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Published ahead of print on April 3, 2008, doi:10.1165/rcmb.2007-0263OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 270-278, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0263OC

Cyclic Strain–Induced HSP27 Phosphorylation Modulates Actin Filaments in Airway Smooth Muscle Cells

Subhendu Chaudhuri1 and Paul G. Smith1

1 Division of Pharmacology and Critical Care, Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio

Correspondence and requests for reprints should be addressed to Subhendu Chaudhuri, M.S., Ph.D., Division of Pharmacology and Critical Care, Department of Pediatrics, Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail: sxc30{at}case.edu


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mechanical stress (cyclic deformational strain) increases proteins of cytoskeletal and contractile domains in airway smooth muscle (ASM) cells in a manner that increases cell contractility. Here we studied the role of HSP27 in strain-induced microfilament formation and stability. Cultured ASM cells showed rapid phosphorylation of HSP27 upon cyclic strain within a few minutes that continued for 30 to 40 minutes. Such increases in HSP27 phosphorylation were abolished with SB 202190, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), but not by PD 98059 (an inhibitor of extracellular regulated kinase), GF109203X (an inhibitor of protein kinase C), or Y27632 (an inhibitor of Rho kinase). Direct activation of RhoA by GTP{gamma}S did not alter the level of HSP27 phosphorylation. Confocal microscopy revealed that cells pre-incubated with SB 202190, and/or Y27632 resulted in disorganization of stress fibers upon strain, unlike PD 98059 and GF 1092030X, suggesting that both p38 MAPK and Rho kinase were necessary for strain-induced microfilament formation. To determine the relationship between HSP27 and RhoA in strain-induced microfilament formation, cells were transfected with various isoforms of HSP27 and RhoA before strain. Co-expression of inactive HSP27 (3A-HSP27) with constitutively active EGF-RhoA (RhoV14) caused diminution of microfilaments compared with constitutive active EGFP-RhoA (RhoV14) alone, suggesting that HSP27 is necessary for microfilament stability. Similarly, expression of phosphomimicking HSP27 (3D-HSP27) was sufficient for retaining microfilament formation even when co-expressed with the dominant-negative RhoA (EGFP-RhoN17). Thus, HSP27 activation is necessary for microfilament stability independently of RhoA activation.

Key Words: HSP27 • p38 MAP kinase • Rho • mechanical strain • smooth muscle cells



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Mechanical stress reorganizes cytoskeletal apparatus in airway smooth muscle. Understanding signaling mechanisms that control these processes might lead to therapeutic means to attenuate smooth muscle hyperresponsiveness.

 
Microfilaments are highly dynamic fibers that must dissolve and reform to control various cellular functions such as adhesion, motility, cytokinesis, contraction/ relaxation, endocytosis, and gene expression. Not only must formation/dissolution be rapid to allow these functions, reorganization must occur in a specific manner that serves the various functions of microfilaments. For example, we have demonstrated a change in configuration of microfilaments as airway smooth muscle (ASM) cells shifted from the proliferative phenotype to the contractile phenotype as a result of mechanical strain (1–4). The microfilaments became more numerous and arranged in parallel bundles perpendicular to the lines of strain. This particular configuration allowed the cells to produce greater force of contraction and became more rigid (3). This latter characteristic might have protected the cell from disruption by repeated stretching. An analogous situation may be prevalent in vivo, where abnormal mechanical stress accompanies airway hyperresponsiveness, smooth muscle hypertrophy, and hyperplasia (5). The cytoskeleton is likely involved in ASM hyperresponsiveness, as recently demonstrated by similar changes to the cytoskeleton after allergen exposure. These changes led to increased contractile function especially when cytoskeletal stabilization was chemically induced (6). Examination of signal transduction pathway(s) contributing to the stress-induced changes in cytoskeleton will therefore enable us to understand the pathogenesis of several diseases. In diseases such as ventilator-induced bronchopulmonary dysplasia, asthma, and hypertensive vasculopathy, prolonged abnormal mechanical stress on smooth muscle tissue is accompanied by smooth muscle hypertrophy and/or hyperplasia as well as increased contractility of the tissue (1). One mechanism for increased contractility and stiffness at the cellular level in these cases might be alterations in cytoskeletal architecture (7).

Determination of signaling pathways that transduce mechanical signals to restructuring of the cytoskeleton is thus important for understanding the mechanisms of stress-induced airway smooth muscle responsiveness. The mitogen-activated protein kinases (MAPKs) in MAPK signaling cascades play a critical role in transduction of extracellular signals to intracellular responses. The three-kinase modules (MKKK-MKK-MAPK) of MAPK pathways transmit signals by sequential phosphorylation from upstream kinase to downstream kinase. The three important members of MAPK signaling cascades are extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK. p38 MAPK and JNK are classical stress-activated kinases activated by a wide variety of signals such as proinflammatory cytokines TNF-{alpha}, IL-1, ultraviolet radiation, lipopolysaccharide (LPS), osmotic stress, and heat shock. ERK1/2, on the other hand, is activated predominantly by mitogens. HSP27 is phosphorylated via p38MAPK-MK2 pathway upon stress (9, 10). HSP27, a member of the {alpha}-crystallin small heat shock family, is present as large oligomeric complexes, and phosphorylation of serine residues (S15, S78, and S82) by the same protein kinase by various stimuli leads to the disaggregation of the complex oligomers into dimers or tetramers (9–15). Although HSP27 is now well documented to be one of the substrates for MK2 in vivo (9–11), there are other kinases that phosphorylate the same serine residues, such as MK5 (16), protein kinase (PK)C {delta} (17), and PKD (18). cGMP-dependent protein kinase, which phosphorylates Thr143, is the exception (19).

The structural consequence of phosphorylation is disaggregation of HSP27, which is thought to control a wide range of biological activities, including actin stabilization and stress protection (20–22, 25, 26). Because HSP27 is involved in such diverse and seemingly unrelated cellular processes, the functional consequence of HSP27 phosphorylation remains an enigma. Studies showed that microfilament remodeling and cell motility require phosphorylation-induced structural changes of HSP27 (20–22, 25). Recent studies with Arg-Gly-Asp–coated microbeads attached to cytoskeleton have documented a definitive role of HSP27 phosphorylation in modulating changes in endothelial cells, as well as the functional integrity of the endothelial cells (23, 24). However, cellular protection may not require disaggregation of HSP27, as observed with a fluorescent HSP27 (EGFP-HSP27) chimera (28) and a nonphosphorylatable mutant form of HSP27 (13).

Activation of both p38 MAPK–MK2 and RhoA–Rho kinase pathways results in actin filament reorganization (4, 20–23). We previously observed that cyclic strain–induced Rho activation increases organization of contractile and noncontractile proteins in ASM cells (4). A recent study shows that cyclic strain activates p38 MAPK in bovine pulmonary endothelial cells (8). Several lines of evidence suggest that p38 MAPK activation by various stimuli leads to HSP27 phosphorylation (9–13, 20–23). This study shows that cyclic strain activates HSP27 via p38 MAPK signaling pathway, and that Rho kinase activation does not contribute to HSP27 phosphorylation as suggested recently (27). The results further demonstrate that strain-induced HSP27 phosphorylation is unaffected by Rho kinase inhibition. Instead, both Rho–Rho kinase and p38 MAPK–MK2-HSP27 signaling pathways contribute independently to the stability of actin cytoskeleton in cells transformed with combination of constitutive active and dominant-negative mutants of RhoA and HSP27.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Specific inhibitors for kinases were from EMD Biosciences, Inc. (La Jolla, CA). Antibiotics, cell culture media, and fluorescent conjugated antibodies were from Invitrogen (Carlsbad, CA). Leupeptin, aprotinin, and TRITC-labeled phalloidin were obtained from Sigma (St. Louis, MO). Supersignal West Pico Chemiluminescent and BCA protein assay kits were purchased from Pierce (Rockford, IL). Nitrocellulose membrane was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Collagenase type 2 was obtained from Worthington (Lakewood, NJ). All other chemicals were from Fisher Scientific (Pittsburgh, PA). Rabbit polyclonal antibody specific for phospho-HSP27, monoclonal antibody against HSP27, and monoclonal antibodies against phosphorylated and nonphosphorylated p38 MAP kinase were from Cell Signaling Technology, Inc. (Beverly, MA). Polyclonal antibodies for phosphorylated and nonphosphorylated ERK1/2, and all horseradish peroxidase–conjugated antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), except anti-actin secondary antibody, which was obtained from Sigma. Alexa Fluor 405 goat anti-rabbit, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 647 goat anti-mouse, and 4',6-diamidino-2'-phenylindole dihydrochloride (DAPI) were purchased from Molecular Probes, Invitrogen (Eugene, OR). Fluoromount-G solution for mounting cover slip on slides was purchased from Southern Biotech (Birmingham, AL). TransIt-Express transfection reagent was from Mirus Bio Corporation (Madison, WI) and FuGENE HD transfection reagent from Roche Applied Science (Indianapolis, IN). Flex 1 collagen-coated culture plates were purchased from Flexcell International Corp. (Hillsborough, NC).

Cell Culture and Mechanical Strain Procedure
Cells were isolated from human trachealis muscle by digesting with collagenase and elastase in the presence of soybean trypsin inhibitor at 37°C as previously described (2). The cells were harvested by centrifugation after filtering through Nytex and grown in Dulbecco's modified Eagle's medium (DMEM/F12 1:1) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ ml streptomycin at 37°C in a humidified atmosphere with 5% CO2 and 95% air. For the purpose of experiments, cells from passages between four and eight were seeded into 6-well collagen I–coated membrane (BioFlex) and allowed to reach 80% confluence. The cells were serum starved for 24 hours before experiments. The Flexcell apparatus was used for the application of mechanical strain. Six-well culture plates were placed on a manifold in the incubator connected to a vacuum source, and subjected to a 10% increase in surface area of the membranes for 2 seconds followed by 2 seconds of relaxation (cyclic deformational strain) with the aid of computer software. The duration of the strain varied according to the experimental conditions as mentioned in the text. The results are representative of more than five independent experiments using various ASM cell lines.

Cell Lysis
Cells were washed twice with ice-cold PBS and lysed with RIPA buffer (in mM: 50 Tris-HCl, 150 NaCl, 1 EDTA, 1 EGTA, 2 PMSF, 1 sodium pyrophosphate; 0.1% SDS, 0.5% Deoxycholate, 1% Triton X100, 10 µg/ ml leupeptin and aprotinin). After incubation on ice for 30 minutes with intermittent mixing, lysates were centrifuged in a microcentrifuge at 4°C for 10 minutes, and supernatant was transferred into a fresh tube for estimation of protein and immunoblotting. Protein concentration was determined by BCA assay method.

Western Blot
Thirty micrograms of proteins from cell lysates were resolved by 4 to 20% gradient gel and transferred to nitrocellulose membrane. Proteins transferred to the membrane were in turn incubated with antigen-specific primary antibody and then with horseradish-conjugated secondary antibody. The membranes were subsequently incubated with chemiluminescence detection reagent for visualization of protein.

Plasmids
Construction of plasmids encoding constitutively active EGFP-tagged RhoA (V 14) and EGFP-tagged dominant-negative RhoA (N 17) was described previously (5). Wild-type and nonphosphorytable HSP27-S15A, S78A, S82A (3A-HSP27) were generously supplied by Dr. W. T. Gerthoffer (University of Nevada School of Medicine) and phosphomimicking HSP27-S15D, S78D, S82D (3D-HSP27) was a kind gift from Dr. S. S. An (Harvard School of Public Health).

Transient Transfection
A day before transfection, cells were seeded in 6-well collagen-coated Bioflex plates at 5 x 105 cells per well and transfected with either TransIt Express or FuGENE HD according to their protocols at a reagent to DNA ratio of 10:2 for TransIT Express and 6:2 for FuGENE 6D. After 24 hours of transfection, cells were serum starved overnight unless stated otherwise before being subjected to strain followed by immunostaining.

Immunostaining
Cells on membranes were washed three times for 5 minutes with PBS after each step. Cells were fixed with 4% paraformaldehyde in PBS for 10 minutes and permeablized for 1 minute with 0.5% Triton X100. The cells were then treated with 3% goat serum for 1 hour. Cells were incubated for 1 hour with primary antibody at 1:400 dilution, and then with fluorescent-conjugated secondary antibodies in blocking buffer (6 µg/ml of Alexa Fluor 647 Goat ant-mouse; Alexa Fluor 405 Goat anti-rabbit). TRITC-Phalloidin (0.4 µg/ml) in PBS was used for staining actin filaments. Nuclei were stained with DAPI (1 µg/ml) for 5 minutes. The stained cells on the membrane were washed with PBS and a portion of the membrane was placed on a slide with cell side facing up. A coverslip was placed with a few drops of Fluromount-G and allowed to dry overnight before imaging. The cells were examined under Zeiss LSM 410 confocal microscope (Zeiss, Thornwood, NY) equipped with a x63 oil objective lens with a 1.4 numerical aperture.

Statistical Analysis
Data are presented as a mean ± SEM., and analyzed by one-way ANOVA to determine the difference between groups. Differences between individual groups within the set were estimated by a multiple comparison test (Tukey's HSD test). A P value of < 0.05 was considered significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Mechanical Strain Phosphorylates HSP27
HSP27 is readily phosphorylated by various stimuli in a wide variety of cells (11). To investigate whether HSP27 is phosphorylated by mechanical strain, we subjected serum-deprived human ASM cells to 10 minutes of strain unless stated otherwise. Proteins from the strained and unstrained cells were separated by SDS-PAGE and analyzed by immunoblotting. Phosphorylation of HSP27 from serum-deprived cells was induced by cyclic strain, resembling that of serum- or sodium arsenite–stimulated cells (Figure 1A). Both of the latter treatments are known to phosphorylate HSP27. RhoA has also been implicated as an activator of HSP27 (27). Interestingly, activation of small GTPase RhoA with 1 mM of nonhydrolyzable GTP analog (GTP{gamma}S) guanosine 5'-O-(3-thiotriposphate) did not phosphorylate HSP27 (Figure 1B).


Figure 1
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  		Figure 1. Cyclic strain induces HSP27 phosphorylation in human airway smooth muscle (ASM) cells. HSP27 phosphorylation by 10-minute strain in serum-deprived cells was compared with cells incubated with 10% serum and 200 µM sodium arsenite. (A) Resolved proteins from lysates were detected by immunoblotting with respective antibodies against HSP27, phosphorylated HSP27, and actin. (B) The effect of RhoA activation on HSP27 phosphorylation in serum-deprived cells after 30 minutes of incubation with 1 mM GTP{gamma}S was compared in cells treated with 10% serum.

 
To understand the time-course of phosphorylation, we subjected ASM cells to strain for various lengths of time. The result shows that the onset of phosphorylation is rapid, being maximal after 10 minutes and showing only a gradual decline to approximately 70% of the maximal value after 40 minutes (Figures 2A and 2B).


Figure 2
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  		Figure 2. Time-dependent phosphorylation of HSP27 by cyclic strain. Serum-starved cells were strained for variable lengths of time (0–40 min). (A) The resolved proteins from cell lysates were detected by Western blotting using antibodies specific for HSP27, phosphorylated HSP27, and actin. Results from immunoblots were quantified by densitometry, and expressed in arbitrary units. Values of phosphoprotein bands were related those of non–phospho-specific protein bands. Results are means ± SEM for 10 independent experiments. *P < 0.05 compared with unstrained cells (B).

 
Identification of Protein Kinases Involved in Strain-Induced HSP27 Phosphorylation
Since a wide range of stimuli activate MAPK signaling cascades in a variety of cells and can mediate HSP27 phosphorylation by multiple kinases, it is important to determine which protein kinase(s) of intracellular signaling pathways is/are responsible in ASM cells for HSP27 phosphorylation in response to cyclic deformational strain. We used specific inhibitors for kinases to assess the contribution of different pathways in strain-induced HSP27 phosphorylation. Pre-incubation of quiescent smooth muscle cells with SB 202190, a relatively specific inhibitor of p38 MAPK, prevented phosphorylation of HSP27 (Figure 3). HSP27 phosphorylation was unaffected by Y27632, a specific inhibitor of Rho kinase. Pre-incubation of smooth muscle cells with GF 109203X, a specific inhibitor of PKC, before subjecting to strain showed no inhibition of HSP27 phosphorylation. Phosphorylation of HSP27 was similarly unaffected in cells pretreated with PD 98059, a specific inhibitor of MKK1 (MEK1), the upstream activator of ERK1 and ERK2. ERK was active constitutively in unstrained cells (Figures 4A and 4B). Cyclic deformational strain, like other stimuli, modestly increased both ERK1 and ERK2 phosphorylation (29, 30), and increased further in cells upon inhibition of p38 MAPK activity by SB 202190. This increment in the level of ERK phosphorylation was reduced to the basal level in cells preincubated with PD98059. ERK activation had no consequence on HSP27 phosphorylation (Figure 4). This modest increment of ERK activation, affected negatively by p38 MAP kinase, was most likely the result of the repression of proliferating activity of ERK in unstimulated cells (31). Taken together, these results demonstrate that in serum-free conditions neither Rho kinase, PKC, nor ERK1/2 of MAPK cascade are involved in HSP27 phosphorylation. Subsequent observations confirmed that strain activates the p38 MAPK signaling pathway and that phosphorylation of p38 MAPK paralleled the phosphorylation of HSP27 in a time-dependent manner (Figure 5).


Figure 3
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  		Figure 3. Assesment of signaling pathways that mediate HSP27 phosphorylation by kinase inhibitors. Serum-deprived cells were incubated with 10 µM of inhibitors for 30 minutes before subjecting to strain for 30 minutes. (A) The proteins from cell lysates were detected by immunoblotting with corresponding specific antibodies: HSP27, phosphorylated HSP27, and actin. Results from immunoblots were quantified by densitometry and expressed in arbitrary units. Values of phosphoprotein bands were related those of non–phospho-specific protein bands. Results are means ± SEM for eight independent experiments. *P < 0.05 compared with unstrained cells; **P < 0.05 when compared with strained cells (B).

 

Figure 4
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  		Figure 4. Extracellular regulated kinase (ERK) does not activate HSP27 upon strain. Quiescent ASM cells were incubated with 10 µM of SB202190, an inhibitor of p38 mitogen-activated protein kinase (MAPK) (lane 3) and 10 µM of MEK inhibitor PD98059 for ERK inactivation (lane 4) for 30 minutes and without inhibitors (lanes 1 and 2). Cells were strained for 30 minutes. (A) Immunoblot of resolved proteins from cell lysates either on 4 to 20% gradient or 10% SDS-PAGE were identified with anti-HSP27, anti–phospho-HSP27, anti-ERK, and anti–phospho-ERK antibodies, respectively. Results from immunoblots were quantified by densitometry and expressed in arbitrary units. Values of phosphoprotein bands were related those of non–phospho-specific protein bands. Results are means ± SEM for eight independent experiments. *P < 0.05 when compared with unstrained cells (B).

 

Figure 5
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  		Figure 5. p38 MAPK is activated by cyclic strain in a time-dependent manner. Serum-starved cells were strained for variable lengths of time (0–40 min). (A) The resolved proteins from cell lysates were detected by immunoblotting using antigen-specific antibodies. Results from immunoblots were quantified by densitometry and expressed in arbitrary units. Values of phosphoprotein bands were related those of non–phospho-specific protein bands. Results are means ± SEM for five independent experiments. *P < 0.05 compared with unstrained cells (B).

 
Influence of HSP27 Phosphorylation on Actin Filament Organization
One of the properties of HSP27 is to bind F-actin at the barbed ends to inhibit microfilament polymerization (20, 25, 32–34). Phosphorylation has been shown to induce disaggregation of oligomeric complexes of HSP27 with subsequent disassociation from actin filaments, which may result in a higher rate of actin polymerization (21). On the other hand, competition with other actin-modifying proteins might provide microfilament stability by reducing actin filament disruption. In an attempt to elucidate whether phosphorylation of HSP27 produced by mechanical strain causes changes in actin dynamics, we pre-incubated the cells with the kinase inhibitors, subjected them to strain for 30 minutes, and subsequently observed microfilament presence by immunostaining. Actin stress fibers and microfilaments were present with and without strain in cells (Figures 6A and 6B). However, when cells were preincubated with SB 202190- and/or Y27632, patchy staining of actin filaments and disruption of stress fibers was noticeable, confirming the importance of HSP27 phosphorylation (Figures 6C and 6D). Consistent with our immunoblotting results, PD 98,059 (inhibitor of ERK1/2) and GF 109203X (inhibitor of PKC) did not affect actin filaments (Figures 6E and 6F).


Figure 6
Figure 6
Figure 6
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  		Figure 6. Investigation of the role of HSP27 in actin filament dynamics upon strain with inhibitor of kinases. Serum-deprived cells were preincubated for 30 minutes with inhibitors before being subjected to 30 minutes of strain cycle. (A) Unstrained cells; (B) strained cells; (C) cells incubated with SB202190; (D) cells treated with Y27632 or both SB 202190 and Y 27632; (E) cells treated with GF109203X and cells treated with PD 98059. Cells were formaldehyde fixed, permeabilized, and immunostained with goat anti-mouse Alexa Fluor 647 (blue) for HSP27, goat anti-rabbit Alexa Fluor 405 (blue) or Alexa Fluor 488 (yellow) for phosphorylated HSP27, and phalloidin-TRITC (red) for F-actin. SB, SB202190; Y, Y27632; PD, PD 98059; GFX, GF109203X. (G) Cell nuclei were visualized by DAPI before and after strain, and HSP27 was stained with rabbit polyclonal antibody followed by Alexa Fluor (yellow)–coupled secondary (anti-rabbit) antibody.

 
There is divergent evidence about the changes in cellular localization of HSP27 after heat shock and other stimuli (28, 35, 36). Some reported observing faster disaggregation of nuclear proteins in cells overexpressing HSP27, which was deemed to be the result of translocation of smaller aggregates of HSP27 from cytoplasm into nucleus after stress of HSP27 (35–37). Others showed with chimeric HSP27 that disaggregation and translocation of HSP27 into nucleus are not necessary for stress protection (28), and may well be cell type specific (36). To understand the reported nucleocytoplasmic translocation of HSP27 as a consequence of stimuli, we immunostained cells for total and phosphorylated HSP27 to delineate the cellular localization during strain in the presence and absence kinase specific inhibitors as mentioned earlier (Figures 6C to 6E). In the absence of strain both unphosphorylated and phosphorylated HSP27 were located in cytoplasm. No observable translocation into the nucleus occurred on strain (Figure 6G). Translocation of HSP27 did not occur on increasing the length of strain (our unpublished observation). This did not come as a surprise because the p38 MAPK–MK2 complex is translocated from nucleus to cytoplasm on activation due to the masking of nuclear localization signal. MK2, in the cytoplasm, then phosphorylates HSP27 among other substrates (38).

To confirm the effect of phosphorylation of HSP27 on actin filament dynamics, we co-expressed nonphosphorylatable mutant of HSP27 (3A-HSP27), where three serine residues were replaced with alanine (S15A, S78A, S82A) with either GFP-tagged constitutively active RhoA (RhoV14) or GFP-RhoV14 alone. Similarly, we expressed phosphomimicking HSP27 (3D-HSP27), where three serine residues were mutated to aspartic, with dominant-negative GFP-RhoN17. This approach allowed us to delineate the relative contribution of HSP27 and RhoA to actin filament remodeling. The cells coexpressing nonphosphorylatable mutant 3A-HSP27 and constitutively active RhoV14 showed reduction in the amount of stress fibers compared with cells expressing RhoV14 alone. The effect was reversed in cells expressing dominant-negative Rho (RhoN17) and phosphomimicking HSP27 (3D-HSP27). The cells co-expressing RhoN17 and wild-type HSP27 retained more stress fibers compared with cells expressing RhoN17 (Figure 7). These results are consistent with the notion that activated HSP27 contributes to actin filament stability in cells exposed to stress, unlike nonphosphorytable mutant of HSP27 (13, 23).


Figure 7
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  		Figure 7. Investigation of microfilament organization in cells cotransfected with isoforms of HSP27and/or GFP-tagged Rho. Cells were seeded a day before transfection and grown in complete medium. Cells were either transfected with constitutive active Rho (RhoV14) or cotransfected with 3A-HSP27, dominant-negative Rho (RhoN17), and/or 3D-HSP27. After 24 hours of incubation, cells were fixed, permeabilized, and stained for F-actin with Phalloidin-TRITC. V14, RhoV14; N17, RhoN17; 3D-HSP27, phosphomimicking HSP27; 3A-HSP27, nonphosphorylatable HSP27; and Wt-HSP27, wild-type HSP27.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our previous studies showed that cyclic strain increases organization of contractile and noncontractile proteins of ASM cells through Rho-mediated signaling pathway (3, 4). Since a wide range of stimuli activate p38 MAP kinase, we examined the relationship between Rho/Rho kinase and p38 MAPK/MK2/HSP27 pathways in strain-induced filament organization and stability. Our results suggest that the two pathways work in parallel but are functionally independent. The present study shows that cyclic strain activates p38 MAPK, leading to phosphorylation of its downstream effector HSP27. The time-course of HSP27 phosphorylation is similar to that of other proteins we have studied in that there is a transient rise over minutes but then an attenuation of this response with increasing time (3, 4).

The temporal association between phosphorylation of cytoskeletal and cytoskeletal-associated proteins and cytoskeletal reorganization suggests that the cytoskeleton responds to mechanical stress by reorganization in a manner that both stabilizes the cell and diminishes the deformation of the cell by stress (39). Accordingly, the cytoskeletal response must vary with duration, direction, and magnitude of the imposed forces. First cytoskeletal structures must dissolve to allow cell movement relative to the direction or magnitude of the stretch. If the stretch is of great enough magnitude that relationships between myosin head regions and actin filaments are disrupted or inhibited from cycling, cell softening and relaxation would follow. With continued stress, cytoskeletal elements must adapt with increased numbers and organization to maintain the ability to withstand and contract against the imposed load. While sustained levels of phosphorylation are not seen with the strain protocols used, cell migration continues such that cells lie perpendicular to the direction of strain. Such cell migration is also dependent on the dissolution and reorganization of the cytoskeleton. We believe that such movement occurs because stretch through the width of the cell causes less deformation than stretch parallel to the length of the cell. In addition to the reorientation of the cell, cell morphology after prolonged strain shows increased numbers and organization of cytoskeletal elements. These morphologic changes in turn appear responsible for increased cell stiffness and enhanced contractility (40). Accordingly, prolonged duration of mechanical stress appears to have the opposite effect on cell mechanics (increased stiffness) to acute length changes. The smooth muscle cell relaxation is well described with acute stretch, either through dissolution of filaments necessary before reorganization of the cell, and or through disruption of cytoskeletal elements and relationships (41).

The conformational change associated with phosphorylation of HSP27 by its upstream activator MK2 is thought to lead the dissociation of HSP27 from the barbed ends of actin filaments, thereby enabling addition of actin monomers (22). The role of other two members in this family, MK3 and MK5, in phosphorylation of HSP27 is uncertain, but MK2 has been shown to phosphorylate HSP27 specifically in vivo (9–12, 26). We, therefore, concluded that the strain-induced activation of p38 MAPK/MK2 signaling pathway leads to HSP27 phosphorylation. This conclusion is supported by the observation that strain-induced phosphorylation is abolished in the presence of SB 202190, a specific inhibitor of p38 MAPK. Moreover, the inhibition of MEK1, the upstream effector of ERK and PKC, did not change the levels of strain-induced HSP27 phosphorylation. These results together with the observed parallel activation of HSP27 and p38 MAPK strongly favor that cyclic strain activates p38 MAPK/MK2/HSP27 signaling pathway in cells.

It is well documented that the pathways of RhoA-Rock/ and Rac/Cdc42-PAK play a critical role in cytoskeletal remodeling through sequential phosphorylation of LIM kinase and cofilin by inhibiting cofilin's depolymerization activity (42, 43). The RhoA–Rho kinase pathway, in addition, maintains a steady level of myosin light-chain (MLC) phosphorylation by MLC kinase through phosphorylation-induced inhibition of myosin phosphatase and thereby increases actomyosin-based contractility. Independent of RhoA, HSP27 has been shown to affect microfilament stability, stress protection, and chaperoning properties (11, 13, 21, 26, 35).

RhoA has recently been shown to mediate cytoskeletal reorganization via HSP27 (27). This obviously requires HSP27 phosphorylation (20), and evokes the possibility of a connection between p38 MAK/MK2 and RhoA/Rho kinase. We, therefore, decided to use inhibitors to study the relative contributions of the two pathways in imposing changes in actin filament organization and levels in response to strain via HSP27. The actin cytoskeleton shows noticeable increased organization in the absence of inhibitor of p38 MAPK upon strain. Consistent with inhibition of phosphorylation of HSP27 by p38 MAPK inhibitor, SB 202190, stress fiber formation induced by activation of p38 MAPK/MK2/HSP27 pathway is also reduced in serum-deprived cells. Reduction in stress fiber formation was more pronounced with the Rho kinase inhibitor Y27632 despite the fact that cyclic strain–induced HSP27 phosphorylation was not affected in quiescent cells. These results indicate that p38 MAPK is the upstream effector of HSP27 phosphorylation in mechanically strained cells. Stabilization of actin filaments by HSP27 that resulted from p38 MAPK activation, therefore, operates independently of the RhoA pathway because of disparate actin filament disruption by the respective inhibitors and inability of Rho kinase inhibitor to attenuate HSP27 phosphorylation. The notion that the two pathways are functionally independent is further strengthened by the observation of the reduction of stress fibers in cells co-transfected with constitutive active Rho and phosphodeficient HSP27 compared with RhoA alone. Furthermore, we observed the presence of more stress fibers in cells co-expressing dominant-negative RhoA and phosphomimicking HSP27 than RhoN17 alone. All these observations are in agreement with views that phosphorylation of HSP27 leads to actin filament stability independent of RhoA activation.

The present work also shows that cyclic strain of 30-minute duration did not alter the localization of unphosphorylated and phosphorylated HSP27 from cytoplasm to nucleus, in contradiction to other studies (28, 35–37). Of course, this observation neither excludes the possibility a cell-specific mechanism nor that nuclear localization is necessary for stress protection as inferred from the experiments with EGFP-HSP27 chimera in human A549 lung carcinoma and murine L929 (lacking endogenous HSP27) cells (28).

While we have no evidence that HSP27 phosphorylation requires RhoA activation, phosphorylated HSP27 may influence the role of actin-binding proteins in actin cytoskeleton dynamics. This involves stabilization, polymerization, and depolymerization of filaments, which are partly controlled by actin-binding proteins. Actin depolymerization factor (ADF/cofilin) and tropomyosin are two such proteins that control filament turnover (42, 43). ADF/cofilin is activated by Rho/ROCK/LIM kinase and Rac/Cdc42-PAK through LIM kinase/cofilin. More recently, the involvement of MK2/LIM kinase has been documented in endothelial cells (44). Thus, activation of LIM kinases by any one of the above-described pathways will have an impact on stress fiber formation and stabilization because phosphorylation of ADF/cofilin by LIMK inhibits actin depolymerization activity. Tropomyosin, on the other hand, binds actin and stabilizes actin stress fibers by stopping access of ADF/cofilin to actin filaments (43). Furthermore, phospho-cofilin binding protein 14-3-3 may play a critical role in cytoskeletal dynamics by binding to phosphorylated HSP27, which in turn leads to the presence of excess phosphorylated cofilin (26, 45, 46). Dephosphorylation of cofilin by cofilin phosphatases (called Slingshot and chronophin) allows cofilin to compete with tropomyosin for binding to the barbed ends (discrete sites), which will counteract the stabilization effect of tropomyosin with depolymerizing activity of ADF/cofilin. In view of the limited change in the organization of actin filaments in cells observed in the present study, the competing effects of cofilin, tropomyosin, 14-3-3, and HSP27 are likely to be detrimental for stress fiber stability and reduction in actin filament dynamics.

In conclusion, HSP27 is phosphorylated by mechanical strain in human airway smooth muscle cells through p38MAPK/MK2 signal transduction pathway. The cumulative disruptive effect of inhibitors (Y27632 and SB202190) on actin filaments suggests that the two signaling pathways, p38MAPK/MK2/HSP27 and Rho/Rho kinase, are mutually exclusive. The prospect of modulation of ADF/cofilin through LIM kinases by both signaling pathways brings a new perspective into MK2/HSP27-induced remodeling of actin filaments. This suggests the need for further studies.


   Acknowledgments
 
The authors thank Drs. W. T. Gerthoffer (University of Nevada School of Medicine) and S. S. An (Harvard School of Public Health) for HSP27 constructs. The authors are also grateful to Dr. M. Burke for critical reading of the manuscript and S. Richer for technical assistance with microscopy.


   Footnotes
 
Originally Published in Press as DOI: 10.1165/rcmb.2007-0263OC on April 3, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form June 11, 2007

Accepted in final form March 6, 2008


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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