AJP - Lung Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 294: L69-L78, 2008. First published November 9, 2007; doi:10.1152/ajplung.00235.2007
1040-0605/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/L69    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Komalavilas, P.
Right arrow Articles by Brophy, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Komalavilas, P.
Right arrow Articles by Brophy, C. M.

The small heat shock-related protein, HSP20, is a cAMP-dependent protein kinase substrate that is involved in airway smooth muscle relaxation

Padmini Komalavilas,1,2 Raymond B. Penn,3 Charles R. Flynn,1,2 Jeffrey Thresher,1 Luciana B. Lopes,1 Elizabeth J. Furnish,1 Manhong Guo,3 Manuel A. Pallero,4 Joanne E. Murphy-Ullrich,4 and Colleen M. Brophy1,2

1Center for Metabolic Biology, Arizona State University, Tempe, and 2Carl T. Hayden VA Medical Center, Phoenix, Arizona; 3Center for Human Genomics, Wake Forest University, Winston-Salem, North Carolina; 4Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 13 June 2007 ; accepted in final form 5 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Activation of the cAMP/cAMP-dependent PKA pathway leads to relaxation of airway smooth muscle (ASM). The purpose of this study was to examine the role of the small heat shock-related protein HSP20 in mediating PKA-dependent ASM relaxation. Human ASM cells were engineered to constitutively express a green fluorescent protein-PKA inhibitory fusion protein (PKI-GFP) or GFP alone. Activation of the cAMP-dependent signaling pathways by isoproterenol (ISO) or forskolin led to increases in the phosphorylation of HSP20 in GFP but not PKI-GFP cells. Forskolin treatment in GFP but not PKI-GFP cells led to a loss of central actin stress fibers and decreases in the number of focal adhesion complexes. This loss of stress fibers was associated with dephosphorylation of the actin-depolymerizing protein cofilin in GFP but not PKI-GFP cells. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, intact strips of bovine ASM were precontracted with serotonin followed by ISO. Activation of the PKA pathway led to relaxation of bovine ASM, which was associated with phosphorylation of HSP20 and dephosphorylation of cofilin. Finally, treatment with phosphopeptide mimetics of HSP20 possessing a protein transduction domain partially relaxed precontracted bovine ASM strips. In summary, ISO-induced phosphorylation of HSP20 or synthetic phosphopeptide analogs of HSP20 decreases phosphorylation of cofilin and disrupts actin in ASM, suggesting that one possible mechanism by which HSP20 mediates ASM relaxation is via regulation of actin filament dynamics.

asthma; actin; protein transduction; cofilin

β-ADRENERGIC RECEPTOR AGONISTS (β-agonists) are widely used for the treatment of bronchospasm associated with acute asthma attacks; however, their molecular mechanisms of action are still not completely defined. β-Agonists stimulate the heterotrimeric G protein Gs, which, in turn, activates adenylyl cyclase, catalyzing the hydrolysis of ATP into cAMP (42). In airway smooth muscle (ASM), elevated cytosolic cAMP levels activate PKA, which phosphorylates several membrane and/or intracellular proteins promoting ASM relaxation and bronchodilation of contracted airways (55). The proposed mechanisms of PKA-induced relaxation include reduction of intracellular Ca2+ levels as well as a reduction of the sensitivity of the contractile apparatus to intracellular calcium (31). Ca2+ desensitization or Ca2+-independent smooth muscle relaxation refers to a decrease in muscle tension at a constant Ca2+ concentration. PKA-mediated phosphorylation of myosin light chain kinase has been attributed to Ca2+ desensitization of the contractile unit (1). It has been proposed that the mechanism of Ca2+ desensitization is via activation of myosin light chain phosphatase (12, 30, 48, 50, 52). However, tonic phase of smooth muscle contraction can occur in the setting where intracellular Ca2+ levels and myosin light chain phosphorylation are at or near basal levels (14, 19, 32, 47). In addition, cyclic nucleotide-dependent smooth muscle relaxation has been shown to be independent of myosin light chain phosphorylation (3, 29, 32, 43, 57). Collectively, these data support the possibility that Ca2+-independent smooth muscle relaxation can occur through mechanisms that are independent of myosin light chain phosphorylation.

Numerous investigations have implicated the cAMP-PKA pathway in regulating the actin cytoskeleton in cultured cells (15, 24, 39, 45). Activation of the PKA pathway has been shown to lead to morphological changes (stellation) that are associated with loss of actin stress fibers. Treatment of cultured rat aortic smooth muscle cells with dibutyryl cAMP leads to rapid, extensive, and reversible changes in their morphology including stellation and disintegration of actin filaments (10). cAMP has been demonstrated to modulate morphology of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway (41). Inhibition of PKA led to increased F-actin content and organization into stress fibers in lung endothelial cells (40). Since tonic contraction of smooth muscle requires an intact actin cytoskeleton (thin filaments), disruption of the actin cytoskeleton is an alternate possible mechanism of smooth muscle relaxation.

A recently identified substrate protein of PKA is the small heat shock-related protein (HSP20) (4, 43). PKA activation leads to increases in the phosphorylation of HSP20 on serine 16 (Ref. 4). HSP20 is highly and constitutively expressed in muscle tissues and shares considerable homology with the family of small heat shock proteins, such as HSP27, myotonic dystrophy kinase binding protein, and the crystallins (28). Agonists that stimulate HSP20 phosphorylation, as well as analogs of phosphorylated HSP20 that contain a transduction domain (allowing for cellular penetration without altering membrane permeability) and the phosphorylated serine 16, induce relaxation of smooth muscle from a variety of different species and types of smooth muscles (4, 17, 18, 43, 53, 56). The mechanism by which HSP20 mediates relaxation is still not well-understood, but several reports have suggested it involves actin cytoskeletal regulation (7, 43, 54, 56). Mesangial cells transfected with wild-type HSP20 had few stress fibers that were localized to the periphery of the cell, whereas mesangial cells transfected with a (phosphorylation-insensitive) Ser16->Ala16 mutant HSP20 had abundant stress fibers throughout the cells that did not change with activation of cAMP pathways (56). Treatment with transducible phospho-HSP20 peptide analogs containing the phosphorylation site and a protein transduction domain led to disruption of actin stress fibers and increases in G-actin in NIH/3T3 cells (16).

Cofilin is an actin-depolymerizing protein that, when phosphorylated, binds to the intracellular scaffolding protein, 14-3-3 (Refs. 5, 21). When displaced from 14-3-3, cofilin becomes dephosphorylated and activated as an actin-depolymerizing protein (20, 38). HSP20 also contains a binding motif for 14-3-3. Using size-exclusion chromatography and chemical cross-linking, Chernik et al. (11) reported direct interaction between human 14-3-3{gamma} and intact phosphorylated HSP20 but not unphosphorylated or mutated (S16D) HSP20 in vitro. Coimmunoprecipitation studies have also shown that 14-3-3 is associated with phospho-cofilin in Swiss 3T3 cells (16). Phospho-cofilin can be displaced from 14-3-3 by spiking the immunoprecipitates with phosphopeptide analogs of HSP20. Hence, one putative mechanism by which phosphorylated HSP20 leads to alterations in cell morphology is via competing with phospho-cofilin for binding to 14-3-3. Taken together, these data suggest that activation of cyclic nucleotide signaling pathways leading to increased phospho-HSP20 may be modulating actin filament dynamics.

To investigate the potential role of HSP20 in PKA-induced ASM relaxation, stable lines of human ASM (HASM) cells expressing green fluorescent protein (GFP) or a GFP chimera of the PKA inhibitory peptide PKI (PKI-GFP; Ref. 22) were generated. These cells were used to determine morphological and biochemical changes in response to PKA stimulation. In addition, intact bovine ASM was used to further elucidate the role of PKA in β-agonist-induced bovine ASM relaxation. Given the established effect of activation of PKA on cytoskeletal dynamics (loss of actin stress fibers), we hypothesized that one of the possible mechanisms of Ca2+-independent smooth muscle relaxation is via a thin filament (actin) regulatory mechanism involving PKA-dependent phosphorylation of HSP20.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Materials. Cell culture reagents including Ham's F-12 media, L-glutamine, BSA, G418, PBS, and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). DTT, HEPES, Trizma base, Triton X-100, formaldehyde, Tween-20, CaCl2, sodium nitroprusside, forskolin (FSK), and isoproterenol (ISO) were purchased from Sigma Chemical (St. Louis, MO). Precast acrylamide gels, SDS, Tris-glycine-SDS buffer (TGS), Tris-glycine (TG), and prestained Precision Plus Protein All Blue Standards were purchased from Bio-Rad (Hercules, CA). Phospho-HSP20 peptides (YARAAARQARAWLRRApSAPLPGLK-COOH) and HSP20 control peptides where the phosphoserine is replaced with alanine (YARAAARQARAWLRRAAAPLPGLK-COOH) were synthesized by UCB Bioproducts-Lonza (Cambridge, MA) and American Peptide (Sunnyvale, CA). Mouse anti-HSP20 antibodies were from Advanced Immunochemical (Long Beach, CA); rabbit anti-phospho Ser16-HSP20 antibodies were generated against the phosphopeptide WLRRApSAPLPGLK (17); mouse anti-vasodilator-stimulated phosphoprotein (VASP) antibodies were from BD Biosciences (San Jose, CA); rabbit anti-PKG antibody was purchased from Stressgen (Victoria, British Columbia, Canada); rabbit anti-actin were from Sigma Aldrich (St. Louis, MO); anti-GAPDH antibodies were obtained from Abcam (Cambridge, MA); rabbit anti-phospho-cofilin-2 (Ser3) antibodies were from Upstate Biotechnology (Charlottesville, VA); and rabbit anti-cofilin-2 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell culture and agonist treatment. HASM lines stably expressing GFP or PKI-GFP were generated by retroviral infection at Wake Forest University and maintained as described previously (22). HASM cultures were grown in Ham's F-12 media (Invitrogen) supplemented with 10% FBS, 25 mM HEPES, 12 mM NaOH, 1.7 mM CaCl2, 100 U/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml G418 (complete media). Cultures were used for 4–5 passages after transfection, and phenotype changes were monitored by the expression of PKG (6). Before treatment with agonists, cells were serum-starved for 24 h in complete media lacking FBS and containing 0.1% BSA. Serum-starved cells plated in 100-mm dishes were stimulated with vehicle (control), 10 µM FSK, or 1 µM ISO for 10 min. Cells were rinsed once with PBS (140 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·7H2O, 1.4 mM KH2PO4, pH 7.4), harvested by scraping, snap-frozen in liquid nitrogen, and stored at –80°C. For stress fiber experiments, cells were plated onto 18-mm cover glasses in 60-mm dishes 1 day before experiment so that they were 65–75% confluent before agonist treatment. Serum-starved cells were treated with agonists, 50 µM phospho-HSP20 peptides or 50 µM HSP20 control peptides (18) as indicated, washed three times in PBS, and fixed for 30 min in 4% (wt/vol) formaldehyde prepared in PBS. Following fixation, cells were washed three times in PBS and then permeabilized in 0.1% (vol/vol) Triton X-100 in PBS for 15 min at room temperature (RT) with gentle rocking and then washed three times in PBS.

Immunofluorescence microscopy and immunoblotting. Formaldehyde-fixed and detergent-permeabilized cells were processed for indirect immunofluorescence microscopy by adding Alexa 568-conjugated phalloidin (1:1,000 in PBS; Invitrogen) and 4,6-diamidino-2-phenylindole (DAPI; 1:1,000) and letting cells gently rock in the dark at RT for 1 h. Following three washes with PBS, labeled cells were viewed using a Zeiss Axiovert 200M epifluorescence microscope (Carl Zeiss, Thornwood, NY), equipped with a Xcite light source, and GFP autofluorescence and Alexa 568-conjugated phalloidin fluorescence images were obtained using filter sets for exciting GFP (excitation 492/18 nm) and Alexa 568 (excitation 572/23 nm) and an AxioCam HR digital camera. Images were acquired and processed using AxioVision 4.5 and Photoshop 7.0 software packages, respectively.

For immunoblot analysis, control and agonist-treated cells were scraped from a 100-mm culture dish into 100-µl UDC buffer (8 M urea, 10 mM DTT, 4% CHAPS), and the samples were rotated for 2 h at RT. Samples were clarified by centrifugation at ~8,000 g for 10 min. Protein concentrations were determined using the Bradford assay (Pierce Chemical, Rockford, IL). Unless indicated otherwise, 20 µg of protein was separated on 4–20% polyacrylamide mini-gels in 1x TGS buffer (25 mM Tris·HCl, pH 8.3, 192 mM glycine, 0.1% wt/vol SDS) at 120 V for 1.5 h. Electrophoretic transfer of proteins from the gels onto polyvinylidene difluoride membranes was carried out in 1x TG buffer (25 mM Tris·HCl, pH 8.3, 192 mM glycine) at 50 V for 12 h at 4°C. The blot was subsequently incubated with one of the following primary antibodies: mouse anti-HSP20 (1:4,000 dilution); rabbit anti-phospho Ser16-HSP20 (1:500); mouse anti-VASP (1:2,000); rabbit anti-PKG (1:1,000); rabbit anti-actin (1:500); anti-GAPDH (1:300); rabbit anti-phospho-cofilin (1:2,000); rabbit anti-cofilin (1:1,000); and either of one corresponding secondary antibodies, Alexa Fluor 680-conjugated affinity-purified goat anti-mouse secondary antibody (Invitrogen) or IRDye800-conjugated affinity-purified goat anti-rabbit secondary antibody (Rockland Scientific, Gilbertsville, PA). Membranes were scanned, and the intensities of selected bands were directly quantified by the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE) as described previously (22).

Focal adhesion measurement. HASM cells were seeded and cultured overnight with transfer to no serum medium (culture medium supplemented with 1 mg/ml BSA) overnight before experimentation. Cells were either untreated or treated with 100 nM Hep I (thrombospondin peptides, positive control), 10 µM FSK, 25 µM phospho-HSP20 peptides, or 25 µM HSP20 control peptides as described above. Cells were fixed and examined (at least 250 cells per condition) for the presence or absence of focal adhesions by interference reflection microscopy. The percentage of cells positive for focal adhesions was determined in a minimum of three independent experiments. A cell was scored positive for focal adhesions if it contained at least five focal adhesions.

Procurement of bovine ASM tissue. Fresh bovine lung was obtained from a local abattoir (Miller's Southwest Processing, Queen Creek, AZ). The tissue was immediately placed in HEPES buffer (10 mM HEPES, pH 7.4; 140 mM NaCl; 4.7 mM KCl; 1.0 mM MgSO4; 1.0 mM NaH2PO4; 1.5 mM CaCl2; and 10 mM glucose) and stored on ice during transfer to the laboratory. Briefly, a secondary airway passage was dissected from bovine lung and cut open longitudinally. The airway was then pinned at each corner to a dissection tray. Epithelium was removed from the smooth muscle tissue by gentle rubbing with a cotton-tipped applicator. A 1-cm wide by 3-cm long strip of ASM (tangential to airflow) was then carefully dissected from the structure of the airway passage. Cross-sectional strips (1.5–2 mm wide) were then cut for use in the muscle bath.

Physiological measurements. Bovine ASM strips were suspended in a muscle bath containing a bicarbonate buffer (120 mM NaCl, 4.7 mM KCl, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM glucose, 1.5 mM CaCl2, and 25 mM Na2HCO3; pH 7.4) and equilibrated with 95% O2-5% CO2 at 37°C. Force measurements were obtained with a Kent Scientific (Litchfield, CT) force transducer (TRN001) interfaced with PowerLab from AD Instruments (Colorado Springs, CO). Data were recorded with Chart software 5.1.1 (AD Instruments). Each strip was washed every 15 min with 37°C bicarbonate buffer for the first hour of equilibration, and the length was progressively adjusted until maximal tension was obtained. The tissue was allowed to equilibrate for an additional 2 h without disturbance before experimentation was initiated. Each strip was initially tested for viability with high extracellular potassium (110 mM KCl with equimolar replacement of NaCl in bicarbonate buffer), the maximal tension obtained was taken as 100%, and tension obtained with agents (agonists, antagonists) was calculated. At the end of the experiments, all strips were washed and contracted with KCl to ensure viability of the tissues.

To determine the role of endogenous HSP20 in bovine ASM relaxation, simultaneous physiological water bath experiments were conducted using identical strips of ASM dissected from the same tissue. These strips were placed in vials of bicarbonate buffer and equilibrated in a physiological water bath with 95% O2-5% CO2 at 37°C for 3 h. The muscle strips were washed with fresh buffer each 15 min for the first hour and then left undisturbed for the subsequent 2 h. Strips were then contracted with 1 µM serotonin. At 5 min after the serotonin dose, one strip was snap-frozen and pulverized in liquid nitrogen, and the other strip was relaxed with 1 µM ISO for 5 min or 10 µM FSK for 5 min. Strips were quick-frozen in the same manner along with an untreated control strip. All samples were stored at –80°C for later analysis using one- or two-dimensional gel electrophoresis.

Determination of HSP20 phosphorylation in bovine ASM tissue. Proteins from frozen muscle strips were extracted in buffer containing 8 M urea, 10 mM DTT, and 4% CHAPS (UDC buffer). The mixtures were vortexed at RT overnight and then centrifuged at 14,000 rpm for 15 min. For one-dimensional separation, equal amounts (20 µg) of proteins were placed in Laemmli sample buffer (Bio-Rad) containing 62.5 mM Tris·HCl, pH 6.8, 25% glycerol, 2% SDS, 5% 2-mercaptoethanol, heated for 5 min at 100°C, and separated by SDS-PAGE as described above. For two-dimensional analysis, 75 µg of extracted proteins were separated by first dimension isoelectric focusing (IEF) using the Protean IEF Cell (Bio-Rad), and second dimension using SDS-PAGE and probed with anti-HSP20 antibodies as described earlier (17).

Statistical analysis. Values are reported as means ± SE. Statistical analysis was performed by unpaired Student's t-test or one-way ANOVA followed by Tukey's test (GraphPad Software, San Diego, CA). The criterion for significance was P < 0.05. Stress values are calculated with the following formula: Stress [newtons (N)/m2] = force (g) x 0.0987/area, where area is equal to the wet weight (milligrams)/length (millimeters at maximal length) divided by 1.055.

RESULTS

Inhibition of PKA inhibits ISO-induced phosphorylation of HSP20 in HASM cells. To determine whether PKA mediates phosphorylation of HSP20 in ASM, HASM cells stably expressing either GFP (control) or the PKI-GFP chimera were generated as described previously (22). VASP phosphorylation by PKA at Ser157 causes a significant mobility shift on one-dimensional SDS-PAGE gels (8) and was used as a marker of intracellular PKA activity in the HASM cells (22). GFP and PKI HASM cells were stimulated with the β-agonist ISO, and the phosphorylation of VASP and HSP20 was determined by immunoblotting. Treatment with 1 µM ISO for 10 min increased VASP phosphorylation in the control GFP cells but not in PKI-GFP cells (Fig. 1, A and B). Similarly, ISO led to increases in the phosphorylation of HSP20 in the control GFP cells but not in PKI-GFP cells (Fig. 1, A and C), suggesting the inhibition of PKA in PKI HASM cells. FSK (10 µM, 10 min), which activates PKA through direct activation of adenylyl cyclase, also led to increases in HSP20 and VASP phosphorylation in GFP cells and not in PKI-GFP cells (Fig. 1, A–C).


Figure 1
View larger version (31K):
[in this window]
[in a new window]

  		Fig. 1. Inhibition of PKA inhibits isoproterenol (ISO)- or forskolin (FSK)-induced phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and small heat shock-related protein HSP20 and dephosphorylation of cofilin in human airway smooth muscle (HASM) cells. HASM cell lines were treated with 1 µM ISO (A; lanes 2 and 4) or 10 µM FSK (A; lanes 6 and 8) for 10 min and snap-frozen. Whole cell lysates (20 µg) of control and ISO- or FSK-treated cells expressing green fluorescent protein (GFP; lanes 1, 2, 5, and 6) or GFP-PKA inhibitory fusion protein (PKI-GFP; lanes 3, 4, 7, and 8) were separated by SDS-PAGE and probed with the indicated antibodies. The positions of VASP and phosphorylated VASP (pVASP) are indicated by arrows on the left. Bands were normalized to actin, and ratios of pVASP-to-VASP (B), pHSP20-to-HSP20 (C), and phospho-cofilin-to-cofilin (D) were determined, n = 4–6; *P < 0.05 with respect to control.

 
PKA inhibition and cofilin dephosphorylation in HASM cells. Cofilin is an actin-depolymerizing protein that is activated upon dephosphorylation by phosphatases such as slingshot and chronophin (20, 38). Phosphorylated cofilin contains a binding motif for the scaffolding protein 14-3-3, and association of cofilin with 14-3-3 protects cofilin from dephosphorylation (21). HSP20 also contains a binding motif for 14-3-3, and binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevents the association of cofilin with 14-3-3 (Ref. 16). Increases in cAMP levels have been demonstrated to cause cofilin dephosphorylation (51). To assess the role of PKA in the regulation of cofilin phosphorylation in ASM, GFP and PKI-GFP HASM cells were treated with either 1 µM ISO for 10 min or 10 µM FSK for 10 min, and phosphorylation of cofilin was analyzed by immunoblotting. FSK or ISO treatment significantly decreased phospho-cofilin levels in control GFP cells but had no effect in PKI-GFP cells (Fig. 1, A and D).

Inhibition of PKA prevents changes in morphology and focal adhesion complexes in HASM cells. To investigate the role of PKA-mediated phosphorylation of HSP20 in the regulation of central actin stress fibers, serum-starved HASM cells were treated with ISO or FSK, and stress fiber formation was analyzed by fluorescence microscopy. Treatment with 1 µM ISO for 30 min or 10 µM FSK for 30 min led to disruption of stress fibers, with only cortical actin remaining, in HASM cells that express GFP alone (Fig. 2, B and C). However, ISO or FSK treatment did not lead to stress fiber disruption in PKI-GFP-expressing cells (Fig. 2, E and F).


Figure 2
View larger version (77K):
[in this window]
[in a new window]

  		Fig. 2. Inhibition of the PKA inhibits ISO- or FSK-mediated stellation and stress fiber disruption in HASM cells. A–F: representative epifluorescence micrographs of HASM control (A and D), 1 µM ISO for 30 min (B and E), or 10 µM FSK for 30 min (C and F) cells expressing GFP (A–C) or PKI-GFP (D–F). Cells grown for 24 h in serum-free medium were treated with the agents and then washed, processed for microscopy, and labeled with Alexa 568-conjugated phalloidin to reveal the actin cytoskeleton and 4,6-diamidino-2-phenylindole (DAPI) to reveal nucleus; bar = 50 µm.

 
Loss of stress fibers has been associated with a loss of focal adhesion complexes (2). To examine the effect of PKA on focal adhesion complexes, HASM cells were treated with FSK (10 µM, 30 min), and loss of focal adhesions were measured using interference reflection microscopy. Hep I peptide (from thrombospondin-1 and -2 with focal adhesion-labilizing activity; Ref. 36) was used as a positive control. Hep I treatment led to decreases in focal adhesions in both GFP and PKI-GFP cells (Fig. 3A). Hep I activates cGMP pathways leading to activation of PKG (37). PKG is not inhibited by PKI. FSK stimulation led to decreases in focal adhesions in GFP cells but not in PKI-GFP cells (Fig. 3A).


Figure 3
View larger version (48K):
[in this window]
[in a new window]

  		Fig. 3. pHSP20 peptide causes stress fiber disruption and focal adhesion (FA) changes in GFP- as well as PKI-GFP-expressing HASM cells. HASM cells were grown as described in MATERIALS AND METHODS. A: cells were serum-starved overnight and either untreated (control) or treated with 100 nM Hep I (thrombospondin peptides, positive control), 10 µM FSK, or 25 µM pHSP20 peptide (HSP20 peptide) containing a protein transduction domain and the active phosphorylation site of HSP20 or HSP20 control peptide (HSP20 control) for 30 min. Cells were fixed and examined (at least 250 cells per condition) for the presence or absence of FA by interference reflection microscopy. The percentage of cells positive for FA was determined in 5 independent experiments (n = 5; *P < 0.05). B–G: representative epifluorescence micrographs of HASM control (B and E), 50 µM pHSP20 peptide (C and F), and 50 µM HSP20 control peptide (D and G) for 24 h in cells expressing GFP (B–D) or PKI-GFP (E–G). Cells grown for 24 h in serum-free medium were treated with the agents and then washed, processed for microscopy, and labeled with Alexa 568-conjugated phalloidin to reveal the actin cytoskeleton and DAPI to reveal nucleus; bar = 50 µm. H: HASM cell lines were untreated (C; lanes 1 and 4) or treated with 50 µM pHSP20 peptide for 30 min or 24 h (lanes 2, 3, 5, and 6) and snap-frozen. Whole cell lysates (20 µg) of control and treated cells expressing GFP (H; lanes 1–3) or PKI-GFP (H; lanes 4–6) were separated by SDS-PAGE and probed with the indicated antibodies. I: densitometric measurements of the bands, which were normalized to GAPDH, and ratio of phospho-cofilin-to-cofilin were determined and plotted along with results obtained from treatment of GFP and PKI-GFP cells with HSP20 control peptides. n = 3; *P < 0.05 with respect to control.

 
To examine the role of HSP20 phosphorylation in the regulation of cell morphology and focal adhesion disassembly, HASM cells were treated with a transducible synthetic phosphopeptide analog of HSP20 containing the active phosphorylation site of HSP20 (serine 16, which is phosphorylated during active relaxation; Ref. 4; WLRRApSAPLPGLK) attached to a protein transduction domain (YARAAARQARA) or a control peptide where phosphoserine is replaced with alanine and stress fiber formations and focal adhesion disassemblies were measured. Treatment with the phospho-HSP20 peptides led to changes in morphology, loss of stress fibers, and decreases in focal adhesion complexes (Fig. 3, A–G) in both GFP- and PKI-GFP-expressing cells. There were no changes in morphology, stress fibers, or focal adhesion complexes in cells treated with the peptide controls. To directly assess the role of phospho-HSP20 peptides in the regulation of cofilin phosphorylation, GFP and PKI-GFP HASM cells were treated with 50 µM phospho-HSP20 peptides or control peptides for either 30 min or 24 h and phosphorylation of cofilin was analyzed by immunoblotting. Phospho-HSP20 peptide treatment significantly decreased phospho-cofilin levels in control GFP as well as in PKI-GFP cells (Fig. 3, H and I). There were no decreases in phospho-cofilin levels in cells treated with the HSP20 control peptides (Fig. 3I). Taken together, these data suggest that the effects of the phospho-HSP20 peptides are downstream of PKA.

Stimulation of HSP20 phosphorylation with ISO in intact bovine ASM. To confirm that phosphorylated HSP20 plays a role in PKA-induced ASM relaxation, isolated intact strips of bovine ASM were equilibrated in a muscle bath. The strips were precontracted with serotonin (5-hydroxytryptamine, 5-HT) and then treated with ISO, with force generation continuously recorded. Parallel strips were similarly treated and snap-frozen, and phosphorylation of HSP20 was subsequently analyzed by immunoblotting. Serotonin treatment led to a dose-dependent increase in force, and ISO induced a dose-dependent relaxation in bovine ASM (Fig. 4, A and B). When related to 110 mM KCl-induced contraction, 0.1, 1, and 10 µM serotonin generated 6.3% ± 1%, 25.1% ± 5.5%, and 62.7% ± 15.4%, respectively. Two-dimensional gel electrophoresis and Western blot analysis demonstrate increases in the phosphorylation of HSP20 in response to ISO stimulation (Fig. 4C).


Figure 4
View larger version (30K):
[in this window]
[in a new window]

  		Fig. 4. HSP20 phosphopeptide treatment leads to relaxation of bovine ASM. Isolated bovine ASM strips were suspended in a muscle bath and equilibrated for 2 h in bicarbonate buffer as described in MATERIALS AND METHODS. The muscle strips were treated with cumulative log doses (0.01–1 µM) of serotonin (Ser) followed by cumulative log doses (0.01–1 µM) of ISO, and force generated was recorded. A representative tracing for force generation with Ser and ISO is shown (A). Cumulative data were obtained when the force was converted to stress, and decrease in stress minus that of the untreated control was converted to a percentage of the maximal Ser (1–5 µM) contraction and plotted against the concentration of ISO (means ± SE, n = 3; *P < 0.05; B). Bovine ASM strips were equilibrated in a water bath and contracted with 1 µM Ser for 5 min, followed by 1 µM ISO for 5 min. The strips were quick-frozen and pulverized, and proteins were analyzed by 2-dimensional (2-D) gel electrophoresis and immunoblotting. Representative section of 2-D blot (pH 4–7) is shown (C) with untreated control, Ser-treated tissue, and Ser + ISO. Arrow indicates phosphorylation of HSP20 (pHSP20). These blots are representative of 3 separate experiments. D: bovine ASM strips were equilibrated in a muscle bath as described above and precontracted with 5 µM Ser. The strips were then relaxed with increasing doses (0.1–2 mM) of pHSP20 peptide (P20 peptide) or HSP20 control peptide analogs. The percent contraction was calculated as above and plotted against HSP20 peptide or control peptide concentration (means ± SE, n = 3; *P = 0.034 at 2 mM P20 peptide compared with 2 mM control peptide).

 
To examine the direct role of HSP20 phosphorylation on ASM relaxation, bovine ASM strips were precontracted with either serotonin (1 or 5 µM) or increasing doses of carbachol (0.1, 0.5, and 5 µM) and treated with phospho-HSP20 peptides or HSP20 control peptides. Transduction of serotonin precontracted strips of bovine ASM with phospho-HSP20 peptides led to a dose-dependent decrease in stress, which was significant at 2 mM phospho-HSP20 peptide (45.7% ± 7.6% compared with 90.0% ± 1.4%), whereas there was no significant decrease in stress with HSP20 control peptides (Fig. 4D). The decreases in stress generated by the phospho-HSP20 peptide with respect to maximum KCl contraction were 41.5% ± 3.6%, 39.2% ± 1.7%, 35.7% ± 1.7%, 29.3% ± 3.2%, and 20.2% ± 2.4% for 0, 0.1, 0.5, 1, and 2 mM phospho-HSP20 peptide, respectively. Similarly, phospho-HSP20 peptides, and not the control peptides, led to a dose-dependent decrease in stress in carbachol precontracted strips (Fig. 5, A–C). However, the peptide analogs did not lead to complete relaxation of bovine ASM. Phospho-HSP20 peptides were more effective on strips contracted with lower concentration of carbachol (0.1 µM) that generated 40–60% of KCl contraction compared with the higher concentration (0.5 or 5 µM) that generated 80–120% of KCl contraction. When bovine ASM was contracted with 0.5 µM carbachol, significant relaxation was achieved only at 4 mM phospho-HSP20 peptide compared with control peptide (75.11% ± 3.5% and 97.63% ± 3.4%, respectively), whereas at 0.1 µM carbachol, significant relaxation was achieved at 3 and 4 mM phospho-HSP20 peptide (55.57% ± 9.7% and 42.5% ± 12.3% for 3 and 4 mM HSP20 peptide compared with 96.9% ± 3.0% and 97.6% ± 3.4% for 3 and 4 mM control peptide). There was no significant difference in the response of the control peptide to 0.1, 0.5, or 5 µM carbachol (data not shown).


Figure 5
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 5. HSP20 phosphopeptide treatment leads to relaxation of carbachol (CCh)-contracted bovine ASM. A and B: bovine ASM strips were equilibrated in a muscle bath as described in Fig. 4 legend and precontracted with increasing doses of CCh ("a" = 0.01 µM, "b" = 0.05 µM, and "c" = 0.1 µM). The strips were then relaxed with increasing doses ("d" = 0.5 mM, "e" = 1 mM, "f" = 2 mM, "g" = 3 mM, and "h" = 4 mM) of P20 peptide analogs or HSP20 control (P20 control) peptide analogs. C: cumulative data obtained from the effect of P20 peptide when bovine ASM strips were contracted with 0.1, 0.5, or 5 µM doses of CCh. The percent contraction was calculated as in Fig. 4 and plotted against pHSP20 peptide or HSP20 control peptide concentration (means ± SE, n = 3 for each concentration of CCh; *P < 0.001 when P20 control is compared with 0.1 µM + 3 and 4 mM P20 peptide and P < 0.05 when P20 control is compared with 0.5 µM + 4 mM P20 peptide).

 
To determine whether the activation of PKA led to changes in cofilin phosphorylation, bovine ASM strips were precontracted with 5 µM serotonin and treated with 1 µM ISO or 10 µM FSK for 5 min, and the phosphorylation of cofilin was analyzed by SDS-PAGE and Western blotting. Activation of PKA by ISO or FSK led to a significant decrease in the phosphorylation of cofilin (Fig. 6, A and B).


Figure 6
View larger version (23K):
[in this window]
[in a new window]

  		Fig. 6. Isoproterenol (IPT) and FSK reduce the levels of phospho-cofilin in bovine ASM. Isolated bovine ASM strips were suspended in a muscle bath and equilibrated for 3 h in bicarbonate buffer as described in MATERIALS AND METHODS. Bovine ASM strips were contracted with 5 µM Ser for 5 min, 5 µM Ser for 5 min followed by 1 µM IPT, or 5 µM Ser for 5 min followed by 10 µM FSK for 5 min. The strips were quick-frozen and pulverized, and the proteins were separated by SDS-PAGE and probed with the indicated antibodies. A shows a representative blot. Bands were normalized to β-actin, and ratio of phospho-cofilin-to-cofilin (B) was determined; n = 3; *P < 0.05 with respect to Ser alone. 5-HT, 5-hydroxytryptamine.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In this investigation, we used cultured HASM cells that expressed a peptide inhibitor of PKA to assess events downstream of PKA signaling. Treatment of HASM cells with activators of PKA led to increases in the phosphorylation of the PKA substrates VASP and HSP20 in cells expressing GFP alone but not in cells expressing PKI-GFP. This was associated with distinct morphological changes, decreases in actin stress fibers, and focal adhesion complexes in the GFP but not in PKI-GFP cells. There were decreases in the phosphorylation of the actin-depolymerizing protein cofilin in the GFP but not in PKI-GFP cells. Hence, one possible mechanism to explain the loss of actin stress fibers and focal adhesions is activation of cofilin. PKA inhibition has been demonstrated earlier in HASM cells expressing PKI, where treatment with ISO or FSK caused only a small percentage of VASP to shift relative to that observed for the GFP-expressing lines, and agonist-stimulated cAMP response element (CRE)-luciferase activity in these cells was abolished (22).

In this study, we demonstrate that the phosphopeptide analogs of HSP20 lead to loss of stress fibers, decreases in focal adhesion complexes, and decreases in cofilin phosphorylation in both GFP- and PKI-GFP-expressing HASM cells. These data suggest that the events upstream of PKA can be bypassed by directly introducing phosphopeptide analogs of at least one of the substrates of PKA and that the mechanism of action may be competition for binding to the scaffolding protein 14-3-3. However, the effect of phospho-HSP20 peptides on cofilin dephosphorylation was more pronounced in PKI-GFP compared with GFP-expressing cells, suggesting that other pathways may also be involved in dephosphorylation of cofilin or that the expression of PKI led to alterations in the expression or activity of other signaling events. ISO-induced actin depolymerization in ASM cells has been reported earlier to be by PKA-dependent as well as PKA-independent pathways involving Src kinase and Gs (24, 25). Cofilin is an actin-depolymerizing protein that, when phosphorylated, binds to the intracellular scaffolding protein, 14-3-3 (Refs. 5, 21). When displaced from 14-3-3, cofilin becomes dephosphorylated and activated as an actin-depolymerizing protein (20, 38). Binding of 14-3-3 protein to phosphopeptide analogs of HSP20 prevents the association of cofilin with 14-3-3 (Ref. 16). It is likely that cyclic nucleotide-dependent relaxation of smooth muscles includes multiple and redundant pathways and mechanisms.

β-Agonist- or nitrovasodilator-induced phosphorylation of HSP20 has been demonstrated to mediate relaxation of vascular smooth muscle from various tissues (4, 17, 18, 43). Phosphorylation of HSP20 also inhibits agonist-mediated contraction, intimal hyperplasia, and platelet aggregation (33, 53, 57). Here, we have also demonstrated that ISO-induced relaxation of intact bovine ASM is associated with phosphorylation of HSP20 (Fig. 4C). Phosphopeptide analogs of HSP20 also relaxed bovine ASM, demonstrating a direct role for HSP20 in ASM relaxation (Figs. 4D and 5). However, the analogs did not completely relax ASM. This may be due to the fact that the phosphopeptide analogs do not contain the complete structure of the HSP20 molecule and hence require higher concentrations than the intact, phosphorylated HSP20 molecule (17). Another possibility is that PKA phosphorylates other proteins to enable more complete relaxation. For example, β2-adrenergic relaxation of ASM has been associated with PKA-dependent and -independent regulation of large-conductance, calcium-activated potassium channels resulting in hyperpolarization as well as mechanisms involving calcium sensitivity of the contractile elements due to activation of myosin light chain phosphatase (31). The effect of ISO in nonhuman airways has also been demonstrated to involve enhanced Ca2+ pump activity to decrease [Ca2+]i and myosin light chain phosphatase activation to decrease Ca2+ sensitivity of the contractile apparatus (27). Activation of PKA by ISO or FSK led to a significant decrease in the phosphorylation of cofilin in bovine ASM. These results suggest that ISO-induced PKA-mediated phosphorylation of HSP20 may induce actin depolymerization through cofilin dephosphorylation in ASM, and this may be one of the mechanisms for Ca2+-independent relaxation of ASM along with the other mechanisms such as Ca2+ desensitization and myosin phosphatase activation.

β-Agonists are the most commonly used therapy for relief of acute bronchospasm in asthmatics. It is widely assumed that β-agonists mediate their effect primarily by increasing cAMP concentration through activation of β2-adrenergic receptor-adenylyl cyclase pathway. Several mediators of inflammation and therapies such as β-agonists themselves can cause alterations in β2-adrenergic receptor responsiveness in a time-dependent and cell-specific manner (42). Agonist-specific desensitization may also be responsible for the loss of prophylactic bronchoprotection and deterioration of asthma control clinically observed with regular use of β-agonists (13, 46, 49). Several investigations have also identified that airway inflammation or cytokine treatment contributes to β2-adrenergic receptor dysfunction and a loss of the relaxant effect of β-agonists in ASM cells, tissues, and in vivo models (9, 23, 34, 35). Adverse effects have been documented with inhaled β2-adrenergic agonist in patients with a genetic polymorphism that results in homozygosity for arginine rather than glycine at amino acid residue 16 of the β2-adrenergic receptor (26). A recent meta-analysis that compares anticholinergics and β2-adrenergic agonists indicated that treatment with β2-adrenergic agonists led to a twofold increase in respiratory deaths (44). Collectively, these studies suggest that regulation of proximal transmembrane signaling events in the β2-adrenergic receptor-Gs-adenylyl cyclase pathway may limit the efficacy of β-agonist therapy. In the present study, we demonstrate that β-agonist-induced relaxation of ASM is associated with increases in the phosphorylation of HSP20. In addition, phosphopeptide analogs of HSP20 linked to a transduction domain can produce the same morphological and physiological responses as do activation of the cAMP-PKA pathway. Thus transducible peptide analogs of HSP20 that act in the same manner as the physiological downstream PKA effector HSP20 represent a potential treatment approach to bronchospasm capable of overcoming problems associated with β2-adrenergic receptor desensitization.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant RO1-HL-58027 and a VA Merit Review Award (C. M. Brophy), National Heart, Lung, and Blood Institute Grant RO1-HL-58506 (R. B. Penn), and Research Facilities Improvement Program Grant C06 RR from the National Center for Research Resources (J. E. Murphy-Ullrich).


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
HSP20 technology was developed at Arizona State University and licensed to Orthologic. P. Komalavilas owns stock of Orthologic and received cash distribution from Arizona Engineered Therapeutics (AzERx) associated with its sale to Orthologic. C. R. Flynn is a consultant for and owns stock of Orthologic. J. Thresher owns stock of Orthologic. E. J. Furnish was a consultant for and owns stock of Orthologic, has one patent issued and several pending, and received royalties from Arizona State University, and received cash distribution from AzERx associated with its sale to Orthologic. C. M. Brophy is a consultant for and owns stock of Orthologic and has patents pending from Arizona State University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Komalavilas, Center for Metabolic Biology, College of Liberal Arts and Sciences, Arizona State University, PO Box 873704, Tempe, AZ 85287-3704 (e-mail: padmini.komalavilas{at}asu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Adelstein RS, Conti MA, Hathaway DR, Klee CB. Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosine 3': 5'-monophosphate-dependent protein kinase. J Biol Chem 253: 8347–8350, 1978.[Abstract/Free Full Text]
  2. Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N, Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275: 1308–1311, 1997.[Abstract/Free Full Text]
  3. Barany M, Barany K. Dissociation of relaxation and myosin light chain dephosphorylation in porcine uterine muscle. Arch Biochem Biophys 305: 202–204, 1993.[CrossRef][Web of Science][Medline]
  4. Beall A, Bagwell D, Woodrum D, Stoming TA, Kato K, Suzuki A, Rasmussen H, Brophy CM. The small heat shock-related protein, HSP20, is phosphorylated on serine 16 during cyclic nucleotide-dependent relaxation. J Biol Chem 274: 11344–11351, 1999.[Abstract/Free Full Text]
  5. Birkenfeld J, Betz H, Roth D. Identification of cofilin and LIM-domain-containing protein kinase 1 as novel interaction partners of 14-3-3 zeta. Biochem J 369: 45–54, 2003.[CrossRef][Web of Science][Medline]
  6. Boerth NJ, Dey NB, Cornwell TL, Lincoln TM. Cyclic GMP-dependent protein kinase regulates vascular smooth muscle cell phenotype. J Vasc Res 34: 245–259, 1997.[Web of Science][Medline]
  7. Brophy CM, Dickinson M, Woodrum D. Phosphorylation of the small heat shock-related protein, HSP20, in vascular smooth muscles is associated with changes in the macromolecular associations of HSP20. J Biol Chem 274: 6324–6329, 1999.[Abstract/Free Full Text]
  8. Butt E, Abel K, Krieger M, Palm D, Hoppe V, Hoppe J, Walter U. cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem 269: 14509–14517, 1994.[Abstract/Free Full Text]
  9. Callaerts-Vegh Z, Evans KL, Dudekula N, Cuba D, Knoll BJ, Callaerts PF, Giles H, Shardonofsky FR, Bond RA. Effects of acute and chronic administration of beta-adrenoceptor ligands on airway function in a murine model of asthma. Proc Natl Acad Sci USA 101: 4948–4953, 2004.[Abstract/Free Full Text]
  10. Chaldakov GN, Nabika T, Nara Y, Yamori Y. Cyclic AMP- and cytochalasin B-induced arborization in cultured aortic smooth muscle cells: its cytopharmacological characterization. Cell Tissue Res 255: 435–442, 1989.[Web of Science][Medline]
  11. Chernik IS, Seit-Nebi AS, Marston SB, Gusev NB. Small heat shock protein Hsp20 (HspB6) as a partner of 14-3-3gamma. Mol Cell Biochem 295: 9–17, 2007.[CrossRef][Web of Science][Medline]
  12. Choudhury N, Khromov AS, Somlyo AP, Somlyo AV. Telokin mediates Ca2+-desensitization through activation of myosin phosphatase in phasic and tonic smooth muscle. J Muscle Res Cell Motil 25: 657–665, 2004.[CrossRef][Web of Science][Medline]
  13. Cockcroft DW, McParland CP, Britto SA, Swystun VA, Rutherford BC. Regular inhaled salbutamol and airway responsiveness to allergen. Lancet 342: 833–837, 1993.[CrossRef][Web of Science][Medline]
  14. Dillon PF, Aksoy MO, Driska SP, Murphy RA. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211: 495–497, 1981.[Abstract/Free Full Text]
  15. Dong JM, Leung T, Manser E, Lim L. cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha. J Biol Chem 273: 22554–22562, 1998.[Abstract/Free Full Text]
  16. Dreiza CM, Brophy CM, Komalavilas P, Furnish EJ, Joshi L, Pallero MA, Murphy-Ullrich JE, von Rechenberg M, Ho YS, Richardson B, Xu N, Zhen Y, Peltier JM, Panitch A. Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics. FASEB J 19: 261–263, 2005.[Abstract/Free Full Text]
  17. Flynn CR, Brophy CM, Furnish EJ, Komalavilas P, Tessier D, Thresher J, Joshi L. Transduction of phosphorylated heat shock-related protein 20, HSP20, prevents vasospasm of human umbilical artery smooth muscle. J Appl Physiol 98: 1836–1845, 2005.[Abstract/Free Full Text]
  18. Flynn CR, Komalavilas P, Tessier D, Thresher J, Niederkofler EE, Dreiza CM, Nelson RW, Panitch A, Joshi L, Brophy CM. Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle. FASEB J 17: 1358–1360, 2003.[Abstract/Free Full Text]
  19. Gerthoffer WT, Murphy RA. Myosin phosphorylation and regulation of cross-bridge cycle in tracheal smooth muscle. Am J Physiol Cell Physiol 244: C182–C187, 1983.[Abstract/Free Full Text]
  20. Gohla A, Birkenfeld J, Bokoch GM. Chronophin, a novel HAD-type serine protein phosphatase, regulates cofilin-dependent actin dynamics. Nat Cell Biol 7: 21–29, 2005.[CrossRef][Web of Science][Medline]
  21. Gohla A, Bokoch GM. 14-3-3 Regulates actin dynamics by stabilizing phosphorylated cofilin. Curr Biol 12: 1704–1710, 2002.[CrossRef][Web of Science][Medline]
  22. Guo M, Pascual RM, Wang S, Fontana MF, Valancius CA, Panettieri RA Jr, Tilley SL, Penn RB. Cytokines regulate beta-2-adrenergic receptor responsiveness in airway smooth muscle via multiple PKA- and EP2 receptor-dependent mechanisms. Biochemistry 44: 13771–13782, 2005.[CrossRef][Web of Science][Medline]
  23. Hakonarson H, Herrick DJ, Serrano PG, Grunstein MM. Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J Clin Invest 99: 117–124, 1997.[Web of Science][Medline]
  24. Hirshman CA, Zhu D, Panettieri RA, Emala CW. Actin depolymerization via the β-adrenoceptor in airway smooth muscle cells: a novel PKA-independent pathway. Am J Physiol Cell Physiol 281: C1468–C1476, 2001.[Abstract/Free Full Text]
  25. Hirshman CA, Zhu D, Pertel T, Panettieri RA, Emala CW. Isoproterenol induces actin depolymerization in human airway smooth muscle cells via activation of an Src kinase and GS. Am J Physiol Lung Cell Mol Physiol 288: L924–L931, 2005.[Abstract/Free Full Text]
  26. Israel E, Chinchilli VM, Ford JG, Boushey HA, Cherniack R, Craig TJ, Deykin A, Fagan JK, Fahy JV, Fish J. Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial. Lancet 364: 1505–1512, 2004.[CrossRef][Web of Science][Medline]
  27. Janssen LJ, Tazzeo T, Zuo J. Enhanced myosin phosphatase and Ca2+-uptake mediate adrenergic relaxation of airway smooth muscle. Am J Respir Cell Mol Biol 30: 548–554, 2004.[Abstract/Free Full Text]
  28. Kato K, Goto G, Inaguma Y, Hasegawa K, Morishita R, Asano T. Purification and characterization of a 20-kDa protein that is highly homologous to alphaB-crystallin. J Biol Chem 269: 15302–15309, 1994.[Abstract/Free Full Text]
  29. Katoch SS. Reversal of endothelin-1-induced contractions by isoproterenol without myosin dephosphorylation in tracheal smooth muscle. Indian J Exp Biol 30: 252–254, 1992.[Medline]
  30. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Okawa K, Iwamatsu A, Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science 273: 245–248, 1996.[Abstract]
  31. Kotlikoff MI, Kamm KE. Molecular mechanisms of beta-adrenergic relaxation of airway smooth muscle. Annu Rev Physiol 58: 115–141, 1996.[CrossRef][Web of Science][Medline]
  32. McDaniel NL, Chen XL, Singer HA, Murphy RA, Rembold CM. Nitrovasodilators relax arterial smooth muscle by decreasing [Ca2+]i and uncoupling stress from myosin phosphorylation. Am J Physiol Cell Physiol 263: C461–C467, 1992.[Abstract/Free Full Text]
  33. McLemore EC, Tessier DJ, Flynn CR, Furnish EJ, Komalavilas P, Thresher JS, Joshi L, Stone WM, Fowl RJ, Brophy CM. Transducible recombinant small heat shock-related protein, HSP20, inhibits vasospasm and platelet aggregation. Surgery 136: 573–578, 2004.[CrossRef][Web of Science][Medline]
  34. Moore PE, Lahiri T, Laporte JD, Church T, Panettieri RA Jr, Shore SA. Selected contribution: synergism between TNF-{alpha} and IL-1β in airway smooth muscle cells: implications for β-adrenergic responsiveness. J Appl Physiol 91: 1467–1474, 2001.[Abstract/Free Full Text]
  35. Motojima S, Yukawa T, Fukuda T, Makino S. Changes in airway responsiveness and beta- and alpha-1-adrenergic receptors in the lungs of guinea pigs with experimental asthma. Allergy 44: 66–74, 1989.[CrossRef][Web of Science][Medline]
  36. Murphy-Ullrich JE, Gurusiddappa S, Frazier WA, Hook M. Heparin-binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity. J Biol Chem 268: 26784–26789, 1993.[Abstract/Free Full Text]
  37. Murphy-Ullrich JE, Pallero MA, Boerth N, Greenwood JA, Lincoln TM, Cornwell TL. Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly. J Cell Sci 109: 2499–2508, 1996.[Abstract]
  38. Niwa R, Nagata-Ohashi K, Takeichi M, Mizuno K, Uemura T. Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin. Cell 108: 233–246, 2002.[CrossRef][Web of Science][Medline]
  39. Ohara O, Nakano T, Teraoka H, Arita H. cAMP negatively regulates mRNA levels of actin and tropomyosin in rat cultured vascular smooth muscle cells. J Biochem (Tokyo) 109: 834–839, 1991.[Abstract/Free Full Text]
  40. Patterson CE, Lum H, Schaphorst KL, Verin AD, Garcia JG. Regulation of endothelial barrier function by the cAMP-dependent protein kinase. Endothelium 7: 287–308, 2000.[Web of Science][Medline]
  41. Pelletier S, Julien C, Popoff MR, Lamarche-Vane N, Meloche S. Cyclic AMP induces morphological changes of vascular smooth muscle cells by inhibiting a Rac-dependent signaling pathway. J Cell Physiol 204: 412–422, 2005.[CrossRef][Web of Science][Medline]
  42. Penn RB, Benovic JL. Regulation of G protein-coupled receptors. In: Handbook of Physiology. Endocrinology. Cellular Endocrinology, edited by Conn PM. Oxford, UK: Oxford Univ. Press, sect. 7, vol. I, 1998, p. 125–164.
  43. Rembold CM, Foster DB, Strauss JD, Wingard CJ, Eyk JE. cGMP-mediated phosphorylation of heat shock protein 20 may cause smooth muscle relaxation without myosin light chain dephosphorylation in swine carotid artery. J Physiol 524: 865–878, 2000.[Abstract/Free Full Text]
  44. Salpeter SR, Buckley NS, Salpeter EE. Meta-analysis: anticholinergics, but not beta-agonists, reduce severe exacerbations and respiratory mortality in COPD. J Gen Intern Med 21: 1011–1019, 2006.[CrossRef][Web of Science][Medline]
  45. Sandau KB, Gantner F, Brune B. Nitric oxide-induced F-actin disassembly is mediated via cGMP, cAMP, and protein kinase A activation in rat mesangial cells. Exp Cell Res 271: 329–336, 2001.[CrossRef][Web of Science][Medline]
  46. Sears MR, Taylor DR, Print CG, Lake DC, Li QQ, Flannery EM, Yates DM, Lucas MK, Herbison GP. Regular inhaled beta-agonist treatment in bronchial asthma. Lancet 336: 1391–1396, 1990.[CrossRef][Web of Science][Medline]
  47. Silver PJ, Stull JT. Regulation of myosin light chain and phosphorylase phosphorylation in tracheal smooth muscle. J Biol Chem 257: 6145–6150, 1982.[Free Full Text]
  48. Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 1325–1358, 2003.[Abstract/Free Full Text]
  49. Spitzer WO, Suissa S, Ernst P, Horwitz RI, Habbick B, Cockcroft D, Boivin JF, McNutt M, Buist AS, Rebuck AS. The use of beta-agonists and the risk of death and near death from asthma. N Engl J Med 326: 501–506, 1992.[Abstract]
  50. Surks HK, Mochizuki N, Kasai Y, Georgescu SP, Tang KM, Ito M, Lincoln TM, Mendelsohn ME. Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Ialpha. Science 286: 1583–1587, 1999.[Abstract/Free Full Text]
  51. Takuma T, Ichida T, Yokoyama N, Tamura S, Obinata T. Dephosphorylation of cofilin in parotid acinar cells. J Biochem (Tokyo) 120: 35–41, 1996.[Abstract/Free Full Text]
  52. Tang DC, Stull JT, Kubota Y, Kamm KE. Regulation of the Ca2+ dependence of smooth muscle contraction. J Biol Chem 267: 11839–11845, 1992.[Abstract/Free Full Text]
  53. Tessier DJ, Komalavilas P, Liu B, Kent CK, Thresher JS, Dreiza CM, Panitch A, Joshi L, Furnish E, Stone W, Fowl R, Brophy CM. Transduction of peptide analogs of the small heat shock-related protein HSP20 inhibits intimal hyperplasia. J Vasc Surg 40: 106–114, 2004.[CrossRef][Web of Science][Medline]
  54. Tessier DJ, Komalavilas P, Panitch A, Joshi L, Brophy CM. The small heat shock protein (HSP) 20 is dynamically associated with the actin cross-linking protein actinin. J Surg Res 111: 152–157, 2003.[CrossRef][Web of Science][Medline]
  55. Thirstrup S. Control of airway smooth muscle tone: II-pharmacology of relaxation. Respir Med 94: 519–528, 2000.[CrossRef][Web of Science][Medline]
  56. Woodrum D, Pipkin W, Tessier D, Komalavilas P, Brophy CM. Phosphorylation of the heat shock-related protein, HSP20, mediates cyclic nucleotide-dependent relaxation. J Vasc Surg 37: 874–881, 2003.[CrossRef][Web of Science][Medline]
  57. Woodrum DA, Brophy CM, Wingard CJ, Beall A, Rasmussen H. Phosphorylation events associated with cyclic nucleotide-dependent inhibition of smooth muscle contraction. Am J Physiol Heart Circ Physiol 277: H931–H939, 1999.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
S. J. Gunst and W. Zhang
Actin cytoskeletal dynamics in smooth muscle: a new paradigm for the regulation of smooth muscle contraction
Am J Physiol Cell Physiol, September 1, 2008; 295(3): C576 - C587.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
294/1/L69    most recent
00235.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Komalavilas, P.
Right arrow Articles by Brophy, C. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Komalavilas, P.
Right arrow Articles by Brophy, C. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.