Iskandar F. Abdullaev

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)

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.

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.

Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a d‐[3H]aspartate (glutamate) release assay to explore the ROS‐dependent regulation of glutamate‐permeable volume‐regulated anion channels (VRACs). Exposure of rat microglia to hypo‐osmotic media stimulated Cl− currents and d‐[3H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H2O2 potently increased swelling‐activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan‐stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin‐insensitive NOX4. Using RT‐PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.

STIM1 controls endothelial barrier function independently of Orai1 and Ca2+ entry.

The calcium sensor STIM1 disrupts the endothelial barrier by coupling the thrombin receptor to the actin cytoskeleton. Breaking the Endothelial Barrier Thrombin is an endogenous ligand that induces vasoconstriction and can also disrupt the barrier formed by blood vessel endothelial cells, which leads to increased vascular permeability and leakage of plasma into the tissue. Using the thrombin-induced decrease in transendothelial resistance in two types of cultured endothelial cells as a model of barrier disruption, Shinde et al. found that the calcium-responsive protein STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA and rearrangement of the actin cytoskeleton, which contribute to loss of cell-cell contact. Surprisingly, this role did not involve various cation channels that are targets of STIM1. How STIM1 couples the thrombin receptor to RhoA remains an open question. Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum–localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release–activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.

Calcium-Activated Potassium Channels BK and IK1 Are Functionally Expressed in Human Gliomas but Do Not Regulate Cell Proliferation

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca2+-activated K+ channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K+ channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca2+-activated K+ channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.

Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction

Multiple Ca2+ release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca2+ signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIδ6 isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nm thrombin maximally increased CaMKIIδ6 activation assessed by Thr287 autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIδ (siCaMKIIδ) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nm thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIδ inhibited 2.5 nm thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIδ6 mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIδ6/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

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

Recent breakthroughs in the store-operated calcium (SOC) entry pathway have identified Stim1 as the endoplasmic reticulum (ER) calcium sensor and Orai1 as the pore forming subunit of the highly calcium selective CRAC channel. Previous studies have suggested that endothelial cell (EC) SOC is encoded by members of the Canonical Transient Receptor Potential (TRPC) channel family, either TRPC1 or TRPC4. Here we show that passive store depletion or receptor activation by thrombin or VEGF activates SOC entry pathway in primary EC with classical SOC pharmacological features. EC possess the archetypical store-depletion activated CRAC current. By amplifying currents in divalent free bath solutions, we show that EC CRAC has similar characteristics to that recorded from RBL cells, namely a similar time course of activation, sensitivity to 2-APB and low concentrations of lanthanides, the same inwardly rectifying I/V relationship, very positive reversal potential, and large sodium currents displaying the typical phenomenon of depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOC entry and CRAC currents in EC which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, complete knockdown of either TRPC1 or TRPC4 proteins had no effect on SOC entry in EC. Smaller CRAC current densities in EC compared to those recorded in RBL cells were due to lower expression of Stim1. Ectopic expression of Stim1 in EC increased their CRAC currents to a size comparable to those in RBL cells. Knockdown of either 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 Stim1. These results are first to establish the requirement of Stim1/Orai1 in the endothelial SOC pathway necessary for proliferation.