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1Department of Pharmacy, 2Dorothy M. Davis Heart and Lung Research Institute, and 3Division of Pulmonary and Critical Care Medicine, The Ohio State University, Columbus, Ohio
Submitted 5 September 2003 ; accepted in final form 30 August 2004
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acute lung injury; phosphatidylinositol 3'-kinase; fibroblast growth factor-7; acute respiratory distress syndrome
PI3K is a vital regulatory protein responsible for maintaining cell viability. PI3K phosphorylates phosphoinositides at their 3'-position that in turn, activate downstream effector molecules. Akt/protein kinase B is a primary downstream target of PI3K intermediates and major component in cell survival (14). Akt mediates its antiapoptotic effect through the phosphorylation and inactivation of multiple downstream targets involved in the regulation of apoptosis. These substrates include two key regulators of the cell death machinery (i.e., BAD and procaspase-9), glycogen synthase kinase-3
(GSK-3), transcriptional factors of the forkhead family, and IKK, a kinase that regulates the NF-
B transcription factor, to name a few. This cascade provides a mechanism utilized by extracellular survival factors to promote cell survival and prevent apoptosis through early signaling events channeled through PI3K/Akt. We believe a similar series of events may occur when lung epithelial cells are exposed to KGF based on several observations that include 1) growth factors activate RTKs and prevent apoptosis via activation of the PI3K/Akt pathway (9, 16, 32); 2) transgenic animals that overexpress KGF have an Akt-dependent, reduced lung epithelial cell apoptosis in hyperoxic conditions (25); and 3) a constitutively active form of Akt introduced intratracheally into the lungs of mice by adenovirus gene transfer techniques protects mice from hyperoxic pulmonary damage and delays death (20).
The lung epithelium is a fundamental barrier that provides protection against the outside environment and restricts fluid accumulation in the airway. Excessive alveolar epithelial cell damage and breakdown is a hallmark of acute respiratory distress syndrome (ARDS). Patients with ARDS rapidly accumulate fluid in the airway and alveolus, resulting in severely compromised ventilation. In addition, this injury can generate maladaptive restoration of the epithelial barrier resulting in fibrotic scarring (2, 3). The pathogenic mechanisms responsible for fluid accumulation and tissue remodeling remain unclear but are believed to involve overattenuation of the inflammatory response and accelerated apoptosis of the epithelium (29). Attenuation of the Fas/Fas ligand (Fas/FasL) pathway has been implicated as one of the primary apoptotic pathways activated in the alveolar epithelium at the onset of ARDS in humans (1, 7, 12). In support of this, Fas-mediated apoptosis of the lung epithelium causes acute lung injury and fibrosis in mice (11, 15, 17) and leads to the development of ARDS and fibrosis in humans (18, 22). Our laboratory has shown that Fas-mediated apoptosis of human lung epithelial cells is modulated by proinflammatory cytokines (5).
Based on these observations we predict that, during localized stress, PI3K and Akt are mobilized by external signals present in the tissue microenvironment, including growth factors, to internally protect the lung epithelium from external apoptotic stimuli that promote an untimely death. Cell preservation becomes a "tug-of-war" between exposure to both pro- and antiapoptotic factors. Specifically, we hypothesized that KGF preserves lung integrity by inhibiting alveolar epithelial cell apoptosis via activation of the Akt survival pathway. To test this we determined that KGF directly activates Akt kinase and that upregulation of kinase activity is linked to inhibition of Fas-mediated apoptosis in A549 cells, a human lung epithelial cell line. We also provide evidence that KGF prevents apoptosis via Akt activation. Based on this, we propose that the PI3K/Akt survival pathway represents a novel proximal therapeutic target that can be exploited to preserve or restore lung function in patients with ARDS.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental conditions for apoptosis and caspase activity measurements.
A549 cells were plated at 80–90% confluence in either 25-cm2 tissue culture flasks (Falcon/Fisher, Pittsburgh, PA) or eight-well glass chamber slides (Fisher). Cells were incubated for 2 days postpassage before further manipulation to allow cells to reach full confluence. After serum starvation, KGF (10–100 ng/ml, R&D Systems) and/or recombinant human IFN-
(250 units/ml; BioSource International, Camarillo, CA) were added to cultures. After 24 h, cells were given a second stimulus of either a Fas cross-linking antibody (FasAb, 10–100 ng/ml clone CH-11; Kamiya Biomedical, Seattle, WA) or an isotype control (10–100 ng/ml IgM; Sigma, St. Louis, MO). Cells were incubated an additional 24 h before analysis for the number of apoptotic cells or caspase activity. A549 cells were also stimulated as described above; however, 30 min before the initial stimulus, cells were given the PI3K inhibitor LY-294002 (10 µM; CalBiochem, San Diego, CA) or an equivalent dose of the solvent DMSO as the vehicle control. After 24 h, cells were given a second dose of the above inhibitors or solvent at half the original dose, and 30 min later the cells were stimulated with the FasAb as described above.
Cell extracts. After the treatment described above, adherent cells were trypsinized and pooled with nonadherent cells present in the original medium. Cells were then pelleted at 300 g for 10 min at room temperature, supernatants were discarded, and the cell pellet was resuspended in 200 µl of 1x KPM complete buffer (2x KPM = 100 mM PIPES, pH 7; 100 mM KCl; 20 mM EGTA; and 3.84 mM MgCl2), 1 mM DTT, 0.1 mM PMSF, 10 µg/ml cytochalasin B, 2 µg/ml chymostatin, 2 µg/ml leupeptin, 2 µg/ml antipain, and 2 µg/ml pepstatin A. Resuspended cells were transferred to 0.5-ml Eppendorf tubes and pelleted again (3,000 rpm for 10 min). Cell pellets were resuspended in 15 µl of 1x KPM complete buffer, frozen in liquid nitrogen, placed in a room temperature water bath until cells had thawed, and then vortexed. The freeze-thaw procedure was performed a total of four times. Samples were then centrifuged at 14,000 rpm for 20 min at 4°C. The resulting supernatant or cell extract was removed to a fresh 0.5-ml Eppendorf tube, and an aliquot was analyzed for protein concentration by the Bradford method (Bio-Rad, Hercules, CA), frozen in liquid nitrogen, and stored at –70°C.
Enzymatic caspase activity measured with amino trifluoro methyl coumarin. For all amino trifluoro methyl coumarin (AFC) preparations, cells (3 x 106 cells) were collected by centrifugation and washed with KPM buffer and lysed by 4 cycles of freeze-thawing as previously described (5). The presence of active caspases was determined by AFC assay using a specific fluoro-substrate as previously described. Lysates were incubated with DEVD-AFC in a cyto-buffer (10% glycerol, 50 mM PIPES pH 7.0, and 1 mM EDTA) containing 1 mM DTT and 20 µM DEVD-AFC (Enzyme Systems Products). The release of free AFC was determined using a Cytofluor 4000 fluorimeter (Perseptive, Framingham, MA; filters: 400 nm for excitation, 505 nm for emission).
Immunohistochemistry. A549 cells were stimulated as described above. Adherent cells were nonenzymatically disadhered and pooled with disadherent cells followed by cytospin preparation of cells onto silane-treated slides. Cells were then rinsed twice with 1x PBS and fixed in ice-cold pure methanol for 30 min at –20°C. After being washed twice with washing buffer (1x PBS and 0.1% Tween 20), wells were blocked with blocking buffer [1x PBS, 1% bovine serum albumin (BSA), and 0.1% Tween 20] for 10 min. Cells were then incubated with the M30 CytoDEATH antibody (Boehringer Mannheim, Indianapolis, IN), a monoclonal antibody that specifically detects caspase-cleaved human cytokeratin-18 (CK-18), and diluted 1:10 in blocking buffer for 1 h at room temperature. Cells were washed twice with washing buffer and then incubated with 10 µg/ml anti-mouse-IgG labeled with fluorescein (Boehringer Mannheim) for 30 min at room temperature in the dark. Cells were again washed twice with washing buffer before incubation with 0.5 µg/ml 4',6-diamidine-2'-phenylindole dihyrochloride (DAPI; Roche Molecular Biochemicals, Indianapolis, IN) for 5 min at room temperature in the dark. Cells were washed three times with washing buffer and allowed to air dry slightly. Antifade (Molecular Probes, Eugene, OR) was added to each slide before addition of coverslip. Cells were considered to be apoptotic if caspase-cleaved CK-18 appeared in the cytoplasm. Specificity was confirmed by comparison against an antibody that recognizes native CK-18 in all cells as well as comparison to an isotype control antibody MOPC21 (Sigma). Apoptotic (M30-positive cells) and total cells (DAPI-stained nuclei) were enumerated by a blinded observer who randomly selected six fields of view per treatment condition. Data are presented as the average percentage of apoptotic cells divided by the total number of cells per viewing area. All results are compared with actinomycin D-treated cells as a positive control. In our experience, identical results are obtained when using the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling assay, thereby confirming the validity of our assay.
Akt in vitro kinase assay.
The dose and time response of Akt activation by KGF was determined. A549 cells were first treated with increasing doses of KGF (0–100 ng/ml). Next, the optimum dose was used to determine the time-dependent response of Akt activation. Initial studies were done with extended time points (0–12 h). From our initial observations, Akt activation occurred within minutes. On the basis of this, all further studies evaluated Akt with time points ranging between 1 and 20 min. Under all conditions, cells were lysed in 1 ml of ice-cold buffer (50 mM Tris·HCl pH 7.5, 0.1% Triton X-100, 1 mM EDTA, pH 8.3, 1 mM EGTA pH 8.0, 50 mM NaF, 10 mM -glycerophosphate, 5 mM sodium pyrophosphate, 10 mg/ml aprotinin, and 10 mg/ml leupeptin, 1 mM sodium vanadate, and 14.2 mM 2-mercaptoethanol) for 15 min. Nuclei were removed by centrifugation, and then cytoplasmic extracts were immunoprecipitated with an immobilized Akt antibody (Cell Signaling Technology, Beverly, MA). The immunoprecipitates were then removed from beads in cold lysis buffer and measured for Akt kinase activity using glycogen synthase kinase-3 (GSK-3, Cell Signaling Technology) as substrate. Proteins and peptides were separated on a 12% SDS-polyacrylamide gel and then transferred onto nitrocellulose membranes. The lower portion of the membrane was immunoblotted with phospho-GSK-3-specific antibody and then subject to autoradiography. The upper portion of the membrane was immunoblotted with an antibody that recognizes total Akt to confirm equal loading and standardize results. We semiquantified densitometry of the phospho-GSK-3 by standardizing each sample to its own total Akt blot and presenting it as a complement to the autoradiography results.
Detection of apoptotic vs. necrotic cells. The Vybrant Apoptosis Assay Kit (V-13243, Molecular Probes) was used to distinguish apoptotic and necrotic cells under our culture conditions. Briefly, at the end of the treatment period cells were washed in cold PBS and then stained per the manufacturer’s recommendations with green fluorescent YO-PRO-1 dye, which enters and stains apoptotic cells only, and propidium iodide, which enters and stains necrotic cells. Cells were immediately prepared for viewing in a cytospin preparation and enumerated under fluorescent microscopy.
Statistical analysis. All data were expressed as means ± SE. Paired t-tests were used for single comparisons (Microsoft Excel; Microsoft, Redmond, WA). For comparisons that involved multiple variables and observations, two- and three-way ANOVA (JMP; SAS Institute, Cary, NC) was used. Having passed statistical significance by ANOVA, individual comparisons were made using the Tukey multiple-comparison test. Statistical significance was defined as a P value < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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resulted in apoptosis in >20% (23.2 ± 8.8%) of the total population of cells (Fig. 1). An isotype control antibody matched to the FasAb did not induce apoptosis (data not shown). Pretreatment with KGF before the addition of IFN- and FasAb significantly decreased the incidence of apoptosis by approximately one-half (12.2 ± 4.9%). Addition of the PI3K inhibitor LY-249002, before KGF, IFN-, and FasAb, increased the number of apoptotic cells to >70% of the total population, even in the presence of KGF (74.5 ± 13.2%). Treatment with LY-249002 alone also resulted in apoptosis, without evidence of necrosis as measured by YO-PRO-1 staining, but approximately threefold less (26.5 ± 7.4%) than that measured in the LY-249002/KGF/IFN-/FasAb treatment group as well as the LY-249002/IFN-/FasAb treatment groups. On the basis of these findings we concluded that the PI3K/Akt survival axis contributes to KGF protection against Fas-mediated apoptosis.
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and anti-Fas antibody compared with baseline levels (Fig. 4). Pretreatment with KGF significantly decreased the amount of caspase activity in cell lysates. Treatment with LY-249002 reversed the protective effect of KGF and significantly increased the amount of caspase-3 activity. This further supports our previous observation that KGF inhibits apoptosis. Consistent with our earlier findings, LY-249002 treatment alone induced caspase activity. Again, this observation is not surprising when considering that the A549 cell line is derived from human adenocarcinoma tissue and that many cancer cells have been found to possess a relatively high basal level of Akt kinase activity (8).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, an activator of the PI3K/Akt signaling axis (5). To expand upon these observations we turned our attention to KGF, a growth factor unique to the epithelium with known protective effects that we reasoned would inhibit alveolar epithelial cell apoptosis through activation of a well-known survival signal transduction pathway. To test this hypothesis we determined that KGF inhibits lung epithelial cell death following Fas activation, a relevant apoptosis pathway in ARDS, and then determined that cellular preservation was dependent on activation of the PI3K/Akt kinase signal transduction pathway. In particular, KGF inhibited apoptosis initiated by IFN- and FasAb as measured by a reduction in caspase-cleaved CK-18. We previously reported that IFN-, a relevant cytokine in the context of acute lung injury, increases the surface expression of Fas on the lung epithelium, thereby increasing the apoptotic response to Fas stimulation (5). The decrease in cytokeratin cleavage correlated directly with a reduction in caspase-3 activity. In agreement with other investigations, induction of Akt kinase activity by KGF was dose- and time-dependent and most effective in maintaining cell viability when activated before exposure to apoptosis-inducing stimuli. The inhibitory effect on apoptosis provided by KGF was at least partially dependent on activation of PI3K. These data support our original hypothesis that KGF preserves lung epithelial cell viability by inhibiting apoptosis and that protection is mediated in part through activation of the PI3K-dependent survival pathway. We predict that PI3K/Akt provides an essential function in the alveolar epithelium as a central survival pathway during intervals of localized stress associated with inflammation and apoptosis. In this scenario, PI3K and Akt are mobilized by external signals present in the tissue microenvironment, including growth factors and cytokines, to internally protect the cell from external apoptotic stimuli that promote an untimely death. Cell preservation then becomes a tug-of-war between external stimuli and the relative timing of exposure to both pro- and antiapoptotic factors. If terminal caspase activation, such as caspase-3, precedes growth factor stimulation, a commitment to death is made that becomes very difficult to reverse. Therefore, therapeutic intervention is complicated by the fact that agents directed at the alveolar epithelium of "at risk" individuals susceptible to developing acute lung injury must be administered before the onset of damage. Our results obtained from A549 cells indicate that KGF provides protection but only when PI3K is active. In the presence of PI3K inhibition, KGF lost the ability to protect cells from apoptosis. Interestingly, a recent investigation conducted in primary rat alveolar type II cells reports that KGF activation was mediated through the extracellular signal-regulated kinase pathway in addition to the PI3K pathway (24). The implication from this as it relates to our findings is that the lung epithelium has redundant signaling mechanisms to convey RTK protection.
Fas, also known as CD95/Apo-1, is constitutively expressed by human alveolar epithelial cells (7, 12) and is believed to play a major role in controlling lung epithelial cell apoptosis during inflammation. Excessive Fas activation in the lung causes significant tissue pathology. Topical administration of FasL to the airway accelerates apoptosis of epithelial cells and causes pulmonary fibrosis in mice (10, 11). Aerosolization of pathogenic bacteria promotes Fas-FasL-dependent increases in permeability and tissue pathology (21). In humans, increased levels of soluble bioactive FasL in bronchoalveolar lavage fluid directly correlate with disease severity in adults with ARDS (1, 22). These observations suggest that Fas-mediated apoptosis of the lung epithelium is an important process in the development of acute lung injury at the onset of ARDS. For this reason, we investigated Fas-mediated apoptosis in A549 cells as a representative model to identify key cellular events that determine cell survival. We investigated external signals that converge upon a common, internal biochemical pathway that facilitates cell survival. We anticipated that identification of biochemical events involved with protection against inflammatory stimuli that promote cell death would reveal a potential treatment strategy to prevent acute lung injury. KGF is a potent survival factor for the lung epithelium and provides protection against many external stimuli. On the basis of our investigation and others, we believe that protection afforded by KGF is at least in part related to activation of the PI3K/Akt pathway and perhaps others. Activation of this survival pathway helps to protect tissue barrier function during inflammatory stress and facilitate wound repair when the barrier is compromised. Several reports demonstrate that KGF is a mitogen that promotes repair by inducing cell proliferation (28, 34), whereas others indicate that KGF modifies the cell phenotype by reducing sensitivity to apoptosis without inducing cell division (31). We found that prior treatment with KGF prevented Fas-mediated death without inducing proliferation. Therefore, we predict that one mechanism by which KGF modifies lung epithelial behavior is through activation of survival pathways that arrest the Fas-induced apoptosis before caspase activation. This would also indicate that prophylactic therapeutic strategies can be directed toward viable, differentiated lung epithelium without a requisite for cell proliferation. This is supported by previous studies demonstrating that growth factor stimulation prevents or inhibits apoptosis via the PI3K/Akt pathway (9, 16, 25, 32).
Our results corroborate previous investigations and further establish a framework to dissect the role of KGF-induced inhibition of apoptosis in the human lung epithelium. Our findings indicate that KGF triggers the PI3K/Akt response and induces cell protection presumably through modulation of intermediate proteins located upstream from terminal caspases. We anticipate that this investigation will help to further identify biochemical events that can prevent acute lung injury as it pertains to the epithelium. This investigation supports the hypothesis that KGF can preserve epithelial cell integrity by inhibiting apoptosis and suggests that protection is mediated in part through activation of the PI3K survival pathway. Therefore, the PI3K/Akt axis may serve as a therapeutic target to preserve epithelial integrity and provide a useful strategy to block apoptosis thereby limiting acute lung injury in humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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