The intermediate filament framework is one of the three cytoskeletal systems in mammalian cells. Its well spread filamentous structure from the nucleus to the plasma membrane is believed to provide protection against various mechanical stresses (1, 2). Intermediate filaments also undergo disassembly/assembly and spatial reorganization in cells in response to external stimulation and during mitosis. The dynamic property of the intermediate filament system plays a fundamental role in mediating changes in cell shape, division, and migration; signaling molecule distribution; and smooth muscle force development (1, 3-7).The dynamic characteristics of the intermediate filament network may be regulated by protein phosphorylation. Vimentin is the most abundant intermediate filament protein in various cell types, including smooth muscle cells (2,5,6). Vimentin phosphorylation in association with vimentin disassembly and spatial reorganization occurs during mitosis or in response to extracellular stimulation (8, 9). In cultured smooth muscle cells, contractile stimulation triggers vimentin phosphorylation at Ser-56 concurrently with vimentin partial disassembly and spatial reorientation (6).The disassembly and spatial reorganization of the vimentin network may regulate the translocation of certain molecules (7, 10, 11). The adapter protein p130 Crk-associated substrate (CAS) 2 has been shown to participate in the signaling processes that regulate smooth muscle contraction and cell migration (12-14). Our recent study has suggested that vimentin phosphorylation and disassembly are related to CAS redistribution during contractile activation of smooth muscle (10). In addition, external stress initiates Rho kinase redistribution associated with vimentin depolymerization in fibroblasts and the translocation of Ca 2ϩ /calmodulin-dependent protein kinase II in differentiated smooth muscle cells, which may be an important event for cell signaling (7, 11).p21-activated kinase (PAK) may be an upstream regulator of the vimentin network (6, 9). In cultured smooth muscle cells, agonist-mediated vimentin phosphorylation at Ser-56 and spatial reorientation of the vimentin network are inhibited by silencing of PAK1, a dominant isoform in smooth muscle (6,15). Additionally, PAK has been implicated in modulating smooth muscle contraction; introduction of an active PAK isoform into smooth muscle potentiates force development at constant intracellular calcium (16). Expression of an inactive PAK1 mutant attenuates migration of cultured smooth muscle cells in response to platelet-derived growth factor (15).In response to external stimulation, PAK undergoes autophosphorylation at Thr-423, which increases PAK activity for substrates (17,18). In addition to the small GTPases Cdc42 and Rac1, the activity of PAK may be regulated by the paxillin kinase linker/PIX (PAK-interacting exchange factor; guanine nucleotide exchange factor) (19 -21). CAS has been shown to interact with the paxillin kinase linker/PIX via CrkII and paxillin (21-* This work was ...
Abstract-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. Key Words: tyrosine kinase Ⅲ actin cytoskeleton Ⅲ contraction Ⅲ vascular smooth muscle Ⅲ adapter protein A ctin cytoskeleton remodeling has recently emerged as an important cellular process mediating smooth muscle contraction. 1-7 A pool of globular actin (G-actin) is stimulated onto filamentous actin (F-actin) in a variety of smooth muscle cells and tissues in response to agonist stimulation. Blockage of actin polymerization by the inhibitors cytochalasin and latrunculin attenuates active force on activation with contractile stimuli whereas myosin regulatory light chain phosphorylation (MRLCP) is not disrupted. 5,8 -10 These studies suggest that actin dynamics and MRLCP are independently regulated, and that both dynamic changes in the actin cytoskeleton and myosin activation are required for force development during contractile stimulation of smooth muscle. 4,8 -11 However, the mechanisms that regulate the actin cytoskeleton in smooth muscle are not completely understood.The tyrosine phosphorylated protein Crk-associated substrate (CAS) has been implicated in the modulation of the actin cytoskeleton in smooth muscle cells as well as in nonmuscle cells including COS-7 cells and NIH3T3 cells. 9,12-14 Downregulation of CAS by antisense dramatically attenu...
Rationale Through largely unknown mechanisms, Ca2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca2+ entry (SOCE) 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 while the platelet-derived growth factor (PDGF) activates the canonical Ca2+ release-activated Ca2+ (CRAC) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca2+-selective channel contributed by Orai1, Orai3 and STIM1 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.
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.
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