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Am J Physiol Lung Cell Mol Physiol 293: L1194-L1207, 2007. First published August 24, 2007; doi:10.1152/ajplung.00453.2006
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Overexpression of human Hsp27 inhibits serum-induced proliferation in airway smooth muscle myocytes and confers resistance to hydrogen peroxide cytotoxicity

Sonemany Salinthone,1 Mariam Ba,1 Lisa Hanson,1 Jody L. Martin,2 Andrew J. Halayko,3 and William T. Gerthoffer1

1Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada; 2Cardiovascular Institute, Loyola University Medical Center, Maywood, Illinois; and 3Department of Physiology, University of Manitoba, Winnipeg, Manitoba, Canada

Submitted 16 November 2006 ; accepted in final form 24 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Airway smooth muscle (ASM) hypertrophy and hyperplasia are characteristics of asthma that lead to thickening of the airway wall and obstruction of airflow. Very little is known about mechanisms underlying ASM remodeling, but in vascular smooth muscle, it is known that progression of atherosclerosis depends on the balance of myocyte proliferation and cell death. Small heat shock protein 27 (Hsp27) is antiapoptotic in nonmuscle cells, but its role in ASM cell survival is unknown. Our hypothesis was that phosphorylation of Hsp27 may regulate airway remodeling by modifying proliferation, cell survival, or both. To test this hypothesis, adenoviral vectors were used to overexpress human Hsp27 in ASM cells. Cells were infected with empty vector (Ad5) or wild-type Hsp27 (AdHsp27 WT), and proliferation and death were assessed. Overexpressing Hsp27 WT caused a 50% reduction in serum-induced proliferation and increased cell survival after exposure to 100 µM hydrogen peroxide (H2O2) compared with mock-infected controls. Overexpression studies utilizing an S15A, S78A, and S82A non-phosphorylation mutant (AdHsp27 3A) and an S15D, S78D, and S82D pseudo-phosphorylation mutant (AdHsp27 3D) showed phosphorylation of Hsp27 was necessary for regulation of ASM proliferation, but not survival. Hsp27 provided protection against H2O2-induced cytotoxicity by upregulating cellular glutathione levels and preventing necrotic cell death, but not apoptotic cell death. The results support the notion that ASM cells can be stimulated to undergo proliferation and death and that Hsp27 may regulate these processes, thereby contributing to airway remodeling in asthmatics.

airway smooth muscle remodeling; proliferation and cytotoxicity; glutathione

ASTHMA IS A DISEASE CHARACTERIZED BY chronic inflammation, airway obstruction, bronchial hyperresponsiveness, and structural changes or remodeling of the airway wall.

The inflammatory and remodeling processes result from a highly complex interaction between various cell types, including inflammatory modulators like eosinophils and macrophages, and structural tissue cells like epithelial cells, fibroblasts, and smooth muscle cells (7, 26). The consequence of remodeling is subbasement membrane thickening, increased collagen deposition, mucosal metaplasia, and airway smooth muscle (ASM) hypertrophy and hyperplasia. The remodeling events lead to increased thickness of the airway wall that underpins excessive narrowing of the airway lumen, resulting in the decreased airflow that characterizes asthma.

Smooth muscle hypertrophy and hyperplasia can be the result of increased cellular migration and proliferation, or reduced cell death via apoptosis or necrosis. Many inflammatory modulators are detected in bronchoalveolar lavage fluid from asthmatic airways and have been shown to induce ASM mitogenesis in vitro (30). The mitogenic stimuli include peptide growth factors such as EGF, insulin-like growth factor, and PDGF, inflammatory cell mediators like {alpha}-thrombin and tryptase, ECM components, contractile agonists such as histamine, and cytokines (reviewed in Refs. 9 and 34). In contrast to the substantial evidence supporting ASM proliferation in asthmatic airways, there is little evidence for ASM apoptosis. Hamann et al. (18) were the first to demonstrate ASM apoptosis by treating cells with an antibody against the Fas receptor. Treatment with salbutamol and neutrophil elastase also induces ASM apoptosis (29). Airway biopsy studies, however, did not detect alterations in markers of apoptosis. Additionally, ASM cells in culture appear to be resistant to serum deprivation-induced apoptosis (unpublished observations). This supports the notion that decreased or abnormal apoptosis or necrosis may contribute to smooth muscle hyperplasia and hypertrophy.

Heat shock protein 27 (Hsp27) is a mammalian phosphoprotein that belongs to a family of small HSPs including Hsp20 and {alpha}B-crystallin. Expression of Hsp27 is induced in many cell types such as neurons, fibroblasts, and epithelial cells in response to heat shock, oxidative stress, and inflammatory mediators (reviewed in Ref. 4). Constitutive expression of Hsp27 is also detected in other cell types including ASM cells. Phosphorylation of Hsp27 is required for ASM cell migration in response to PDGF (19). Hsp27 also promotes proliferation and survival in other cell types including vascular smooth muscle (6, 45).

Our hypothesis was that Hsp27 participates in airway remodeling by regulating ASM proliferation and survival. To test this hypothesis, we surveyed the effect of several mitogenic and proapoptotic treatments known to induce proliferation and death in nonmuscle cells. Newborn calf serum (NCS) was the only stimulus to sufficiently induce ASM proliferation. TNF{alpha} and a combination of EGF and FGF did not. Sodium nitroprusside (SNP), H2O2, and Fas antibody were sufficient inducers of smooth muscle cell death. No evidence of apoptosis was observed, suggesting cell death resulted from necrotic cell death. Overexpression of wild-type Hsp27 decreased proliferation and increased cell survival. Blocking the p38 MAPK pathway, therefore preventing Hsp27 phosphorylation by MK2, did not have any effect on ASM mitogenesis or death. The results support the notion that Hsp27 may regulate ASM cell proliferation and survival, thereby contributing to airway remodeling in asthmatics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Sodium arsenite (NaAsO2), SNP, and all other tissue culture reagents were purchased from Sigma (St. Louis, MO). Hydrogen peroxide (H2O2) was purchased from Fisher Scientific (Houston, TX). SB-239063 was a generous gift from Glaxo Smith Kline. Antibodies for cyclooxygenase-1 (COX-1), caspase-3, p38 MAP kinase, and I{kappa}B{alpha} were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Phosphorylated p38 MAP kinase selective antibody was purchased from Cell Signaling Technology (Beverly, MA). Antibodies for the Fas receptor and phosphorylated histone were obtained from Upstate Biotechnology (Lake Placid, NY). Nonselective human Hsp27 antibody and Ser15-specific, Ser78-specific, and Ser82-specific phosphorylation antibodies were purchased from Stressgen (Victoria, BC, Canada). The cell titer 96 Aqueous One solution cell proliferation assay and lactate dehydrogenase (LDH) assay kits were purchased from Promega (Madison, WI). The glutathione (GSH) assay kit was obtained from Cayman Chemical (Ann Harbor, MI). The HEK-293 packaging cell line was purchased from Microbix (Toronto, ON, Canada).

Cell culture. Primary human bronchial smooth muscle cells (hBSMCs) were kindly provided by Dr. Andrew Halayko (Manitoba, Canada). hBSMC cultures were prepared as previously described from healthy segments of second-to-third generation mainstem bronchi obtained from patients undergoing lung resection surgery (18, 36). All procedures were approved by the Human Research Ethics Board (University of Manitoba). All hBSMCs were grown to passages 6–7 in a humidified 5% CO2 atmosphere chamber at 37°C in M199 medium supplemented with 10% NCS, 2 mM glutamine, 2 ng/ml FGF, and 0.5 ng/ml EGF. Media was changed every 3 days. All treatments were performed when cells were 95% confluent. HEK-293 packaging cells used for viral propagation were grown on DMEM media, supplemented with 10% fetal bovine serum. Viral infections were performed in M199 + 0.1% NCS, 2 mM glutamine, 2 ng/ml FGF, 0.5 ng/ml EGF, and ITS+ Premix (BD Biosciences). Infected cells were maintained in the same medium.

Adenoviral constructs and infections. Adenoviruses expressing wild-type Hsp27 (AdHsp27 WT), non-phosphorylation mutant (AdHsp27 3A), and a phosphorylation mutant form of Hsp27 (AdHsp27 3D) were prepared as previously described (27). The AdHsp27 WT contains an insert coding for human wild-type Hsp27. The AdHsp27 3A contains a form of human Hsp27 wherein the three serines known to be phosphorylated (Ser15, Ser78, and Ser82) have been mutated to alanines and was made as described previously (19). The AdHsp27 3D construct was kindly provided by Dr. Jody Martin (Loyola Univ., Maywood, IL) and contains serine to glutamic acid mutations at Ser15, Ser78, and Ser82. Transgene expression was driven by the immediate-early cytomegalovirus (CMV)-promoter, inserted into the E1 region of an E1-deleted human adenovirus type 5. Adenovirus vectors were propagated from the HEK-293 packaging cell line as previously described (17). Titers were calculated by infecting HEK-293 cells with serial dilutions of adenoviral constructs by plaque overlay assay. After 1 h of infection, cells were overlaid with a mixture of 1% agarose with 2x MEM + 5% yeast extract. Plaque-forming units (PFU) were counted 4 days after infection. Smooth muscle cells were infected with adenoviral vectors at a multiplicity of infection (MOI) of 20 PFU/cell. Media was removed, and cells were infected with virus diluted in a minimal volume of M199 + 0.1% NCS. Infected cells were incubated at 37°C for 1 h, with manual shaking occurring every 15 min. M199 + 0.1% NCS was then added to appropriate volume. Cells were incubated at 37°C for 3 days before experiments. Media was aspirated out and replaced with fresh M199 + 0.1% NCS every other day. For mock infection, cells were treated exactly as above, except with no virus added to the M199 + 0.1% NCS.

Immortalized tetracycline-inducible hBSMC lines. Low-passage (3–5) hBSMCs were immortalized by retroviral gene transfer. Briefly, RetroPak PT67 packaging cells (Clontech) were transfected with the retroviral vector pLXIN containing human telomerase reverse transcriptase (hTERT), and the resulting viral-containing supernatant was used to infect hBSMC between passages 3 and 4. Stable cell populations were selected with 200 µg/ml G418 (GIBCO). The retroviral gene expression system RevTet-Off (Clontech) was used to establish inducible Hsp27 hBSMC lines. In the absence of tetracycline or doxycycline, transcription was activated by binding of the tetracycline-controlled transactivator (tTA) to the tet-response element (TRE) upstream of the CMV promoter. The retroviral vector pRevTet-Off containing the tTA was modified to express the puromycin selection marker instead of neomycin because the immortalized hBSMC already expressed a neomycin marker. The neomycin gene was removed by restriction digestion with AscI and partial digestion with NcoI. A puromycin resistance gene was digested from pLPCX (Clontech) with AscI and NcoI and ligated into the partially digested pRevTet-Off vector to create pRevTet-Off (puro). pRevTet-Off (puro) was used to create the parental cell line hBSMC-tTA. RetroPak PT67 packaging cells (Clontech) were transfected with pRevTet-Off (puro), and the resulting virus-containing supernatant was used to infect immortalized hBSMC. Stable cell populations were selected for 2–4 days in the presence of puromycin (1 µg/ml). This cell line served as a control and parental cell line for infection with retroviral pRevTRE Hsp27 WT and Hsp27 3A mutant. RetroPak PT67 cells were transfected with pRevTRE Hsp27 WT or the 3A mutant. Stable hBSMC-tTA cells were infected with the virus-containing supernatant and selected for ~5 days in the presence of 1 mg/ml hygromycin. Stable cell populations were then propagated in complete media plus 50 ng/ml doxycycline. Induction of Hsp27 overexpression was performed by removing doxycycline containing media and replating in serum-free media. Cells were washed twice with PBS before trypsinizing and at 12 and 24 h after replating. This was necessary to remove all traces of doxycycline. Upregulation of Hsp27 expression was verified by SDS-PAGE analysis of proteins and Western blotting.

Cell proliferation. To measure proliferation, cells were trypsinized, plated in six-well plates at 69,000 cells/well, and arrested for 24 h with M199 plus 0.1% NCS media. Mitogenesis was stimulated with 10% NCS, 10 ng/ml TNF{alpha}, or a combination of EGF and FGF (0.5 and 2 ng/ml, respectively). Cells were then incubated at 37°C for 0, 2, or 3 days. Media was aspirated, and cells were lifted by trypsinization. Five hundred microliters each of the cell suspension was diluted in 10 ml of hypotonic solution (Fisher Scientific, Santa Clara, CA) and counted using a Coulter counter (Fullerton, CA).

For Hsp27 overexpression experiments, cells were either infected or not infected with empty vector (Ad5), AdHsp27 WT, AdHsp27 3D, or AdHsp27 3A mutant for 3 days and then replated into six-well plates at 69,000 cells/well. Mitogenesis was stimulated with 10% NCS for 0, 2, or 3 days, and cells were counted as above.

For p38 MAPK inhibition experiments, confluent cells were trypsinized and plated into six-well plates at 69,000 cells/well and concomitantly arrested in M199 plus 0.1% NCS for 24 h. Cells were pretreated with 10 µM SB-239063 for 30 min and stimulated with M199 plus 10% NCS and 10 µM SB-239063 for 0, 2, or 3 days. Drugs and media were refreshed daily. Cells were then harvested and counted as described.

Cell cycle analysis by flow cytometry. Tet-Off regulated hBSM cell lines were grown to confluence in M199 plus 10% NCS with 50 ng/ml doxycycline. Cells were trypsinized and replated at 50% confluence in M199 plus 0.1% NCS minus doxycycline for 3 days. Cells were then treated with M199 media plus 10% NCS minus doxycycline and incubated at 37°C for 2 or 3 days. Media was removed, and cells were washed two times in PBS. Cells were trypsinized, centrifuged, and resuspended in hypotonic solution containing 14.3 µg/ml RNase A, 0.01% Triton X-100, 20 µg/ml propidium iodide, and 0.11 mg/ml trisodium citrate in PBS. Cells were incubated for 2 h at 37°C. Cells were centrifuged at 4,000 g for 5 min and resuspended in 500 µl of hypotonic solution. Forward light scatter, side light scatter, and PI fluorescence data were collected using a flow cytometer (Beckman Coulter XL-MCL, Fullerton, CA). Sample analysis was performed using FlowJo software (Tree Star, Ashland, OR) to obtain cell cycle distribution of propidium iodide-positive cells.

MK2 activity assay. To verify the efficacy of SB-239063 in inhibiting p38 MAP kinase, we assayed activity of MAP kinase-activated protein kinase 2 (MAPKAP kinase 2), which is activated by p38 MAP kinase. MAPKAP kinase 2 was assayed by extracting the enzyme from cultured cells stimulated with serum for 30 min in 30 mM MOPS, 80 mM beta-glycerolphosphate, 2 mM EGTA, 0.1 mM Na3VO4, 25 mM MgCl2, 40 mM KCl, 0.1 mM NaF, 1 mM AEBSF, and 1 µM leupeptin. Protein concentrations were determined using the bicinchoninic acid method using bovine serum albumin as the standard. Kinase activity was assayed at 30°C for 30 min in 60 µl of final volume containing 25 mM MOPS (pH 7.2), 25 mM beta-glycerophosphate, 15 mM MgCl2, 1 mM EGTA, 0.1 mM NaF, 1 mM Na3VO4, 4 mM dithiothreitol, 10 µg protein from cell extracts, 10 µCi of [{gamma}-32P]ATP (250 µM), and 0.15 mg/ml recombinant human Hsp27. Recombinant human Hsp27 was expressed in Escherichia coli and purified by DEAE chromatography as previously described (24). Phosphorylated Hsp27 was resolved by 12% acrylamide SDS-PAGE, and phosphorylation levels were visualized and quantitated with a Bio-Rad model GS-525 Molecular Imager and Molecular Analyst software.

Cell viability assay. hBSM cells were infected with Ad5 empty vector, AdHsp27 WT, AdHsp27 3D, or AdHsp27 3A for 3 days, trypsinized, and plated in 96-well plates at 10,000 cells/well for 24 h. Uninfected control cells were processed in the same manner. After a total of 4 days postinfection, cells were treated with varying concentrations of H2O2, Fas antibody, SNP, or NaAsO2. The MTS cell proliferation assay was then performed using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay following the manufacturer's protocol (Promega, Madison, WI). Briefly, media was removed and replaced with 100 µl of fresh M199 media plus 0.1% NCS. Twenty microliters of CellTiter 96 Aqueous One Solution Reagent was added and incubated at 37°C for 3 h. The production of formazan was then measured by measuring absorbance at 490 nm using a Bio-Rad model 680 microtiter plate reader (Hercules, CA). The absorbance for the uninfected control samples was defined as 100% survival, and absorbance of treated samples was expressed as a percentage of the nontreated control.

To determine the effect of p38 MAPK inhibition on proliferation, confluent cells were trypsinized and replated into 96-well plates at 10,000 cells/well in M199 plus 0.1% NCS for 24 h. Arrested cells were pretreated with 10 µM SB-239063 for 30 min and then exposed to H2O2 for 24 h. Cells were then used for MTS assay as described above.

Caspase-3 cleavage. Confluent hBSMCs were arrested for 24 h and then treated with 100 µM H2O2 for various times. Media were removed, and cells were washed three times with ice-cold PBS. Cells were scraped in extraction buffer containing 60 mM Tris·HCl, 2% SDS, 10% glycerol, 1 µM leupeptin, 1 mM EGTA, 1 mM Na2EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), and 5 mM NaF and processed for immunoblotting as described below.

Immunocytochemistry. Confluent hBSMC were trypsinized and plated on glass coverslips at 50,000 cells/10 cm2 in M199 plus 0.1% NCS for 24 h. Cells were stimulated with H2O2 for 24 h. Media was removed, and cells were washed three times with PBS. Cells were fixed for 15 min with the addition of 4% paraformaldehyde in PBS at room temperature. The cells were washed three times for 5 min each with PBS. Cells were permeabilized by incubation with 0.2% Triton-X 100 diluted in PBS, pH 7.4, at room temperature for 20 min, followed by washing in PBS. Nonspecific antibody binding was blocked by incubating coverslips in 2% bovine serum albumin in PBS for 1 h. Primary antibodies were incubated overnight at 4°C. Rabbit anti-phospho histone H2B IgG primary antibody was diluted in 2% BSA plus 0.1% Tween 20. Coverslips were then washed four times in PBS for 5 min each. Cells were incubated with secondary antibody at room temperature for 1 h. Alexa 488-conjugated goat anti-rabbit IgG secondary antibodies were diluted in 2% BSA + 0.1% Tween 20 at 1:600 dilution. Cells were washed four times for 5 min each and then incubated in 1 µM 4'-6-diamidino-2-phenylindole diluted in PBS for 5 min. Cells were washed four times, and coverslips were mounted with Aqua-Mount (Fisher Scientific, Fair Lawn, NJ). Fluorescently labeled cells were viewed on a Nikon Eclipse 800 microscope (Melville, NY). Images were collected using SimplePCI software (Compix, Cranberry Township, PA) and a Spot RT color charge-coupled device camera (Diagnostic Instruments, Sterling, MI).

DNA fragmentation. hBSMCs were plated in six-well plates and grown to 80–90% confluence in M199 plus 10% NCS and then growth arrested in 0.1% M199 for 24 h. Cells were treated with H2O2 for 24 h, and genomic DNA was extracted using the Wizard Genomic Kit (Promega) following the manufacturer's protocol. Briefly, treated cells were washed in PBS, scraped in 200 µl of sterile PBS, and centrifuged at 14,000 g for 10 s. The supernatant was decanted, and 100 µl of Nucleic Acid Lysis Buffer was added to lyse the cells. RNase solution (0.5 µl) was added to the mixture and incubated at 37°C for 20 min to digest any RNA present. To remove protein, 33.3 µl of Protein Precipitation solution was added and chilled on ice for 5 min. The sample was centrifuged at 14,000 g for 4 min, and the supernatant was transferred to a new tube. DNA was precipitated in 100 µl of isopropanol and washed in 70% ethanol. The ethanol was aspirated, and the DNA was resuspended in DNA rehydrate solution. DNA fragments were separated by loading 5 µg of DNA on a 2% agarose gel and then stained in ethidium bromide (1 µg/ml). The gel was visualized under UV light using an Alpha Imager 2200 camera and software (Alpha Innotech, San Leandro, CA).

LDH assay. Confluent hBSMCs were trypsinized, plated into 96-well plates in M199 plus 0.1% NCS at 10,000 cells/well, and then incubated at 37°C for 24 h. Cells were then treated with varying concentrations of H2O2 for 24 h. Media was used to assay for LDH release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit following the manufacturer's protocol (Promega). Fifty microliters of treated cell media was transferred to a 96-well plate. Reconstituted substrate mix was then added to each well (50 µl) and incubated for 30 min at room temperature in the dark. LDH converts tetrazolium salt into a red formazan product with the help of an electron acceptor (diaphorase). Termination of the reaction was achieved by adding 50 µl of stop solution into each well. Absorbance was recorded at 490 nm. Maximum LDH release was arbitrarily set at 100%, and all other samples were expressed as percentages thereof.

I{kappa}B degradation. hBSMCs were grown to 80% confluence and were then either infected or not infected with 20 MOI Ad5 or AdHsp27 WT for 3 days in M199 plus 0.1% NCS. Cells were then treated with 100 µM H2O2 for various times. Media was removed, and cells were washed three times with cold PBS. Total protein was extracted in extraction buffer and processed for immunoblotting as described.

GSH assay. hBSMCs were grown to 80% confluence and were infected or not infected with 20 MOI Ad5 or AdHsp27 WT for 3 days. Cells were trypsinized, plated into 96-well plates in M199 plus 0.1% NCS, and incubated at 37°C for 24 h. Cells were either treated or not treated with 100 µM H2O2 for 24 h. Media was aspirated, and cells were washed with PBS. Five percent metaphosphoric acid solution was added to each well. Cells were lysed by freezing at –80°C and thawing at 37°C. The process was performed three times. GSH levels were measured using a GSH assay kit (Cayman Chemical, Ann Arbor, MI). Samples of cell lysate (50 µl) were transferred to a new 96-well plate. Assay cocktail mix (150 µl) containing MES buffer, reconstituted cofactor mixture, reconstituted enzyme mixture, 5,5'-dithio-bis-2-nitrobenzoic acid, and water was added to each well. The plate was incubated in the dark at room temperature for 25 min. The absorbance was recorded at 405 nm. The concentration of GSH was determined by extrapolation from the GSH standard curve that was processed concomitantly with all cell lysate samples.

Immunoblot analysis. Total protein was extracted from hBSMC to measure caspase-3, COX-1, Hsp27, phosphorylated Hsp27, and I{kappa}B expression. Treated or nontreated cells were washed with cold PBS and immediately lysed with extraction buffer containing 60 mM Tris·HCl, 2% SDS, 10% glycerol, 1 µM leupeptin, 1 mM EGTA, 1 mM Na2EDTA, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, and 5 mM NaF. Cellular extracts were sonicated for 20 min and centrifuged at 10,000 g for 20 min at 4°C. The supernatants were used to assay for protein expression. Protein concentrations were determined by the bicinchoninic acid method using bovine serum albumin as the standard. Total protein extracts were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose paper and blocked in a 50:50 solution of Odyssey Blocking Buffer (LI-COR, Lincoln, NE) and 1x PBS for 1 h. Protein detection was achieved by labeling with anti-human caspase-3 (sc-1226, 1:2,000), COX-1 (sc-1752, 1:15,000), Hsp27 (spa 800, diluted 1:10,000), phospho-Ser15, phosphor-Ser78, or phospho-Ser82 Hsp27 phosphorylation-specific antibodies (each diluted 1:5,000) or I{kappa}B{alpha} (SC-371, diluted 1:5,000) primary antibodies diluted in a 50:50 solution of blocking buffer and PBS + 0.1% Tween 20 for 1 h. The membranes were washed four times with PBS + 0.1% Tween 20 and then incubated with anti-goat, anti-rabbit, or anti-mouse Alexa Fluor 680-conjugated secondary antibody (Molecular Probes) diluted 1:100,000 in a 50:50 solution of blocking buffer and PBS + 0.1% Tween 20 for another hour. The membranes were washed as previously described, rinsed with PBS, and viewed with the Odyssey Infrared Imaging System (LI-COR).

Statistical analysis. Statistical analysis was performed by post hoc testing with the Student-Newman-Keuls method or a paired t-test using SigmaStat software (Jandel Scientific, San Rafael, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Overexpression of wild-type and mutant Hsp27 in hBSMC. In human ASM cells, Hsp27 is constitutively expressed, and endogenous levels are not altered upon treatment with a variety of stimuli (19). Therefore, to determine the role of Hsp27 in modulation of smooth muscle proliferation and survival, an overexpression strategy was utilized. Recombinant adenoviral vectors were used at 20 MOI to overexpress wild-type Hsp27 (AdHsp27 WT) and phosphorylation-deficient mutant Hsp27 (AdHsp27 3A) in 80% confluent hBSMCs. All experiments were conducted 3 days postinfection, at which time media was aspirated, cells were washed in ice-cold PBS, and protein lysates were harvested. Proteins from these clarified lysates were separated via SDS-PAGE, and blots were probed with human Hsp27 antibody. Bands from resulting blots were analyzed via densitometry. Figure 1A shows immunoblots of Hsp27 demonstrating overexpression 3 days postinfection. COX-1 expression was used for loading control since the expression of COX-1 does not change in ASM cells under a variety of conditions (38). Densitometric analysis revealed an approximately fourfold overexpression of AdHsp27 WT and roughly a threefold increase in AdHsp27 3A transgene expression (Fig. 1B) compared with empty vector and mock-infected cells, which showed significant levels of endogenous Hsp27.


Figure 1
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  		Fig. 1. Heat shock protein 27 (Hsp27) overexpression in human bronchial smooth muscle cells (hBSMCs). Adenoviral vectors were used to overexpress an empty vector control (Ad5), wild-type Hsp27 (AdHsp27 WT), or Hsp27 phosphorylation mutant (AdHsp27 3A) in hBSMCs at 20 multiplicity of infection (MOI) for 3 days. A: Western blots using anti-Hsp27 antibody (1:10,000 dilution) illustrate Hsp27 transgene overexpression. B: densitometry analysis was performed on immunoblots to quantify Hsp27 expression. Data were normalized to mock-infected controls. Hsp27 wild-type and mutant exhibited a 4-fold and 2.5-fold, respectively, increase in transgene expression. N = 3 donors. *Significantly different from unstimulated controls. P < 0.05. COX-1, cyclooxygenase-1.

 
Survey of mitogens. Many inflammatory mediators, such as cytokines like TNF{alpha} and IL-1beta, and peptide growth factors, are mitogenic to ASM cells (3, 21, 47). To induce proliferation in hBSMCs, cells were plated at 69,000 cells/well (~35% confluent) and concomitantly arrested for 24 h. Cells were then stimulated with either 10 ng/ml TNF{alpha}, a combination of EGF and FGF (0.5 and 2 ng/ml, respectively), or 10% serum (NCS) for 0, 2, or 3 days. Cells were trypsinized and counted using the Coulter counter. Results indicated that neither TNF{alpha} nor a combination of EGF and FGF stimulation induced hBSMC proliferation (Fig. 2). Ten percent serum induced a doubling in smooth muscle cells after 3 days of treatment, resulting in a final cell density of ~120,000 cells/well.


Figure 2
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  		Fig. 2. Serum-induced airway smooth muscle (ASM) cells. hBMSCs were seeded at 69,000 cells/well and simultaneously arrested for 24 h. Cells were stimulated with 10 ng/ml TNF{alpha} and EGF and FGF or 10% serum for 0, 2, and 3 days. TNF{alpha} and EGF and FGF did not stimulate hBSMC growth. There was a doubling in cell number after 3 days of serum stimulation. N = 3 donors in duplicate. *Significantly different from unstimulated controls. P < 0.05.

 
Effect of hsp27 on serum-induced hBSMC proliferation. We then used 10% serum as the stimulus to test the hypothesis that Hsp27 expression levels could alter ASM proliferation rate. hBSMCs were mock-infected or infected at 20 MOI for 3 days with one of two adenoviruses, the empty vector (Ad5) or AdHsp27 WT. Proliferation was stimulated with 10% serum for 0, 2, or 3 days. Cells were counted, and growth data were expressed as fold change of unstimulated cells (Fig. 3A). As expected, infection with Ad5 resulted in some initial decrease in proliferation, suggesting that the infection process itself affects growth. There was only a 1.5-fold increase in growth after a 2-day stimulation compared with mock-infected cells. However, a twofold increase in growth was observed after 3 days, similar to mock-infected cells. In contrast, cells overexpressing wild-type Hsp27 did not proliferate significantly after serum stimulation (Fig. 3A). These data suggest a role for Hsp27 in modulation of ASM cell proliferation.


Figure 3
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  		Fig. 3. Inhibition of smooth muscle cell proliferation by Hsp27. A: cells were mock-infected or infected with 20 MOI of Ad5 empty vector or AdHsp27 WT for 3 days. Cells were trypsinized and plated at 69,000 cells/well and then stimulated with 10% serum for 0, 2, and 3 days. Growth was expressed as fold-change of unstimulated samples. Cells overexpressing wild-type Hsp27 resulted in very little growth compared with mock-infected or Ad5-infected cells. B: cells were infected with Ad5, Hsp27 WT, or Hsp27 3A and stimulated with serum for 3 days. Hsp27 WT inhibited proliferation, whereas Hsp27 3A did not. C: cells were infected with 40 MOI of viruses and stimulated with 10% serum for 3 days. Data indicate that increasing Hsp27 3A expression does not inhibit proliferation. D: cells were infected with Ad5 or AdHsp27 3D phosphorylation mutant and stimulated with serum for 3 days. Hsp27 3D inhibits proliferation, suggesting that Hsp27 phosphorylation may mediate Hsp27 inhibition of cell proliferation in ASM cells. N = 4–5 donors in duplicate. *Significantly different from mock-infected and Ad5-infected controls. P < 0.05.

 
The effect of hsp27 phosphorylation on cell proliferation. Human Hsp27 undergoes stress-induced phosphorylation on Ser15, Ser78, and Ser82 by activated MK2 (23). Changes in Hsp27 phosphorylation regulate the polymer size of Hsp27, chaperone functions, and antiapoptotic effects in nonmuscle cells (reviewed in Ref. 8). In addition, stimulation of ASM cells with 10% serum resulted in phosphorylation of endogenous Hsp27 at Ser15, Ser78, and Ser82 (Fig. 4C). To determine whether inhibition of hBSMC proliferation is a function of the phosphorylation state of Hsp27, cells were infected with phosphorylation-deficient mutant Hsp27 in which serines at positions 15, 78, and 82 were mutated to alanines. Infected cells were then trypsinized, plated, and stimulated with 10% serum for 0, 2, or 3 days. Cells were counted, and data were expressed as fold change of the cell count at day 0 (Fig. 3B). Overexpression of mutant Hsp27 had no effect on serum-induced proliferation, which was in contrast to the inhibitory effect of wild-type Hsp27, suggesting the effect of Hsp27 on smooth muscle proliferation is phosphorylation dependent. Since the expression of Hsp27 3A is less than Hsp27 WT, we also conducted an experiment whereby ASM cells were infected with 40 and 80 MOI of viruses to determine if the results obtained were due to differences in protein expression. As shown in Fig. 3C, overexpression of AdHsp27 3A at 40 MOI did not inhibit ASM cell proliferation compared with Ad5-infected cells. A similar result was obtained with infection at 80 MOI (results not shown). To further confirm the observation that Hsp27 phorphorylation may be important in regulating smooth muscle cell proliferation, the pseudophosphorylated mutant (AdHsp27 3D) where the three serines were mutated to glutamic acid residues to mimic phosphorylation was overexpressed, and the effect on proliferation was measured. Consistent with the results obtained with AdHsp27 WT overexpression, AdHsp27 3D also inhibited ASM cell proliferation (Fig. 3D). Collectively, these data suggest that the inhibition of smooth muscle proliferation by Hsp27 is mediated in part by its phosphorylation state.


Figure 4
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  		Fig. 4. Effect of p38 MAPK on hBSMC proliferation. A: crude extracts of cells stimulated with 10% serum in the absence or presence of SB-239063 or vehicle control were used to assay for Hsp27 phosphorylation. Stimulation resulted in an ~2-fold increase in Hsp27 phosphorylation compared with unstimulated control. Treatment with SB-239063 attenuated serum-induced Hsp27 phosphorylation to basal levels. *P < 0.05 compared with stimulated samples; #P < 0.05 compared with stimulated samples minus SB-239063. B: hBSMCs were seeded at 69,000 cells/well and arrested for 24 h. Cells were pretreated with 10 µM SB-239063 or vehicle control for 30 min and then stimulated with 10% serum. Both DMSO- and SB-239063-treated samples exhibited an ~2.5-fold increase in proliferation. C: arrested confluent hBSMCs were pretreated with DMSO vehicle control or 10 µM SB-239063 for 30 min before stimulation with 10% serum for 30 min. Total protein was extracted and used for immunoblot analysis for Hsp27 phosphorylation. Serum stimulated Hsp27 phosphorylation at Ser15, Ser78, and Ser82. SB-239063 blocked phosphorylation at Ser15 and Ser78. N = 4 donors.

 
The effect of p38 MAPK on serum-induced hBSMC proliferation. Because Hsp27 is a downstream effector of the p38 MAPK signaling cascade, we sought to elucidate the role of p38 MAPK in serum-induced smooth muscle proliferation. Hsp27 phosphorylation depends on activation of p38 MAPK and subsequent activation of MK2. We used the second generation p38 MAPK inhibitor SB-239063 to block p38 activation (41). The efficacy of the drug was verified using an MK2 kinase assay whereby cell-free phosphorylation of Hsp27 is measured. Confluent, growth-arrested hBSMCs were treated with 10% serum with or without SB-239063 for 30 min. Crude protein lysates were harvested and used to phosphorylate purified recombinant human Hsp27. Figure 4A demonstrated an ~1.8-fold increase in MK2 activity upon stimulation with 10% serum compared with unstimulated control. Pretreatment with 10 µM SB-239063 reduced MK2 activity to below basal levels, verifying the efficacy of SB-239063 to inhibit p38 MAPK in hBSMCs. This was further confirmed by Western blot analysis checking for inhibition of endogenous Hsp27 phosphorylation. Pretreatment with 10 µM SB-239063 inhibited serum-induced phosphorylation of Hsp27 at Ser15 and Ser78 (Fig. 4C).

To define the role of p38 MAPK in hBSMC proliferation, cells were plated at 69,000 cells/well and arrested in low-serum media for 24 h. Cells were treated with 10 µM SB-239063 or vehicle control for 30 min, stimulated with 10% serum for 0, 2, and 3 days, and then counted. Since SB-239063 is subject to degradation, drug and media were refreshed each day. The results obtained for vehicle control demonstrated a doubling in cell number after 2 days of serum stimulation (Fig. 4B). By day 3, there was a fivefold increase in cell growth compared with unstimulated control. Pretreatment with SB-239063 did not result in inhibition in cell proliferation as observed with cells that were overexpressing Hsp27 WT. Similar to vehicle control, there was a doubling in cell population after stimulation with serum for 2 days. There was also a fivefold increase in proliferation after 3 days. Results indicated that blocking p38 MAPK did not have an effect on serum-stimulated smooth muscle cell proliferation, suggesting that the p38 MAPK signaling cascade may not be involved in the inhibitory effect of Hsp27 on serum-induced smooth muscle proliferation. This observation was unexpected, but not surprising since the p38 MAPK pathway has been shown by many investigators to not regulate cellular proliferation.

Hsp27 does not inhibit hBSMC proliferation by halting cell cycle progression. To determine if Hsp27 inhibits hBSMC proliferation by inhibiting cell cycle progression, Tet-off stable cell lines overexpressing wild-type and mutant Hsp27 were used. Hsp27 cell lines were grown to confluence in media plus 50 ng/ml doxycycline. Transcriptional activation of Hsp27 was examined 7 days after removal of doxycycline, which allows the tTA to bind to the TRE upstream of the CMV promoter, therefore driving transcription of Hsp27. For cell cycle analysis, the cell lines were grown to confluence in the presence of doxycycline. Cells were trypsinized, plated at 50% confluence, and concomitantly arrested in M199 plus 0.1% NCS without doxycycline for 4 days. Cells were then stimulated with 10% serum for 2 or 3 days and then harvested for flow cytometry as described. Using FlowJo software for data analysis, the cell cycle profile for each cell line was obtained (data not shown). There was no significant difference in the distribution of cells in various phases of the cell cycle between tTA control and Hsp27 WT or 3A cell lines. The data suggest that Hsp27 does not inhibit smooth muscle proliferation by halting cell cycle progression once cells enter the cell cycle and that Hsp27 may have an effect on the rate of proliferation.

Survey of cell death inducers. We next addressed the hypothesis that Hsp27 has antiapoptotic effects in airway smooth muscle similar to those reported in nonmuscle cells (8). An alternate hypothesis was that Hsp27 affected necrotic cell death as in vascular smooth muscle cells (6). To study the effect of Hsp27 on cell survival, we first conducted a survey of chemicals that would induce hBSMC death. H2O2, SNP, NaAsO2, and Fas antibody all induce apoptosis in both muscle and nonmuscle cell types. We tested these agents on confluent hBSMCs seeded in 96-well plates at 10,000 cells/well and growth arrested for 24 h. Cell death was stimulated with varying concentrations of H2O2, SNP, Fas antibody, or NaAsO2 for 24 h. Viability was assessed using the MTS assay, which measures the integrity of the mitochondria, following the manufacturer's protocol. The absorbance signal of untreated cells was defined as 100% survival, and the data for treated samples were expressed as percentages of untreated controls. Figure 5, A–C, demonstrated the effectiveness of H2O2, SNP, and Fas antibody induction of smooth muscle cell death at 24 h. At 100 µM H2O2, there was 50% death, and the amount of killing was increased to 70% with 200 µM H2O2. At maximum concentration, SNP and Fas antibody resulted in ~55% and 50% cell death, respectively. NaAsO2, however, was not effective in stimulating cell death, even at the highest concentration used (Fig. 5D).


Figure 5
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  		Fig. 5. Induction of smooth muscle cell death. hBSMCs were plated onto a 96-well plate at 10,000 cells/well and then treated with varying concentrations of H2O2 (A), sodium nitroprusside (SNP) (B), Fas antibody (C), and NaAsO2 (D) for 24 h. Viability was assessed using the MTS assay where tetrazolium is broken down to formazan. Untreated samples were arbitrarily designated as 100% survival, and treated samples were expressed as percentages of untreated controls. Results indicated that H2O2, SNP, and Fas antibody can each induce significant cell death after 24 h. In contrast, NaAsO2 was ineffective. N = 4 donors in triplicate.

 
Protective effect of hsp27 on hBSMC death. To determine whether Hsp27 could modify smooth muscle cell survival, hBSMCs were mock-infected or infected at 80% confluence with Ad5 or AdHsp27 WT at 20 MOI for 3 days. Cells were trypsinized and plated into 96-well plates and incubated at 37°C for 24 h. Various concentrations of H2O2, SNP, and Fas antibody were used to induce cell death, and MTS assay was performed 24 h after treatment with H2O2. Survival was expressed as percentages of unstimulated control, which was arbitrarily designated as 100% survival. Hsp27 overexpression resulted in ~25–30% increase in survival at 100 µM H2O2 compared with mock-infected cells (Fig. 6A). There was no significant difference in viability at 50 or 200 µM H2O2 (data not shown). In contrast, there was no difference in viability between mock-infected, Ad5 or AdHsp27 WT-infected cells after treatment with either SNP or Fas antibody (data not shown).


Figure 6
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  		Fig. 6. Effect of Hsp27 overexpression on smooth muscle viability. A: hBSMCs were infected with Ad5, wild-type Hsp27, or Hsp27 3A and plated onto 96-well plates 72 h postinfection. Cells were then treated with H2O2 24 h later. Viability was assessed using MTS assay. A: cells overexpressing Hsp27 wild-type and Hsp27 3A resulted in increased viability, suggesting that Hsp27 may provide protection against H2O2 cytotoxicity. N = 5 donors in triplicate. Significantly different from mock-infected and Ad5-infected controls; P < 0.05. B: arrested cells were either treated or not treated with the p38 MAPK inhibitor SB-239063 for 30 min and then stimulated with H2O2. The MTS cell viability assay was performed, and viability was expressed as percentage of unstimulated controls. N = 4 donors in triplicate. Significantly different compared with untreated samples. C: cells were infected with Ad5 or AdHsp27 3D and stimulated with H2O2 for 24 h. AdHsp27 did not provide cellular protection against H2O2. D: cells were either pretreated with DMSO control or 10 µM SB-239063 for 30 min and then stimulated with 100 µM H2O2 for 30 min. Phosphorylation of Hsp27 was determined by immunoblotting. H2O2 phosphorylated Hsp27 at Ser15, Ser78, and Ser82. SB-239063 inhibited phosphorylation of Ser15 and Ser78. N = 4 donors.

 
We then tested the notion that the degree of protection against H2O2-induced cell death might be enhanced by increasing Hsp27 overexpression levels. hBMSCs were infected with 0, 20, 40, or 80 MOI AdHsp27 WT for 3 days at 37°C. Cells were trypsinized, plated, and treated in the same fashion as previously described. Similar to previous observations, infection with AdHsp27 WT at 20 MOI resulted in ~50% increase in survival when stimulated with 100 µM H2O2 (data not shown). Interestingly, cells infected with 40 or 80 MOI of AdHsp27 WT were not any more resistant to H2O2 than cells infected at 20 MOI. This suggests that the degree of overexpression achieved at 20 MOI was sufficient for maximum protection. Collectively, the overexpression data provide evidence for Hsp27 enhancement of smooth muscle cell survival after oxidative stress.

Measurement of apoptosis markers. H2O2 induces both apoptotic and necrotic cell death depending on the cell type. To determine whether H2O2-induced hBSMC death is through apoptosis or necrosis, several apoptotic markers were measured after H2O2 exposure. Activation of the caspase cascade, specifically caspase-3, is a marker of early apoptotic events. Upon activation, caspase-3 gets cleaved at the DEVD amino acid sequence, giving rise to p20 and p11 cleavage products. To measure caspase-3 activation, confluent cells were arrested for 24 h and then stimulated with 100 µM H2O2 for various times. Total protein was extracted and separated on 10% SDS-PAGE gel. Immunoblots were probed with caspase-3 antibody that recognized the p20 subunit of caspase-3. Band intensity of procaspase-3 (inactive form) was quantitated and expressed as percentages of untreated samples. Results showed that H2O2 treatment did not induce caspase-3 cleavage at any time point studied (Fig. 7A).


Figure 7
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  		Fig. 7. Measurement of apoptosis markers. A: arrested hBSMCs were treated with 100 µM H2O2 for 24 h. Total protein was extracted, and caspase-3 cleavage experiments were performed by immunoblot analysis. H2O2 did not induce caspase-3 cleavage in hBSMCs. B: cells were plated on glass coverslips and arrested for 24 h before stimulation with H2O2. Panels a–d show results with airway smooth muscle cells. Panels e–h show results with HeLa cells. Hydrogen peroxide did not induce histone H2B phosphorylation as seen with colcemid-treated HeLa-positive controls (arrow). C: Lactate dehydrogenase (LDH) assay was performed on H2O2-stimulated cells to measure LDH release. Data were expressed as fold change LDH release. N = 3–4 donors; LDH assay was performed in triplicate. *Significantly different compared with untreated samples. P < 0.05.

 
Histone H2B phosphorylation is another apoptosis marker. Histone H2B, along with histones H2A, H3, and H4, form nucleosomes that are associated with chromatin. Phosphorylation of H2B occurs at the stage in apoptosis when nuclear DNA is cleaved by caspase-associated DNase enzymes (1). To determine if H2O2 induces histone phosphorylation, hBSMCs were seeded onto glass coverslips at 50,000 cells/well and growth arrested in M199 media plus 0.1% NCS for 24 h. Cells were stimulated with 100 µM H2O2 for 24 h and then fixed with paraformaldehyde. After fixation, cells were permeabilized, stained with anti-phosphophorylated histone antibody, and visualized under fluorescent microscopy. Figure 7B, top, is a montage of images displaying hBSMCs stained with Alexa 488 anti-rabbit secondary antibody. Unstimulated hBSMCs displayed some background staining, in which the fluorescent intensity was similar to H2O2-treated cells. The data suggest that H2O2 does not induce histone phosphorylation. For this experiment, Hela cells were used as positive controls. Histone phosphorylation in Hela cells was induced with 100 ng/ml colcemid to inhibit microtubule polymerization and mitotic spindle formation. Similar to the results obtained for hBSMCs, there was some background staining of untreated control cells. As expected, colcemid-stimulated Hela cells displayed nuclear staining with a punctate staining pattern indicative of histone phosphorylation (Fig. 7B).

DNA fragmentation is a late endpoint measurement of apoptosis. To assess if H2O2 increased DNA fragmentation, hBSMCs were grown to confluence and then growth arrested for 24 h. Genomic DNA was harvested from treated and nontreated cells, and DNA was separated on 2% agarose gel, stained with ethidium bromide, and visualized under UV irradiation. There was no evidence of DNA fragmentation in untreated samples, nor did treatment with 50 or 100 µM H2O2 induce DNA fragmentation (data not shown).

The apparent lack of effect of H2O2 on caspase-3 cleavage, histone phosphorylation, and DNA fragmentation suggests that H2O2-induced ASM cell death is not through apoptosis, but probably necrosis. To test this hypothesis, we assayed release of LDH from cells treated with H2O2. LDH release is commonly used to evaluate the presence and degree of muscle cell damage. The normal plasma membrane is impermeable to LDH, but damage to the cell membrane results in a change in the membrane permeability and subsequent leakage of LDH into the extracellular fluid. Confluent hBSMCs were growth arrested and treated with 0, 50, 100, and 200 µM H2O2 for 24 h. Media was removed and used for measurement of LDH release. Figure 7C shows a significant increase in LDH release into the cell culture medium with increasing concentrations of H2O2. These data, together with the negative data obtained for measurement of apoptosis markers, demonstrates that H2O2 induces necrosis in hBSMCs.

Mechanism of cytoprotection by hsp27 in hBSMCs. Phosphorylation of Hsp27 regulates its oligomerization and functions as a chaperone of denatured proteins and as a regulator of actin filament formation. Hsp27 phosphorylation is also implicated in cellular protection against stress in many cell types, including cardiac (42) and renal epithelial cells (11). This information, together with data showing phosphorylation of endogenous Hsp27 at Ser15, Ser78, and Ser82 by H2O2 (Fig. 6D), led to the hypothesis that the phosphorylation of Hsp27 is necessary for cellular protection against H2O2. To test this hypothesis, hBSMCs were infected with Ad5, AdHsp27 WT, or AdHsp27 3A for 3 days. Cells were trypsinized, plated onto 96-well plates, and stimulated with H2O2 for 24 h, and MTS assay was performed on treated cells. Cells infected with the Ad5 empty vector exhibited an ~55% decrease in viability upon stimulation with 100 µM H2O2 compared with unstimulated control (Fig. 6A). Approximately 90% of cells overexpressing Hsp27 WT were still viable after stimulation with H2O2. Overexpression of Hsp27 WT inhibited cell death by ~40% compared with cells infected with the Ad5 empty vector control. Cells overexpressing the non-phosphorylation mutant, Hsp27 3A, exhibited a 35% decrease in viability. Similar to wild-type Hsp27, cells overexpressing Hsp27 3A were also less sensitive to H2O2 cytotoxicity. The data suggest that cytoprotection by Hsp27 may not be dependent on its phosphorylation state. This is confirmed by data indicating that blockade of p38 MAPK signaling with a chemical inhibitor that inhibits the phosphorylation of endogenous Hsp27 at Ser15 and Ser78 did not provide protection against H2O2-induced cell death (Fig. 6B). Overexpression of the Hsp27 3D phosphorylation mutant also did not provide protection against H2O2 (Fig. 6C) compared with Ad5-infected cells, further substantiating our observations.

It is reported that Hsp27 can inhibit cell death by enhancement of NF-{kappa}B activity (32). Thus, we investigated whether Hsp27 protection was mediated by NF-{kappa}B signaling. To pursue this, we measured I{kappa}B protein degradation, which is an inhibitory protein of NF-{kappa}B. Degradation of I{kappa}B permits NF-{kappa}B translocation to the nucleus to activate transcription. Mock-infected, Ad5 or AdHsp27 WT-infected hBSMCs were stimulated with 100 µM H2O2 for 0 and 30 min, and protein was extracted for immunoblot analysis of I{kappa}B expression. Stimulated mock-infected cells did not result in reduced I{kappa}B expression as measured by densitometry of band intensity, which would suggest degradation of I{kappa}B (Fig. 8A). Similarly, neither Ad5-infected controls nor AdHsp27 WT-infected cells exhibited reduced I{kappa}B expression upon stimulation with H2O2. This suggests that Hsp27 did not provide protection against H2O2 in hBSMCs by degrading I{kappa}B to promote NF-{kappa}B translocation and activation of gene transcription of antiapoptotic genes.


Figure 8
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  		Fig. 8. Mechanism of Hsp27 protection against H2O2. hBSMCs were either not infected or infected with Ad5 or wild-type HSP27. Cells were then treated with 100 µM H2O2. A: total protein was extracted and probed for I{kappa}B expression with anti-I{kappa}B (1:500 dilution). Data collected are depicted in graphical format. Results indicate that Hsp27 did not enhance I{kappa}B degradation. B: concentration of total GSH (tGSH) in cells overexpressing wild-type Hsp27 upregulated glutathione levels by 2.5-fold when stimulated with H2O2 compared with untreated samples. N = 3 donors in duplicate. *Significantly different compared with untreated samples; #significantly different compared with stimulated mock-infected or Ad5-infected cells. P < 0.05.

 
Filomeni et al. (14) and Will et al. (46) demonstrated that Hsp27 inhibits cellular apoptosis and necrosis by regulating GSH levels. This led to the hypothesis that inhibition of H2O2-induced cell death by Hsp27 in ASM cells was mediated by intracellular GSH levels. To test this hypothesis, cells overexpressing Hsp27 WT or mock-infected and Ad5-infected cells were either treated or not treated with 100 µM H2O2 for 24 h, and GSH assay was performed as described in MATERIALS AND METHODS. Data were expressed as micromolar concentration of total GSH in cell extracts as calculated from GSH standard curves. In Fig. 8B, there was ~2 µM total GSH measured in the mock-infected sample. As expected, treatment with H2O2 completely depleted GSH levels below the minimum detectable concentration. Similar results were obtained for Ad5-infected samples, with ~1.8 µM total GSH detected in untreated cells. GSH levels decreased to ~0.3 µM upon stimulation with H2O2. Overexpression of wild-type Hsp27 also resulted in similar results in unstimulated samples, with 2.1 µM total GSH. Stimulated samples, however, resulted in an increase to ~5 µM GSH. Furthermore, Hsp27 overexpression upregulated GSH levels compared with mock-infected or Ad5-infected cells. These data supported the hypothesis that Hsp27 inhibited H2O2-induced necrotic cell death by modulation of GSH levels in hBSMCs.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Inflammation promotes structural changes or remodeling observed in the asthmatic airway, resulting in the classic narrowing of the lumen and obstruction of airflow. Humans with fatal asthma have been reported to exhibit greater degrees of luminal narrowing and ASM mass when compared with asthmatic patients that experienced nonasthma-related death (reviewed in Ref. 34). There is evidence supporting the notion that ASM hypertrophy and hyperplasia is attributed to increased smooth muscle proliferation and/or resistance to cell death. However, the regulatory components mediating these processes have not been completely identified, nor have feasible therapeutic targets been identified to inhibit BSMC proliferation.

This investigation sought to define the role of Hsp27 in ASM mitogenesis and survival. Many substances produced by resident airway cells or infiltrating inflammatory cells such as peptide growth factors, cytokines, and reactive oxygen and nitrogen species are capable of inducing either cellular proliferation or apoptosis or necrosis (reviewed in Ref. 34). The expression of Hsp27 is inducible by these molecules, leading to altered function in many cell types. For instance, Hsp27 induction by NaAsO2 in cultured astrocytes reduced cellular sensitivity to hydrogen peroxide-stimulated cell death (12).

The effect of Hsp27 on ASM proliferation was determined using an adenoviral-mediated gene delivery system. To do this, initial studies were performed to identify compounds that would induce cellular proliferation. Although TNF{alpha}, 10% serum, and a combination of FGF and EGF were used to stimulate hBSMC growth, FBS was the only stimulus proven to be mitogenic under the conditions we used in our studies. Consequently, for all experiments, myocytes were stimulated with serum. The effect of TNF{alpha} on ASM growth is somewhat controversial. There is evidence that TNF{alpha} does not have a mitogenic effect on ASM cells. Others, however, reported that TNF{alpha} can induce proliferation at low picomolar concentrations (reviewed in Ref. 2). EGF and FGF have been shown to be proliferative in many cell types. It is possible that EGF and FGF act synergistically with other factors to induce cell growth. For example, the addition of EGF and FGF to 10% serum resulted in a three- to fourfold higher cell density than 10% serum alone (16).

Cells overexpressing wild-type Hsp27 exhibited an approximately twofold reduction in cell growth after 3 days of serum treatment. This is in agreement with work by Kindas-Mugge et al. (22) and Horman et al. (20) demonstrating reduced proliferation rate in Hsp27 overexpressing cell lines. Cells that were infected with the Ad5 empty vector displayed a slight decrease in growth compared with mock-infected cells. This was not surprising since adenoviruses are known to inhibit cell growth (13). Although there are limitations to using adenoviruses for cell growth studies, the considerable and significant differences in proliferation between wild-type-expressing cells vs. the Ad5-infected cells demonstrate that the inhibition of ASM growth is due to overexpression of Hsp27.

Under steady-state conditions, Hsp27 exists predominantly as a large oligomeric structure of up to 800 kDa. During stress response, Hsp27 is phosphorylated by MK2, which is a downstream target of p38 MAPK. Phosphorylation of Hsp27 leads to reorganization of the oligomeric unit to smaller dimers and tetramers, thereby altering protein function (reviewed in Ref. 8). To determine if phosphorylation was necessary for Hsp27 inhibition of ASM cell growth, a phosphorylation mutant was used. In contrast to the effects of wild-type Hsp27, overexpression of the S15A, S78A, and S82A non-phosphorylation mutant did not inhibit cell proliferation. This agrees with studies by Horman et al. (20) where Hsp27 phosphorylation was induced by 12-O-tetradecanoylphorbol-13-acetate and TNF{alpha} leading to inhibition of MCF-7 cell growth. We further confirmed our results by overexpressing the phosphorylation mutant (Hsp27 3D), which also inhibited serum-induced proliferation consistent with the observations made with wild-type Hsp27.

Our data suggest that Hsp27 phosphorylation may play a role in the regulation of ASM cell growth. Since Hsp27 is phosphorylated by a downstream effector of p38 MAPK, we investigated the role of p38 in ASM cell proliferation. To pursue this, p38 activation was blocked by a chemical inhibitor, SB-239063, before stimulation with serum. Control cells exhibited a fivefold increase in proliferation upon stimulation with 10% serum for 3 days. Blocking the activation of p38 MAPK did not alter proliferation compared with the control, suggesting that p38 does not mediate proliferation. This is consistent both with our observation that S15A, S78A, and S82A Hsp27 mutants had no effect on myocyte proliferation and reports that p38 MAPK signaling is primarily involved in stress response and not mitogenesis.

Cellular growth in an organism is carefully controlled by regulating the different and successive phases of the cell cycle, which are G0/G1, S, and G2/M. Gap 1 (G1) is the interval between mitosis and DNA replication that is characterized by cell growth. The transition that occurs at the restriction point (R) in G1 commits the cell to the proliferative cycle. If the conditions that signal this transition are not present, the cell exits the cell cycle and enters G0, a nonproliferative phase during which growth, differentiation, and apoptosis occur. Replication of DNA occurs during the synthesis (S) phase, which is followed by a second gap phase (G2) during which growth and preparation for cell division in the M phase occurs. HSPs have been implicated in regulation of the cell cycle. Galea-Lauri and colleagues (15) demonstrated a 10% increase in U-937 cells in the G1 phase when Hsp90 was knocked down. The introduction of the murine homolog of Hsp27 (Hsp25) cDNA into murine L929 fibroblasts stimulated the accumulation of mRNA for the cell cycle inhibitory protein, p21 (33). Consequently, we investigated the role of Hsp27 in regulation of the cell cycle in ASM cells. Stable cell lines expressing wild-type and mutant Hsp27 were arrested without doxycycline for 4 days and then stimulated with serum for 3 days. The results obtained showed no differences in population distribution between the Hsp27-expressing cell lines compared with control in G0/G1 or S phase. This suggests that Hsp27 does not regulate smooth muscle proliferation by halting cell cycle progression and that other mechanisms of growth regulation are involved.

Clearly, our data provided evidence for Hsp27 modulation of ASM cell growth. Since regulation of growth and death are often controlled by the same modulator, we sought to define the role of Hsp27 in regulation of smooth muscle cell survival. Cells typically die either by apoptosis (programmed cell death) or necrosis where the cell membrane is ruptured, releasing cellular content into the external environment. Initial experiments were performed to identify cell death inducers. The compounds chosen were shown to induce apoptosis and/or necrosis in many different cell types. For instance, the nitric oxide donor (SNP) induces necrosis and apoptosis in chromaffin cells (43). Lautrette et al. (25) demonstrated Fas-induced apoptosis in Jurkat T leukemia cell line, and Hamann et al. (18) reported similar effects in human ASM cells. NaAsO2 and H2O2 treatment resulted in cellular cytotoxicity and death in colorectal adenocarcinoma and hepatocellular carcinoma cell lines as well as Hela cells (31) and astrocytes (12). Similar to the reported literature, SNP, H2O2, and antibody against the Fas receptor were sufficient to induce hBSMC death. NaAsO2, however, did not kill hBSMC cells. Although this was unexpected, it was not unprecedented. Pi and colleagues (35) showed that long-term exposure of cultured human keratinocytes to low-level arsenite caused resistance to apoptosis. In addition, exposure of cultured astrocytes to 50 µM NaAsO2 resulted in induction of HSPs that provided protection against cell death (12). Collectively, this suggests that the pro- or antiapoptotic effect of NaAsO2 is dependent on the concentration used and the cell types under study.

Induction of HSPs is a cellular response against stress-induced cell damage and death. The present overexpression studies with wild-type Hsp27 showed attenuated H2O2-induced cell death in hBSMCs, indicating that Hsp27 provides protection against H2O2 cytotoxicity. Overexpression of the non-phosphorylation mutant (3A) also provided protection, suggesting that the phosphorylation of Hsp27 may not be necessary. However, overexpression studies utilizing the phosphorylation mimic mutant (Hsp27 3D) resulted in no protection against H2O2, indicating that Hsp27 phosphorylation may mediate its protective response.

Since Hsp27 provided protection against H2O2, we investigated the mechanism of H2O2-induced cell death. Cellular apoptosis and necrosis can be induced by the same pathophysiological exposures. Markers of apoptosis include caspase-3 cleavage and subsequent activation, histone phosphorylation, and DNA fragmentation (reviewed in Ref. 40). Our results showed that H2O2 did not induce caspase-3 cleavage, histone phosphorylation, or DNA fragmentation. The negative data obtained for apoptotic markers suggest that H2O2 stimulated cell death via necrosis. To confirm this, we measured LDH release. Although there were slight increases in LDH release into the medium after stimulation with 100 µM H2O2, the amount of release was not consistent with the viability assays. The contrasting data is attributed to the assays themselves. The MTS assay measures the ability of the mitochondria to degrade the formazan compound into a product that is then measured. If the integrity of the mitochondria is compromised, which suggests a loss in viability, the formazan compound will not be converted to the measured product. This occurs early in the cell death process. The LDH assay, on the other hand, measures membrane integrity, which is a later event.

The mechanisms by which Hsp27 is proposed to inhibit cell death are numerous and varied. As reviewed by Beere (5), Hsp27 prevents cell death by regulating the apoptotic pathway, by activation of prosurvival signals, by stabilization of the actin cytoskeleton, or by regulating GSH levels. Because we found H2O2 induced necrosis in ASM cells, this led to the hypothesis that protection by Hsp27 is mediated by activation of the prosurvival transcription factor NF-{kappa}B. To test this, we assayed for I{kappa}B degradation, which would release NF-{kappa}B and enable it to translocate to the nucleus, thereby initiating transcription of prosurvival genes. Overexpression of wild-type Hsp27 did not induce I{kappa}B degradation in hBSMCs, suggesting that Hsp27 inhibition of cell death is mediated by another mechanism or mechanisms.

Glutathione or GSH is a tripeptide composed of glutamate, cysteine, and glycine amino acid residues that performs a variety of important physiological and metabolic functions in mammalian cells. It serves as a reductant, is conjugated to drugs to make them more soluble, is involved in amino acid transport across cell membrane, is a cofactor for some enzymatic reactions, and aids in the rearrangement of protein disulfide bonds (reviewed in Ref. 44). There is ample evidence that implicates GSH depletion in cell death (10) and that mechanisms that prevent GSH depletion increase survival (39). Hsp27 maintains or enhances GSH levels in several cell types and thereby decreases sensitivity to apoptosis and necrosis (4, 28). To determine whether Hsp27 provided protection against H2O2 in hBMSCs by regulating GSH levels, a GSH assay was performed. Treatment with H2O2 in mock-infected and Ad5-infected cells resulted in a decrease in GSH levels. Overexpression of wild-type Hsp27 upregulated GSH levels by 2.5-fold compared with untreated controls. The data are consistent with Hsp27 inhibition of hBSMC death being mediated at least in part by maintaining GSH levels. The maintenance of intracellular GSH levels allows for the defense against toxic oxygen radicals that may be harmful to the cell. The identity of specific target proteins whose functions are preserved by reduction or gluthionylation is unclear at this point. Nor is it obvious exactly how cell membrane patency and cell viability is preserved.

To summarize, we provide evidence that modulation of ASM proliferation and survival is sensitive to levels of expression of Hsp27 and to the phosphorylation status of Hsp27. Although the conclusions drawn are from overexpression studies, and is somewhat limited by no experimentation with reduced endogenous Hsp27, the information obtained is still valuable and provides insight to the role of Hsp27 in ASM cells. The inhibition of cell proliferation by Hsp27 overexpression may reflect a role of this protein in favoring a non-growing, contractile phenotype. This is consistent with the relatively abundant expression of Hsp27 in differentiated smooth muscle tissue (24). The protective effect of Hsp27 against oxidative cell damage may be important in both the differentiated contractile state as well as in proliferating cells exposed to chemical stressors such as toxic oxygen and toxic nitrogen species. This latter observation could be beneficial to healthy individuals but may contribute to pathogenesis in asthmatics if it promotes an inappropriate degree of cell survival. Therefore, future studies are necessary to provide a clearer understanding of the molecular mechanisms of Hsp27 modulation of smooth muscle cell function.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-48183 and HL-077726.


   ACKNOWLEDGMENTS
 
The technical assistance of Shanti Rawat and Margaret Elorza is gratefully acknowledged. Helpful discussion of the manuscript was provided by Cherie Singer.

Present address of W. T. Gerthoffer: Dept. of Biochemistry & Molecular Biology, Univ. of South Alabama, College of Medicine, 307 University Blvd., Mobile, AL 36688-0002.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. T. Gerthoffer, Dept. of Biochemistry & Molecular Biology, Univ. of South Alabama, College of Medicine, 307 University Blvd., Mobile, AL 36688-0002 (e-mail: wgerthoffer{at}usouthal.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
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 MATERIALS AND METHODS
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 DISCUSSION
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