Rajender K. Motiani

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.

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.

Orai3 is an estrogen receptor α-regulated Ca2+ channel that promotes tumorigenesis

Store‐operated Ca2+ entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca2+‐selective conductance involved in cellular proliferation and migration. We recently described up‐regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α‐expressing (ERα+) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ~63%) and protein (by ~44%) with no effect on Orai1. ERα knockdown decreases Orai3‐mediated SOCE (by ~43%) and the corresponding Ca2+ release‐activated Ca2+ (CRAC) current (by ~42%) in ERα+ MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca2+ influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE‐dependent phosphorylation of extracellular signal‐regulated kinase (ERK1/2; by ~44%) and focal adhesion kinase (FAK; by ~46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ~49%). Significantly, Orai3 knockdown selectively decreased anchorage‐independent growth (by ~58%) and Matrigel invasion (by ~44%) of ERα+ MCF7 cells with no effect on ERα– MDA‐MB231 cells. Moreover, Orai3 knockdown inhibited ERα+ cell tumorigenesis in immunodeficient mice (~66% reduction in tumor volume). These data establish Orai3 as an ERα‐regulated channel and a potential selective therapeutic target for ERα+ breast cancers.—Motiani, R. K., Zhang, X., Harmon, K. E., Keller, R. S., Matrougui, K., Bennett, J. A., Trebak, M. Orai3 is an estrogen receptor α‐regulated Ca2+ channel that promotes tumorigenesis. FASEB J. 27, 63–75 (2013). www.fasebj.org

Store-Independent Orai1/3 Channels Activated by Intracrine LeukotrieneC4: Role in Neointimal Hyperplasia

Rationale: Through largely unknown mechanisms, Ca2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca2+ entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca2+ entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown. Objective: The goal of this study was to determine the agonist-evoked Ca2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. Methods and Results: Ca2+ imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca2+ release-activated Ca2+ channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca2+-selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation. Conclusions: These results demonstrate that distinct native Ca2+-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

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.

miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat

AIMS Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. METHODS AND RESULTS MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. CONCLUSION Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.

Inhibition of interleukin‐1β–induced matrix metalloproteinases 1 and 13 production in human osteoarthritic chondrocytes by prostaglandin D2

OBJECTIVE To investigate the effects of prostaglandin D2 (PGD2) on interleukin-1beta (IL-1beta)-induced matrix metalloproteinase 1 (MMP-1) and MMP-13 expression in human chondrocytes and the signaling pathways involved in these effects. METHODS Chondrocytes were stimulated with IL-1 in the presence or absence of PGD2, and expression of MMP-1 and MMP-13 proteins was evaluated by enzyme-linked immunosorbent assay. Messenger RNA (mRNA) expression and promoter activity were analyzed by real-time reverse transcription-polymerase chain reaction and transient transfections, respectively. The role of the PGD2 receptors D prostanoid receptor 1 (DP1) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) was evaluated using specific agonists and antibody-blocking experiments. The contribution of the cAMP/protein kinase A (PKA) pathway was determined using cAMP-elevating agents and PKA inhibitors. RESULTS PGD2 decreased in a dose-dependent manner IL-1-induced MMP-1 and MMP-13 protein and mRNA expression as well as their promoter activation. DP1 and CRTH2 were expressed and functional in chondrocytes. The effect of PGD2 was mimicked by BW245C, a selective agonist of DP1, but not by 13,14-dihydro-15-keto-PGD2, a selective agonist of CRTH2. Furthermore, treatment with an anti-DP1 antibody reversed the effect of PGD2, indicating that the inhibitory effect of PGD2 is mediated by DP1. The cAMP-elevating agents 8-Br-cAMP and forskolin suppressed IL-1-induced MMP-1 and MMP-13 expression, and the PKA inhibitors KT5720 and H89 reversed the inhibitory effect of PGD2, suggesting that the effect of PGD2 is mediated by the cAMP/PKA pathway. CONCLUSION PGD2 inhibits IL-1-induced production of MMP-1 and MMP-13 by chondrocytes through the DP1/cAMP/PKA signaling pathway. These data also suggest that modulation of PGD2 levels in the joint may have therapeutic potential in the prevention of cartilage degradation.