Mohamed Trebak

Stim1 and Orai1 Mediate CRAC Currents and Store-Operated Calcium Entry Important for Endothelial Cell Proliferation

Recent breakthroughs in the store-operated calcium (Ca2+) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca2+ sensor and Orai1 as the pore forming subunit of the highly Ca2+-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca2+ release-activated Ca2+ current (ICRAC), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na+ currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and ICRAC in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and ICRAC. Ectopic expression of Stim1 in ECs increased their ICRAC to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

Evidence for STIM1- and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: role in proliferation and migration

The identity of store‐operated calcium (Ca2+) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orail as essential components of ICRAC, a highly Ca2+‐selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called “synthetic”) display SOCE with classic features, namely inhibition by 2‐aminoethoxydiphenyl borate, ML‐9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of ICRAC. Protein knockdown of either STIM1 or Orail in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orail knockdown reduced ICRAC in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up‐regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show ICRAC in VSMCs and resolve a long‐standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.— Potier, M., Gonzalez, J. C., Motiani, R. K., Abdullaev, I. F., Bisaillon, J. M., Singer, H. A., Trebak, M. Evidence for STIM1‐ and Orai1‐ dependent store‐operated calcium influx through IC RAC in vascular smooth muscle cells: role in proliferation and migration. FASEB J. 23, 2425–2437 (2009)

The non-excitable smooth muscle: Calcium signaling and phenotypic switching during vascular disease

Calcium (Ca2+) is a highly versatile second messenger that controls vascular smooth muscle cell (VSMC) contraction, proliferation, and migration. By means of Ca2+ permeable channels, Ca2+ pumps and channels conducting other ions such as potassium and chloride, VSMC keep intracellular Ca2+ levels under tight control. In healthy quiescent contractile VSMC, two important components of the Ca2+ signaling pathways that regulate VSMC contraction are the plasma membrane voltage-operated Ca2+ channel of the high voltage-activated type (L-type) and the sarcoplasmic reticulum Ca2+ release channel, Ryanodine Receptor (RyR). Injury to the vessel wall is accompanied by VSMC phenotype switch from a contractile quiescent to a proliferative motile phenotype (synthetic phenotype) and by alteration of many components of VSMC Ca2+ signaling pathways. Specifically, this switch that culminates in a VSMC phenotype reminiscent of a non-excitable cell is characterized by loss of L-type channels expression and increased expression of the low voltage-activated (T-type) Ca2+ channels and the canonical transient receptor potential (TRPC) channels. The expression levels of intracellular Ca2+ release channels, pumps and Ca2+-activated proteins are also altered: the proliferative VSMC lose the RyR3 and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase isoform 2a pump and reciprocally regulate isoforms of the ca2+/calmodulin-dependent protein kinase II. This review focuses on the changes in expression of Ca2+ signaling proteins associated with VSMC proliferation both in vitro and in vivo. The physiological implications of the altered expression of these Ca2+ signaling molecules, their contribution to VSMC dysfunction during vascular disease and their potential as targets for drug therapy will be discussed.

A Novel Native Store-operated Calcium Channel Encoded by Orai3 SELECTIVE REQUIREMENT OF Orai3 VERSUS Orai1 IN ESTROGEN RECEPTOR-POSITIVE VERSUS ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS

Store-operated calcium (Ca2+) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca2+ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca2+ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca2+-selective Ca2+ release-activated Ca2+ current (ICRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca2+ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER−) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER+ breast cancer cells represent the first example where the native SOCE pathway and ICRAC are mediated by Orai3. Future studies implicating Orai3 in ER+ breast cancer progression might establish Orai3 as a selective target in therapy of ER+ breast tumors.

Endoplasmic Reticulum Stress Is Involved in Cardiac Damage and Vascular Endothelial Dysfunction in Hypertensive Mice

Objective—Cardiac damage and vascular dysfunction are major causes of morbidity and mortality in hypertension. In the present study, we explored the beneficial therapeutic effect of endoplasmic reticulum (ER) stress inhibition on cardiac damage and vascular dysfunction in hypertension. Methods and Results—Mice were infused with angiotensin II (400 ng/kg per minute) with or without ER stress inhibitors (taurine-conjugated ursodeoxycholic acid and 4-phenylbutyric acid) for 2 weeks. Mice infused with angiotensin II displayed an increase in blood pressure, cardiac hypertrophy and fibrosis associated with enhanced collagen I content, transforming growth factor-&bgr;1 (TGF-&bgr;1) activity, and ER stress markers, which were blunted after ER stress inhibition. Hypertension induced ER stress in aorta and mesenteric resistance arteries (MRA), enhanced TGF-&bgr;1 activity in aorta but not in MRA, and reduced endothelial NO synthase phosphorylation and endothelium-dependent relaxation (EDR) in aorta and MRA. The inhibition of ER stress significantly reduced TGF-&bgr;1 activity, enhanced endothelial NO synthase phosphorylation, and improved EDR. The inhibition of TGF-&bgr;1 pathway improved EDR in aorta but not in MRA, whereas the reduction in reactive oxygen species levels ameliorated EDR in MRA only. Infusion of tunicamycin in control mice induced ER stress in aorta and MRA, and reduced EDR by a TGF-&bgr;1–dependent mechanism in aorta and reactive oxygen species–dependent mechanism in MRA. Conclusion—ER stress inhibition reduces cardiac damage and improves vascular function in hypertension. Therefore, ER stress could be a potential target for cardiovascular diseases.

Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration

We recently demonstrated that thapsigargin-induced passive store depletion activates Ca(2+) 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 Ca(2+) 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 Ca(2+) channels activated by PDGF in VSMC remains unknown. Here we show that inhibitors of store-operated Ca(2+) entry (Gd(3+) and 2-aminoethoxydiphenyl borate at concentrations as low as 5 microM) prevent PDGF-mediated Ca(2+) entry in cultured rat aortic VSMC. Protein knockdown of STIM1, Orai1, and PDGF receptor-beta (PDGFRbeta) impaired PDGF-mediated Ca(2+) influx, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Scratch wound assay showed that knockdown of STIM1, Orai1, or PDGFRbeta inhibited PDGF-mediated VSMC migration, but knockdown of STIM2, Orai2, and Orai3 was without effect. STIM1, Orai1, and PDGFRbeta 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 Ca(2+) entry and migration in VSMC and are upregulated in vivo during vascular injury and provide insights linking PDGF to STIM1/Orai1 during neointima formation.

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

Ryanodine Receptor Blockade Reduces Amyloid-β Load and Memory Impairments in Tg2576 Mouse Model of Alzheimer Disease

In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP695), or APP harboring the Swedish double mutation (APPswe) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.

Mechanisms of Phospholipase C-Regulated Calcium Entry

In a variety of cell types, activation of phospholipase C-linked receptors results in the generation of intracellular Ca2+ signals comprised of components of both intracellular Ca2+ release, and enhanced entry of Ca2+ across the plasma membrane. This entry of Ca2+ occurs by either of two general mechanisms: the release of stored Ca2+ can activate, by an unknown mechanism, store-operated channels in the plasma membrane, a process known as capacitative calcium entry. Alternatively, second messengers generated at the plasma membrane can activate Ca2+ channels more directly, a non-capacitative calcium entry process. This review summarizes current knowledge of the underlying signaling mechanisms and the nature of the channel molecules responsible for these two general categories of regulated Ca2+ entry.

Interleukin-10 Released by CD4+CD25+ Natural Regulatory T Cells Improves Microvascular Endothelial Function Through Inhibition of NADPH Oxidase Activity in Hypertensive Mice

Objective—We previously demonstrated that a reduced number of CD4+CD25+-regulatory T cells (Tregs) was associated with microvascular dysfunction in hypertension. However, the underlying mechanism by which Tregs regulate vascular endothelial function remains unknown. Methods and Results—Control and interleukin (IL)-10−/− knockout mice were infused with angiotensin II (400 ng/kg/min) for 2 weeks (hypertensive [HT] and HT-IL-10−/−). Endothelium-dependent relaxation (EDR) in response to acetylcholine was significantly reduced in mesenteric resistance artery (MRA) from HT and HT-IL-10−/− compared with control and IL-10−/− mice. Importantly, the incubation of MRA from HT mice with the conditioned media of cultured Tregs, isolated from control mice, reduced NADPH oxidase activity and improved EDR, whereas no effect was observed in MRA from control mice incubated with the same media. These effects were reversed when MRAs were preincubated with IL-10 antibody or IL-10 receptor antagonist, whereas incubation with transforming growth factor-&bgr; receptor antagonist had no effect. The transfer of cultured Tregs, isolated from control mice, into HT-IL-10−/− mice reduced systolic blood pressure (SBP) and NADPH oxidase activity and improved EDR in MRA compared with untreated HT-IL-10−/− mice. In vivo treatment of HT mice with IL-10 (1000 ng/mouse) significantly reduced SBP and NADPH oxidase activity and improved EDR in MRA compared with untreated HT mice. The transfer of cultured Tregs, isolated from IL-10−/− mice, into HT mice did not reduce SBP or NADPH oxidase activity or improve EDR. The incubation of MRA from HT mice with apocynin improved EDR, whereas NADPH oxidase substrate attenuated EDR in MRA from control mice, which was reversed with exogenous IL-10. Conclusion—These data demonstrate that IL-10 released from Tregs attenuates NADPH oxidase activity, which is a critical process in the improvement of microvascular endothelial function in hypertension, suggesting that Tregs/IL-10 could be a therapeutic target for treatment of vasculopathy in hypertension.