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Activation of immunological pathways and disturbances of extracellular matrix (ECM) dynamics are important contributors to the pathogenesis of chronic kidney diseases. Glomerular mesangial cells (MCs) are critical for homeostasis of glomerular ECM dynamics. Interleukin-6 (IL-6) can act as a pro/anti-inflammatory agent relative to cell types and conditions. This study investigated whether IL-6 influences ECM protein production by MCs and the regulatory pathways involved. Experiments were carried out in cultured human MCs (HMCs) and in mice. We found that overexpression of IL-6 and its receptor decreased the abundance of fibronectin and collagen type IV in MCs. ELISA and immunoblot analysis demonstrated that thapsigargin [an activator of store-operated Ca2+ entry (SOCE)], but not the endoplasmic reticulum stress inducer tunicamycin, significantly increased IL-6 content. This thapsigargin effect was abolished by GSK-7975A, a selective inhibitor of SOCE, and by silencing Orai1 (the channel protein mediating SOCE). Furthermore, inhibition of NF-κB pharmacologically and genetically significantly reduced SOCE-induced IL-6 production. Thapsigargin also stimulated nuclear translocation of the p65 subunit of NF-κB. Moreover, MCs overexpressing IL-6 and its receptor in HMCs increased the content of the glucagon-like peptide-1 receptor (GLP-1R), and IL-6 inhibition of fibronectin was attenuated by the GLP-1R antagonist exendin 9–39. In agreement with the HMC data, specific knockdown of Orai1 in MCs using the targeted nanoparticle delivery system in mice significantly reduced glomerular GLP-1R levels. Taken together, our results suggest a novel SOCE/NF-κB/IL-6/GLP-1R signaling pathway that inhibits ECM protein production by MCs.

Extracellular calcium ions support synaptic activity but also reduce excitability of central neurons. In the present study, the effect of calcium on excitability was explored in cultured hippocampal neurons. CaCl2 injected by pressure in the vicinity of a neuron that is bathed only in MgCl2 as the main divalent cation caused a depolarizing shift in action potential threshold and a reduction in excitability. This effect was not seen if the intracellular milieu consisted of Cs+ instead of K-gluconate as the main cation or when it contained ruthenium red, which blocks release of calcium from stores. The suppression of excitability by calcium was mimicked by caffeine, and calcium store antagonists cyclopiazonic acid or thapsigargin blocked this action. Neurons taken from synaptopodin-knockout mice show significantly reduced efficacy of calcium modulation of action potential threshold. Likewise, in Orai1 knockdown cells, calcium is less effective in modulating excitability of neurons. Activation of small-conductance K (SK) channels increased action potential threshold akin to that produced by calcium ions, whereas blockade of SK channels but not big K channels reduced the threshold for action potential discharge. These results indicate that calcium released from stores may suppress excitability of central neurons. NEW & NOTEWORTHY Extracellular calcium reduces excitability of cultured hippocampal neurons. This effect is mediated by calcium-gated potassium currents, possibly small-conductance K channels. Release of calcium from internal stores mimics the effect of extracellular calcium. It is proposed that calcium stores modulate excitability of central neurons.

The short-term effect of high-glucose (HG) treatment on store-operated Ca2+entry in mesangial cells (MCs) is not well-known. The aim of the present study was to determine whether and how HG treatment for a short period altered protein abundance of Orai1, the channel mediating store-operated Ca2+entry in MCs. Rat and human MCs were exposed to HG (25 mM) for 2, 4, 8, and 24 h, and the abundance of Orai1 protein was significantly decreased at the time points of 8 and 16 h. Consistently, HG treatment for 8 h significantly reduced store-operated Ca2+entry in rat MCs. However, HG treatment for the same time periods did not alter the levels of Orai1 transcript. Cycloheximide, a protein synthesis inhibitor, did not affect the HG-induced decrease of Orai1 protein, suggesting a posttranslational mechanism was involved. However, the HG effect on Orai1 protein was significantly attenuated by MG132 (a ubiquitin-proteasome inhibitor) and NH4Cl (a lysosomal pathway inhibitor). Furthermore, HG treatment for 8 h stimulated ubiquitination of Orai1 protein. We further found that polyethylene glycol-catalase, an antioxidant, significantly blunted the HG-induced reduction of Orai1 protein. In support of involvement of reactive oxygen species in the HG effects, hydrogen peroxide (H2O2) itself significantly decreased abundance of Orai1 protein and increased the level of ubiquitinated Orai1. Taken together, these results suggest that a short-term HG treatment decreased abundance of Orai1 protein in MCs by promoting the protein degradation through the ubiquitination-proteasome and –lysosome mechanisms. This HG-stimulated posttranslational mechanism was mediated by H2O2.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca2+homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine C2C12muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca2+regulatory channels, including sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca2+release-activated Ca2+channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca2+regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.

Our previous study demonstrated that the abundance of extracellular matrix proteins was suppressed by store-operated Ca2+entry (SOCE) in mesangial cells (MCs). The present study was conducted to investigate the underlying mechanism focused on the transforming growth factor-β1 (TGF-β1)/Smad3 pathway, a critical pathway for ECM expansion in diabetic kidneys. We hypothesized that SOCE suppressed ECM protein expression by inhibiting this pathway in MCs. In cultured human MCs, we observed that TGF-β1 (5 ng/ml for 15 h) significantly increased Smad3 phosphorylation, as evaluated by immunoblot. However, this response was markedly inhibited by thapsigargin (1 µM), a classical activator of store-operated Ca2+channels. Consistently, both immunocytochemistry and immunoblot showed that TGF-β1 significantly increased nuclear translocation of Smad3, which was prevented by pretreatment with thapsigargin. Importantly, the thapsigargin effect was reversed by lanthanum (La3+; 5 µM) and GSK-7975A (10 µM), both of which are selective blockers of store-operated Ca2+channels. Furthermore, knockdown of Orai1, the pore-forming subunit of the store-operated Ca2+channels, significantly augmented TGF-β1-induced Smad3 phosphorylation. Overexpression of Orai1 augmented the inhibitory effect of thapsigargin on TGF-β1-induced phosphorylation of Smad3. In agreement with the data from cultured MCs, in vivo knockdown of Orai1 specific to MCs using a targeted nanoparticle small interfering RNA delivery system resulted in a marked increase in abundance of phosphorylated Smad3 and in nuclear translocation of Smad3 in the glomerulus of mice. Taken together, our results indicate that SOCE in MCs negatively regulates the TGF-β1/Smad3 signaling pathway.

Ca(2+)entry into the cell via store-operated Ca(2+)release-activated Ca(2+)(CRAC) channels triggers diverse signaling cascades that affect cellular processes like cell growth, gene regulation, secretion, and cell death. These store-operated Ca(2+)channels open after depletion of intracellular Ca(2+)stores, and their main features are fully reconstituted by the two molecular key players: the stromal interaction molecule (STIM) and Orai. STIM represents an endoplasmic reticulum-located Ca(2+)sensor, while Orai forms a highly Ca(2+)-selective ion channel in the plasma membrane. Functional as well as mutagenesis studies together with structural insights about STIM and Orai proteins provide a molecular picture of the interplay of these two key players in the CRAC signaling cascade. This review focuses on the main experimental advances in the understanding of the STIM1-Orai choreography, thereby establishing a portrait of key mechanistic steps in the CRAC channel signaling cascade. The focus is on the activation of the STIM proteins, the subsequent coupling of STIM1 to Orai1, and the consequent structural rearrangements that gate the Orai channels into the open state to allow Ca(2+)permeation into the cell.

Cardiac c-kit+progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit+progenitor cells is not well understood. The present study investigates the functional store-operated Ca2+entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca2+influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit+progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca2+signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca2+signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit+cells.

Calcium (Ca2+) regulates a plethora of cellular functions including hallmarks of cancer development such as cell cycle progression and cellular migration. Receptor-regulated calcium rise in nonexcitable cells occurs through store-dependent as well as store-independent Ca2+entry pathways. Stromal interaction molecules (STIM) and Orai proteins have been identified as critical constituents of both these Ca2+influx pathways. STIMs and Orais have emerged as targets for cancer therapeutics as their altered expression and function have been shown to contribute to tumorigenesis. Recent data demonstrate that they play a vital role in development and metastasis of a variety of tumor types including breast, prostate, cervical, colorectal, brain, and skin tumors. In this review, we will retrospect the data supporting a key role for STIM1, STIM2, Orai1, and Orai3 proteins in tumorigenesis and discuss the potential of targeting these proteins for cancer therapy.

The present study was carried out to investigate if hepatic nuclear factor (HNF)4α contributed to the high glucose-induced increase in stromal interacting molecule (STIM)1 protein abundance in glomerular mesangial cells (MCs). Western blot and immunofluorescence experiments showed HNF4α expression in MCs. Knockdown of HNF4α using a small interfering RNA approach significantly increased mRNA expression levels of both STIM1 and Orai1 and protein expression levels of STIM1 in cultured human MCs. Consistently, overexpression of HNF4α reduced expressed STIM1 protein expression in human embryonic kidney-293 cells. Furthermore, high glucose treatment did not significantly change the abundance of HNF4α protein in MCs but significantly attenuated HNF4α binding activity to the Stim1 promoter. Moreover, knockdown of HNF4α significantly augmented store-operated Ca2+entry, which is known to be gated by STIM1 and has recently been found to be antifibrotic in MCs. In agreement with those results, knockdown of HNF4α significantly attenuated the fibrotic response of high glucose. These results suggest that HNF4α negatively regulates STIM1 transcription in MCs. High glucose increases STIM1 expression levels by impairing HNF4α binding activity to the Stim1 promoter, which subsequently releases Stim1 transcription from HNF4α repression. Since the STIM1-gated store-operated Ca2+entry pathway in MCs has an antifibrotic effect, inhibition of HNF4α in MCs might be a potential therapeutic option for diabetic kidney disease.

Orai1 interacts with transient receptor potential protein of the canonical subfamily (TRPC4) and contributes to calcium selectivity of the endothelial cell store-operated calcium entry current ( ISOC). Orai1 silencing increases sodium permeability and decreases membrane-associated calcium, although it is not known whether Orai1 is an important determinant of cytosolic sodium transitions. We test the hypothesis that, upon activation of store-operated calcium entry channels, Orai1 is a critical determinant of cytosolic sodium transitions. Activation of store-operated calcium entry channels transiently increased cytosolic calcium and sodium, characteristic of release from an intracellular store. The sodium response occurred more abruptly and returned to baseline more rapidly than did the transient calcium rise. Extracellular choline substitution for sodium did not inhibit the response, although 2-aminoethoxydiphenyl borate and YM-58483 reduced it by ∼50%. After this transient response, cytosolic sodium continued to increase due to influx through activated store-operated calcium entry channels. The magnitude of this sustained increase in cytosolic sodium was greater when experiments were conducted in low extracellular calcium and when Orai1 expression was silenced; these two interventions were not additive, suggesting a common mechanism. 2-Aminoethoxydiphenyl borate and YM-58483 inhibited the sustained increase in cytosolic sodium, only in the presence of Orai1. These studies demonstrate that sodium permeates activated store-operated calcium entry channels, resulting in an increase in cytosolic sodium; the magnitude of this response is determined by Orai1.

The present study was conducted to determine whether and how store-operated Ca2+entry (SOCE) in glomerular mesangial cells (MCs) was altered by high glucose (HG) and diabetes. Human MCs were treated with either normal glucose or HG for different time periods. Cyclopiazonic acid-induced SOCE was significantly greater in the MCs with 7-day HG treatment and the response was completely abolished by GSK-7975A, a selective inhibitor of store-operated Ca2+channels. Similarly, the inositol 1,4,5-trisphosphate-induced store-operated Ca2+currents were significantly enhanced in the MCs treated with HG for 7 days, and the enhanced response was abolished by both GSK-7975A and La3+. In contrast, receptor-operated Ca2+entry in MCs was significantly reduced by HG treatment. Western blotting showed that HG increased the expression levels of STIM1 and Orai1 in cultured MCs. A significant HG effect occurred at a concentration as low as 10 mM, but required a minimum of 7 days. The HG effect in cultured MCs was recapitulated in renal glomeruli/cortex of both type I and II diabetic rats. Furthermore, quantitative real-time RT-PCR revealed that a 6-day HG treatment significantly increased the mRNA expression level of STIM1. However, the expressions of STIM2 and Orai1 transcripts were not affected by HG. Taken together, these results suggest that HG/diabetes enhanced SOCE in MCs by increasing STIM1/Orai1 protein expressions. HG upregulates STIM1 by promoting its transcription but increases Orai1 protein through a posttranscriptional mechanism.

Sirolimus (rapamycin) is used in drug-eluting stent strategies and proved clearly superior in this application compared with other immunomodulators such as pimecrolimus. The molecular basis of this action of sirolimus in the vascular system is still incompletely understood. Measurements of cell proliferation in human coronary artery smooth muscle cells (hCASM) demonstrated a higher antiproliferative activity of sirolimus compared with pimecrolimus. Although sirolimus lacks inhibitory effects on calcineurin, nuclear factor of activated T-cell activation in hCASM was suppressed to a similar extent by both drugs at 10 μM. Sirolimus, but not pimecrolimus, inhibited agonist-induced and store-operated Ca2+ entry as well as cAMP response element binding protein (CREB) phosphorylation in human arterial smooth muscle, suggesting the existence of an as-yet unrecognized inhibitory effect of sirolimus on Ca2+ signaling and Ca2+-dependent gene transcription. Electrophysiological experiments revealed that only sirolimus but not pimecrolimus significantly blocked the classical stromal interaction molecule/Orai-mediated, store-operated Ca2+ current reconstituted in human embryonic kidney cells (HEK293). A link between Orai function and proliferation was confirmed by dominant-negative knockout of Orai in hCASM. Analysis of the effects of sirolimus on cell proliferation and CREB activation in an in vitro model of arterial intervention using human aorta corroborated the ability of sirolimus to suppress stent implantation-induced CREB activation in human arteries. We suggest inhibition of store-operated Ca2+ entry based on Orai channels and the resulting suppression of Ca2+ transcription coupling as a key mechanism underlying the antiproliferative activity of sirolimus in human arteries. This mechanism of action is specific for sirolimus and not a general feature of drugs interacting with FK506-binding proteins.

Store-operated Ca2+entry (SOCE) is critical for Ca2+signaling in nonexcitable cells; however, its role in the regulation of cardiomyocyte Ca2+homeostasis has only recently been investigated. The increased understanding of the role of stromal interaction molecule 1 (STIM1) in regulating SOCE combined with recent studies demonstrating the presence of STIM1 in cardiomyocytes provides support that this pathway co-exists in the heart with the more widely recognized Ca2+handling pathways associated with excitation-contraction coupling. There is now substantial evidence that STIM1-mediated SOCE plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo, and there is growing support for the contribution of SOCE to Ca2+overload associated with ischemia/reperfusion injury. Here, we provide an overview of our current understanding of the molecular regulation of SOCE and discuss the evidence supporting the role of STIM1/Orai1-mediated SOCE in regulating cardiomyocyte function.

Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca2+/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.

Store-operated Ca2+entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca2+-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca2+-independent phospholipase A2(iPLA2β, or PLA2G6) in activation of cat-SOC current ( Icat-SOC), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA2β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA2β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.

Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau.

Previous studies in mouse pulmonary arterial smooth muscle cells (PASMCs) showed that cannonical transient receptor potential channel TRPC1 and stromal interaction molecule 1 (STIM1) mediate the sustained component of capacitative Ca2+entry (CCE), but the molecular candidate(s) that mediate the transient component of CCE remain unknown. The aim of the present study was to examine whether Orai1 mediates the transient component of CCE through activation of STIM1 in mouse PASMCs. In primary cultured mouse PASMCs loaded with fura-2, cyclopiazonic acid (CPA) caused a transient followed by a sustained rise in intracellular Ca2+concentration ([Ca2+]i). The transient but not the sustained rise in [Ca2+]iwas partially inhibited by nifedipine. The nifedipine-insensitive transient rise in [Ca2+]iand the increase in Mn2+quench of fura-2 fluorescence caused by CPA were both reduced in cells treated with Orai1 siRNA. These responses to CPA were further reduced in cells treated with Orai1 and STIM1 small interfering (si)RNA. Moreover, overexpression of STIM1 enhanced the rise in [Ca2+]iand the increase in Mn2+quench of fura-2 fluorescence caused by CPA, and these responses were reduced in cells treated with Orai1 siRNA. RT-PCR revealed Orai1 and STIM1 mRNAs, and Western blot analysis identified Orai1 and STIM1 proteins in mouse PASMCs. Furthermore, Orai1 was found to coimmunoprecipitate with STIM1, and the precipitation level of Orai1 was increased in cells subjected to store-depletion. Immunostaining revealed colocalization of Orai1 and STIM1 proteins, and the colocalization of these proteins was more apparent after store-depletion. These data provide direct evidence that the transient component of CCE is mediated by Orai1 channel as a result of STIM1 activation in mouse PASMCs.

Store-operated Ca2+ entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca2+ from the transverse tubule into the junctional cleft during periods of depleting Ca2+ release. In dystrophic muscle fibers, SOCE may be a source of Ca2+ overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca2+ release. To examine this, Ca2+ in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca2+ from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca2+ transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca2+ concentrations ([Ca2+]SR). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca2+ into the fiber for an equivalent depletion of [Ca2+]SR via SOCE, it remains unclear whether this is deleterious.

We recently demonstrated that thapsigargin-induced passive store depletion activates Ca2+ entry in vascular smooth muscle cells (VSMC) through stromal interaction molecule 1 (STIM1)/Orai1, independently of transient receptor potential canonical (TRPC) channels. However, under physiological stimulations, despite the ubiquitous depletion of inositol 1,4,5-trisphosphate-sensitive stores, many VSMC PLC-coupled agonists (e.g., vasopressin and endothelin) activate various store-independent Ca2+ entry channels. Platelet-derived growth factor (PDGF) is an important VSMC promigratory agonist with an established role in vascular disease. Nevertheless, the molecular identity of the Ca2+ channels activated by PDGF in VSMC remains unknown. Here we show that inhibitors of store-operated Ca2+ entry (Gd3+ and 2-aminoethoxydiphenyl borate at concentrations as low as 5 μM) prevent PDGF-mediated Ca2+ entry in cultured rat aortic VSMC. Protein knockdown of STIM1, Orai1, and PDGF receptor-β (PDGFRβ) impaired PDGF-mediated Ca2+ influx, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Scratch wound assay showed that knockdown of STIM1, Orai1, or PDGFRβ inhibited PDGF-mediated VSMC migration, but knockdown of STIM2, Orai2, and Orai3 was without effect. STIM1, Orai1, and PDGFRβ mRNA levels were upregulated in vivo in VSMC from balloon-injured rat carotid arteries compared with noninjured control vessels. Protein levels of STIM1 and Orai1 were also upregulated in medial and neointimal VSMC from injured carotid arteries compared with noninjured vessels, as assessed by immunofluorescence microscopy. These results establish that STIM1 and Orai1 are important components for PDGF-mediated Ca2+ entry and migration in VSMC and are upregulated in vivo during vascular injury and provide insights linking PDGF to STIM1/Orai1 during neointima formation.

Ca+release-activated Ca2+(CRAC) channels are activated when free Ca2+concentration in the intracellular stores is substantially reduced and mediate sustained Ca2+entry. Recent studies have identified Orai1 as a CRAC channel subunit. Here we demonstrate that passive Ca2+store depletion using the inhibitor of the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin (TG), enhances the surface expression of Orai1, a process that depends on rises in cytosolic free Ca2+concentration, as demonstrated in cells loaded with dimethyl BAPTA, an intracellular Ca2+chelator that prevented TG-evoked cytosolic free Ca2+concentration elevation. Similar results were observed with a low concentration of carbachol. Cleavage of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor, synaptosomal-assiciated protein-25 (SNAP-25), with botulinum neurotoxin A impaired TG-induced increase in the surface expression of Orai1. In addition, SNAP-25 cleaving by botulinum neurotoxin A reduces the maintenance but not the initial stages of store-operated Ca2+entry. In aggregate, these findings demonstrate that store depletion enhances Orai1 plasma membrane expression in an exocytotic manner that involves SNAP-25, a process that contributes to store-dependent Ca2+entry.

calcium release-activated currents (CRAC) were first described in the early 1990s and shown to carry out key cellular functions. As examples, CRAC channels mediate the depolarizing effect of muscarinic receptor activation in pancreatic β cells ([1][1]) and promote cell cycle progression

Platelet-derived growth factor (PDGF) and its receptor are known to be substantially elevated in lung tissues and pulmonary arterial smooth muscle cells (PASMC) isolated from patients and animals with pulmonary arterial hypertension. PDGF has been shown to phosphorylate and activate Akt and mammalian target of rapamycin (mTOR) in PASMC. In this study, we investigated the role of PDGF-mediated activation of Akt signaling in the regulation of cytosolic Ca2+ concentration and cell proliferation. PDGF activated the Akt/mTOR pathway and, subsequently, enhanced store-operated Ca2+ entry (SOCE) and cell proliferation in human PASMC. Inhibition of Akt attenuated the increase in cytosolic Ca2+ concentration due to both SOCE and PASMC proliferation. This effect correlated with a significant downregulation of stromal interacting molecule (STIM) and Orai, proposed molecular correlates for SOCE in many cell types. The data from this study present a novel pathway for the regulation of Ca2+ signaling and PASMC proliferation involving activation of Akt in response to upregulated expression of PDGF. Targeting this pathway may lead to the development of a novel therapeutic option for the treatment of pulmonary arterial hypertension.

Ca2+ entry through store-operated channels (SOCs) in the plasma membrane plays an important role in regulation of vascular smooth muscle contraction, tone, and cell proliferation. The C-type transient receptor potential (TRPC) channels have been proposed as major candidates for SOCs in vascular smooth muscle. Recently, two families of transmembrane proteins, Orai [also known as Ca2+ release-activated Ca2+ channel modulator (CRACM)] and stromal interacting molecule 1 (STIM1), were shown to be essential for the activation of SOCs mainly in nonexcitable cells. Here, using small interfering RNA, we show that Orai1 plays an essential role in activating store-operated Ca2+ entry (SOCE) in primary cultured proliferating human aortic smooth muscle cells (hASMCs), whereas Orai2 and Orai3 do not contribute to SOCE. Knockdown of Orai1 protein expression significantly attenuated SOCE. Moreover, inhibition of Orai1 downregulated expression of Na+/Ca2+ exchanger type 1 (NCX1) and plasma membrane Ca2+ pump isoform 1 (PMCA1). The rate of cytosolic free Ca2+ concentration decay after Ca2+ transients in Ca2+-free medium was also greatly decreased under these conditions. This reduction of Ca2+ extrusion, presumably via NCX1 and PMCA1, may be a compensation for the reduced SOCE. Immunocytochemical observations indicate that Orai1 and NCX1 are clustered in plasma membrane microdomains. Cell proliferation was attenuated in hASMCs with disrupted Orai1 expression and reduced SOCE. Thus Orai1 appears to be a critical component of SOCE in proliferating vascular smooth muscle cells, and may therefore be a key player during vascular growth and remodeling.