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The Ca2+ release-activated Ca2+ (CRAC) current is a major signaling event in non-excitable cells whereby Ca2+ store depletion activates Ca2+ entry across the plasma membrane from the extracellular space. Stromal interaction molecule 1 (STIM1) and Orai1 proteins are the key molecular players of the CRAC channel. Previous studies have linked activity of this channel to many physiological functions, and dysregulation of the CRAC channel has been associated with various diseases. In the absence of inducible tissue-specific knockout mice, in vivo knockdown studies examining the endogenous function of CRAC channel proteins, STIM1 and Orai1, are a challenge. In this chapter, we describe a lentiviral delivery system of shRNA-mediated gene silencing that has proven successful in studying the endogenous CRAC channel in vivo.

The multifunctional Ca2+/calmodulin-dependent protein kinase II δ-isoform (CaMKIIδ) promotes vascular smooth muscle (VSM) proliferation, migration, and injury-induced vascular wall neointima formation. The objective of this study was to test if microRNA-30 (miR-30) family members are endogenous regulators of CaMKIIδ expression following vascular injury and whether ectopic expression of miR-30 can inhibit CaMKIIδ-dependent VSM cell function and neointimal VSM hyperplasia induced by vascular injury. The CaMKIIδ 3′UTR contains a consensus miR-30 binding sequence that is highly conserved across species. A significant decrease in miR-30 family members and increase in CaMKIIδ2 protein expression, with no change in CaMKIIδ mRNA expression, was observed in medial layers of VSM 7 days post-injury. In vitro, overexpression of miR-30c or miR-30e inhibited CaMKIIδ2 protein expression by ~50% in cultured rat aortic VSM cells, and inhibited VSM cell proliferation and migration. In vivo, lenti-viral delivery of miR-30c into injured rat carotid arteries prevented the injury-induced increase in CaMKIIδ2. Furthermore, neointima formation was dramatically inhibited by lenti-viral delivery of miR-30c in the injured medial smooth muscle. These studies define a novel mechanism for regulating CaMKIIδ expression in VSM and provide a new potential therapeutic strategy to reduce progression of vascular proliferative diseases, including atherosclerosis and restenosis.

Transient receptor potential canonical subfamily, member 7 (TRPC7) is the most recently identified member of the TRPC family of Ca(2+)-permeable non-selective cation channels. The gene encoding the TRPC7 channel plasma membrane protein was first cloned from mouse brain. TRPC7 mRNA and protein have been detected in cell types derived from multiple organ systems from various species including humans. Gq-coupled protein receptor activation is the predominant mode of TRPC7 activation. Lipid metabolites involved in the phospholipase C (PLC) signaling pathway, including diacylglycerol (DAG) and its precursor the phosphatidylinositol-4,5-bisphosphate (PIP2), have been shown to be direct regulators of TRPC7 channel. TRPC7 channels have been linked to the regulation of various cellular functions however, the depth of our understanding of TRPC7 channel function and regulation is limited in comparison to other TRP channel family members. This review takes a historical look at our current knowledge of TRPC7 mechanisms of activation and its role in cellular physiology and pathophysiology.

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

Orai (Orai1, Orai2, and Orai3) proteins form a family of highly Ca2+-selective plasma membrane channels that are regulated by stromal-interacting molecules (STIM1 and STIM2); STIM proteins are Ca2+sensors located in the membrane of the endoplasmic reticulum. STIM and Orai proteins are expressed in vascular and airway smooth muscle and constitute the molecular components of the ubiquitous store-operated Ca2+entry pathway that mediate the Ca2+release-activated Ca2+current. STIM/Orai proteins also encode store-independent Ca2+entry pathways in smooth muscle. Altered expression and function of STIM/Orai proteins have been linked to vascular and airway pathologies, including restenosis, hypertension, and atopic asthma. In this review we discuss our current understanding of Orai proteins and the store-dependent and -independent signaling pathways mediated by these proteins in vascular and airway smooth muscle. We also discuss the current studies linking altered expression and function of Orai proteins with smooth muscle-related pathologies.

Transient receptor potential canonical subfamily, member 7 (TRPC7) is the most recently identified member of the TRPC family of Ca(2+)-permeable non-selective cation channels. The gene encoding the TRPC7 channel plasma membrane protein was first cloned from mouse brain. TRPC7 mRNA and protein have been detected in cell types derived from multiple organ systems from various species including humans. Gq-coupled protein receptor activation is the predominant mode of TRPC7 activation. Lipid metabolites involved in the phospholipase C (PLC) signaling pathway, including diacylglycerol (DAG) and its precursor the phosphatidylinositol-4,5-bisphosphate (PIP2), have been shown to be direct regulators of TRPC7 channel. TRPC7 channels have been linked to the regulation of various cellular functions however, the depth of our understanding of TRPC7 channel function and regulation is limited in comparison to other TRP channel family members. This review takes a historical look at our current knowledge of TRPC7 mechanisms of activation and its role in cellular physiology and pathophysiology.

The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott–Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.

Airway smooth muscle (ASM) is a key target cell in allergen-induced asthma known to contribute to airway hyperresponsiveness (AHR) and chronic airway remodeling. Changes in ASM calcium homeostasis have been shown to contribute to AHR although the mechanisms and Ca(2+) signal effectors are incompletely understood. In the present study, we tested the function of ASM multifunctional protein kinase Ca(2+)/calmodulin-dependent kinase II (CaMKII) isoforms CaMKIIδ and CaMKIIγ in allergen-induced AHR and airway remodeling in vivo. Using a murine model of atopic asthma, we demonstrate that CaMKIIδ protein is upregulated in ASM derived from ovalbumin (OVA)-treated animals compared to controls. A genetic approach to conditionally knock out smooth muscle CaMKIIδ and CaMKIIγ in separate Cre-loxp systems was validated, and using this loss-of-function approach, the function of these CaMKII isoforms was tested in ovalbumin (OVA)-induced airway remodeling and AHR. OVA treatment in control mice had no effect on ASM remodeling in this model of AHR, and CaMKIIδ knockouts had no independent effects on ASM content. However, at 1 day post-final OVA challenge, OVA-induced AHR was eliminated in the CaMKIIδ knockouts. OVA-induced peribronchial inflammation and bronchoalveolar lavage fluid (BALF) levels of the Th2 cytokine IL-13 were significantly decreased in the CaMKIIδ knockouts. Unexpectedly, we found increased peribronchial eosinophils in the smooth muscle CaMKIIδ knockouts compared to control animals at 1 day post-final challenge, suggesting that lack of ASM CaMKIIδ delays the progression of AHR rather than inhibiting it. Indeed, when AHR was determined at 7 days post-final OVA challenge, CaMKIIδ knockouts showed robust AHR while AHR was fully resolved in OVA-challenged control mice. These in vivo studies demonstrate a role for smooth muscle CaMKIIδ in promoting airway inflammation and AHR and suggest a complex signaling role for CaMKIIδ in regulating ASM function. These studies confirm the diverse roles of ASM cells as immune effectors that control AHR and call for further studies into CaMKIIδ-mediated signaling in ASM cells during disease.

Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and cell mitosis. In this report, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of PAK activation). Treatment with PAK led to disassembly of wild type vimentin filaments, but not vimentin S56A (alanine substitution at serine-56) mutant filaments, as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS, an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of the vimentin S56A mutant in cells inhibited the increase in phosphorylation at Ser-56 and ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly), and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation as compared to cells expressing wild type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing S56A vimentin mutant. Expression of vimentin S56A mutant depressed PAK phosphorylation at Thr-423 induced by 5-HT. The expression of the vimentin S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.

Rationale: Through largely unknown mechanisms, Ca 2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca 2+ entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca 2+ 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 Ca 2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. Methods and Results: Ca 2+ imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca 2+ release-activated Ca 2+ channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca 2+ -selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca 2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC 4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC 4 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 Ca 2+ -selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC 4 acts through hitherto unknown intracrine mode to elicit store-independent Ca 2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Airway smooth muscle cell (ASMC) remodeling contributes to the structural changes in the airways that are central to the clinical manifestations of asthma. Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+) entry pathway in rat primary ASMC that is pharmacologically reminiscent of SOCE. Molecular knockdown of STIM1 and Orai1 proteins inhibited PDGF-activated Ca(2+) entry in these cells. Whole-cell patch clamp recordings revealed the activation of Ca(2+) release-activated Ca(2+) (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.