Store-operated Ca Entry Research Articles

STIM1 functionally couples to transient receptor potential ankyrin 1 contributing to nociception

Abstract STIM1 is a calcium sensor that can sense calcium level changes in the endoplasmic reticulum (ER) and respond to extracellular stimuli. We have reported that STIM1 is expressed in nociceptors. However, its functional significance remains unclear. Here, we show that STIM1 plays an important role in sensing cold, chemical, and noxious mechanical stimuli in both male and female mice. We found that activation of transient receptor potential ankyrin 1 (TRPA1) triggers ER Ca2+ release, STIM1 translocation, and store-operated Ca2+ entry (SOCE). Immunostaining and western blot results reveal that TRPA1 is expressed in the ER. In addition, STIM1 deficiency in the primary sensory neurons reduces cold-, allyl isothiocyanate (TRPA1 agonist)-, and bradykinin-induced Ca2+ entry and nociception. Moreover, intraplantar injection of thapsigargin, an ER Ca2+–ATPase inhibitor, evokes nociception and increases pain hypersensitivity, which is significantly attenuated in STIM1 conditional knockout or L3/L4 dorsal root ganglia STIM1 knockdown mice. Mechanistic studies demonstrate that STIM1-mediated SOCE increases neuronal excitability and decreases potassium channel Kv4-mediated outward currents in small to medium-sized dorsal root ganglion neurons, which is abolished by inhibiting the mitogen-activated protein kinase/extracellular receptor kinase pathway. Our findings demonstrate that STIM1 acts as a transducer of nociception and uncover a novel link between STIM1 and TRPA1ER. Our study also provides new insights into TRPA1-mediated nociception.

Store-operated calcium entry facilitates LPS-induced superoxide anion-dependent macrophage extracellular traps.

Macrophage extracellular traps (METs) represent a recently discovered complex defence mechanism that is distinct from phagocytosis and involves the release of DNA and antibacterial proteins. They play an important role in pathogen removal, and calcium ions (Ca2+) have also been reported to be involved. In the present study, we identified METotic cells using digitonin as an alternative to Triton X-100, coupled with immunofluorescence staining using lamin antibodies. The limited permeability of digitonin ensures exclusive intranuclear antibody labelling of MET cells, therefore providing a straightforward and intuitive differentiation method. We found that under lipopolysaccharide stimulation, macrophages undergo store-operated Ca2+ entry (SOCE) to facilitate Ca2+ influx. Elevation of cytoplasmic Ca2+ levels by SOCE promotes the generation of superoxide anions by NADPH oxidase (NOX), ultimately leading to METosis. In summary, our study strengthens the role of Ca2+ in NOX-dependent METosis, which differs from previous studies focusing on Ca2+ in the NOX-independent pathway. Our research reveals that Ca2+-mediated regulation of NOX plays a crucial role in METosis, especially in SOCE, and provides novel ideas for future research.

Campari2 genomic interrogation of homeostatic calcium activity identifies TIM1 as a negative regulator of T cell function.

Calcium signals regulate crucial cellular functions yet many genes coding for Ca2+handling proteins remain unknown as their identification relies on low-throughput single-cell approaches. Here we describe a method to measure Ca2+ activity using CaMPARI2, flow cytometry and pooled genome interrogation. CAMPARI2 screen (CaMP-Screen) identified enhancers and inhibitors of homeostatic Ca2+ activity, highlighting a predominant role for store-operated Ca2+ entry (SOCE) and lipid signalling pathways. Genes reducing basal Ca2+ activity were linked to Prader Willy syndrome, T cell dysfunction, and deafness. Silencing of HAVCR1 gene, coding for T cell transmembrane immunoglobulin and mucin (TIM1), enhanced Ca2+ signals in T cells and promoted signaling under resting but not after TCR engagement. Our findings establish CaMP-Screen as an efficient detector of low-amplitude Ca2+ signals and identify new genes associated to pathologies that regulate Ca2+ homeostasis, reporting TIM1 as a negative regulator of Ca2+ signals driving T cell function.

Resting Ca2+ fluxes protect cells from fast mitochondrial fragmentation, cell stress responses, and immediate transcriptional reprogramming

Homeostatic calcium ion (Ca2+) fluxes between the endoplasmic reticulum, cytosol, and extracellular space occur not only in response to cell stimulation but also in unstimulated cells. Using murine astrocytes as a model, we asked whether there is a signaling function of these resting Ca2+ fluxes. The data showed that endoplasmic reticulum (ER) Ca²⁺ depletion, induced by sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) inhibition, resulted to prolonged Ca²⁺ influx and mitochondrial fragmentation within 10 to 30 min. This mitochondrial fragmentation could be prevented in Ca2+-free medium or by inhibiting store-operated Ca2+ entry (SOCE). Similarly, attenuation of STIM proteins, which are vital ER Ca2+ sensors, protected mitochondrial morphology. On the molecular level, ER Ca2+ depletion, achieved either by removing extracellular Ca2+ or through acute SERCA inhibition, led to changes in gene expression of about 13% and 41% of the transcriptome within an hour, respectively. Transcriptome changes were associated with universal biological processes such as transcription, differentiation, or cell stress. Strong increase in expression was observed for the transcription factor ATF4, which is under control of the kinase PERK (EIF2AK3), a key protein involved in ER stress. Corroborating these findings, PERK was rapidly phosphorylated in Ca2+-free medium or after acute pharmacological inhibition of SOCE. In summary, resting, homeostatic Ca2+ fluxes prevent immediate-early cell stress and transcriptional reprogramming.

1828-P: STIM1 Deficiency Impairs Glucose Regulation by Altering Insulin Granules, Mitochondria, and Endoplasmic Reticulum Ultrastructure in Beta Cells

Introduction and Objective: Impairments in endoplasmic reticulum (ER) calcium (Ca2+) homeostasis are associated with β cell dysfunction and the development of diabetes. Store-operated Ca2+ entry (SOCE) replenishes ER Ca2+ stores via plasma membrane Ca2+ channels that are regulated by the ER Ca2+ sensor stromal interaction molecule 1 (STIM1). We have previously shown that female mice with β cell-specific STIM1 (STIM1Δβ) deletion exhibit reduced β cell mass, increased α cell mass, and diminished β cell maturity markers. Furthermore, RNAseq analysis on islets from control and STIM1Δβ female mice revealed 55 significantly modulated pathways, including “mitochondrial dysfunction” and “endocytosis.” Here, we sought to determine the effect of STIM1 deletion on mitochondrial function, insulin granule maturation, and ER architecture. Methods: Mitochondrial function was evaluated using morphology analysis and a RIP-promotor controlled-Mito CEPIA. Insulin granule maturation was determined using syncollin-dsRedE5TIMER (a fluorescent time construct) and TEM analysis. Lastly, expansion microscopy (ExM) and single molecule localization microscopy (SMLM) was used to determine ER architecture. Results: We found abnormal morphology and impaired mitochondrial Ca2+ levels in STIM1 KO compared to WT INS-1 cells. Analysis with syncollin-dsRedE5TIMER showed accumulation of old insulin granules in STIM1 KO cells, and TEM revealed a significant increase in the ratio of immature to mature granules and a reduction in granule halo size in islets from STIM1Δβ female mice. Lastly, ExM and SMLM revealed a significant reduction in tubule diameter in STIM1 KO cells under ER stress, and STIM1 KO cells exhibited further decreases in tubule diameter in ER regions distal to the nucleus. Conclusion: Our findings link STIM1 loss to alterations in mitochondrial morphology, insulin granule maturation, and ER ultrastructure that may impair β cell function. Disclosure T. Kono: None. M. McLaughlin: None. P. Sohn: None. P. Krishnan: None. W. Wu: None. C. Lee: None. F. Huang: None. M. Slak Rupnik: None. C. Evans-Molina: Advisory Panel; DiogenX. Research Support; Bristol-Myers Squibb Company, Lilly Diabetes. Advisory Panel; Isla Technologies, Neurodon. Research Support; Neurodon. Funding U.S. Department of Veterans Affairs Merit Award (I01BX001733)

Functional characterization of the store-operated calcium entry pathway in naked mole-rat cells.

Naked mole-rats (NMRs, Heterocephalus glaber) are highly unusual rodents exhibiting remarkable adaptations to their subterranean habitat and resistance to developing various age-related diseases such as those related to abnormal cell proliferation or cancer, neurodegeneration and inflammation. In other rodents, as well as humans, a ubiquitous Ca2+ influx pathway, namely the store-operated Ca2+ entry (SOCE), has been implicated in all these diseases. SOCE is triggered by intracellular Ca2+ store depletion resulting in interaction of Stim proteins with Orai proteins, the putative homologues of which appear to be present in the NMR genome, but no functional characterization of SOCE in NMRs has yet been conducted. In this study, we provide the first functional and pharmacological characterization of SOCE in NMR using both excitable and non-excitable cells.

Store-operated Ca2+ entry is involved in endothelium-to-mesenchymal transition in lung vascular endothelial cells.

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor β1 (TGF-β1), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVECs). An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca2+ signaling is involved in EndMT. In this study, we tested the hypothesis that TGF-β1-induced EndMT in human LVEC is Ca2+-dependent. Treatment of LVEC with TGF-β1 for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1, and enhancement of store-operated Ca2+ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca2+ with EGTA or BAPTA-AM, respectively, abolished EndMT in response to TGF-β1. Moreover, EGTA diminished TGF-β1-induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-β-mediated increase in SNAI1/2 and EndMT but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-β-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca2+ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca2+ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of precapillary pulmonary hypertension.NEW & NOTEWORTHY EndMT has been reported to contribute to the pathogenesis of PAH. In this study, we aimed to determine the role of Ca2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β1 requires store-operated Ca2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time, we demonstrated that TGF-β1-induced EndMT is a Ca2+-dependent event, whereas inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β1.

STIM2β is a Ca2+ signaling modulator for the regulation of mitotic clonal expansion and PPARG2 transcription in adipogenesis.

Intracellular Ca2+ is crucial in the regulation of adipocyte lipid metabolism and adipogenesis. In this study, we aimed to investigate the regulation mechanism of intracellular Ca2+ levels ([Ca2+]i) during adipocyte differentiation. We found that the expression of stromal interaction molecule 2 beta (STIM2β), which is the inhibitor of store-operated Ca2+ entry (SOCE), is upregulated throughout the differentiation process. Evaluation of [Ca2+]i in 3 T3-L1 and primary stromal vascular fraction (SVF) cells revealed that the basal Ca2+ level is downregulated after differentiation. Knockout (KO) of STIM2β in 3T3-L1 and primary SVF cells showed increased [Ca2+]i, indicating the involvement of STIM2β in the regulation of [Ca2+]i during adipogenesis. We further evaluated the function of STIM2β-mediated [Ca2+]i in early and terminal differentiation of adipogenesis. Analysis of cell proliferation rate during mitotic clonal expansion (MCE) in wild-type and STIM2β KO 3T3-L1 cell lines revealed that a larger population of KO cells underwent G1 arrest, suggesting that reduced [Ca2+]i by STIM2β induces MCE. Additionally, ablation of STIM2β increased differentiation efficiency, with more lipid accumulation and rapid transcriptional activation of adipogenic genes, especially proliferator-activator receptor γ2 (PPARG2). We found that PPARG2 transcription is regulated by store-operated calcium entry (SOCE) downstream transcription factors, confirming that increased [Ca2+]i by STIM2β ablation promotes PPARG2 transcription during adipogenesis. Additionally, STIM2β KO mice showed hypertrophic adipose tissue development. Our data suggest that STIM2β-mediated [Ca2+]i plays a pivotal role in the regulation of mitotic clonal expansion and PPARG2 gene activation and provides evidence that MCE is not a prerequisite process for terminal differentiation during adipogenesis.

Myotubularin related protein 7, a novel STIM1 binding protein.

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor in the endoplasmic reticulum (ER) membrane. The protein plays a crucial role in store-operated Ca2+ entry (SOCE) by transducing ER Ca2+ depletion signals to Ca2+ release-activated Ca2+ channel protein 1 (ORAI1) at the plasma membrane. Myotubularin related protein 7 (MTMR7) is a lipid phosphatase that dephosphorylates phosphoinositides. Using yeast two-hybrid analysis, immunoprecipitation and fluorescence microscopy, we discovered that MTMR7 interacts with STIM1 at the ER. These observations identified MTMR7 as a novel STIM1-binding protein that bridges myotubularins and phosphoinositide signaling with SOCE. Our research revealed a novel link between Ca2+ signaling and phosphoinositide biology, positioning MTMR7 as a potential marker or drug target for SOCE related human pathophysiology.

Parity Dependent Differential Uterine Oxytocin Response: A Prominent Role for ORAI Channels

Introduction: Pregnancy permanently affects the health and function of the uterus, specifically the intracellular Ca 2+ management, as proven by the differential response to oxytocin in virgin and proven breeder rats we have previously described. Hypothesis: Here, we propose that store operated Ca 2+ entry (SOCE) channels, ORAI and TRPC channels, contribute to the parity-dependent modulation of the uterine contractile responses to oxytocin. Methods: Motility experiments were conducted on uterine strips from virgin (V) and proven breeder (PB) non pregnant female 18-week-old rats euthanized at proestrus. Dose response curves to oxytocin (10E-10 to 10E-5 M) in the presence of the highly specific ORAI blocker synta66 (1 µM) and TRPC3 blocker Pyr3 (1 µM) were recorded. Area under the curve (AUC), frequency, and amplitude of contractions were measured. T-tests and ANOVA two ways were used for statistical analysis. Results: With syntax 66, previously observed differential response to oxytocin was greatly attenuated. The, the curve parameters (minimum, maximum, range of increase, and EC50) measured for AUC and frequency were similar (P>0.05). The EC50 of the amplitude curve was smaller for PB, while both minimum and maximum of the curve were greater than in V samples. In all our earlier experiments the amplitude response was stronger in V strips. Conversely, preconditioning with Pyr3 had a much smaller effect. The maximal AUC response to oxytocin was indeed similar in V and PB, however the minimum and range recorded resembled those observed in the absence of inhibitors. The amplitude and frequency curves followed the trends observed in the absence of inhibitors. Conclusions: ORAI channels, not TRPC3, appear to significantly contribute to the differential response of virgin and proven breeder rats to oxytocin. Further investigation of other TRPC channels will clarify the overall role of SOCE in this phenomenon. This study was supported by a Kenneth Suarez Fellowship to VG and an MWU intramural fund to MP. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Organellar Ca2+ shuttling and store-operated Ca2+ entry during nutrient starvation

Study Objective and Hypothesis: Macroautophagy is a process in which unwanted cellular components are packaged and delivered to the lysosome for degradation to maintain homeostasis. It is strongly activated by nutrient depletion (starvation), which induces an increase in intracellular Ca2+ (starvation-induced Ca2+ signal, SICS) that is predicted to initiate many Ca2+-dependent activities of autophagy. Despite the importance of SICS, its regulation is unclear. We hypothesized that the endoplasmic reticulum (ER) and the lysosome are linked sources of starvation-induced Ca2+ release, leading to store-operated Ca2+ entry (SOCE). We also hypothesized and that the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel is involved in the release of Ca2+ during starvation. Methods: SICR and SICS were measured by monitoring changes in intracellular Ca2+ concentration with the fura-2 Ca2+ indicator upon removal of extracellular nutrients in the absence or presence, respectively, of extracellular Ca2+. The roles of the ER, lysosome, and TRPML1 as components of SICR were assessed using pharmacological inhibitors and activators of the SERCA pump and TRPML1. Role of SOCE in SICS was tested with pharmacological inhibition or molecular truncation of the stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that is critical component of SOCE activation. Results: TRPML1 activation using agonist MLSA1 in the absence of extracellular Ca2+ triggered a small Ca2+ release signal; this effect is abolished by pre-depletion of ER Ca2+, indicating that the main source of lysosomal Ca2+ release via TRPML1 is ER Ca2+. Following starvation-induced Ca2+ release (SICR), TRPML1 agonist MLSA1 no longer triggers a Ca2+ release signal, indicating that lysosomal Ca2+ is a contributing source of SICR. Consistently, overexpression of TRPML1 enhances the kinetics and amplitude of SICS in the presence of extracellular Ca2+. Heterologous expression of STIM1, the critical molecular switcher of SOCE, increases SICS magnitude by 5-fold. This enhancement is abolished by expression of a STIM1 in which the intra-ER Ca2+-binding loop has been truncated. SICS is also strongly inhibited by pretreatment with STIM1 inhibitor ML-9. Conclusion: Our data support a model in which the ER and lysosome are linked sources of Ca2+ release during nutrient starvation, whose depletion leads to activation of store-operated Ca2+ entry. The data also suggest that TRPML1-mediated Ca2+ release contributes to the dynamics of Ca2+ release upon nutrient starvation. NIH grant HL173818 to Q-KT This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Stabilization of Podocyte Actin Cytoskeleton by SMPDL3b Attributed to Inhibition of Store-Operated Ca2+ Entry by Choline

Our recent studies demonstrate that deletion of the Smpdl3b gene, encoding sphingomyelin phosphodiesterase acid-like 3b (SMPDL3b), induces minimal change disease-like podocytopathy in mice. However, the underlying molecular mechanisms remain unclear. While SMPDL3b has been traditionally regarded as a lipid-modifying enzyme regulating sphingolipid metabolism, emerging evidence challenges this view, showing that SMPDL3b lacks enzymatic activity against sphingolipids. High-resolution structural analysis indicates that SMPDL3b preferentially cleaves CDP-choline, leading to subsequent production of choline, an intracellular messenger. Based on these findings, we hypothesize that SMPDL3b-dependent CDP-choline cleavage and subsequent choline production play a pivotal role in regulating podocyte actin cytoskeleton. To test our hypothesis, we isolated podocytes from WT/WT and Smpdl3b-/- mice for primary culture. Using LC-MS/MS, we observed significantly reduced CDP-choline levels and remarkably elevated choline concentrations in Smpdl3b-/- podocytes compared to WT/WT podocytes. Immunofluorescent staining of F-actin and paxillin (focal adhesion marker) revealed that Smpdl3b gene deletion disrupted actin cytoskeleton and markedly reduced focal adhesion in podocytes, which was reversed by choline and phosphocholine, but not CDP-choline treatment. Atomic force microscopy confirmed that reduced cellular elasticity in Smpdl3b-/- podocytes was restored by choline and phosphocholine, but not CDP-choline. The therapeutic effects of phosphocholine were blocked by pre-treatment with Lansoprazole (phosphocholine phosphatase inhibitor), implicating choline as the active product. Mechanistically, Smpdl3b deletion increased stromal interaction molecule 1 (STIM1)-Orai1 interaction and store-operated Ca2+ entry (SOCE) in podocytes, which were substantially attenuated by choline and phosphocholine but unaffected by CDP-choline. Lansoprazole pre-treatment similarly blocked phosphocholine’s effects, further supporting the role of choline. These findings suggest that SMPDL3b regulates podocyte actin cytoskeleton stability via CDP-choline cleavage and choline production, which suppress Orai1 channel-mediated SOCE and stabilize the actin cytoskeleton. This study is supported by NIH grants DK054927 and DK120491. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Adipocyte Septin-7 attenuates obesogenic adipogenesis and promotes lipolysis to prevent obesity.

The white adipose tissue (WAT) expansion plays a significant role in the development of obesity. Cytoskeletal remodeling directly impacts adipogenic program, however, the precise mechanism remains poorly understood. Here, we identified a crucial role of Septin-7 (SEPT7), a cytoskeleton component, in the regulation of diet-induced processes of adipogenesis, lipogenesis, and lipolysis in WAT. A high-fat diet (HFD)-induced obesity model was constructed using mice with inducible adipocyte-specific SEPT7 deficiency. The impact of SEPT7 on adipocyte morphology, cell number and metabolism capacity were evaluated with immunofluorescence, isoproterenol induced lipolysis assay, glucose tolerance test and insulin tolerance test. Adipocyte mTmG reporter line was established to trace invivo adipogenesis. The preadipocyte 3T3-L1 cell was induced for exploring role of SEPT7 in adipocyte differentiation. qRT-PCR and Western-blot were used to investigate the expression of PPARγ, C/EBPα, and HSL in 3T3-L1 cell with siRNA-mediated SEPT7 knockdown. SEPT7 expression was greatly induced in obesogenic human and murine adipocytes. Mice lacking SEPT7 in mature white adipocytes demonstrated defective differentiation of preadipocyte into mature adipocytes when fed HFD resulting in larger adipocytes, increased WAT inflammation and reduced lipolysis, which leading to increased WAT mass, liver fat accumulation and impaired glucose tolerance. Mechanistically, we identified SEPT7 restrains store-operated Ca2+ entry (SOCE) and regulates adipocyte adipogenesis and lipolysis by targeting PPARγ, C/EBPα and HSL. We demonstrated that SEPT7 negatively regulates adipogenesis while promotes lipolysis and its repression drives WAT expansion and impaired metabolic health.

Orai1 Participates in Coronary Artery Dysfunction Caused by Hypertension via Regulating Smooth Muscle Cell Phenotype Transformation.

Hypertension plays a critical role in the development of vascular remodeling and atherosclerosis. STIM/Orai1 proteins mediate store-operated Ca2+ entry (SOCE), which is one of the cellular Ca2+ signaling machinery involved in the pathological process of cardiovascular remodeling. However, the role and mechanism of Orai1/Orai1 mediated SOCE in coronary artery dysfunction caused by hypertension remain incompletely elucidated. The present study aimed to investigate the role of the Orai1/NFAT/calcineurin signaling pathway in hypertension-induced coronary vasoconstriction impairment utilizing spontaneous hypertension rats (SHRs) and coronary arterial smooth muscle cells (CASMCs) exposed to high hydrostatic pressure (180 mmHg). Here, we found that agonists (5-HT, U46619, and ET-1) induced coronary artery constriction that was significantly reduced in SHRs compared with Wistar rats. The SOCE inhibitors SKF96365 and 2-APB also significantly inhibited coronary artery constriction in both SHRs and Wistar rats; only the inhibitory effect of low concentrations (50 μM) of 2-APB on SHRs was weaker than that of Wistar rats. Hypertension/high hydrostatic pressure (180 mmHg) induced phenotypic transformation of CASMCs, with an increase in the expression of STIM1/Orai1, Calcineurin-NFAT2, and the synthetic phenotypic marker protein OPN, and a decrease in the contractile phenotypic marker protein SMMHC. The intervention of Orai1/Orai1 mediated SOCE (overexpression with ad-Orai1, inhibition of SOCE channel with BTP2 or downregulation with Orai1 siRNA) regulated STIM1, Calcineurin-NFAT2 expression, and contraction/synthesis phenotypic markers. Together, these findings suggest that hypertension leads to coronary vascular dysfunction via the upregulation of Orai1, which is required for the phenotypic transformation of VSMCs by activating the Calcineurin-NFAT signaling pathway.

Store-operated Ca2+ entry contributes to the ASM phenotype transition in asthma

Aim of the study Phenotype modulation of airway smooth muscle cells (ASMC), characterized by a shift toward a more proliferative and synthetic phenotype from contractile cells, plays a crucial role in airway remodeling in asthma. STIM1 and Orai1, key components of store-operated Ca2+ entry (SOCE), have been demonstrated to enhance ASMC proliferation and migration. This study investigated the impact of STIM1/Orai1-mediated SOCE on ASMC phenotype transition and extracellular matrix (ECM) deposition in asthma. Materials and Methods The ASMCs were treated with PDGF-BB and SOCE inhibitors. Immunocytochemistry staining, enzyme-linked immunosorbent assay, and western blot assay were employed to detect the ASMC’s proliferation as well as the expressions of contractile proteins, inflammatory cytokines and ECM. Moreover, the effect of SOCE repression in ECM deposition were evaluated in an asthmatic mouse model. Results ASMCs from airways of mice were treated with PDGF-BB to induce the ‘proliferative/synthetic’ phenotype. We observed elevated expressions of STIM1 and Orai1 in phenotype-switched ASMCs, along with enhanced SOCE. SKF-96365 and RO2959, which target of STIM1/Orai1, could significantly inhibit SOCE activation in ASMCs. Moreover, these SOCE inhibitors mitigated the elevated proliferation rate, decreased the secretion of inflammatory cytokines and restored the reduced levels of contractile proteins in phenotype-switched ASMCs induced by PDGF-BB. Furthermore, we observed that PDGF-BB-induced ‘proliferative/synthetic’ ASMCs exhibited increased production of ECM components, including collagen I and fibronectin, as well as metalloproteinases (MMPs) such as MMP2 and MMP9, all of which were effectively inhibited by SKF-96365 and RO2959. In vivo experiments also demonstrated that SOCE inhibitors decreased ECM deposition and MMPs production in the asthmatic mouse model. Conclusions These findings underscored the significant role of STIM1/Orai1-mediated SOCE in ASMC phenotype modulation and its impact on the excessive ECM deposition driven by ASMCs. Thus, our findings suggest that STIM1/Orai1-mediated SOCE may contribute to airway remodeling in asthma.

Vasodilatory Effect of n-Butanol Extract from Sanguisorba officinalis L. and Its Mechanism.

The dried root of Sanguisorba officinalis L. (commonly known as Diyu) has been studied for its various pharmacological effects, including its antibacterial, antitumor, antioxidant, and anti-inflammatory activities. In the present study, primary cultured vascular endothelial cells (HUVECs) and isolated phenylephrine-precontracted rat thoracic aortic rings were examined to investigate the possible mechanism of a butanol extract of Diyu (BSO) in its vascular relaxant effect. HUVECs treated with BSO produced a significantly higher amount of nitric oxide (NO) compared to the control. However, its production was inhibited by pretreatment with NG-nitro-L-arginine methylester (L-NAME) or wortmannin. BSO also increased the phosphorylation levels of endothelial nitric oxide synthase (eNOS) and Akt. In the aortic ring, BSO relaxed PE-precontracted rat thoracic aortic rings in a concentration-dependent manner. The absence of the vascular endothelium significantly attenuated BSO-induced vasorelaxation. The non-selective NOS inhibitor, L-NAME, and the selective inhibitor of soluble guanylyl cyclase (sGC), 1H-[1,2,4]-oxadiazolo-[4,3-α]-quinoxalin-1-one (ODQ), dramatically inhibited the BSO-induced relaxation effect of the endothelium-intact aortic ring. Ca2+-free buffer and intracellular Ca2+ homeostasis regulators (TG, Gd3+, and 2-APB) inhibited BSO-induced vasorelaxation. In Ca2+-free Krebs solution, BSO markedly reduced PE-induced contraction. Vasodilation induced by BSO was significantly inhibited by wortmannin, an inhibitor of Akt. Pretreatment with the non-selective inhibitor of Ca2+-activated K+ channels (KCa), tetraethylammonium (TEA), significantly attenuated the BSO-induced vasorelaxant effect. Furthermore, BSO decreased the systolic blood pressure and heart rate in a concentration-dependent manner in rats. In conclusion, BSO induces vasorelaxation via endothelium-dependent signaling, primarily through the activation of the PI3K-Akt-eNOS-NO signaling pathway in endothelial cells, and the activation of the NO-sGC-cGMP-K⁺ channels pathway in vascular smooth muscle cells. Additionally, store-operated Ca2+ entry (SOCE)-eNOS pathways and the inhibition of Ca2⁺ mobilization from intracellular stores contribute to BSO-induced vasorelaxation.

Novel Compounds Target Aberrant Calcium Signaling in the Treatment of Relapsed High-Risk Neuroblastoma.

High-risk neuroblastoma (HRNB) is an extracranial solid pediatric cancer. Despite the plethora of treatments available for HRNB, up to 65% of patients are refractory or exhibit an initial response to treatment that transitions to therapy-resistant relapse, which is invariably fatal. A key feature that promotes HRNB progression is aberrant calcium (Ca2+) signaling. Ca2+ signaling is regulated by several druggable channel proteins, offering tremendous therapeutic potential. Unfortunately, many of the Ca2+ channels in HRNB also perform fundamental functions in normal healthy cells, hence targeting them increases the potential for adverse effects. To overcome this challenge, we sought to identify novel Ca2+ signaling pathways that are observed in HRNB but not normal non-cancerous cells with the hypothesis that these novel pathways may serve as potential therapeutic targets. One Ca2+ signaling pathway that is deregulated in HRNB is store-operated Ca2+ entry (SOCE). SOCE relays the release of Ca2+ from the endoplasmic reticulum (ER) and Ca2+ influx via the plasma membrane and promotes cancer drug resistance by regulating transcriptional programming and the induction of mitochondrial Ca2+ (mtCa2+)-dependent signaling. mtCa2+ signaling is critical for cellular metabolism, reactive oxygen production, cell cycle, and proliferation and has a key role in the regulation of cell death. Therefore, a dynamic interplay between ER, SOCE, and mitochondria tightly regulates cell survival and apoptosis. From a library of synthesized novel molecules, we identified two structurally related compounds that uniquely disrupt the dynamic interplay between SOCE, ER, and mitochondrial signaling pathways and induce cell death in HRNB. Our results revealed that compounds 248 and 249 activate distinct aberrant Ca2+ signals that are unique to relapsed HRNB and could be exploited to induce mtCa+ overload, a novel calcium influx current, and subsequent cell death. These findings establish a potential new pathway of calcium-mediated cell death; targeting this pathway could be critical for the treatment of refractory and relapsed HRNB.

Anoctamin 9 determines Ca2+ signals during activation of T-lymphocytes.

Activation of T-cells is initiated by an increase in intracellular Ca2+, which underlies positive and negative regulation. Because the phospholipid scramblase and ion channel ANO9 (TMEM16J) was shown previously to regulated Ca2+ signals in renal epithelial cells, we asked whether ANO9 demonstrates a similar regulation in T-cells. We used measurements of the intracellular Ca2+ concentration to examine the effects of ANO9 on intracellular Ca2+ signaling and demonstrated expression of ANO9 and its effects on cellular and molecular parameters. ANO9 was found to be expressed in human lymphocytes, including the Jurkat T-lymphocyte cell line and mouse lymphocytes. ANO9 has been shown to affect intracellular Ca2+ signals in renal epithelial cells. Here we demonstrate the essential role of ANO9 during initiation of intracellular Ca2+ signals in Jurkat T-cells and isolated mouse lymphocytes. ANO9 is essential for the initial rise in intracellular Ca2+ due to influx of extracellular Ca2+ through store-operated ORAI1 Ca2+ entry channels. ANO9 is indispensable for T-cell function, independent on whether cells are activated by stimulation of the T-cell receptor with CD3-antibody or by PMA/phytohemagglutinin. Upon activation of T-cells and formation of the immunological synapse, ANO9 recruits the Ca2+-ATPase (PMCA) to the plasma membrane, which is supported by the scaffolding protein discs large 1 (DLG1). PMCAs maintain low Ca2+ levels near ORAI1 channels thereby suppressing Ca2+-inhibition of ORAI1 and thus retaining store-operated Ca2+ entry (SOCE). It is suggested that ANO9 has a role in interorganelle communication and regulation of cellular protein trafficking, which probably requires its phospholipid scramblase function.

Ca2+ Signaling in Cardiac Fibroblasts: An Emerging Signaling Pathway Driving Fibrotic Remodeling in Cardiac Disorders.

Cardiac fibrosis is a scarring event that occurs in the myocardium in response to multiple cardiovascular disorders, such as acute myocardial infarction (AMI), ischemic cardiomyopathy, dilated cardiomyopathy, hypertensive heart disease, inflammatory heart disease, diabetic cardiomyopathy, and aortic stenosis. Fibrotic remodeling is mainly sustained by the differentiation of fibroblasts into myofibroblasts, which synthesize and secrete most of the extracellular matrix (ECM) proteins. An increase in the intracellular Ca2+ concentration ([Ca2+]i) in cardiac fibroblasts is emerging as a critical mediator of the fibrogenic signaling cascade. Herein, we review the mechanisms that may shape intracellular Ca2+ signals involved in fibroblast transdifferentiation into myofibroblasts. We focus our attention on the functional interplay between inositol-1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) and store-operated Ca2+ entry (SOCE). In accordance with this, InsP3Rs and SOCE drive the Ca2+ response elicited by Gq-protein coupled receptors (GqPCRs) that promote fibrotic remodeling. Then, we describe the additional mechanisms that sustain extracellular Ca2+ entry, including receptor-operated Ca2+ entry (ROCE), P2X receptors, Transient Receptor Potential (TRP) channels, and Piezo1 channels. In parallel, we discuss the pharmacological manipulation of the Ca2+ handling machinery as a promising approach to mitigate or reverse fibrotic remodeling in cardiac disorders.

STIM1 and Endoplasmic Reticulum-Plasma Membrane Contact Sites Oscillate Independently of Calcium-Induced Calcium Release.

Calcium (Ca²⁺) release from intracellular stores, Ca²⁺ entry across the plasma membrane, and their coordination via store-operated Ca²⁺ entry (SOCE) are critical for receptor-activated Ca²⁺ oscillations. However, the precise mechanism of Ca²⁺ oscillations and whether their control loop resides at the plasma membrane or intracellularly remain unresolved. By examining the dynamics of stromal interaction molecule 1 (STIM1)-an endoplasmic reticulum (ER)-localized Ca²⁺ sensor that activates the Orai1 channel on the plasma membrane for SOCE-and in mast cells, we found that a significant proportion of cells exhibited STIM1 oscillations with the same periodicity as Ca²⁺ oscillations. These cortical oscillations, occurring in the cell's cortical region and shared with ER-plasma membrane (ER-PM) contact sites proteins, were only detectable using total internal reflection fluorescence microscopy (TIRFM). Notably, STIM1 oscillations could occur independently of Ca²⁺ oscillations. Simultaneous imaging of cytoplasmic Ca²⁺ and ER Ca²⁺ with SEPIA-ER revealed that receptor activation does not deplete ER Ca²⁺, whereas receptor activation without extracellular Ca²⁺ influx induces cyclic ER Ca²⁺ depletion. However, under such nonphysiological conditions, cyclic ER Ca²⁺ oscillations lead to sustained STIM1 recruitment, indicating that oscillatory Ca²⁺ release is neither necessary nor sufficient for STIM1 oscillations. Using optogenetic tools to manipulate ER-PM contact site dynamics, we found that persistent ER-PM contact sites reduced the amplitude of Ca²⁺ oscillations without alteration of oscillation frequency. Together, these findings suggest an active cortical mechanism governs the rapid dissociation of ER-PM contact sites, thereby control amplitude of oscillatory Ca²⁺ dynamics during receptor-induced Ca²⁺ oscillations.