Articles in PresS. Am J Physiol Lung Cell Mol Physiol (September 3, 2004). doi:10.1152/ajplung.00309.2003
Keratinocyte Growth Factor induces Akt kinase activity and inhibits Fas-mediated apoptosis in A549 lung epithelial cells
Shenying Bao12, Yijie Wang2, Patricia Sweeney 1, Alpana Chaudhuri1, Andrea I. Doseff2,3, Clay B. Marsh2,3, and Daren L. Knoell1, 2
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Department of Pharmacy, 2Dorothy M. Davis Heart and Lung Research Institute, and the 3 Division of Pulmonary and Critical Care Medicine The Ohio State University, Columbus, OH 43210 Running Head: FGF-7 inhibits Fas-mediated apoptosis
Correspondence should be addressed to: Dr. Daren L. Knoell, F.C.C.P Associate Professor of Pharmacy and Internal Medicine Director of the Lung Cell Isolation Program the Davis Heart and Lung Research Institute 500 W. 12th Ave., Parks Hall Columbus, OH 43210 (W) 614-292-0075 (F) 614-292-1335
[email protected]
Copyright © 2004 by the American Physiological Society.
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Abstract: Adult respiratory distress syndrome (ARDS) is a syndrome characterized by the rapid influx of protein-rich edema fluid into the airspaces. The magnitude of alveolar epithelial cell injury is a key determinant of disease severity and an important predictor of patient outcome. The alveolar epithelium is positioned at the interface of the host response in the initiation, progression and recovery phase of the disease. Keratinocyte Growth Factor (KGF) is a potent survival factor unique to the epithelium that promotes lung epithelial cell survival, accelerates wound closure, and reduces fibrosis. We therefore hypothesized that KGF preserves lung function by inhibiting apoptosis through activation of a signal transduction pathway responsible for cell survival. To test this hypothesis we determined that KGF inhibits death following Fas activation, a relevant apoptosis pathway, and then determined that cell survival is mediated through activation of the PI3K/Akt kinase signal transduction pathway. We found that KGF induces a dose- and time-dependent increase in Akt kinase activity and as expected, activation of Akt via KGF is PI3K-dependent. KGF inhibited Fas induced apoptosis as measured by a reduction in apoptotic cells and caspase-3 activity. This investigation supports our original hypothesis that KGF protects the lung epithelium by inhibiting apoptosis and that protection occurs through activation of PI3K/Akt-mediated cell survival pathway. Our results are in agreement with other reports that identify the PI3K/Akt axis as a key intracellular pathway in the lung epithelium that may serve as a therapeutic target to preserve epithelial integrity during inflammation. Key Words: acute lung injury, phosphatidylinositol 3’ kinase (PI3K), FGF-7, adult respiratory distress syndrome (ARDS)
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Introduction Keratinocyte growth factor (KGF), also known as FGF-7, is a member of the fibroblast growth factor (FGF) family and is produced exclusively by fibroblasts. Unlike most FGFs that have broad mitogenic activity, KGF is specific for the epithelium(6, 26) through interaction with the receptor tyrosine kinase (RTK), FGF receptor IIIb (FGFRIIIb)(23). Topical administration of KGF to the airway reduces lung injury caused by hyperoxia and toxic compounds, facilitates epithelial repair following radiationinduced DNA damage(28, 34), and protects against permeability defects in human airway epithelial cells exposed to oxidants(30, 31). KGF binding to FGFRIIIb induces receptor dimerization at the cell surface and autophosphorylation of tyrosine residues located on the cytoplasmic portion of the receptor complex(13). The functional role of tyrosine autophosphorylation is to provide docking sites for downstream signal transduction molecules. Many of these interactions occur with proteins such as the p85 adapter subunit phosphatidylinositol 3’ kinase (PI3K), that possess Src homology 2 (SH2) domains. PI3K translocates to the receptor via SH2 binding to the consensus pYXXM motif located on the cytoplasmic region of the receptor, where pY represents a phosphorylated tyrosine residue(27). A tyrosine residue (Tyr619) exists in the intracellular domain of FGFIIIb with a methionine at the +3 position in the cytoplasmic domain of FGFRIIIb thereby providing a substrate for interaction with PI3K. Once bound to the receptor, the p110 catalytic subunit of PI3K activates its kinase activity. 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 (PKB) is a primary downstream target of PI3K
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intermediates and major component in cell survival(14). Akt mediates its anti-apoptotic 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-3 (GSK-3 ), transcriptional factors of the forkhead family, and IKK, a kinase that regulates the NF-kB 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 over-express KGF have an Aktdependent, 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 over-attenuation of the inflammatory response
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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 pro-inflammatory 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 anti-apoptotic 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
Cell culture: A549 cells, a human lung epithelial cell line, were purchased from the American Type Culture Collection (Rockville, MD)(19).
Cells were cultured under
standard conditions in F-12 nutrient mixture (HAM) (Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco BRL) (complete growth medium). A549 cells were passaged at 80-90% confluence using 0.25% trypsin, 1 mM EDTA (Gibco BRL). At the onset of each experiment, cultures were serum-starved for a period of 48 hours before addition of any factors to eliminate background signal noise contributed by serum containing medium.
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 8-well glass chamber slides (Fisher). Cells were incubated for 2 days post-passage before further manipulation to allow cells to reach full confluence. Following serum starvation, KGF (10 to 100 ng/ml, R&D Systems) and/or rhIFN- (250 U/ml) (BioSource International, Camarillo, CA) were added to cultures. After 24 hours, cells were given a second stimulus of either a Fas-crosslinking antibody (clone CH-11, Kamiya Biomedical Company, Seattle, WA) (10 – 100 ng/ml) or an isotype control (IgM, Sigma, St. Louis, MO) (10 – 100 ng/ml). Cells were incubated an additional24 hours prior to analysis for the number of apoptotic cells or caspase activity. A549 cells were also stimulated as described above, however, 30 minutes prior to the initial stimulus, cells
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were given the phosphotidalinositol 3-kinase inhibitor, LY294002 (10 µM) (CalBiochem, San Diego, CA) or an equivalent dose of the solvent DMSO as the vehicle control. After 24 hours, cells were given a second dose of the above inhibitors or solvent at half the original dose, and 30 minutes later the cells were stimulated with the Fas-crosslinking antibody as described above.
Cell Extracts: Following the treatment described above, adherent cells were trypsinized and pooled with nonadherent cells present in the original medium. Cells were then pelleted at 300 x g for 10 minutes 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 pepstatain A.
Resuspended cells were transferred to 0.5 ml
eppendorf tubes and pelleted again (3,000 rpm for 10 minutes).
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 minutes at 4MC. 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 –70MC.
Enzymatic caspase activity measured with amino trifluoro methyl coumarin: For all amino trifluoro methyl coumarin preparations, cells (3 x 106 cells) were collected by
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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 amino trifluoro methyl coumarin assay (AFC) 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, 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 Company, Framingham, MA. Filters: excitation; 400 nm, emission; 505 nm).
Immunohistochemistry: A549 cells were stimulated as described above. Adherent cells were nonenzymatically disadhered and pooled with disadherent cells followed by cytopsin preparation of cells onto silane treated slides. Cells were then rinsed twice with 1x PBS and fixed in ice-cold pure methanol for 30 minutes at –20MC. After washing twice with washing buffer (1x PBS and 0.1% Tween 20), wells were blocked with blocking buffer (1x PBS, 1% BSA and 0.1% Tween 20) for 10 minutes. Cells were then incubated with the M30 CytoDEATH antibody (Boehringer Mannheim, Indianapolis, IN), a monoclonal antibody that specifically detects caspase cleaved human cytokeratin18 (CK-18), diluted 1:10 in blocking buffer for 1 hour 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 minutes at room temperature in the dark. Cells were again washed twice with washing buffer prior to incubation with 0.5 µg/ml
4',6-Diamidine-2'-phenylindole
dihyrochloride
(DAPI)
(Roche
Molecular
Biochemicals, Indianapolis, IN) for 5 minutes at room temperature in the dark. Cells
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were washed 3 times with washing buffer and allowed to air dry slightly. Anti-fade (Molecular Probes, Eugene, Oregon) was added to each slide prior to addition of cover slip. 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 that randomly selected six fields of view per treatment condition. Data is presented as the average percent of apoptotic cells divided by the total number of cells per viewing area. All results are compared to actinomycin-D treated cells as a positive control. Based on our experience, identical results are obtained when using the TUNEL 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 to 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 to 12 hours). From our intitial observations, Akt activation occurred within minutes. Based on this, all further studies evaluated Akt with time points ranging between 1 to 20 minutes. Under all conditions, cells were lysed in 1 ml ice-cold buffer (50mM Tris-HCl pH 7.5, 0.1% Triton X-100, 1mM EDTA, pH 8.3, 1mM EGTA pH 8.0, 50mM NaF, 10mM
-
glycerophosphate, 5mM sodium pyrophosphate, 10mg/ml aprotinin and 10mg/ml leupeptin, 1mM sodium vanadate, 14.2 mM beta mercaptoethanol) for 15 min. Nuclei were removed by centrifugation and then cytoplasmic extracts were immunoprecipitated
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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-3 peptide (GSK-3, Cell Signaling Technology, Beverly, MA) as substrate. Proteins and peptides were separated on a 12% SDS-polyacrylamide gel, then transferred onto nitrocellulose membranes. The lower portion of the membrane was immunoblotted with phospho-GSK3 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. Densitometry of the phospho-GSK3 was semi-quantified by standardizing each sample to its own total Akt blot and presented as a compliment to the autoradiography results.
Detection of Apoptotic versus Necrotic Cells. The Vybrant® Apoptosis Assay Kit (V13243, Molecular Probes, Eugene, OR) 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 only enters and stains apoptotic cells, and propidium iodide, which enters and stains necrotic cells. Cells were immediately prepared for viewing in a cytospin preparation and enumerated using flurorescent microscopy.
Statistical Analysis. All data were expressed as mean + SEM. 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;
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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
KGF Prevents Apoptosis. Caspase processing of a key structural intermediate filament protein unique to epithelial cells, cytokeratin 18 (CK18), was used to quantify cellular apoptosis. CK18 has been identified as a substrate for caspases 3, 6, and 7 during epithelial cell apoptosis(4). Activation of Fas with a anti-Fas cross-linking antibody in conjunction with IFN resulted in apoptosis in over twenty percent (23.2 + 8.8 %) of the total population of cells (Figure 1). An isotype control antibody matched to the Fas-cross linking antibody did not induce apoptosis (data not shown). Pretreatment with KGF prior to 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, LY249002, prior to KGF, IFN and FasAb increased the number of apoptotic cells to over seventy percent of the total population, even in the presence of KGF (74.5 + 13.2 %). Treatment with LY249002 alone also resulted in apoptosis, without evidence of necrosis as measured by YOPRO-1 staining, but approximately three-fold less (26.5 + 7.4 %) than that measured in
the
LY249002/KGF/IFN /FasAb
treatment
group
as
well
as
the
LY249002/IFN /FasAb treatment groups. Based on these findings we concluded that the PI3K/Akt survival axis contributes to KGF protection against Fas-mediated apoptosis.
KGF induces Akt kinase activity.
KGF has previously been shown to promote lung
epithelial cell survival but the mechanisms by which this occurs are not fully understood. Conflicting reports indicate that KGF protects the lung epithelium via mitogen-dependent (28, 34) as well as -independent events(31). KGF did not induce proliferation in A549
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cells as measured by a common cell proliferation assay (data not shown) thereby indicating that mitogenic activity is not responsible for cell survival. In order to inhibit apoptosis, we reasoned that KGF preserves cell viability through activation of Akt, a key survival factor in mammalian cells. To establish this we first measured the Akt kinase activity in A549 lung epithelial cells in response to increasing doses of KGF. Confluent cultures of serum starved A549 cells were treated with recombinant KGF for 15 minutes at increasing doses and then total cell lysates were analyzed for Akt in vitro kinase activity. Our findings indicate that A549 cells cultured in 10% bovine serum albumin (BSA) have a relatively high constitutive level of Akt kinase activity that is reduced approximately 2-fold with serum starvation (Figure 2a). The high constitutive level of kinase activity can likely be attributed to growth factors present in serum and the fact that the cell line is of cancer origin. Importantly, in the absence of serum, KGF treatment alone restored and elevated Akt kinase activity to levels observed in BSA positive conditions in a dose-dependent manner. Increasing concentrations of KGF proportionally induced the level of Akt kinase activity with maximum stimulation at 4.5-fold above serum-starved baseline levels at a concentration of 100 ng/ml.
Next, we evaluated the
time course of Akt kinase induction by KGF. Using a concentration of 100 ng/ml, we found that KGF reproducibly generated a peak in Akt kinase activity within 20 minutes of exposure (Figure 2b). We did not find additional modulation in kinase function following evaluation of extended time points (data not shown).
Akt Induction by KGF requires PI3K. We expected that KGF-mediated receptor tyrosine kinase activity would result in the recruitment and activation of p85 and p110 PI3K,
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leading to activation of Akt. In order to confirm this we evaluated KGF’s ability to induce Akt kinase function in the presence of the PI3K inhibitor LY249002. Inhibition of PI3K by LY249002 resulted in a substantial reduction of Akt kinase activity following treatment with KGF in serum-starved A549 lung epithelial cells when compared to DMSO treatment alone (Figure 3). This effect was highly reproducible and observed under optimal conditions for growth factor stimulation following 20 minutes of exposure. We also consistently showed a moderate decrease in KGF-induced Akt kinase activity following treatment with the vehicle DMSO alone. This is not entirely unexpected since DMSO has antioxidant properties and Akt function is linked to oxidant activation(33).
KGF induced Akt activation inhibits caspase 3. The intracellular apoptotic cascade that ensues following Fas receptor stimulation requires activation of the “initiator” caspase-8 followed by activation of “executioner” caspases including caspase-3. Our previous result demonstrated that CK-18, a caspase-3 substrate, is enzymatically processed following Fas receptor activation and that cleavage is inhibited by exposure to KGF. Based on this we reasoned that the preservation of lung epithelial cells by KGF is linked to down-modulation of caspase-3 activation by the PI3K/Akt signaling axis. Consistent with previous data, the level of caspase-3 activity increased in cells treated with the combination of IFN
and anti-Fas antibody when compared to baseline levels.
Pretreatment with KGF significantly decreased the amount of caspase activity in cell lysates. Treatment with LY249002 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,
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LY249002 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
Previously, we reported that apoptosis of the lung epithelium is controlled by inflammatory cytokines and that caspase activation is inhibited by interleukin-1 , 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 dependant 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 prior to exposure to apoptosis inducing stimuli.
The inhibitory effect on apoptosis
provided by KGF was at least partially dependent on activation of PI3K. This data supports 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.
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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 anti-apoptotic 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 reported that KGF activation was mediated through the extracellular signalregulated kinase (ERK) 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 receptor tyrosine kinase 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).
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Aerosolization of pathogenic bacteria promotes Fas-Fas ligand dependent increases in permeability and tissue pathology(21). In humans, increased levels of soluble bioactive FasL in broncho-alveolar 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. Based on 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 prior to caspase activation. This would also indicate that prophylactic therapeutic
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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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Acknowledgements:
This research was supported by grants to D.L.K. from the NIH (HL56336) and to C.B.M (HL63800, HL6108, HL67176, HL70294).
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Figure Legends:
Figure 1. KGF Inhibits Fas-Mediated Apoptosis. Confluent layers of A549 cells were incubated in serum-free conditions and exposed to KGF (10-100 ng/ml), IFN (250 U/ml), or a combination of KGF and IFN . Twenty-four hours later cells were treated with a Fas-cross linking antibody (FasAb) to initiate apoptosis. Additionally, KGF was administered 12 hours after IFN and FasAb. The following day cells were fixed and stained with M30 antibody and DAPI. Apoptotic cells were enumerated by standard fluorescent microscopy. IFN augmented Fas-mediated death which was significantly inhibited by KGF pretreatment (* P < 0.05). Administration of KGF after induction of apoptosis failed to protect cells which is not shown. Addition of the PI3K inhibitor, LY249002, prior to KGF, IFN and FasAb reversed the protection afforded by KGF and potentiated apoptosis (* P < 0.05). Treatment with LY249002 alone also resulted in apoptosis but approximately three-fold less than that measured in the LY249002/KGF/IFN /FasAb and LY249002/IFN /FasAb treatment groups.
Figure 2. KGF Induces Akt kinase in A549 cells. We next determined if KGF could induce Akt kinase activity in A549 cells by incubating confluent layers of epithelial cells with KGF for 15 minutes followed by immunoprecipitation of total Akt and analysis of induced kinase activity using GSK-3 as substrate. The level of kinase activity (Panel A) was standardized against total Akt content in each sample (Panel B) and these results are represented by densitometric analysis (Panel C). Our results demonstrate that A549 cells have a constitutively high level of Akt kinase activity under serum containing growth
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conditions that is reduced by approximately one-half following overnight serum starvation. Under serum starved conditions, Akt kinase activity was fully restored and exceded baseline levels by KGF treatment alone in a dose and time dependent manner (data obtained from one experiment and representative of five separate experiments). (* P < 0.05 when compared to serum-starved cells)
Figure 3. KGF Induces Akt Kinase in a PI3K dependent manner. A549 cells were exposed to an optimum dose of KGF (100 ng/ml for 20 minutes) under serum starved conditions. Similar to previous results a significant increase in Akt activity above that of serum-starved baseline conditions was observed. Pretreatment with the PI3K inhibitor LY294002 substantially reduced Akt kinase activity as measured by Akt-dependent phosphorylation of GSK-3 (data obtained from one experiment and representative of five separate experiments).
Figure 4. KGF Inhibits Fas-mediated Caspase-3 Activity. Confluent layers of A549 cells were treated as previously described. Whole cell lysates were isolated and then measured for active caspase-3 which was standardized by total protein concentration in each sample. Our results are consistent with data from previous experiments and demonstrate that KGF treatment resulted in a reduction in caspase-3 activation following exposure to IFN and FasAb. Addition of the PI3K inihibitor, LY294002, further enhanced caspase-3 activity and prevented the ability of KGF to inhibit caspase-3 activation.
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