by blocking the sodium/calcium exchanged, TRP channels, and ZIP7. Cation store depletion regulated Zn²⁺ influx. [Zn²⁺]_o stimulation resulted in the phosphorylation of PKC substates, MLC, and β3 integrin. Platelet activation via GPVI or Zn²⁺ resulted in ZIP7 phosphorylation in a casein kinase 2-dependent manner and initiated elevations of [Zn²⁺]_i that were sensitive to the inhibition of Orai1, ZIP7, or IP₃R-mediated pathways. These data indicate that platelets detect and respond to changes in [Zn²⁺]_o via influx into the cytosol through TRP channels and the NCX exchanger. Platelet activation results in the externalization of ZIP7, which further regulates Zn²⁺ influx. Increases in [Zn²⁺]_i contribute to the activation of cation-dependent enzymes. Sensitivity of Zn²⁺ influx to thapsigargin indicates a store-operated pathway that we term store-operated Zn²⁺ entry (SOZE). These mechanisms may affect platelet behavior during thrombosis and hemostasis. Full article

The systemic effects of physical activity are mediated by the release of IL-6 and other myokines from contracting muscle. Although the release of IL-6 from muscle has been extensively studied, the information on the cellular mechanisms is fragmentary and scarce, especially regarding the role of Ca²⁺ signals. The aim of this study was to characterize the role of the main components of Ca²⁺ signals in human skeletal muscle cells during IL-6 secretion stimulated by the Ca²⁺ mobilizing agonist ATP. Primary cultures were prepared from surgical samples, fluorescence microscopy was used to evaluate the Ca²⁺ signals and the stimulated release of IL-6 into the medium was determined using ELISA. Intracellular calcium chelator Bapta, low extracellular calcium and the Ca²⁺ channels blocker La³⁺ reduced the ATP-stimulated, but not the basal secretion. Secretion was inhibited by blockers of L-type (nifedipine, verapamil), T-type (NNC55-0396) and Orai1 (Synta66) Ca²⁺ channels and by silencing Orai1 expression. The same effect was achieved with inhibitors of ryanodine receptors (ryanodine, dantrolene) and IP3 receptors (xestospongin C, 2-APB, caffeine). Inhibitors of calmodulin (calmidazolium) and calcineurin (FK506) also decreased secretion. IL-6 transcription in response to ATP was not affected by Bapta or by the T channel blocker. Our results prove that ATP-stimulated IL-6 secretion is mediated at the post-transcriptional level by Ca²⁺ signals, including the mobilization of calcium stores, the activation of store-operated Ca²⁺ entry, and the subsequent activation of voltage-operated Ca²⁺ channels and calmodulin/calcineurin pathways. Full article