Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCα

Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 ± 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 ± .9% decreases in cell length ( n = 4)]. Furthermore, in 3D cells, translocation of both PKCα and of RhoA was greatly enhanced and resulted in a greater association of PKCα-RhoA in the membrane fraction. In 3G transfected cells, PKCα and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCα-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCα and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCα-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.

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Phosphorylated HSP27 modulates the association of phosphorylated caldesmon with tropomyosin in colonic smooth muscle

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00350.2005 ◽

2006 ◽

Vol 291 (4) ◽

pp. G630-G639 ◽

Cited By ~ 24

Author(s):

Sita Somara ◽

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Thin Filament ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Contractile Proteins ◽

Concomitant Increase ◽

Thin Filament Regulation ◽

Hsp 27 ◽

Actomyosin Interaction

Thin-filament regulation of smooth muscle contraction involves phosphorylation, association, and dissociation of contractile proteins in response to agonist stimulation. Phosphorylation of caldesmon weakens its association with actin leading to actomyosin interaction and contraction. Present data from colonic smooth muscle cells indicate that acetylcholine induced a significant association of caldesmon with PKCα and sustained phosphorylation of caldesmon at ser789. Furthermore, acetylcholine induced significant and sustained increase in the association of phospho-caldesmon with heat-shock protein (HSP)27 with concomitant increase in the dissociation of phospho-caldesmon from tropomyosin. At the thin filament level, HSP27 plays a crucial role in acetylcholine-induced association of contractile proteins. Present data from colonic smooth muscle cells transfected with non-phospho-HSP27 mutant cDNA indicate that the absence of phospho-HSP27 inhibits acetylcholine-induced caldesmon phosphorylation. Our results further indicate that the presence of phospho-HSP27 significantly enhances acetylcholine-induced sustained association of phospho-caldesmon with HSP27 with a concomitant increase in acetylcholine-induced dissociation of phospho-caldesmon from tropomyosin. We thus propose a model whereby upon acetylcholine-induced phosphorylation of caldesmon at ser789, the association of phospho-caldesmon (ser789) with phospho-HSP27 results in an essential conformational change leading to dissociation of phospho-caldesmon from tropomyosin. This leads to the sliding of tropomyosin on actin thus exposing the myosin binding sites on actin for actomyosin interaction.

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Signaling pathways mediating gastrointestinal smooth muscle contraction and MLC20 phosphorylation by motilin receptors

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00305.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. G23-G31 ◽

Cited By ~ 52

Author(s):

Jiean Huang ◽

Huiping Zhou ◽

Sunila Mahavadi ◽

Wimolpak Sriwai ◽

Vijay Lyall ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Kinase Inhibitor ◽

Rho Kinase ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Dominant Negative ◽

Phosphatase Inhibitors ◽

Sustained Contraction ◽

Pi Hydrolysis

The signaling cascades initiated by motilin receptors in gastric and intestinal smooth muscle cells were characterized. Motilin bound with high affinity (IC50 0.7 ± 0.2 nM) to receptors on smooth muscle cells; the receptors were rapidly internalized via G protein-coupled receptor kinase 2 (GRK2). Motilin selectively activated Gq and G13, stimulated Gαq-dependent phosphoinositide (PI) hydrolysis and 1,4,5-trisphosphate (IP3)-dependent Ca2+ release, and increased cytosolic free Ca2+. PI hydrolysis was blocked by expression of Gαq minigene and augmented by overexpression of dominant negative RGS4(N88S) or GRK2(K220R). Motilin induced a biphasic, concentration-dependent contraction (EC50 = 1.0 ± 0.2 nM), consisting of an initial peak followed by a sustained contraction. The initial Ca2+-dependent contraction and myosin light-chain (MLC)20 phosphorylation were inhibited by the PLC inhibitor U-73122 and the MLC kinase inhibitor ML-9 but were not affected by the Rho kinase inhibitor Y27632 or the PKC inhibitor bisindolylmaleimide. Sustained contraction and MLC20 phosphorylation were RhoA dependent and mediated by two downstream messengers: PKC and Rho kinase. The latter was partly inhibited by expression of Gαq or Gα13 minigene and abolished by coexpression of both minigenes. Sustained contraction and MLC20 phosphorylation were partly inhibited by Y27632 and bisindolylmaleimide and abolished by a combination of both inhibitors. The inhibition reflected phosphorylation of two MLC phosphatase inhibitors: CPI-17 via PKC and MYPT1 via Rho kinase. We conclude that motilin initiates a Gαq-mediated cascade involving Ca2+/calmodulin activation of MLC kinase and transient MLC20 phosphorylation and contraction as well as a sustained Gαq- and Gα13-mediated, RhoA-dependent cascade involving phosphorylation of CPI-17 by PKC and MYPT1 by Rho kinase, leading to inhibition of MLC phosphatase and sustained MLC20 phosphorylation and contraction.

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Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction

AJP Cell Physiology ◽

10.1152/ajpcell.00253.2008 ◽

2008 ◽

Vol 295 (3) ◽

pp. C576-C587 ◽

Cited By ~ 233

Author(s):

Susan J. Gunst ◽

Wenwu Zhang

Keyword(s):

Extracellular Matrix ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Actin Polymerization ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Mechanical Tension ◽

Muscle Tissues ◽

Cytoskeletal Dynamics

A growing body of data supports a view of the actin cytoskeleton of smooth muscle cells as a dynamic structure that plays an integral role in regulating the development of mechanical tension and the material properties of smooth muscle tissues. The increase in the proportion of filamentous actin that occurs in response to the stimulation of smooth muscle cells and the essential role of stimulus-induced actin polymerization and cytoskeletal dynamics in the generation of mechanical tension has been convincingly documented in many smooth muscle tissues and cells using a wide variety of experimental approaches. Most of the evidence suggests that the functional role of actin polymerization during contraction is distinct and separately regulated from the actomyosin cross-bridge cycling process. The molecular basis for the regulation of actin polymerization and its physiological roles may vary in diverse types of smooth muscle cells and tissues. However, current evidence supports a model for smooth muscle contraction in which contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins at the membrane, and proteins within this complex orchestrate the polymerization and organization of a submembranous network of actin filaments. This cytoskeletal network may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. Better understanding of the physiological function of these dynamic cytoskeletal processes in smooth muscle may provide important insights into the physiological regulation of smooth muscle tissues.

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Insulin acutely increases agonist-induced airway smooth muscle contraction in human and rat

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00232.2020 ◽

2021 ◽

Author(s):

Becky J. Proskocil ◽

Gina N. Calco ◽

Zhenying Nie

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Contraction ◽

Intracellular Calcium ◽

Mrna Expression ◽

Airway Smooth Muscle ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Rat Tracheal Smooth Muscle ◽

Isolated Rat

Obesity increases incidence and severity of asthma but the molecular mechanisms are not completely understood. Hyperinsulinemia potentiates vagally induced bronchoconstriction in obese rats. Since bronchoconstriction results from airway smooth muscle contraction, we tested whether insulin changed agonist-induced airway smooth muscle contraction. Obesity prone and resistant rats were fed a low-fat diet for 5 weeks and treated with insulin (Lantus, 3 units/rat s.c.) 16 h before vagally induced bronchoconstriction was measured. Ex vivo, contractile responses to methacholine were measured in isolated rat tracheal rings and human airway smooth muscle strips before and after incubation (0.5 - 2 h) with 100 nM insulin or 13.1 nM insulin like growth factor-1 (IGF-1). M2 and M3 muscarinic receptor mRNA expression was quantified by qRT-PCR and changes in intracellular calcium were measured in response to methacholine or serotonin in isolated rat tracheal smooth muscle cells treated with 1 µM insulin. Insulin, administered to animals 16 h prior, potentiated vagally induced bronchoconstriction in both obese prone and resistant rats. Insulin, not IGF-1, significantly increased methacholine-induced contraction of rat and human isolated airway smooth muscle. In cultured rat tracheal smooth muscle cells, insulin significantly increased M2, not M3, mRNA expression and enhanced methacholine- and serotonin-induced increase in intracellular calcium. Insulin alone did not cause an immediate increase in intracellular calcium. Thus, insulin, acutely potentiated agonist-induced increase in intracellular calcium and airway smooth muscle contraction. These findings may explain why obese individuals with hyperinsulinemia are prone to airway hyperreactivity and give insights into future targets for asthma treatment.

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HSP27 phosphorylation and interaction with actin-myosin in smooth muscle contraction

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00141.2001 ◽

2002 ◽

Vol 282 (5) ◽

pp. G894-G903 ◽

Cited By ~ 61

Author(s):

Khalil N. Bitar

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Signal Transduction Pathway ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Thin Filament Regulation ◽

Kinase C ◽

Hsp27 Phosphorylation ◽

Myosin Interaction

We have investigated the role of heat shock protein 27 (HSP27) phosphorylation and the association of HSP27 with contractile proteins actin, myosin, and tropomyosin. Smooth muscle cells were labeled with [32P]orthophosphate. C2-ceramide (0.1 μM), an activator of protein kinase C (PKC), induced a sustained increase in HSP27 phosphorylation that was inhibited by calphostin C. C2-ceramide-induced (0.1 μM) sustained colonic smooth muscle cell contraction was accompanied by significant increases in the association of HSP27 with tropomyosin and in the association of HSP27 with actin. The significant increases occurred at 30 s after stimulation and were sustained at 4 min. Contraction was also associated with strong colocalization of HSP27 with tropomyosin and with actin as observed after immunofluorescent labeling of tropomyosin, actin, and HSP27 followed by confocal microscopy. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that phosphorylation of HSP27 could affect myosin association with actin. In conclusion 1) HSP27 phosphorylation appears to be necessary for reorganization of HSP27 inside the cell and seems to be directly correlated with the PKC signal transduction pathway, and 2) agonist-induced phosphorylation of HSP27 modulates actin-myosin interaction through thin-filament regulation of tropomyosin.

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Role of p47phox in regulating Cdc42GAP, vimentin, and contraction in smooth muscle cells

AJP Cell Physiology ◽

10.1152/ajpcell.00324.2009 ◽

2009 ◽

Vol 297 (6) ◽

pp. C1424-C1433 ◽

Cited By ~ 20

Author(s):

Qing-Fen Li ◽

Dale D. Tang

Keyword(s):

Hydrogen Peroxide ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Contraction ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Regulatory Subunit ◽

Ros Generation ◽

Vitro Assay

Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47phox, a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47phox, but not plasmids for luciferase shRNA, inhibited the expression of p47phox. ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47phox shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47phox-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47phox. The knockdown of p47phox inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47phox subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47phox-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.

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Paraoxon Attenuates Vascular Smooth Muscle Contraction through Inhibiting Ca2+Influx in the Rabbit Thoracic Aorta

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/829190 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Shouhong Zhou ◽

Liying Liu ◽

Xuhong Yang ◽

Shujin Wu ◽

Gengrong Chen

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Muscle Contraction ◽

Thoracic Aorta ◽

Muscle Cells ◽

Smooth Muscle Contraction ◽

Inhibitory Effect ◽

Aortic Smooth Muscle ◽

Vascular Smooth Muscle Contraction

We investigated the effect of paraoxon on vascular contractility using organ baths in thoracic aortic rings of rabbits and examined the effect of paraoxon on calcium homeostasis using a whole-cell patch-clamp technique in isolated aortic smooth muscle cells of rabbits. The findings show that administration of paraoxon (30 μM) attenuated thoracic aorta contraction induced by phenylephrine (1 μM) and/or a highK+environment (80 mM) in both the presence and absence of thoracic aortic endothelium. This inhibitory effect of paraoxon on vasoconstrictor-induced contraction was abolished in the absence of extracellularCa2+, or in the presence of theCa2+channel inhibitor, verapamil. But atropine had little effect on the inhibitory effect of paraoxon on phenylephrine-induced contraction. Paraoxon also attenuated vascular smooth muscle contraction induced by the cumulative addition of CaCl2and attenuated an increase of intracellularCa2+concentration induced byK+in vascular smooth muscle cells. Moreover, paraoxon (30 μM) inhibited significantly L-type calcium current in isolated aortic smooth muscle cells of rabbits. In conclusion, our results demonstrate that paraoxon attenuates vasoconstrictor-induced contraction through inhibitingCa2+influx in the rabbits thoracic aorta.

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