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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.

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 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.