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Published ahead of print on August 21, 2003, doi:10.1165/rcmb.2003-0019OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 379-387, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0019OC

Rho Kinase Inhibition Initiates Apoptosis in Human Airway Epithelial Cells

Michael Moore, Bertha A. Marroquin, Wendy Gugliotta, Roberta Tse and Steven R. White

Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois

Address correspondence to: Steven R. White, M.D., University of Chicago, Section of Pulmonary and Critical Care Medicine, 5841 S. Maryland Ave., MC 6076, Chicago, IL 60637. E-mail: swhite{at}medicine.bsd.uchicago.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Disruption of the actin cytoskeleton elicits profound changes in cell survival and function. The actin cytoskeleton is regulated in a hierarchical manner by Rho GTPases. Rho kinase, a downstream effector of RhoA, regulates the formation of stress fibers and focal adhesions. Disruption of the actin cytoskeleton causes apoptosis in airway epithelial cells. To examine further the relation of cytoskeletal integrity and apoptosis, we tested whether inhibition of Rho kinase would elicit apoptosis in airway epithelial cells. Inhibition with either Y-27632 or HA1077 induced membrane ruffling and loss of actin stress fibers, and apoptosis in airway epithelial cells that was blocked by inhibiting caspase function or by inhibiting protein synthesis. Cells overexpressing constitutively active Rho kinase, but not native Rho kinase, were resistant to Rho kinase inhibitor–induced stress fiber disruption and apoptosis. Inhibition of Rho kinase disrupted actin stress fibers but did not induce apoptosis in 3T3 cells. We demonstrate that Rho kinase inhibition induces airway epithelial cell apoptosis associated with changes in actin filament integrity. Our data suggest that Rho kinase may be a regulator of early initiation of apoptosis.

Abbreviations: N-acetyl-(Asp-Glu-Val)-3-amino-4-oxobutaonic acid, Ac-DEVD-cho • N-acetyl-(Tyr-Val-Ala-Asp)-3 amino-4-oxobutaonic acid, Ac-YVAD-cho • 7-amino-4-trifluoromethylcoumarin, AFC • bovine pituitary extract, BPE • cytochalasin D, CYD • benzyloxycarbonyl-Asp-Glu-Val-Asp, (Z-DEVD) • dimethyl sulfoxide, DMSO • ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, EGTA • fetal calf serum, FCS • (5-Isoquinolinesulfonyl) homopiperazine, 2HCl, HA-1077 • monoclonal antibody, mAb • phosphate-buffered saline, PBS • sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE • terminal deoxynucleotidyl transferase mediated dUTP biotin nick end-labeling, TUNEL • 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, Z-Val-Ala-Asp-fluoromethylketone, Z-VAD-fmk


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is a highly conserved and regulated physiologic mechanism responsible for the elimination of unwanted cells at various stages of development, injury, and repair (1, 2). The execution phase of apoptosis is characterized by morphologic changes that include cell shrinkage, chromatin condensation, membrane blebbing, and DNA cleavage (1). In most cells, apoptotic signals trigger actin cytoskeletal changes that result in release from the extracellular matrix, membrane blebbing, and condensation into apoptotic bodies in a sequential fashion (3). These events correlate with loss of stress fibers and cortical actin reorganization, actino-myosin contraction of the actin ring, and dissolution of polymerized actin, respectively (3). Actin reorganization and degradation has been viewed as a later consequence occurring only after a cell has committed to an apoptotic death. However, there is increasing evidence that perturbations of the actin cytoskeleton itself can initiate events that commit a cell to apoptosis. Loss of actin-based, integrin mediated cell–matrix adhesion results in apoptosis in epithelial cells and other cell types (4). Direct disruption of the actin cytoskeleton with cytochalasin D (CYD) or jasplakinolide induces apoptosis of airway epithelial cells (5), HL-60 cells (6), endothelial cells (7), EL4 T lymphoma cells (8), and NIH3T3 cells (8).

Actin is an important functional and structural protein common to all eukaryotic cells. The actin cytoskeleton is an essential scaffold integrating multiple cellular processes like cell–cell communications, cell–matrix interactions, and intracellular signaling (9). Globular actin (G-Actin) monomers are polymerized into filamentous actin (F-actin) in a dynamic, highly regulated process. This is controlled, in part, by the small Rho GTPases (10, 11). The Rho GTPases RhoA, Rac1, and cdc42 trigger the reorganization of the actin cytoskeleton and are responsible for the formation of stress fibers and focal adhesions complexes, lamellipodia and membrane ruffles, and filopdia, respectively (12, 13). Activation of Rho kinase, a downstream target of RhoA (14, 15), has been found to be a key regulator of multiple cell functions including the formation of stress fibers and focal adhesions (15, 16).

Rho GTPases may also regulate cell survival and apoptosis. Overexpression of Rac or the small GTPase cdc42 in a neuron cell line induced apoptosis, and expression of dominant negative mutants of these proteins protected cells against apoptosis induced by withdrawal of neuron growth factor (17). When expressed in Jurkat T cells, an activated form of cdc42 induces apoptosis (18). A dominant-negative mutant of Rac1 (N17) can efficiently abrogate tumor necrosis factor-{alpha}–induced apoptosis in U937 cells (19). A recent report suggests that an inhibitor of RhoA, lovastatin, one of the family of HMG-CoA reductase inhibitors, can induce apoptosis in intestinal epithelial cells, a process that is reversed either by blocking protein synthesis or by preventing translocation of RhoA to the cell membrane (20). Rho kinases also may regulate apoptosis via their control of actin filament integrity (7, 21), a process that may be separate from RhoA activation.

We have previously reported that either inhibition of actin filament elongation with CYD or aggregation of filaments with jasplakinolide elicits apoptosis of airway epithelium (5). We hypothesized that perturbations of filament integrity may be an early initiating event in apoptosis. Rho kinase, as one regulator of filament integrity, might also have a role in cell survival. To test this hypothesis, we examined whether inhibition of Rho kinase could initiate airway epithelial cell apoptosis. We demonstrate that pharmacologic inhibition of Rho kinase disrupts the actin cytoskeleton and elicits apoptosis, whereas expression of a dominant-positive Rho kinase prevents apoptosis induced either by Rho kinase inhibitors or by CYD. Our data suggest that the actin cytoskeleton functions as a checkpoint in the regulation of apoptosis and that perturbations in actin filament integrity can initiate apoptosis, with a survival signal mediated via Rho kinase.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Streptomycin, L-glutamine, penicillin, cytochalasin D, cycloheximide, and anti-actin monoclonal antibody (mAb) were purchased from Sigma, Inc. (St. Louis, MO). Human bronchial airway epithelial medium (as defined below) was purchased from Clonetics, Inc. (Walkersville, MD). Fetal calf serum (FCS) was purchased from Hyclone (Logan, UT) and was heat-inactivated before use. Rhodamine-labeled phalloidin was purchased from Molecular Probes (Eugene, OR). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) biotin nick end-labeling (TUNEL) TACS II fluorescent assay kits and DePsipher kits for mitochrondrial membrane depolarization assay were purchased from Trevigen, Inc. (Rockville, MD). The caspase inhibitor peptides Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), N-acetyl-(Asp-Glu-Val)-3 amino-4-oxobutaonic acid (Ac-DEVD-cho), and N-acetyl-(Tyr-Val-Ala-Asp)-3 amino-4-oxobutaonic acid (Ac-YVAD-cho), as well as the Rho kinase inhibitors (R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y-27632), and (5-Isoquinolinesulfonyl)homopiperazine, 2HCl (HA-1077), were purchased from CN Biosciences, Inc. (La Jolla, CA). Max Efficiency DH5{alpha} competent cells were obtained from Life Technologies, Inc. (Rockville, MD). All other reagents were obtained from Sigma, Inc. and were of the highest quality available.

Cell Culture
The use of human airways was approved by the University of Chicago Institutional Review Board. Primary airway epithelial cells were harvested from transplant donor lungs designated for research as described previously (5). Cells were grown in BEGM medium (Clonetics, Inc., Walkersville, MD) containing 5 mg/ml insulin, 0.5 µg/ml hEGF, 10 mg/ml transferrin, 6.5 µg/ml triiodothyrinine, 0.5 mg/ml hydrocortisone, 0.5 mg/ml epinephrine, and 13 mg/ml BPE. Cells were used at passage 1. The cell line 1HAEo-, a gift of Dieter Gruenert (University of Vermont, Burlington, VT), are SV40-transformed human airway epithelial cells that have cell surface markers similar to primary airway basal epithelial cells (22). NIH 3T3 fibroblasts were obtained from the ATCC. Cell lines were grown on collagen-IV coated chamber slides in Dulbecco's modified essential medium containing 10% FCS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin G. All cells were incubated at 37°C in 5% CO2 and were used when ~ 80–90% confluent. Cells were kept in 10% FCS during all experiments to prevent confounding of apoptosis results by withdrawal of needed growth factors. Agents or their appropriate vehicle controls were added, and cells were incubated for 24 h. At the conclusion of experiments, chamber slides were washed once in fresh medium and fixed in 10% neutral buffered formalin.

Stable Transfections
Expression vectors pEF-BOS-myc containing Rho kinase/Longest and Rho kinase/CAT were generously provided by Kozo Kaibuchi (Nara Institute of Science and Technology, Ikoma, Japan). Plasmids were transformed in competent cells and purified using a QIAfilter Plasmid Maxi Kit (Qiagen Sciences Inc., Valencia, CA). The pEF-BOS-myc empty vector was created by excising the Rho Kinase longest cDNA by BAMHI digestion. 1HAEo- cells grown to 80% confluence (~ 500,000 cells) were co-transfected with 3 µg of total DNA, 15 µl of lipofectamine (Life Technologies, Inc.), and 1,200 µl of OptiMEM (Life Technologies) in 6-well plates for 5 h at 37°C followed by replacement with fresh media. Co-transfections were performed with pcDNA3.1(+) vector (Invitrogen Corp., Carlsbad, CA) in a 5:1 ratio. After 48 h, cells were selected with 300 µg/ml neomycin. Subclones were selected based on Rho kinase/Longest or Rho kinase/CAT expression as determined by Northern blot using the appropriate cDNA from each plasmid as a probe. The pEF-BOS-myc empty vector subclone was selected based on its ability to survive in neomycin.

Assay for Cell DNA Nicking
Apoptotic cells were assessed using a method described previously (5). Apoptotic cells in fixed monolayers were demonstrated by labeling free 3'-hydroxyl groups of DNA using a Trevigen TUNEL fluorescent assay kit. Slides were counterstained with 5 U/ml rhodamine-phalloidin in PBS for 30 min, washed three times in PBS, and then stained with 1 mM Hoechst 33,258 in 95% ethanol for 45 s. Representative images were collected using a 12-bit cooled charge-coupled device camera (Photometrics, Inc., Tucson, AZ) connected to a Nikon fluorescence microscope. TUNEL-positive nuclei and Hoechst-stained nuclei were counted in each image as the area of the nuclei in pixels after visual thresholding and exclusion of extraneous positive pixels using Spectrum IP software (IP Labs, Vienna, VA) on a Macintosh computer. TUNEL-positive cells were expressed as the percentage of the thresholded area of the TUNEL-stained image divided by the thresholded area of the Hoechst-stained image. The TUNEL counts of two fields in the same well were averaged to produce a single N. Previous experiments (5) demonstrated a high correlation to manual counting and demonstrated that changes in cell shape or morphology alone did not alter significantly the ability to detect apoptotic nuclei.

Actin Stress Fiber Imaging
Cell monolayers were stained with rhodamine-labeled phalloidin, after which representative images were collected with a Micromax 1300Y 12-bit thermoelectric cooled digital camera (Roper Scientific, Inc., Trenton, NJ) connected to a Zeiss Axiovert 100 TV fluorescence microscope. Images were collected in Slidebook (Intelligent Imaging Innovations, Inc., Denver, CO).

Extraction of F-Actin and G-Actin
We have described this method previously (5). After interventions, cells were washed twice with PBS and then once with CSK buffer (0.01 M [1,4-piperazinebis (ethane sulfonic acid)], 0.3 M sucrose, 0.025 NaCl, 1 mM ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid [EGTA], and 5 mM MgCl2). Slides then were incubated for 5 min at room temperature in 500 µl CSK buffer containing 1% Triton X-100. Supernatants were collected and centrifuged at 108,000 x g for 50 min at 25°C. Pellets containing noncytoskeletal F-actin were suspended in 500 µl of 8 M urea each and stored at -70°C. Supernatants were precipitated in 2.5 ml methanol overnight at -70°C, and then centrifuged at 10,000 x g for 1 h at 4°C. Pellets containing G-actin were suspended in 500 µl 8 M urea each and stored at -70°C. The remaining cell material on the slides was washed once with CSK, collected in 8 M urea, vortexed briefly, and centrifuged for 5 min at 14,000 rpm. Supernatants containing cytoskeletal F-actin were stored at -70°C. Samples were separated on a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) minigel and transferred to nitrocellulose membranes. Beginning cell numbers in each experiment were equal, and equal volumes of extracted protein were loaded in each lane. Immunodetection was performed using a chemiluminescence protocol (5).

Northern Blot Analysis
Total RNA from samples of cultured cells was isolated using a Qiagen RNeasy Mini Kit with DNase treatment according to directions. Total RNA was resolved by electrophoresis on 1% agarose-formaldehyde gels and transferred to Hybond Nylon membranes (Amersham-Pharmacia, Buckinghamshire, UK) in 10x saline sodium citrate (SSC). Hybridization probes were generated as a gel-purified restriction fragments from plasmid and labeled with Redivue [{alpha}-32P] CTP using Rediprime II (Amersham-Pharmacia). Hybridization was performed overnight at 42°C using standard procedures. The membranes were washed twice for 15 min at RT with 2x SSC/0.1% SDS, and twice for 15 min at 60°C with 0.2x SSC/0.1% SDS. Membranes were exposed for 3–24 h at -70°C to Kodak X-omat AR film.

Caspase 3 Activity Assay
Spectrofluorometric assays of proteolytic activity were performed using a kit (ApoAlert; BD Biosciences, Palo Alto, CA) according to directions. This assay detects the emission shift of 7-amino-4-trifluoromethylcoumarin (AFC) after cleavage of substrate from the caspase-3 target, benzyloxycarbonyl-Asp-Glu-Val-Asp (z-DEVD) (23). After interventions, cell lysates were collected in chilled lysis buffer, clarified by centrifugation at 4°C for 10 min, and incubated for 30 min in reaction buffer containing 10 mM dithiothreitol and 1:50 (vol:vol) dimethylsulfoxide. Samples then were incubated with z-DEVD-AFC at 37°C for 1 h. Liberation of AFC was measured in a microplate spectrofluorimeter with excitation 400 nm, emission 505 nm. Some samples were incubated concurrently with caspase-3 inhibitor to quench activity. Activity was compared with a standard curve generated at the same time using AFC, and expressed finally as pM AFC generated per µg protein per min referenced to the time 0 control AFC generation.

Data Analysis
Data are expressed as the mean ± SEM. Differences were treated by ANOVA (F-test) followed by Fisher's protected least significant difference test, and were considered significant when P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rho Kinase Inhibition by Y-27632 Induces Apoptosis
When a cell commits to apoptosis, actin is reorganized and subsequently degraded in the effector phase of cell death (3). Rho-GTPases such as Rac1, cdc42, and RhoA dynamically regulate actin filament morphology and induce lamellipodia, filopdia, and stress fibers, respectively (reviewed in Ref. 10). We asked whether these GTPases might also modulate initiation of apoptosis by virtue of their regulating effects on these filaments. As a first step to answer this question, we asked whether inhibition of Rho kinase, a downstream effector of RhoA, could initiate both disruption of actin filament integrity and apoptosis. Cells were treated with one of two pharmacologic inhibitors: either Y-27632 (24, 25) or HA-1077 (24, 26) for up to 24 h. Treatment was associated with loss of actin stress fibers, membrane ruffling, and cytoplasmic contraction as demonstrated with rhodamine phalloidin staining (Figures 1A and 1B). In contrast to actin filament disruption induced by CYD (5), Western blot demonstrated no accumulation of G-actin or noncytoskeletal F-actin with minimal change in cytoskeletal F-actin pool over 6 h (Figure 1C). Pharmacologic inhibition of Rho kinase also induced apoptosis of 1HAEo- cells. Cells grown to ~ 80% confluence were treated with Y-27632 or HA1077 for 24 h. Apoptosis, as measured by TUNEL assay and confirmed by nuclear morphology on Hoechst stain, increased in a concentration-dependent manner for both agents (Figures 1D and 1E). Concentrations > 100 µM were associated with substantial detachment of cells from slides; because of secondary necrosis the question of whether detachment was caused by apoptosis or a toxic effect could not be reliably answered, and such concentrations were not further used. Apoptosis induced by inhibition of Rho kinase was also time-dependent. Cells grown to ~ 80% confluence were treated with 10 µM Y-27632 for up to 24 h, after which cells were fixed and apoptosis was determined using the TUNEL assay. Apoptosis was significantly greater 8 h after treatment started and increased to 24 h, the longest time point measured (Figure 1F).








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  		Figure 1. Disruption of actin stress fibers by Rho kinase inhibition. (A) 1HAEo- monolayers grown on glass slides treated with 1.0–100 µM Y-27632 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (B) 1HAEo- monolayers grown on glass slides treated with 1.0–100 µM HA-1077 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (C) Cells were grown in 6-well plates and treated with 10 µM Y-27632 for 1–12 h, then protein lysates were processed to obtain cytoskeletal F-actin, non-cytoskeletal F-actin, and G-actin compartments. Protein compartments were then resolved by SDS-PAGE and labeled with an anti-actin antibody. (D) 1HAEo- cells treated with various concentrations of Y-27632 for 24 h, and cell death was determined by TUNEL fluorescent assay. *P = 0.003 versus control; n = 8 experiments. (E) 1HAEo- cells treated with various concentrations of HA-1077 for 24 h, and cell death was determined by TUNEL fluorescent assay. *P = 0.001 versus control; n = 6 experiments. (F) 1HAEo- cells treated with 10 µM Y-27632 for up to 24 h, and cell death was determined by TUNEL fluorescent assay. *P = 0.04; {dagger}P = 0.001; §P = 0.0001 versus control (n = 8 experiments).

 
We next tested the effects of Rho kinase inhibition in primary human bronchial airway epithelial cells. Treatment was associated with disruption of actin filament morphology and loss of stress fibers as demonstrated with rhodamine phalloidin staining (Figures 2A and 2B). Pharmacologic inhibition of Rho kinase with Y-27632 in the same concentrations used in the cell line experiments also induced apoptosis of human bronchial airway epithelial cells over 24 h (Figures 2C and 2D).






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  		Figure 2. Disruption of primary epithelial cell actin stress fibers by Rho kinase inhibition. (A) Primary airway epithelial cell monolayers grown on glass slides treated with 1.0–100 µM Y-27632 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (B) Primary airway epithelial cell monolayers grown on glass slides treated with 1.0–100 µM HA-1077 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (C) Primary airway epithelial cells treated with various concentrations of Y-27632 for 24 h, and cell death was determined by TUNEL fluorescent assay. *P = 0.005, {dagger}P = 0.0007; §P < 0.0001 versus control (n = 4 experiments). (D) Primary airway epithelial cells treated with various concentrations of HA-1077 for 24 h, and cell death was determined by TUNEL fluorescent assay. *P = 0.005, {dagger}P = 0.0007; §P < 0.0004 versus control (n = 4 experiments).

 
We then asked whether apoptosis induced by Rho kinase inhibition was cell specific. NIH 3T3 cells have prominent actin stress fibers, and Y-27632 treatment was associated with dose-dependent cell contraction and loss of actin stress fibers (data not shown). However, treatment with Y-27632 did not elicit apoptosis: after treatment for 24 h with 100 µM Y-27632, the proportion of TUNEL-positive cells was 0.4 ± 0.1% versus 0.1 ± 0.0% for control (n = 6, P = NS). Positive controls done at the same time with 6 µM staurosporine showed characteristic TUNEL-positive nuclei as well as cell lifting (27) (data not shown).

Y-27632–Induced Apoptosis Requires Activation of Caspases
We have previously shown that inhibiting caspases blocked CYD-induced cell death without preventing disruption of actin filament morphology (5). To determine if caspase cleavage after inhibition of Rho kinase was downstream of actin filament disruption, we treated 1HAEo- cells for 24 h with Y-27632 plus 1–100 µM of either the caspase-3 inhibitor Ac-DEVD-cho or the caspase-1 inhibitors Z-VAD-fmk or Ac-YVAD-cho. Concurrent treatment with either Ac-DEVD-cho or Ac-YVAD-cho blocked Y-27632-induced apoptosis completely, and concurrent treatment with Z-VAD-fmk blocked Y-27632–induced apoptosis substantially (Figure 3A). In contrast, caspase inhibition did not prevent either the disruption of actin filament morphology or the loss of stress fibers (data not shown), similar to our previous data examining actin filament disruption with CYD.






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  		Figure 3. Effect of caspase inhibition on Y-27632 treated cells. (A) Effect of caspase inhibitors. 1HAEo- cell monolayers grown on glass slides were treated with 1.0–100 µM Y-27632 in the presence of 100 µM Ac-YVAD-cho, AC-DEVD-cho, or Z-VAD-fmk for 24 h, then fixed. Cell death was determined by TUNEL fluorescent assay. No points were significantly different from control in each panel (n = 4 in each panel). (B) Caspase-3 activity. 1HAEo- cell monolayers grown on collagen-IV coated plates were treated with 10 µM Y-27632 for 2–24 h, after which caspase-3 activity was determined in protein lysates using a fluorimetric assay for AFC generation from substrate. Samples collected after 24 h treatment were split; one portion was incubated with caspase-3 inhibitor before addition of AFC (right-hand bar) to demonstrate specificity of protease activity (n = 1 experiment).

 
As further confirmation of the role of caspases, we then examined activation of caspase-3 after treatment with Y-27632. 1HAEo- cells were treated for 2–24 h with 10 µM Y-27632, after which protein lysates were evaluated for caspase-3 activity. Activity as measured by generation of AFC was very low in untreated cells (~ 0.3 µM), increased within 2 h of treatment, and was still increased after 24 h of treatment (Figure 3B). Using the sample generated at the 24 h time point, AFC generated could be quenched completely by addition of caspase-3 inhibitor to the assay immediately before addition of the z-DEVD-AFC substrate, demonstrating specific protease activity for the assay.

Dominant Active Rho Kinase–Overexpressing Cells Show Resistance to Actin Filament Disruption and Apoptosis
We then asked whether cells that express a constitutively active Rho kinase (Rho kinase/CAT) would be resistant to actin cytoskeleton derangement and apoptosis elicited by either Y-27632 or HA-1077. Expression of Rho kinase/CAT was confirmed by Northern blot in subclones used for these experiments (data not shown). Rho kinase/CAT cells in the absence of a Rho kinase inhibitor exhibited prominent stress fibers compared with the 1HAEo- cell line (Figures 4A and 4B). In the presence of 1 or 10 µM Y-27632 or HA-1077, Rho kinase/CAT cells were resistant to actin cytoskeletal disruption and maintained normal stress fibers. Treatment with 100 µM elicited a contracted morphology with prominent spindle shapes and loss of stress fibers (Figures 4A and 4B). In contrast to cells lacking this construct, treatment of this cell line with either Y-27632 or HA-1077 for 24 h did not elicit significant cell death (Figures 4C and 4D). Apoptosis in the empty vector transfected control cell line in these experiments was similar to that seen in wild-type cells in previous experiments. Overexpression of native Rho kinase (Rho kinase/longest) did not prevent the loss of stress fibers after treatment with either Y-27632 or HA-1077 (data not shown), but conferred some protection from apoptosis after treatment with either Y-27632 (Figure 4C) or HA-1077 (Figure 4D).






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  		Figure 4. Expression of constitutively active Rho kinase abrogates cell death. (A) 1HAEo- cell monolayers stably transfected with Rho kinase/CAT grown on glass slides treated with Y-27632 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (B) 1HAEo- cell monolayers stably transfected with Rho kinase/CAT grown on glass slides treated with HA-1077 for 24 h, then fixed. Cells were stained with rhodamine-labeled phalloidin. Bar represents 20 µm. Arrows indicate actin stress fibers. (C) 1HAEo- cell monolayers stably transfected with either Rho kinase/CAT (squares), Rho kinase/longest (diamonds), or empty vector control (circles) grown on glass slides were treated with 1.0–100 µM Y-27632 for 24 h, then fixed. Cell death was determined by TUNEL fluorescent assay. {dagger}P = 0.002; §P < 0.0001 versus control in each group (n = 4–10 experiments in each group). (D) 1HAEo- cell monolayers stably transfected with either Rho kinase/CAT (squares), Rho kinase/longest (diamonds), or empty vector control (circles) grown on glass slides were treated with 1.0–100 µM HA-1077 for 24 h, then fixed. Cell death was determined by TUNEL fluorescent assay. *P = 0.01, {dagger}P = 0.003 versus control in each group (n = 4 – 6 experiments in each group).

 
Rho Kinase Inhibition and CYD-Induced Apoptosis Is Mediated by the Actin Cytoskeleton
Prevention of actin filament elongation by CYD induces apoptosis of airway epithelial cells (5). We hypothesized that actin disruption and apoptosis induced by either CYD or Rho kinase inhibition might be controlled by the same signaling pathway. To test this, cells were treated with 0.5 µg/ml CYD, 10 µM Y-27632, or both for 5–24 h. Y-27632–induced apoptosis was similar to that elicited by CYD (Figures 5A and 5B). We then tested whether cells expressing a constitutively active Rho kinase (Rho kinase/CAT) were resistant to actin disruption and apoptosis induced by CYD. Similar to previous experiments, Rho kinase/CAT cells were treated with CYD for 24 h. As was seen with Rho kinase/CAT cells treated with Y-27632, actin morphology was preserved in the presence of CYD (data not shown). Similarly, constitutively active Rho kinase overexpressing cells were resistant to CYD-induced apoptosis at concentrations < 1 µg/ml (Figure 5B). In additional experiments, we examined whether Rho kinase/CAT cells were resistant to apoptosis induced by jasplakinolide, a cell-permeable monocyclic peptide that binds to the same sites on F-actin as phalloidin and induces actin polymerization and aggregation (28), and elicits apoptosis in airway epithelium (5). Treatment with 0.3 µM jasplakinolide for 24 h did not elicit significant apoptosis (2.4 ± 0.4% of cells, n = 6) compared with untreated Rho kinase/CAT cells (2.2 ± 0.7%, n = 8, P = NS). In contrast, similar treatment in 1HAEo- cells transfected with the empty vector elicited lifting and apoptosis in > 90% of cells (n = 8).




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  		Figure 5. Rho kinase inhibition-induced and CYD-induced apoptosis is mediated by the actin cytoskeleton. (A) 1HAEo- cell monolayers grown on glass slides were treated with 0.5 µg/ml CYD, 10 µM Y-27632, or both for 5 h (left) or 24 h (right), then fixed. Cell death was determined by TUNEL fluorescent assay. P values are as indicated (n = 5–9 experiments in each group). (B) 1HAEo- cell monolayers stably transfected with Rho kinase/CAT grown on glass slides were treated with 0.03–1 µg/ml CYD for 24 h, then fixed. Cell death was determined by TUNEL fluorescent assay. *P = 0.002 versus control (n = 6 experiments in each group).

 
De Novo Protein Synthesis Is Required for Rho Kinase Inhibition-Induced Apoptosis
We then asked whether new protein synthesis was required to manifest the effect of Rho kinase inhibition, as apoptosis may be affected differentially by protein synthesis inhibition, depending on cell type and context. 1HAEo- cells were concurrently treated with Y-27632 and cycloheximide for 24 h. Cell death was abrogated completely by cycloheximide at a concentration of 10 µg/ml (Figure 6).



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  		Figure 6. De novo protein synthesis is required for Rho kinase inhibition-induced apoptosis. 1HAEo- cell monolayers grown on glass slides were treated with various doses of CHX ± 30 µM Y-27632 for 24 h, then fixed. Cell death was determined by TUNEL fluorescent assay. *P <= 0.0003 versus control; {dagger}P = 0.002 versus Y-27632 alone (n = 4 in each group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of Rho GTPases such as RhoA, Rac, and cdc42 have substantial effects on actin organization and remodeling in cells. These functions in turn have been implicated in cell movement, axonal guidance, cytokinesis, and changes in cell morphology and polarity (10). Rho kinase, an immediate downstream effector of RhoA, is a Ser/Thr kinase in the myotonic dystrophy family of kinases and has been implicated in the regulation on actin-based cell processes (15, 16). Rho kinase activation has been implicated in membrane blebbing and sequestration of fragmented DNA in the later, commitment phase of apoptosis (29). The effect of Rho GTPases on the early initiator phase of apoptosis is not known. Disruption of actin filament integrity either by blocking end-capping with cytochalasin D, or by aggregating filaments and disrupting three-dimensional structure directly with jasplakinolide, can initiate apoptosis (5), and both RhoA and Rho kinase have been associated with induction of (17–21) or protection from (30) apoptosis. Given the effects of Rho GTPases on actin filament structure, we asked whether Rho kinase similarly could modulate the initiation of apoptosis in airway epithelial cells.

We demonstrate that inhibition of Rho kinase disrupts the actin cytoskeleton and induces apoptosis in human airway epithelial cells. Treatment with two different inhibitors elicited concentration-dependent loss of actin stress fibers associated with membrane ruffles and membrane contraction. Apoptosis was demonstrated by TUNEL assay and by changes in nuclear morphology. Both Y-27632, which contains a pyridine moiety (24), and HA-1077, isoquinolinesulfonic acid derivative (31), interfere in an ATP-dependent manner with Rho kinase (31). This raises the possibility that each may interact with the ATP binding site on other protein kinases (32). However, phosphorylation of the Ser/Thr kinases nuclear factor-{kappa}B, ERK, and PI3 kinase/Akt are not inhibited by Y-27632 in concentrations similar to that used in this study (7). In a recent report, Li and coworkers found that inhibition of Rho kinase with either Y-27632 or HA-1077 caused human umbilical vein endothelial cell apoptosis that was blocked by cycloheximide (7). Concurrent treatment with cycloheximide prevented apoptosis induced by Y-27632 in airway epithelial cells in our experiments. These data suggest that new protein synthesis is required for the effect of Rho kinase inhibition in airway epithelial cells and in endothelial cells.

Expression of a dominant-active Rho kinase, Rho kinase/CAT, bypassed inhibition of Rho kinase and prevented both cytoskeletal disruption and apoptosis. In contrast, overexpressing native Rho kinase conferred only some protection. Combined with the inhibitor data, the prevention of apoptosis by the dominant active Rho kinase demonstrates clearly that Rho kinase mediates a survival signal in airway epithelial cells.

The modulating effects of Rho kinase inhibition on apoptosis and survival are cell type specific, whereas the modulating effects on the actin cytoskeleton are not. This survival function is similar to that noted in endothelial cells (7). In contrast to the effect of Rho kinase inhibition on airway epithelial cells, which elicited both actin stress fiber disruption and cell death, treatment of NIH 3T3 cells with Y-27632 disrupted actin stress fibers but did not lead to apoptosis. Thymocytes from C3 transferase-transgenic mice that lack RhoA function have survival defects (33). Treatment of rats in vivo with Y-27632 increased apoptosis in vascular smooth muscle and prevented neointima formation (34). In contrast, expression of a constitutively active Rho kinase did not prevent either membrane blebbing or apoptosis induced with tumor necrosis factor-{alpha} in 3T3 or Jurkat cells, but was dispensable for caspase activation and cytochrome c release (29, 35). Indeed, caspase cleavage of ROCK-1 induced membrane blebbing (29) via myosin light chain phosphorylation (35). Thus, inhibition of the RhoA/Rho kinase pathway has differing effects on survival and apoptosis depending on cell type.

The actin cytoskeleton has been increasingly investigated as a key modulator in the transduction of apoptotic signals. We have shown previously that disrupting the cytoskeleton by either filament aggregation by jasplakinolide or prevention of filament elongation by CYD induced apoptosis in airway epithelial cells (5). Similar results have been obtained in other cell types, including HL-60 (6) and HUVEC (7). Both CYD and jasplakinolide induce apoptosis in a cell type–specific manner and occasionally act in opposition to each other (6, 8). Although it is increasingly clear that actin filament morphology has an important role in determining cell death or survival, the exact downstream signaling mechanisms are unknown. Here we present evidence that the Rho kinase pathway may be one important mediator of signaling after actin filament disruption that may initiate apoptosis. Along with the effect of Rho kinase activation on membrane blebbing and sequestration of fragmented DNA in the commitment phase of apoptosis, our data and that of Li and colleagues (7) suggest that Rho kinase may have multiple roles in apoptotic initiation and commitment.

We treated cells with both CYD and Y-27632 as we hypothesized that actin disruption and apoptosis induced by either CYD or Rho kinase inhibition might be controlled by the same signaling pathway. Rho kinase activity may be impaired either by direct inactivation or by changing its localization on the actin scaffold. Overexpression of Rho kinase/CAT attenuated CYD induced apoptosis significantly. This suggests that inhibition or disruption of Rho kinase may be one mechanism by which CYD, and similar agents that act upon the cytoskeleton such as jasplakinolide, disrupt structural integrity and induce cell death. In addition, changes in localization of survival pathways on the actin scaffold may be one important mechanism by which physical or environmental stimuli might induce cell death. Overexpression of a constitutively active Rho kinase may then prevent death either by propagating a survival signal or by preserving the actin scaffold.

In summary, we demonstrate a role for Rho kinase in the regulation of airway epithelial cell survival. Apoptosis is induced by inhibition of Rho kinase, involves caspases, and is associated with signs of disrupted actin filament morphology, including loss of stress fibers. Apoptosis induced by Rho kinase inhibitors may be bypassed by expression of the activated kinase, and may involve death signaling pathways common to apoptotic signals elicited by actin disruption. These findings further demonstrate a role for actin filament integrity in airway epithelial cell survival.


   Acknowledgments
 
The authors thank Steve Sun and Paul Kogut for their technical assistance. They also thank Kozo Kaibuchi, Nagoya University, Nagoya, Japan, for the Rho kinase/Longest and Rho kinase/CAT cDNA used in this study. This work was supported by grants HL-60531 and HL-63300 from the National Heart, Lung and Blood Institute, and by an institutional National Research Service Award (HL-07605).

Received in original form January 15, 2003

Received in final form August 4, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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