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. Author manuscript; available in PMC: 2008 Jan 1.
Published in final edited form as: Am J Physiol Lung Cell Mol Physiol. 2006 Sep 22;292(1):L240–L248. doi: 10.1152/ajplung.00199.2006

Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase upon acetylcholine activation of airway smooth muscle

Ruping Wang 1, Qing-Fen Li 1, Yana Anfinogenova 1, Dale D Tang 1,✉
PMCID: PMC1769421  NIHMSID: NIHMS12389  PMID: 16997882

Abstract

The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, stimulation of tracheal smooth muscle strips with acetylcholine (ACh) resulted in the increase in ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly) in association with force development. Activation with ACh also induced vimentin phosphorylation at Ser-56 as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips upon contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The decrease in distribution of CAS in cytoskeletal vimentin elicited by ACh was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble vimentin to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated the dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during acetylcholine activation of tracheal smooth muscle tissues.

Keywords: Intermediate filaments, CAS, cytoskeleton, smooth muscle, contraction

Introduction

The cytoskeletal systems of smooth muscle cells are comprised of myofilaments, microtubules, and the intermediate filament network. It is well established that myofilaments containing thick (myosin) filaments and thin (actin) filaments are major components that participate in force development (9). Disruption of microtubules by nocodazole potentiates high potassium-mediated vascular smooth muscle contraction by increasing intracellular calcium concentration (18). We have recently shown that the downregulation of vimentin (a major intermediate filament protein in airway and vascular smooth muscle) in tracheal smooth muscle tissues diminishes contractile responses to agonist stimulation (35). Flow-induced mechanical responses of mesenteric resistance arteries are also reduced in vimentin knockout mice (10).

The adapter protein p130 Crk-associated substrate (CAS) is a major member of CAS family proteins that has been proposed to serve as docking sites for other proteins in integrin-mediated signaling transduction (4). CAS has been shown to participate in the signaling cascades that regulate force development in smooth muscle (17, 29). The downregulation of CAS in smooth muscle depresses active force development by inhibiting actin polymerization, a cellular process that is important for contractile force development in smooth muscle (2, 11, 26, 28, 32, 37). However, the interaction of CAS with the vimentin framework in mammalian cells in general and in smooth muscle in particular is not well understood.

Vimentin intermediate filaments are capable of binding to certain signaling molecules in mammalian cells. In differentiated smooth muscle cells, Ca2+/calmodulin-dependent kinase IIγ associates with the vimentin network. Rho kinase colocalizes predominantly with the filamentous vimentin network in fibroblasts. Disassembly and/or reorganization of the vimentin cytoskeleton induce the redistribution of these molecules, which has been implicated in regulating cell functions (15, 20).

External stimulation induces vimentin filament disassembly/assembly in cultured smooth muscle cells, chromaffin cells, human fibroblasts, rat RVF-SM cells, and BHK-21 fibroblasts (5, 19, 22, 25). The physiologic significance of vimentin disassembly in response to growth factor stimulation has been proposed to facilitate cell division possibly via spatial reorganization of the vimentin network (16, 34). Remodeling of vimentin filaments has also been shown to be associated with the enhanced migration of promyelocytic leukemia cells during differentiation (1).

The vimentin network may be regulated by vimentin phosphorylation and dephosphorylation on serine/threonine residues (5). P21-activaed kinase (PAK, a serine/theronine kinase) has been shown to be able to phosphorylate vimentin in in vitro studies (8, 25). In cultured smooth muscle cells, agonist stimulation induces vimentin disassembly and vimentin phosphorylation at serine-56 (Ser-56), which are inhibited by silencing of PAK1, a dominant isoform in smooth muscle (3, 25).

The aim of this study was to evaluate the association of CAS with the vimentin network in smooth muscle in response to stimulation with acetylcholine (ACh), a well-known airway smooth muscle contractile agonist. Our results suggest that ACh activation triggers the dissociation of CAS from the vimentin cytoskeleton, which may be mediated through phosphorylation and disassembly of vimentin by PAK.

MATERIALS AND METHODS

Preparation of smooth muscle tissues

Mongrel dogs (20–25 kg) were anesthetized with pentobarbital sodium (30 mg/kg, i.v.) and quickly exsanguinated. All experimental protocols were approved by the Institutional Animal Care and Usage Committee. A 15 cm segment of extra-thoracic trachea was immediately removed and placed at room temperature in physiological saline solution (PSS) containing 110 mM NaCl, 3.4 mM KCl, 2.4 mM CaCl2, 0.8 mM MgSO4, 25.8 mM NaHCO3, 1.2 mM KH2PO4, and 5.6 mM glucose. The solution was aerated with 95%O2-5%CO2 to maintain a pH of 7.4. Rectangular strips of tracheal muscle 0.6–0.8 mm in width, 0.2–0.3 mm in thickness and 9–10 mm in length were dissected from the trachea after removal of the epithelium and connective tissue layer.

Measurement of force development in smooth muscle

Each muscle strip was placed in PSS at 37°C in a 25-ml organ bath and attached to a Grass force transducer that had been connected to a Gould recorder or a computer with A/D converter (Grass). At the beginning of each experiment, muscle strips were stretched to the reference muscle length (9–10 mm). After 10–20 min equilibrium they were stimulated with 10−5M ACh repeatedly until contractile responses and passive tension reached stable. In untreated muscle tissues, an average passive tension was 0.2 g while an average active force was 4 g.

For experiments associated with antisense, oligodeoxynucleotides (ODNs) dissolved in Tris-EDTA buffer were introduced into muscle strips according to experimental procedures described previously (27, 28, 31). Muscle strips were then incubated for 2 days with ODNs in Dulbecco’s Modified Eagle Medium (DMEM). The strips were returned to PSS at 37°C in 25-ml organ baths and stretched to the corresponding reference muscle length. Following repeated stimulation with ACh, each contractile response and passive tension was compared with corresponding preincubation value. For analysis of protein expression and phosphorylation, muscle strips were frozen using liquid N2-cooled tongs, and then pulverized under liquid N2 using a mortar and pestle.

Analysis of soluble vimentin/insoluble vimentin ratio

The amount of soluble and insoluble vimentin was evaluated by modification of the method previously described (16, 25, 34). Briefly, smooth muscle strips were homogenized in a buffer containing 1% Nonidet P-40, 10% glycerol, 20 mM Hepes, pH 7.6, 150 mM NaCl, 2 mM sodium orthovanadate, 2 mM molybdate, 2 mM sodium pyrophosphate and protease inhibitors (2mM benzamidine, 0.5 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride). The homogenates were immediately incubated in the same buffer at 37°C for 30 min. The soluble (disassembled) and insoluble (assembled) fractions were collected after centrifugation at 5,200 rpm, 4°C for 30 min, and separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were probed with vimentin antibody (1:10000 dilution, BD Biosciences, clone RV202) followed by horseradish peroxidase (HRP)-conjugated anti-mouse Ig (Amersham Life Sciences) (25). Immunocomplex on the membranes was reacted with enhanced chemiluminescence (ECL) substrate (Supersignal West Dura Extended Duration Substrate, Pierce). The ECL signals on the immunoblots were detected and analyzed using a Fuji LAS3000 luminescent image system. The ratio of soluble vimentin/insoluble vimentin was determined after scanning densitometry of immunoblots.

Assessment of vimentin phosphorylation at Ser-56

Pulverized muscle strips were mixed with 100 μl of extraction buffer containing 20 mM Tris-HCl at pH 7.4, 2% Triton X-100, 0.2% SDS, 2 mM EDTA, phosphatase inhibitors (2 mM sodium orthovanadate, 2 mM molybdate, and 2 mM sodium pyrophosphate) and protease inhibitors (2 mM benzamidine, 0.5 mM aprotinin and 1 mM phenylmethylsulfonyl fluoride). Each sample was kept on ice for 1 h and then centrifuged for the collection of supernatant. Muscle extracts were boiled in sample buffer (1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl (pH 6.8), 10% glycerol and 0.01% bromophenol blue) for 4 min and separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane, after which the membrane was blocked with 2% gelatin for 1 h and probed with sitespecific, state-dependent antibody for vimentin Ser-56 (custom made by SynPep Inc., Dublin, CA; the synthetic phosphopeptide sequence: Ser-Leu-Tyr-Ala-Ser-phosphoSer56-Pro-Gly-Gly-Ala-Tyr-Cys; antibody dilution: 1:1000) followed by horseradish peroxidase (HRP)-conjugated anti-rabbit Ig (ICN Biomedicals, Inc., Irvine, CA) (25). Proteins were visualized by ECL. The membrane was stripped and reprobed with monoclonal vimentin antibody (BD Bioscience, 1:10000 dilution) followed by HRP-conjugated anti-mouse Ig (Amersham Life Sciences) to normalize for minor differences of protein loading. Changes in protein phosphorylation were expressed as a magnitude increase over levels of phosphorylation in unstimulated muscle strips.

Analysis of CAS association with cytoskeletal vimentin

Insoluble vimentin was collected from smooth muscle tissues by using the method aforementioned. The equal amount of cytoskeletal vimentin was separated by 10% SDS-PAGE, and was transferred to nitrocellulose membranes. The membranes were cut into two parts; the upper part was probed with monoclonal p130CAS antibody (1:2000 dilution, BD Biosciences, clone 24). The lower part of the membrane was blotted with vimentin antibody (1:10000 dilution). The ratio of CAS to vimentin was calculated after densitometrical analysis of immunoblots.

In vitro kinase assay

Wild type vimentin and vimentin S56A (alanine substitution at Ser-56) were produced and purified by using the methods previously described (14, 25). Purified wild type vimentin or the vimentin mutant S56A (0.1mg/ml) was incubated with 2 μg/ml activated PAK (Upstate) for 30 min in a buffer containing 20 mM Hepes (pH 7.5), 60 mM NaCl, 2 mM MgCl2, 5 mM EGTA and 100 μM ATP. High NaCl (150 mM) was then added into the reaction mix followed by incubation at 37°C for 1 h to initiate filament formation. The mixture was treated with 0.1% glutaraldehyde at room temperature for 30 min to stabilize vimentin filaments (14, 25).

Immunoprecipitation

CAS or vimentin was immunoprecipitated from muscle extracts using previously described method with minor modification (23, 25). Briefly, muscle extracts containing equal amounts of protein were precleared for 30 min with 50 μl of 10% protein ASepharose. The precleared extracts were collected after centrifugation at 13,200 rpm for 2 min, and were incubated with monoclonal antibodies against CAS or vimentin overnight and then incubated with 125 μl of a 10% suspension of protein A-Sephasose beads conjugated to rabbit anti-mouse IgG for 2.5 h. Immunocomplexes were washed four times in Tris-buffered saline containing 150 mM NaCl and 50 mM Tris (pH 7.6). All procedures of immunoprecipitation were performed at 4°C.

Far-western analysis

CAS immunoprecipitates were resolved by SDS-PAGE followed by membrane transfer. The membranes were incubated with vimentin and its mutant that had been treated with active PAK (see above) for 2 h at room temperature and then were probed with use of vimentin antibody.

Cell dissociation and immunofluorescence analysis

Smooth muscle cells were freshly dissociated from tracheal smooth muscle tissues according to the experimental procedures previously described (35). The cells were fixed for 10 min in 4% paraformaldehyde, and were then washed three times in Tris-buffered saline (50 mM Tris, 150 mM NaCl, and 0.1% NaN3) followed by permeabilization with 0.2% Triton X-100 for 5 min. Cells were then incubated with CAS monoclonal antibody for 45–60 min at 37°C. Cells were then washed and incubated with a secondary antibody conjugated to either Alexa 546 fluoroprobe (Molecular Probes, Eugene, OR) for 30 min at 37°C. The cellular localization of fluorescently labeled proteins was viewed under laser scanning confocal microscopy (Zeiss Meta 510) using a 63x oil immersion objective. The fluorescence of Alexa 546-labeled protein (red) was excited with a helium/neon laser at 543 nm and emissions were collected at 565–615 nm.

Image analysis for protein localization was carried out using the previously described method with minor modification (35). By using LSM5 analysis software (Zeiss), the pixel intensity was quantified for three to four line scans across the periphery of cells. Ratios of pixel intensity at the cell edge to pixel intensity at the cell interior were determined for each line scan as follows: ratios of the average maximal pixel intensity at the cell periphery to minimal pixel intensity in the cell interior. The ratios of pixel intensity at the cell border to that in the cell interior for all the line scans performed on a given cell were averaged to obtain a single value for the ratio of each cell.

Loading of oligodeoxynucleotides and organ culture

Antisense ODNs were used to selectively suppress PAK1 expression in canine tracheal smooth muscle based on cDNA sequence of human PAK1 (NCBI accession number, NM 002576). The sequence of PAK1 antisense is 5′-GGAGGGGCTGGGGGTTTGTC-3′, sequence of PAK1 sense, 5′-GACAAACCCCCAGCCCCTCC-3′. According to sequence matching results obtained from The National Center for Biotechnology Information, these sequences are not homologous to sequences of any other contractile proteins or cytoskeletal proteins. The antisense molecule targets to a region of mRNA that is unique to PAK1. The phosphorothioate ODNs were synthesized and purified by Invitrogen Corporation, Carlsbad, CA, USA. The ODNs were introduced into the smooth muscle strips by chemical loading (also referred to as reversible permeabilization) using methods we have previously described (27, 30, 31).

Determination of protein expression

Protein expression of ODN-treated muscle strips was assessed by immunoblot analysis. Briefly, muscle extracts were separated by SDS-PAGE followed by transfer. The membrane was then cut into two parts for immunoblotting of different proteins. The upper part of the membrane was blocked with 5% milk for 1 h and probed with monoclonal antibody against myosin light chain kinase (30) followed by horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Amersham). The lower part of the membrane was reacted with a polyclonal antibody against PAK1 (Cell Signaling, Beverly, MA, USA) followed by HRP-conjugated anti-rabbit IgG (ICN). Proteins were visualized by chemiluminescence and quantified by scanning densitometry. Densitometric values of PAK and myosin light chain kinase were determined for sense-treated and antisense-treated strips and normalized to those of no ODN-treated strips. The ratios of these proteins were calculated to verify that changes in protein expression were selective for PAK.

Statistical analysis

All statistical analysis was performed using Prism 4 software (GraphPad Software, San Diego, CA). Comparison among multiple groups was performed by one-way analysis of variance followed by post test (Tukey’s multiple comparison test). Differences between pairs of groups were analyzed by Student-Newman-Keuls test or Dunn’s method. Values of n refer to the number of experiments used to obtain each value. P < 0.05 was considered to be significant.

Results

The ratio of soluble vimentin to insoluble vimentin is increased in smooth muscle strips in response to stimulation with ACh

To determine whether vimentin disassembly occurs during contractile activation, tracheal smooth muscle strips were stimulated with 10−5M ACh for 1–10 min, or they were not stimulated. The ratio of soluble vimentin to insoluble vimentin of these muscle strips was assessed by the method of fractionation (16, 25, 34).

Stimulation with ACh resulted in vimentin partial disassembly in smooth muscle tissues. The amount of soluble vimentin was increased in smooth muscle strips upon ACh stimulation, whereas the level of insoluble vimentin was decreased in response to contractile activation (Fig. 1A). The increase in the soluble/insoluble vimentin ratio was obvious 1–10 min after muscarinic activation (Fig. 1B, n = 5), which is closely associated with the increase in active force (Fig. 1C, n = 10).

Figure 1. Acetylcholine (ACh) stimulation results in the increase in the ratio of soluble vimentin to insoluble vimentin in smooth muscle.

Figure 1

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(A) Immunoblot illustrating the effects of ACh stimulation on the amount of soluble (supernatant) and insoluble (pellet) vimentin (Vim). Tracheal smooth muscle strips were stimulated with 10−5M ACh for 1–10 min, or they were not stimulated. Supernatant (S) and pellet (P) fractions were separated and assessed by immunoblot analysis (See Materials and Methods). C, unstimulated smooth muscle. (B) The ratio of soluble vimentin to insoluble vimentin was assessed by scanning densitometry of immunoblots for the fractions (n = 5). (C) ACh stimulation (10−5M) leads to the increase in active force development. Force is expressed as percentage of contractile response to 10−5M ACh for 5 min (n = 10). All values are means ± SE. *Significantly higher values compared to unstimulated muscle strips (P < 0.05).

Vimentin undergoes phosphorylation at Ser-56 in smooth muscle strips in response to stimulation with ACh

Vimentin dynamics may be regulated by its phosphorylation on serine/threonine positions. We have previously shown that vimentin gets phosphorylated on Ser-56 in cultured smooth muscle cells in response to serotonin stimulation (25). To determine whether muscarinic activation induces vimentin phosphorylation in smooth muscle tissues, tracheal smooth muscle strips were stimulated with 10−5M ACh for 1–10 min. Vimentin phosphorylation on Ser-56 was determined by immunoblot analysis using phospho-vimentin (Ser-56) antibody. Unstimulated muscle strips were also frozen for vimentin phosphorylation.

ACh stimulation of tracheal smooth muscle resulted in the enhancement of vimentin phosphorylation at Ser-56. The phosphorylation levels in smooth muscle strips in response to stimulation with ACh for 1–10 min were significantly higher than the phosphorylation level in unstimulated smooth muscle strips (Fig. 2, P < 0.05, n = 4).

Figure 2. Vimentin undergoes phosphorylation on Ser-56 in smooth muscle elicited by ACh.

Figure 2

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(A) Representative immunoblots show that vimentin phosphorylation at Ser-56 is increased during ACh stimulation for 1–10 min. Blots of extracts of unstimulated muscle strips and ACh-stimulated muscle strips were probed using phospho-vimentin antibody [p-Vim (Ser-56)], stripped and reprobed by use of vimentin antibody (Vim). C, unstimulated smooth muscle. (B) Vimentin phosphorylation is quantified as multiples of phosphorylation levels obtained in unstimulated muscle strips. The values represent means ± SE (n = 4). *Significantly higher phosphorylation levels than the level of unstimulated muscle strips (P < 0.05).

CAS binds to unphosphorylated vimentin in vitro

The vimentin framework binds to CamKII in smooth muscle cells and Rho kinase in fibroblasts (15, 20). The adapter protein CAS has been shown to regulate active force development in arterial smooth muscle (17, 29). Our prior studies have demonstrated that Ser-56 on vimentin is a major phosphorylation site mediated by PAK; phosphorylation at this residue triggers vimentin fiber disassembly (14, 25). To evaluate whether CAS associates with vimentin filaments and whether vimentin phosphorylation at Ser-56 influences its binding to CAS in vitro, far-western analysis was performed. Purified wild type vimentin and the non-phosphorylatable vimentin S56A (alanine substitution at Ser-56) mutant were treated with active PAK followed by the addition of high sodium solution to facilitate filament assembly (14, 25). Blots of CAS immunoprecipitates from tracheal smooth muscle strips were reacted with the phosphorylated or unphosphorylated proteins and were detected by using vimentin antibody.

Treatment with PAK led to Ser-56 phosphorylation of wild type vimentin, but not vimentin S56A mutant (14, 25). CAS is able to bind to unphosphorylated (assembled) vimentin, but not phosphorylated (disassembled) vimentin. Phosphorylated wild type vimentin by PAK was not associated with immobilized CAS immunoprecipitates on blots. In contrast, nonphosphorylatable vimentin S56A mutant interacted with CAS immunoprecipitates on the blots (Fig. 3).

Figure 3. Far-western analysis for the interaction of CAS with vimentin in vitro.

Figure 3

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CAS immunoprecipitates from muscle extracts were resolved by SDS-PAGE followed by membrane transfer. Lane 1, the membrane was probed with use of CAS antibody. This result demonstrates that CAS immunoprecipitates are immobilized on the membrane. Lane 2, the membrane was incubated with wild type vimentin that had been treated with active PAK and then was probed with use of vimentin antibody. No vimentin was found to associate with CAS immobilized on the membrane, indicating that phosphorylated vimentin is not able to bind CAS in vitro. Lane 3, the membrane was incubated with the vimentin mutant S56A (alanine substitution at Ser-56) that had been treated with active PAK, and then was probed with use of vimentin antibody. The vimentin S56A mutant was found to interact with CAS on the membrane, suggesting that non-phosphorylatable vimentin is able to bind CAS in vitro. The immunoblots are representative of three experiments.

The amount of CAS in insoluble vimentin fraction is reduced in smooth muscle strips elicited by ACh

We assessed the association of CAS with vimentin filaments in the context of smooth muscle tissues. Soluble and insoluble (cytoskeletal) vimentin from unstimulated smooth muscle strips was separated by SDS-PAGE, and blots of the fractions were probed with antibodies against CAS and vimentin. CAS was found in the fraction of both insoluble vimentin and soluble vimentin (Fig. 4A). Quantification analysis showed that 25% of total CAS was associated with insoluble vimentin fraction (Fig. 4B, n = 5). However, CAS was not associated with soluble vimentin; no CAS was found in vimentin immununoprecipitates from muscle extracts containing soluble vimentin. Nor did ACh stimulation result in the change in the amount of CAS in vimentin immunoprecipitates (Fig. 4C).

Figure 4. The adapter protein CAS can be found in the cytoskeletal vimentin fraction.

Figure 4

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(A) Representative immunoblots illustrating the presence of CAS in insoluble (cytoskeletal) vimentin. Blots of supernatant (S) and pellet (P) fractions from unstimulated smooth muscle strips were probed with CAS antibody and vimentin antibody. (B) The amount of CAS on the immunoblots from each fraction is quantified after scanning densitometry analysis. The relative amount of CAS in the soluble fraction is expressed as soluble CAS/total CAS (soluble CAS + insoluble CAS) × 100; the amount of insoluble CAS, insoluble CAS/total CAS × 100. All values are means ± SE (n = 5). (C) Blots of vimentin immunoprecipitates from muscle extracts containing soluble vimentin were detected with CAS antibody and vimentin antibody. CAS was not detectable in vimentin immunoprecipitates in unstimualted and ACh-stimulated muscle extracts, indicating that CAS does not associate with soluble vimentin. The immunoblots are representative of three experiments.

We also assessed the effects of ACh stimulation on the association of CAS with cytoskeletal vimentin in smooth muscle. Treatment with ACh led to the decrease in the amount of CAS associated with cytoskeletal vimentin (Fig. 5A). The ratios of CAS to vimentin were statistically lower in smooth muscle tissues in response to ACh stimulation than in unstimulated muscle strips (Fig. 5B, n = 4, P < 0.05).

Figure 5. The level of CAS in cytoskeletal vimentin in response to ACh stimulation is reduced in smooth muscle strips.

Figure 5

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Equal amount of insoluble vimentin from unstimulated and ACh-stimulated smooth muscle strips were separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were probed with use of CAS antibody and vimentin antibody. (A) Immunoblots showing the effects of ACh activation on the amount of CAS in the vimentin fraction. (B) The ratio of CAS to vimentin in the stimulated muscles is normalized to that in unstimulated muscle strips. *Significantly lower ratio of CAS/vimentin in stimulated muscle strips as compared to unstimulated tissues (P < 0.05). Values are means ± SE (n = 4).

Peripheral localization of CAS is increased in freshly dissociated smooth muscle cells in response to ACh stimulation

We also assessed the effects of stimulation with ACh on the subcellular distribution of CAS. Unstimulated and ACh stimulated (10 μM, 5 min) smooth muscle cells were immunostained for CAS and analyzed under confocal fluorescence microscope.

Exposure of smooth muscle cells to ACh caused a redistribution of CAS from the cytoplasm to the cortical region. CAS staining was found primarily in the myoplasm of unstimulated smooth muscle cells. In response to stimulation with ACh, peripheral distribution of CAS was increased while fluorescence intensity of the protein was decreased in the cell interior (Fig. 6A). However, a fraction of total CAS was still observed in the cytoplasm of stimulated cells (Fig. 6A). Ratios of pixel intensity at the cell periphery to that at the cell interior were two-fold higher in ACh-stimulated cells than in unstimulated cells (Fig. 6B, n = 18, P < 0.05).

Figure 6. Stimulation with ACh leads to the increase in peripheral distribution of CAS in freshly dissociated smooth muscle cells.

Figure 6

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Unstimulated and ACh stimulated (10μM, 5 min) cells freshly dissociated from tracheal smooth muscle tissues were immunostained for CAS and analyzed under confocal fluorescence microscope. (A) Without ACh stimulation, CAS was detected in the myoplasm as well as at the cell border. Cell border-associated distribution of CAS was increased in cells in response to ACh stimulation. The arrow indicates a single line scan to quantify pixel intensity for each cell. (B) Protein distribution in cells is expressed as ratios of pixel intensity at the cell periphery to ratios of pixel intensity at the cell interior. Each mean value was obtained from an average of 3–4 line scans in each of 18 unstimulated and 18 stimulated cells from 3 experiments. *Significantly different from the value of unstimulated cells (P < 0.05).

Downregulation of PAK with antisense inhibits the decrease in the association of CAS with cytoskeletal vimentin during ACh stimulation

PAK has been shown to be an upstream regulator of the vimentin network (25). To evaluate whether PAK affects the interaction of CAS with cytoskeletal vimentin, tracheal smooth muscle strips that had been treated with PAK antisense or sense ODNs or not treated with ODNs were stimulated with 10−5 M ACh for 5 min, or they left unstimulated. The association of CAS with insoluble vimentin of these muscle strips was then assessed.

We first verified the effectiveness of PAK antisense treatment. PAK expression was lower in muscle strips treated with antisense ODNs than in strips treated with sense ODNs or no ODNs (Fig. 7A). Myosin light chain kinase (MLCK) was similar in antisense-treated and sense-treated strips and in muscle tissues not treated with ODNs (Fig. 7A). The normalized ratios of PAK versus MLCK in antisense-treated tissues were 0.28 ± 0.04, which was significantly lower than those in sense-treated muscles (1.07 ± 0.07) and in no-ODN-treated strips (1.00 ± 0.00) (mean ± SE, n = 4, P < 0.01).

Figure 7. PAK downregulation attenuates the dissociation of CAS from insoluble vimentin elicited by ACh.

Figure 7

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(A) Representative immunoblots illustrating PAK1 downregulation by antisense in smooth muscle strips. Blots of protein extracts from tracheal smooth muscle tissues that had been treated with antisense or sense ODNs, or with no ODNs were detected with antibodies against PAK1 and myosin light kinase (MLCK). The amount of PAK1 was lower in antisense-treated strips than in muscles treated with no ODNs or sense ODNs. Similar amounts of MLCK, a key enzyme in smooth muscle, were detected in all 3 groups of muscle strips. (B) Smooth muscle strips that had been treated with PAK antisense (AS) or sense (S), or with no ODNs were stimulated with 10−5M ACh for 5 min (black bars), or they were not stimulated (open bars). The ratios of CAS/vimentin were then determined. CAS/vimentin ratios in smooth muscle strips with various treatments are normalized to the ratio obtained from unstimulated muscle strips not treated with ODNs. All values represent means ± SE. * Significantly lower ratios in stimulated tissues than in corresponding unstimulated strips (n = 4, P < 0.05).

The downregulation of PAK inhibited the decrease in the CAS/vimentin ratio in muscle tissues induced by ACh. As compared to muscle strips not treated with ODNs, treatment with PAK antisense or sense ODNs did not affect the ratio of CAS to insoluble vimentin in unstimulated muscle strips. In muscle strips treated with no ODNs or sense ODNs, ACh stimulation resulted in a significant decrease in the CAS/vimentin ratio (Fig. 7B, n = 4, P < 0.05). However, the ratio of CAS to vimentin upon stimulation with ACh was not decreased in tissues treated with antisense when compared to corresponding unstimulated muscles (Fig. 7B, n = 4).

Depletion of PAK attenuates the increase in vimentin phosphorylation at Ser-56, disassembly and active force in smooth muscle strips elicited by ACh

We speculated that the effects of PAK on the association of CAS with cytoskeletal vimentin stem from vimentin phosphorylation and disassembly mediated by PAK. Tracheal smooth muscle strips that had been treated with PAK antisense or sense ODNs or not treated with ODNs were stimulated with 10−5 M ACh for 5 min, or they were not stimulated. Vimentin phosphorylation at Ser-56 and the soluble/insoluble vimentin ratios of these muscle strips were then evaluated.

Although vimentin phosphorylation in unstimulated muscle strips was not affected by the treatment with PAK antisense ODNs, vimentin phosphorylation at Ser-56 in response to ACh stimulation was significantly lower in PAK antisense-treated tissues than in sense-treated or no-ODN-treated muscle strips (Fig. 8, A and B, n = 4, P < 0.05).

Figure 8. Vimentin phosphorylation at Ser-56, the ratio of soluble vimentin to insoluble vimentin and active force induced by ACh are diminished in PAK-deficient muscle tissues.

Figure 8

Figure 8

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Smooth muscle strips that had been incubated with PAK antisense (AS) or sense (S), or not treated with ODNs were stimulated with 10−5M ACh for 5 min, or they were left unstimulated. Vimentin phosphorylation at Ser-56, soluble/insoluble vimentin ratios and active force were then determined. (A) Representative immunoblots illustrating the effects of PAK downregulation on vimentin phosphorylation at Ser-56. US, unstimulated. (B) Vimentin phosphorylation is quantified as multiples of phosphorylation levels obtained in unstimulated muscle strips not treated with ODNs. Open bars, unstimulated; black bars, ACh stimulated. * Significantly higher phosphorylation levels in stimulated muscles than in corresponding unstimulated muscles (n = 4, P < 0.05). ** Significantly lower vimentin phosphorylation upon ACh stimulation in muscle strips treated with antisense ODNs than in muscles not treated ODNs or in muscles treated with sense ODNs (n = 4, P < 0.05). (C) The effects of PAK depletion on ratios of soluble vimentin over insoluble vimentin. Open bars, unstimulted; black bars, ACh stimulated. * Significantly greater ratios of soluble vimentin/insoluble vimentin in stimulated muscles than in corresponding unstimulated strips (P < 0.05, n = 4). ** Significantly lower ratios of soluble vimentin/insoluble vimentin upon ACh stimulation in tissues treated with antisense than in tissues not treated ODNs or in muscle strips treated with sense ODNs (n = 4, P < 0.05). (D) Mean active force in response to 10−5M ACh was quantified as percent of ACh-induced force in each strip before incubation. Asterisk indicates significantly lower response as compared to muscles treated with sense ODNs or not treated with ODNs (n = 8, P < 0.05). All values are means ± SE.

Similarly, treatment with PAK antisense or sense ODNs did not affect the ratio of soluble vimentin/insoluble vimentin in unstimulated muscle strips; however, the soluble vimentin/insoluble vimentin ratio upon stimulation with ACh was significantly lower in muscle strips treated with antisense than in tissues treated with no ODNs or sense ODNs (Fig. 8C, n = 4, P < 0.05).

PAK downregulation also inhibited active force in response to ACh stimulation. Contractile force of tracheal smooth muscle strips was compared before and after the treatment with PAK sense or antisense for 2-day incubation (26, 32, 35). Although active force in muscle strips treated with sense oligonucloetides and in strips not treated with ODNs was not suppressed after the 2-day incubation, contractile response in antisense-treated tissues was significantly reduced to 21% of preincubation force (n = 8, P < 0.01) (Fig. 8D). There were no differences in passive tension among the three groups of strips.

Discussion

The type III intermediate filament protein vimentin is required for active force development in smooth muscle (10, 35). The adapter protein CAS participates in the signaling cascades that regulate smooth muscle contraction (17, 29). Our present results demonstrate that approximately 25% of total CAS binds to the vimentin framework in smooth muscle tissues, and that acetylcholine activation induces the disassociation of CAS from the vimentin cytoskeleton, which may be regulated through vimentin phosphorylation and disassembly by PAK. We propose that the PAK-mediated dissociation of CAS from cytoskeletal vimentin may be an important part of cellular mechanisms that regulate active force development in smooth muscle.

Recent studies indicate that vimentin filaments continuously exchange between a small disassembled faction and a large assembled fraction in certain cell types. External stimulation has been shown to induce vimentin disassembly in a variety of cultured cells including smooth muscle cells (5, 19, 25). Vimentin disassembly has been implicated in the regulation of cell mitosis and migration (1, 16). In this report, ACh stimulation resulted in the increase in the ratio of soluble vimentin/insoluble vimentin and active force development, suggesting that vimentin undergoes disassembly in association with contractile force in smooth muscle in response to agonist activation. The ratios of soluble vimentin/insoluble vimentin in unstimulated muscles were approximately 0.1, indicating that 10% of total vimentin is in disassembled form. The ratios of soluble vimentin/insoluble vimentin during ACh stimulation for 5–10 min were increased to approximately 0.25, implying that 20% of total vimentin is soluble (25).

How the dynamic change in vimentin filaments may affect force development in smooth muscle tissues is currently unknown. The adapter protein CAS has been reported to participate in the signaling process that mediates smooth muscle contractility (17, 29). In this report, we found that CAS was able to bind to cytoskeletal vimentin in in vitro studies as assessed by far-western analysis as well as in smooth muscle tissues as evaluated by the fractionation method. The amount of CAS associated with cytoskeletal vimentin was attenuated during muscarinic activation. These results indicate that vimentin disassembly during contractile activation may induce the release of CAS from the vimentin cytoskeleton, which may allow more CAS to be involved in the signaling cascades that mediate smooth muscle contraction (17, 29).

Our previous studies have shown that CAS is able to regulate the activation of the actinregulatory protein profilin and actin dynamics in smooth muscle (17, 29). It has been well documented that actin polymerization occurs in tracheal smooth muscle strips in response to ACh stimulation. The remodeling of the actin cytoskeleton has emerged as an essential event during contractile stimulation of smooth muscle (2, 26, 30, 32, 37). Actin dynamics in smooth muscle tissues is believed to occur in the cortical region of cells (37). In the present study, activation of smooth muscle with ACh initiated the redistribution of CAS from the myoplasm to the peripheral area. Thus, it is probable that the released CAS from the vimentin cytoskeleton may translocate to the cell border facilitating cortical actin polymerization and force development in smooth muscle.

Vimentin partial disassembly may also mediate changes in the cytoskeletal systems, which might modulate contractile responses. Vimentin filaments in differentiated smooth muscle cells display a well-spread network that extends from the nuclear membrane to the plasma membrane (15, 25). The membrane-associated anchoring of intermediate filaments is believed to occur at the desmosome, an intercellular junction (6, 13, 21). Vimentin filament disassembly in response to stimulation with soluble factors is related to the spatial reorganization of the vimentin network in various cell types (16, 25, 34). Vimentin downregulation disrupts desmosomal organization and suppresses smooth muscle contraction (35). Therefore, the spatial reorientation of vimentin filaments in smooth muscle could strengthen the linkage of vimentin filaments to the desmosome, which may promote intercellular mechanical force transmission (13, 25).

We then sought to understand the mechanisms that regulate the unique association of CAS with the vimentin network. In vitro studies have shown that PAK directly catalyzes vimentin phosphorylation (8, 25). The expression of constitutively active PAK induces vimentin phosphorylation in COS-7 cells (8). The silencing of PAK attenuates vimentin phosphorylation in cultured smooth muscle cells during serotonin stimulation (25). In the present study, PAK downregulation depressed the dissociation of CAS from cytoskeletal vimentin in response to ACh activation. The results lead us to suggest that PAK is a pivotal upstream regulator for the association of CAS with the vimentin network upon contractile activation of smooth muscle.

PAK may regulate the interaction of CAS with cytoskeletal vimentin and force development by affecting vimentin phosphorylation and disassembly in smooth muscle tissues. In BHK-21 fibroblasts, treatment with the protein phosphatase inhibitor calyculin-A induces vimentin phosphorylation in concert with the disassembly of vimentin polymers into soluble tetramers (5). In COS-7 cells expressing active form of PAK, the vimentin framework displays a granulate-like structure (disassembly) (8). In this report, PAK depletion diminished vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin during the muscarinic activation. Active force development was also inhibited in the PAK-deficient tissues. As mentioned above, the dissociation of CAS from cytoskeletal vimentin in response to ACh stimulation was attenuated in the PAK-deficient tissues. Thus, we propose that the contractile stimulation may induce vimentin phosphorylation at Ser-56 and disassembly via PAK. The partial disassembly of the vimentin framework may facilitate the release of vimentinassociated CAS, which may be involved in the induction of actin polymerization and force development (2, 26, 29, 32, 37) (Fig. 9).

Figure 9. Proposed mechanism.

Figure 9

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Contractile stimulation with ACh may trigger vimentin phosphorylation at Ser-56 and vimentin partial disassembly via PAK, which may initiate the dissociation of CAS from the vimentin network. The “released” CAS may participate in the signaling processes (such as actin polymerization) that regulate force development in smooth muscle.

Vimentin phosphorylation may be mediated by several other kinases in vitro and/or other cell types. Although protein kinase A (PKA) is able to phosphorylate vimentin in in vitro biochemical studies (5), it is unlikely that ACh exposure activates PKA in smooth muscle. The activation of PKA by β agonists leads to airway smooth muscle relaxation (11, 24), while ACh stimulation induces force generation in the smooth muscle. Protein kinase C (PKC) and Rho kinase may be activated in smooth muscle in response to ACh stimulation. However, inhibition of PKC or Rho kinase do not inhibit vimentin phosphorylation at Ser-56 (a critical site for smooth muscle function) although several other phosphorylation sites may be mediated by these two kinases (7, 12, 25, 33). In addition, CamKII may mediate phosphorylation of vimentin on several residues other than Ser-56 in vitro (5, 33). Finally, cyclin-dependent kinase 1 and pololike kinase 1 have been reported to mediate vimentin phosphorylation during mitosis of U251 cells and T24 cells (36). Although there is currently no evidence that these two kinases can be activated in differentiated cells, we do not rule out the possibility that ACh stimulation may activate these two kinases, which might mediate vimentin phosphorylation at Ser-56 in smooth muscle tissues.

Summary

ACh stimulation of tracheal smooth muscle strips induces disassembly of cytoskeletal vimentin and vimentin phosphorylation at Ser-56. More importantly, ACh activation leads to the decrease in the distribution of CAS in the vimentin cytoskeleton in concert with active force development. CAS translocates to the peripheral area from the myoplasm upon contractile stimulation. The downregulation of PAK inhibits the decrease in the association of CAS with cytoskeletal vimentin and active force probably by attenuating vimentin phosphorylation at this residue and its disassembly. We conclude that the PAK-mediated CAS release from the vimentin network may be an essential cellular process upon muscarinic activation of smooth muscle tissues.

Acknowledgments

This work was supported by National Heart, Lung, and Blood Institute Grant HL-75388, and an American Heart Association Scientist Development Grant, and Indiana Showalter Foundation (to D.D.T.). The authors thank Amy M. Spinelli and Taoying Huang for their assistance.

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