José C. González-Cobos

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.

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.

TRPC channels in smooth muscle cells.

Transient receptor potential canonical (TRPC) proteins constitute a family of seven (TRPC1-7) nonselective cation channels within the wider TRP superfamily. TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 channels are expressed in vascular smooth muscle cells from human vessels of all calibers and in smooth muscle from organs such as the uterus and the gastrointestinal tract. TRPC channels have recently emerged as important players in the control of smooth muscle function. This review will focus on the retrospective analysis of studies proposing contributions of TRPC channels to native calcium entry pathways in smooth muscle and to physiological and pathophysiological responses with emphasis on the vascular system.

Airway smooth muscle STIM1 and Orai1 are upregulated in asthmatic mice and mediate PDGF-activated SOCE, CRAC currents, proliferation, and migration

Airway smooth muscle cell (ASMC) remodeling contributes to the structural changes in the airways that are central to the clinical manifestations of asthma. Ca2+ signals play an important role in ASMC remodeling through control of ASMC migration and hypertrophy/proliferation. Upregulation of STIM1 and Orai1 proteins, the molecular components of the store-operated Ca2+ entry (SOCE) pathway, has recently emerged as an important mediator of vascular remodeling. However, the potential upregulation of STIM1 and Orai1 in asthmatic airways remains unknown. An important smooth muscle migratory agonist with major contributions to ASMC remodeling is the platelet-derived growth factor (PDGF). Nevertheless, the Ca2+ entry route activated by PDGF in ASMC remains elusive. Here, we show that STIM1 and Orai1 protein levels are greatly upregulated in ASMC isolated from ovalbumin-challenged asthmatic mice, compared to control mice. Furthermore, we show that PDGF activates a Ca2+ entry pathway in rat primary ASMC that is pharmacologically reminiscent of SOCE. Molecular knockdown of STIM1 and Orai1 proteins inhibited PDGF-activated Ca2+ entry in these cells. Whole-cell patch clamp recordings revealed the activation of Ca2+ release-activated Ca2+ (CRAC) current by PDGF in ASMC. These CRAC currents were abrogated upon either STIM1 or Orai1 knockdown. We show that either STIM1 or Orai1 knockdown significantly inhibited ASMC proliferation and chemotactic migration in response to PDGF. These results implicate STIM1 and Orai1 in PDGF-induced ASMC proliferation and migration and suggest the potential use of STIM1 and Orai1 as targets for ASMC remodeling during asthma.

Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels

ABSTRACT We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca2+ entry and Ca2+ release-activated Ca2+ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca2+ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca2+ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca2+ entry pathways.

Leukotriene-C4 Synthase, a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels, Is Required for Neointimal Hyperplasia

Background: LTC4S is required for Orai1/Orai3 LRC channel activation. However, the role of LTC4S in neointimal hyperplasia is unknown. Results: LTC4S knockdown inhibited neointimal hyperplasia. Akt phosphorylation was decreased in injured arteries, and knockdown of Orai3 or LTC4S restored Akt phosphorylation. Conclusion: LTC4S is required for neointimal hyperplasia. Significance: LTC4S is a potential therapeutic target for vascular occlusive diseases. Leukotriene-C4 synthase (LTC4S) generates LTC4 from arachidonic acid metabolism. LTC4 is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC4 was also a cytosolic second messenger that activated store-independent LTC4-regulated Ca2+ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC4S in neointima formation remains unknown. Here we show that LTC4S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC4S protein expression in VSMCs. Inhibition of LTC4S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC4S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC4S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC4S or Orai3 was prevented by shRNA. We conclude that LTC4S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.

What Role for Store-Operated Ca2+ Entry in Muscle?

Store‐operated Ca2+ entry (SOCE) is a receptor‐regulated Ca2+ entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca2+ stores through receptor‐mediated production of inositol 1,4,5‐trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca2+ is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25 nm) to interact with Orai1 channels and activate Ca2+ entry. Ca2+ entry through store‐operated Ca2+ (SOC) channels, originally thought to mediate the replenishment of Ca2+ stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long‐term cell functions such as proliferation, growth, and migration. SOCE has also been proposed to contribute to short‐term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

The universal second messenger Ca controls numerous physiological and pathophysiological cell processes (Clapham, 2007; Cahalan and Chandy, 2009; Hogan et al., 2010). Orai channels contribute Ca entry pathways through either store-dependent, Ca release–activated Ca (CRAC) channels (encoded by Orai1) (Putney, 1990; Hoth and Penner, 1992; Feske et al., 2006; Vig et al., 2006; Zhang et al., 2006), or store-independent, arachidonic acid (AA)-regulated Ca (ARC; Mignen and Shuttleworth, 2000; Mignen et al., 2008) and leukotriene C4 (LTC4)-regulated Ca (LRC; Gonzalez-Cobos et al.,

Store-Independent Orai1/3 Channels Activated by Intracrine LeukotrieneC4Novelty and Significance

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.