Regulation of PDGFR-alpha in rat pulmonary myofibroblasts by staurosporine

Pamela M. Lindroos, Yi-Zhe Wang, Annette B. Rice, and James C. Bonner

Laboratory of Pulmonary Pathobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Upregulation of the platelet-derived growth factor (PDGF) receptor-alpha (PDGFR-alpha ) is a mechanism of myofibroblast hyperplasia during pulmonary fibrosis. We previously identified interleukin (IL)-1beta as a major inducer of the PDGFR-alpha in rat pulmonary myofibroblasts in vitro. In this study, we report that staurosporine, a broad-spectrum kinase inhibitor, upregulates PDGFR-alpha gene expression and protein. A variety of other kinase inhibitors did not induce PDGFR-alpha expression. Staurosporine did not act via an IL-1beta autocrine loop because the IL-1 receptor antagonist protein did not block staurosporine-induced PDGFR-alpha expression. Furthermore, staurosporine did not activate a variety of signaling molecules that were activated by IL-1beta , including nuclear factor-kappa B, extracellular signal-regulated kinase, and c-Jun NH2-terminal kinase. However, both staurosporine- and IL-1beta -induced phosphorylation of p38 mitogen-activated protein kinase and upregulation of PDGFR-alpha by these two agents was inhibited by the p38 inhibitor SB-203580. Finally, staurosporine inhibited basal and PDGF-stimulated mitogenesis over the same concentration range that induced PDGFR-alpha expression. Collectively, these data demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells and possibly for evaluating the role of the PDGFR-alpha as a growth arrest-specific gene.

pulmonary fibrosis; protein kinase C; p38 mitogen-activated protein kinase; interleukin-1beta ; platelet-derived growth factor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PLATELET-DERIVED GROWTH FACTOR (PDGF) is a potent mitogen for connective tissue cells that plays a critical role in lung development (6, 34) and has been implicated in the etiology of pulmonary fibrosis (25, 26). PDGF exists as a disulfide-linked dimer of two polypeptide chains, A and B, that form PDGF-AA, PDGF-BB, or PDGF-AB (9). Two PDGF receptor (PDGFR) subtypes (alpha  and beta ) have different affinities for the three isoforms. PDGFR-beta can only interact with B chain-containing isoforms, whereas PDGFR-alpha can bind all three isoforms (32). Dimerization of these receptor subtypes occurs in response to interaction with PDGF ligands followed by receptor tyrosine kinase phosphorylation and interaction with a variety of signal transduction molecules (for a review, see Ref. 9).

Recent studies (5, 20) have shown that the PDGFR-alpha is inducible in rat models of pulmonary fibrosis, and this represents a potential mechanism of mesenchymal cell hyperplasia that could contribute to the progression of the disease. Upregulation of the PDGFR-alpha in vitro in rat pulmonary myofibroblasts and rat osteoblasts is stimulated by interleukin (IL)-1beta (7, 22, 27, 35), and induction of PDGFR-alpha renders these cell types hyperresponsive to the mitogenic and chemotactic effects of PDGF. These observations are consistent with several studies (27, 30, 33) that showed that maximal responses to PDGF-AB or -BB require coexpression of PDGFR-alpha and PDGFR-beta . Wang et al. (36) recently reported that IL-1beta -induced upregulation of the PDGFR-alpha gene requires the activation of p38 mitogen-activated protein (MAP) kinase.

The microbial alkaloid staurosporine is a broad-spectrum protein kinase inhibitor that has been documented as a useful tool for the study of apoptosis and growth arrest (4, 11, 14, 41). Interestingly, the PDGFR-alpha gene has been identified as a growth arrest-specific gene, and accumulation of the PDGFR-alpha gene product has been suggested to facilitate the exiting of cells from growth arrest after stimulation with PDGF (21). In this study, we report that staurosporine upregulates the PDGFR-alpha gene over the same concentration range (1-100 nM) that causes growth arrest in rat pulmonary myofibroblasts. The mechanism of staurosporine-induced PDGFR-alpha upregulation was not related to inhibition of protein kinase (PK) C activity or inhibition of receptor tyrosine kinase activity. In contrast to IL-1beta , staurosporine did not cause activation of nuclear factor-kappa B (NF-kappa B) or the MAP kinases c-Jun NH2-terminal kinase (JNK) or extracellular signal-regulated kinase (ERK). However, staurosporine and IL-1beta caused phosphorylation of p38 MAP kinase, and upregulation of the PDGFR-alpha was inhibited by the p38 MAP kinase inhibitor SB-203580. These data are consistent with the idea that the PDGFR-alpha is a growth arrest-specific gene that is regulated via the activation of p38 MAP kinase.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. The protein kinase inhibitors staurosporine, genistein, tyrphostin AG-1296, bisindolylmaleimide I, GF-109203X, Ro -31-8220, and H-89 were purchased from Calbiochem (La Jolla, CA). The rat cDNA probe for the PDGFR-alpha was the generous gift of Dr. Yutaka Kitami (Ehime University, Ehime, Japan). PDGF isoforms (AA, AB, and BB), IL-1beta , and the IL-1 receptor antagonist protein (IRAP) were purchased from R&D Systems (Minneapolis, MN). 125I-PDGF-AA (specific activity of 125 µCi/µg) was from Biomedical Technologies (Stoughton, MA). Antibodies to mouse PDGFR-alpha and human PDGFR-beta were from Upstate Biotechnology (Lake Placid, NY); the swine anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody was from DAKO (Santa Barbara, CA). The phosphotyrosine monoclonal PY20 and the goat anti-mouse HRP-conjugated secondary antibodies were from Transduction Laboratories (Lexington, KY). An antibody specific for the phosphorylated form of p38 MAP kinase was from New England Biolabs. [gamma -32P]ATP was from Amersham (Arlington Heights, IL). JNK and ERK kits were purchased from Stratagene (La Jolla, CA). NF-kappa B consensus double-strand oligonucleotide and poly(dI-dC) · poly(dI-dC) were purchased from Promega (Madison, WI). Fischer 344 rat pulmonary myofibroblasts were isolated and characterized as described previously (10). TRI Reagent was from Molecular Research Center (Cincinnati, OH). Immobilon-S membranes were purchased from Millipore (Bedford, MA). The Prime-It II random-primer labeling kit was from Stratagene.

125I-PDGF-AA binding assay. Myofibroblasts in 24-well plates were grown to confluence in 10% fetal bovine serum-Dulbecco's modified Eagle's medium (FBS-DMEM) and then rendered quiescent for 24 h in serum-free defined medium (SFDM; Ham's F-12 medium with HEPES, CaCl2, and 0.25% BSA supplemented with an insulin-transferrin-selenium mixture from Boehringer Mannheim, Indianapolis, IN) for 24 h. Cultures were chilled to 4°C, rinsed in ice-cold binding buffer (Ham's F-12 with HEPES, CaCl2, and 0.25% BSA), and exposed to 1 ng/ml of 125I-PDGF-AA for 3-4 h at 4°C on an oscillating platform in the absence and presence of an excess of cold PDGF-AA. Cells were then rinsed three times in ice-cold binding buffer, solubilized in 1% Triton X-100, 0.1% BSA, and 0.1 M NaOH, and cell-associated radioactivity was measured with a gamma counter. Total binding was measured with 125I-PDGF-AA alone, and nonspecific binding was measured in parallel wells with 125I-PDGF-AA plus a 500-fold excess of nonradioactive PDGF-AA. Specific binding was defined as the difference between total and nonspecific binding.

Northern blot analysis. Total RNA was isolated with TRI Reagent. Twenty micrograms of each sample were electrophoresed in 1% agarose-2 M formaldehyde gels and capillary transferred onto Immobilon-S membranes. A rat cDNA probe for the PDGFR-alpha was labeled with [alpha -32P]dCTP with a Prime-It II random-primer labeling kit. The autoradiographic signal was visualized with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).

Western blot analysis. Cells were washed with PBS and 250 µl of lysis buffer [50 mM Tris · HCl; 1% Triton X-100; 150 mM NaCl; 1 mM EGTA; 1 mM phenylmethylsulfonyl fluoride (PMSF); 0.25% sodium deoxycholate; 1 µg/ml each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF] were added to cover the surface of the attached cells for 20 min at 0-4°C. Extracts were collected without scraping and stored at -70°C. Twenty microliters of each sample were mixed with 5 µl of reducing sample buffer and boiled for 5 min before electrophoresis in a 2-15% Tris-glycine SDS-polyacrylamide gel for 2 h at 130 V and 30 mA. The protein on the gel was transferred to a nitrocellulose membrane (Hybond, Amersham). The membrane was blocked with 3% milk-PBS for 1 h before the addition of a rabbit anti-mouse PDGFR-alpha antibody or a rabbit anti-human PDGFR-beta antibody (at a dilution of 1:500) overnight. After being washed three times with PBS-Tween, a secondary HRP-conjugated swine anti-rabbit antibody was added for 1.5 h at a dilution of 1:2,000. For measurement of autophosphorylation on tyrosine, cell lysates were collected as described above for PDGFR Western blotting. The membranes were blocked with 3% milk for 1 h and then incubated for 24 h at 4°C with a 1:500 dilution of anti-phosphotyrosine (PY20) monoclonal antibody. The membranes were washed three times with PBS-Tween and then incubated with a 1:2,000 dilution of goat anti-mouse IgG-HRP for 90 min. For measurement of inhibitor of NF-kappa B (Ikappa B-alpha ), cell lysates were collected with scraping, and electrophoresed on 12% Tris-glycine-EDTA (TGE) gels, and membranes were incubated with a rabbit anti-human Ikappa B-alpha antibody. For measurement of p38 MAP kinase activation, an anti-phospho-p38 antibody (New England Biolabs) was used at a dilution of 1:1,000. The secondary antibody for Ikappa B-alpha or p38 MAP kinase Western blots was a 1:2,000 dilution of HRP-swine anti-rabbit IgG (DakoPatts, Carpinteria, CA). After a thorough washing with PBS-Tween, all Western blots were developed with an enhanced chemiluminescence luminol kit (Amersham).

Electrophoretic mobility shift assay. Nuclear extracts were prepared as follows. Cells were washed with PBS, trypsinized, and centrifuged at 1,500 rpm for 10 min at 4°C. Cell pellets were resuspended in 400 µl of buffer A [10 mM HEPES, pH 7.9; 2 mM MgCl2; 10 mM KCl; 1.0 mM dithiothreitol (DTT); 1.0 mM PMSF; 5 µg/ml each of aprotinin, pepstatin, and leupeptin; and 0.1% Triton X-100], incubated for 15 min on ice, vortexed for 15 s, and centrifuged for 10 min at 14,000 rpm. Pelleted nuclei were resuspended in 40 µl of buffer C [20 mM HEPES, pH 7.9, 25% (vol/vol) glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1.0 mM DTT, 1.0 mM PMSF, and 5 µg/ml each of aprotinin and leupeptin], incubated for 30 min on ice, and centrifuged for 10 min at 14,000 rpm. Supernatants were diluted with 20 µl of buffer D [20 mM HEPES, pH 7.9, 20% (vol/vol) glycerol, 50 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF] and stored at -80°C. Protein concentrations were determined by the Bradford assay. Three micrograms of nuclear extract were incubated in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-Cl, pH 7.5, and 0.5 mg/ml of poly(dI-dC) · poly(dI-dC)] with [gamma -32P]ATP-labeled NF-kappa B oligonucleotide in a total reaction volume of 20 µl for 20 min at room temperature. Samples were electrophoresed on 6% polyacrylamide gels (0.5× Tris-glycine) with 0.5× Tris-glycine as a running buffer. Gels were dried and exposed to film at -80°C for 2-16 h.

Immunoprecipitation of ERK and phosphorylated heat- and acid-stable protein substrate-1 kinase assay. ERK activity in myofibroblast cell lysates was measured by the ability of these lysates to phosphorylate phosphorylated heat- and acid-stable protein substrate-1 (PHAS-1). Cells grown to confluence in 75-cm2 tissue culture flasks were rendered quiescent in SFDM for 24 h. After 30 min of treatment with the agent of interest, the cells were placed on ice, washed twice with PBS, and scraped off with 800 µl of lysate buffer consisting of 50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 20 µg/ml each of aprotinin, leupeptin, and pepstatin. Lysates were clarified by centrifugation at 13,000 rpm for 10 min, and protein concentrations were determined by Bradford assay. Immunoprecipitation was performed by incubating 200 µl of lysate with 2 µg of anti-ERK 2 (p42) antibody for 2 h and then adding 20 µl of protein A agarose (Santa Cruz Biotechnology). After an overnight incubation at 0-4°C with end-over-end mixing, the immune complex was recovered by centrifugation and washed three times with lysis buffer and one time with 250 mM HEPES (pH 7.4), 10 mM MgCl2, and 200 µM Na3VO4. Immune complex kinase assays were performed with a MAP kinase assay kit (Stratagene) according to the manufacturer's instructions; briefly, the ERK pellets were resuspended in Stratagene reaction buffer containing 120 µg of PHAS-1 substrate along with 3-5 µCi of [32P]ATP in a final volume of 180 µl. Kinase reactions took place for 30 min at room temperature and were stopped by the addition of 4× SDS-PAGE reducing sample buffer and by boiling for 10 min. ERK-PHAS samples were resolved on 4-20% PAGE gels, dried, and autoradiographed.

JNK assay. Cell lysates were collected as described in Immunoprecipitation of ERK and PHAS-1 kinase assay for the ERK assay. JNK was immunoprecipitated from 200 µl of lysate by first incubating with 2 µg of an anti-JNK-1 (p46) polyclonal IgG (Santa Cruz Biotechnology) for 3 h and then adding 20 µl of protein A agarose for an overnight incubation at 0-4°C with end-over-end mixing. The immune complex was recovered by centrifugation and washed three times with lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 1 mM PMSF, and 20 µg/ml each of aprotinin, leupeptin, and pepstatin) and one time with JNK kinase buffer (20 mM HEPES, pH 7.9, 15 mM MgCl2, 1 mM DTT, 100 µM Na3VO4, and 25 mM beta -glycerophosphate). The pellet was resuspended in 180 µl of kinase buffer containing 30 µg of glutathione S-transferase-c-Jun(1-79) (Stratagene), 100 µM ATP, and 3-5 µCi of [32P]ATP. The reaction was allowed to proceed for 30 min at room temperature and was terminated by the addition of SDS loading buffer and boiling for 10 min. Phosphorylated glutathione S-transferase-c-Jun(1-79) was resolved on a 12% SDS-polyacrylamide gel and then autoradiographed.

[3H]thymidine incorporation assay. Cells were grown to confluence with 10% FBS-DMEM in 24-well tissue culture plates (2-cm2 wells) and then rendered quiescent for 24 h with SFDM containing 0.5% FBS. The cells were treated with fresh 0.5% FBS-SFDM containing staurosporine (1-100 nM) or DMSO vehicle for 24 h at 37°C. PDGF-AA, -AB, or -BB (50 ng/ml) or medium alone containing no growth factor was spiked into the medium along with 5 µCi/ml of [3H]thymidine (Amersham) for 24 h. The cells were washed with Ham's F-12 medium at 25°C, placed on ice, and incubated with 0.5 ml/well of 5% trichloroacetic acid for 10 min. After three washes with ice-cold distilled water, solubilization was performed with 0.5 ml/well of 0.2 N NaOH containing 0.1% SDS for 30 min on an oscillating platform. One hundred microliters of each sample were added to 1 ml of Ecolume (Costa Mesa, CA), and radioactivity was measured with a liquid scintillation counter.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Staurosporine induction of PDGFR-alpha expression. Staurosporine caused a dose-dependent increase in PDGFR-alpha mRNA as determined by Northern blot analysis (Fig. 1A) and upregulated PDGFR-alpha protein as determined by Western blot analysis (Fig. 1B). Moreover, staurosporine also caused upregulation of functional cell surface PDGFR-alpha in a concentration-dependent manner as determined by 125I-PDGF-AA binding assays (Fig. 1C). The concentration range of staurosporine used in these experiments (1-100 nM) did not cause significant cytotoxicity as determined by trypan blue exclusion staining or detachment of cells after 24 h in culture (data not shown).


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Staurosporine (Stauro) upregulated platelet-derived growth factor (PDGF) receptor-alpha (PDGFR-alpha ) mRNA and protein in rat pulmonary myofibroblasts. Confluent, quiescent myofibroblasts were exposed to increasing concentrations of Stauro or DMSO vehicle for 24 h. A: PDGFR-alpha mRNA expression. Left: representative PDGFR-alpha Northern blot and rRNAs. Right: scanning densitometry showing multiple of increase of PDGFR-alpha relative to the 28s rRNA signal for 3 separate experiments. B: Western blot analysis. Left: protein levels of PDGFR-alpha and PDGFR-beta after 24 h of Stauro treatment. Right: scanning densitometry showing relative multiple of increase of PDGFR-alpha and PDGFR-beta protein expression in 3 separate experiments. C: 125I-PDGF-AA binding performed on intact cell monolayers. cpm, Counts/min. Data are means ± SE of 3 experiments, each assayed in triplicate. Significantly different from control: *P < 0.05; **P < 0.01.

Staurosporine-induced upregulation of PDGFR-alpha is not due to PKC or PKA inhibition. The effect of staurosporine on PDGFR-alpha expression was not mimicked by more specific inhibitors of PKC (Ro-31-8220, GF-109203X, or calphostin C; Table 1). Furthermore, these inhibitors did not block IL-1beta -induced upregulation of PDGFR-alpha . To further rule out the involvement of PKC isozymes, cells were treated for 24 h with 0.1-10 µM phorbol 12-myristate 13-acetate to downregulate PKC activity. However, phorbol 12-myristate 13-acetate depletion of PKC activity had no effect on the increase in 125I-PDGF-AA binding caused by staurosporine or IL-1beta (data not shown). Staurosporine is also a known inhibitor of PKA (10). Treatment of cells with the PKA inhibitor H-89 did not induce PDGFR-alpha expression (Table 1). Moreover, H-89 did not affect IL-1beta induction of PDGFR-alpha , further supporting the idea that this kinase is not involved in PDGFR-alpha regulation.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of staurosporine and specific inhibitors of PKC and PKA on PDGFR-alpha expression

PDGFR-alpha upregulation by staurosporine is not due to an IL-1beta autocrine loop. Because IL-1beta is a potent inducer of PDGFR-alpha in rat pulmonary myofibroblasts (19), we investigated whether staurosporine was inducing PDGFR-alpha via an IL-1beta autocrine loop. Pretreatment of myofibroblasts for 2 h with 2 µg/ml of IRAP before addition of 100 nM staurosporine had no effect on the increase in 125I-PDGF-AA binding (Fig. 2). As expected, IRAP completely blocked the upregulation caused by IL-1beta . These data confirmed that staurosporine was not causing upregulation of PDGFR-alpha by stimulating the production of IL-1beta by the myofibroblasts.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 2.   Stauro did not induce PDGFR-alpha via an interleukin (IL)-1beta autocrine loop. Confluent, quiescent cultures were pretreated with IL-1 receptor agonist protein (IRAP; 2 µg/ml) or medium alone and incubated with 100 nM Stauro or 2 ng/ml of IL-1beta for 24 h. Data are means ± SE of 3 experiments, each assayed in triplicate. Both IL-1beta and Stauro increased specific binding of 125I-PDGF-AA. IRAP blocked the increase in 125I-PDGF-AA binding induced by IL-1beta but not by Stauro. **P < 0.01.

Staurosporine-induced PDGFR-alpha expression does not require NF-kappa B activation. The activation of the transcription factor NF-kappa B is involved in the IL-1beta -mediated induction of a number of genes (1, 2, 13, 17, 18). Moreover, a recent report (24) has suggested that both IL-1beta and staurosporine activate NF-kappa B, thereby increasing IL-2 production in EL4 thymoma cells. We sought to determine if activation of NF-kappa B was a common mechanism whereby IL-1beta and staurosporine induce PDGFR-alpha . Using electrophoretic mobility shift assays, we clearly showed that staurosporine did not activate NF-kappa B, whereas IL-1beta was a strong activator of NF-kappa B (Fig. 3A). Also, Ikappa B-alpha Western blot analysis showed that staurosporine did not cause degradation of cytosolic Ikappa B-alpha , whereas IL-1beta caused complete degradation of Ikappa B-alpha within 30 min (Fig. 3B).


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 3.   IL-1beta , but not Stauro, activated nuclear factor (NF)-kappa B and caused degradation of inhibitor of NF-kappa B (Ikappa B-alpha ). Confluent, quiescent myofibroblasts were treated with 10 ng/ml of IL-1beta or 100 nM Stauro for 30 min before nuclear extract preparation and analysis of NF-kappa B binding activity by electrophoretic mobility shift assay (EMSA) or collection of cell lysates for detection of Ikappa B-alpha degradation by Western blot analysis. A, left: representative EMSA experiment showing binding of radiolabeled NF-kappa B oligonucleotide to nuclear protein that was induced by IL-1beta treatment but not by Stauro treatment. Free probe, sample containing only the radiolabeled NF-kappa B oligonucleotide. Right: representative experiment showing blocking of radiolabeled NF-kappa B probe binding to nuclear protein with a 30-fold excess of nonradioactive cold probe. B: representative Western blots showing the transient degradation of Ikappa B-alpha after IL-1beta treatment but no degradation of Ikappa B-alpha after Stauro treatment. ', Min. Data represent 3 separate experiments.

PDGFR-alpha increase by staurosporine is not due to inhibition of PDGFR tyrosine kinase activity. Because staurosporine is known to inhibit PDGFR tyrosine kinase activity (31), we determined if staurosporine-induced PDGFR-alpha expression was caused by inhibition of PDGFR autophosphorylation (i.e., initiation of a feedback mechanism involving synthesis of new PDGFR). Cells were pretreated with staurosporine or two other receptor tyrosine kinase inhibitors, genistein and tyrphostin AG-1296, and then stimulated with PDGF isoforms for 5 min. PDGF-AB and PDGF-BB induced strong autophosphorylation of PDGFR, whereas PDGF-AA did not cause autophosphorylation because of the constitutively low expression of this receptor. All three receptor tyrosine kinase inhibitors blocked autophosphorylation by >80% (Fig. 4A). However, genistein and tyrphostin AG-1296 did not upregulate 125I-PDGF-AA binding to intact myofibroblast monolayers (Fig. 4B), indicating that inhibition of PDGFR tyrosine kinase activity did not account for upregulation of PDGFR-alpha by staurosporine.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4.   Genistein and tyrphostin inhibited PDGF-stimulated autophosphorylation but did not upregulate 125I-PDGF-AA binding sites. Myofibroblast cultures were pretreated with 10 nM Stauro, 100 nM genistein, or 100 nM tyrphostin for 1 h before exposure to PDGF isoforms. Cell lysates were collected, and Western blot analysis was performed with a phosphotyrosine antibody (PY20). A: all 3 tyrosine kinase inhibitors suppressed PDGFR autophosphorylation in response to PDGF-AB and PDGF-BB. Note that PDGF-AA did not cause autophosphorylation due to the very low constitutive levels of the PDGFR-alpha . +, Presence; -, absence. Nos. at right, molecular mass. B: Stauro, but not tyrphostin or genistein, increased specific binding of 125I-PDGF-AA to cultured rat pulmonary myofibroblasts. Data are means ± SE of 3 experiments, each assayed in triplicate. **P < 0.01 compared with no addition.

Staurosporine-induced upregulation of PDGFR-alpha is due to activation of p38 MAP kinase. We previously reported (23) that IL-1beta activates JNK-1 and ERK in rat pulmonary myofibroblasts. Therefore, IL-1beta was used as a known activator of these MAP kinases and was compared with staurosporine in kinase assays. Although IL-1beta clearly activated JNK-induced phosphorylation of c-Jun- and ERK-induced phosphorylation of PHAS-1, staurosporine had no effect on these kinases (Fig. 5). However, Western blot analysis with a phospho-p38 MAP kinase antibody demonstrated that staurosporine and IL-1beta activated p38 MAP kinase in a time-dependent manner (Fig. 6A). Moreover, staurosporine-induced upregulation of 125I-PDGF-AA binding was inhibited >70% by pretreatment with 20 µM p38 inhibitor SB-203580 (Fig. 6B).


View larger version (56K):
[in this window]
[in a new window]
 
Fig. 5.   Stauro did not activate c-Jun NH2-terminal kinase (JNK)-induced phosphorylation of c-Jun or extracellular signal-regulated kinase (ERK)-induced phosphorylation of phosphorylated heat- and acid-stable protein substrate-1 (PHAS-1). Confluent, quiescent myofibroblasts were treated with 10 ng/ml of IL-1beta or 100 nM Stauro for 30 or 60 min before collection of cell lysates for JNK or ERK kinase assays. IL-1beta was a potent activator of both kinases, whereas Stauro did not activate JNK or ERK.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 6.   Stauro activated p38 mitogen-activated protein kinase (MAPK), and upregulation of 125I-PDGF-AA binding by Stauro is blocked by the p38 MAPK inhibitor SB-203580. A: confluent myofibroblasts were treated with 100 nM Stauro (top) or 10 ng/ml of IL-1beta (bottom) for the indicated times before collection of cell lysates for Western blotting with an antibody specific for phosphorylated p38 MAPK. Data represent 3 experiments. B: pretreatment of cells with 20 µM SB-203580 (SB) for 1 h significantly inhibited Stauro-induced upregulation of 125I-PDGF-AA binding by >75%. Data are means ± SE of 3 separate experiments. **P < 0.01.

Staurosporine inhibits PDGF-stimulated mitogenesis. Lindroos et al. (22) previously reported that upregulation of the PDGFR-alpha by IL-1beta renders myofibroblasts hyperresponsive to the mitogenic effects of PDGF (22). However, staurosporine inhibited basal thymidine incorporation (0.5% FBS-DMEM with no PDGF) as well as mitogenesis stimulated by all three PDGF isoforms (Fig. 7). Maximal inhibition of basal mitogenesis was observed at 10 nM staurosporine, whereas maximal inhibition of PDGF-stimulated mitogenesis was observed at 50 nM staurosporine. The concentration range used to inhibit mitogenesis (1-100 nM) in these experiments was the same as that used to upregulate the PDGFR-alpha (Fig. 1). No significant cytotoxicity was observed in these experiments by trypan blue nuclear exclusion with as much as 100 nM staurosporine (data not shown).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   Stauro inhibited PDGF-stimulated mitogenesis of rat lung myofibroblasts. Confluent cultures of myofibroblasts in 24-well plates were rendered quiescent for 24 h in serum-free defined medium and then treated with increasing concentrations of Stauro or vehicle (DMSO) for 24 h at 37°C. PDGF isoforms (50 ng/ml) or medium alone (0.5% FBS-DMEM) were then added to the wells along with 5 µCi/ml of [3H]thymidine for 24 h. PDGF-stimulated and basal [3H]thymidine uptake were inhibited in a concentration-dependent manner by Stauro.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we report that staurosporine, a broad-spectrum protein kinase inhibitor, strongly induces PDGFR-alpha . Whereas staurosporine inhibits a variety of protein kinases, p38 MAP kinase was phosphorylated in response to staurosporine treatment, and upregulation of the PDGFR-alpha was due, at least in part, to activation of p38 MAP kinase as determined by inhibition of staurosporine-induced PDGFR-alpha expression with SB-203580. Two members of the MAP kinase family, JNK and ERK, were not activated by staurosporine. Wang et al. (36) recently reported that IL-1beta -induced upregulation of the PDGFR-alpha requires p38 MAP kinase activation. Thus p38 MAP kinase appears to be a common signaling intermediate required for induction of the PDGFR-alpha gene by either staurosporine or IL-1beta .

We initially speculated that staurosporine-induced upregulation of the PDGFR-alpha could be due to PKC inhibition because other investigators (12) have reported that overexpression of PKC-alpha caused suppression of PDGFR-alpha in Swiss/3T3 fibroblasts. However, other more specific inhibitors of PKC (i.e., Ro-31-8220, GF-109203X, and calphostin C) did not mimic the staurosporine effect on PDGFR-alpha expression. PKA inhibition by staurosporine was also excluded as a possible mediator because the more specific PKA inhibitor H-89 had no effect on PDGFR-alpha expression. Moreover, the ability of staurosporine to block tyrosine phosphorylation of the PDGFR and thereby activate a possible feedback loop for increased PDGFR-alpha synthesis was ruled out because other receptor tyrosine kinase inhibitors (genistein and tyrphostin AG-1296) did not upregulate PDGFR-alpha .

Other investigators have shown that staurosporine can induce various biological responses via the activation of MAP kinases. A recent study by Xiao et al. (38) showed that staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils is dependent on the activation of p38 MAP kinase and ERK. Yao et al. (39) found that staurosporine activated a novel JNK isoform, but not JNK-1 or ERK, in rat PC-12 cells, and this contributed to neurite outgrowth in these cells. Although we observed that p38 MAP kinase is involved in staurosporine-induced PDGFR-alpha expression, we did not detect activation of either ERK or JNK after treatment of rat pulmonary myofibroblasts with staurosporine. In contrast, IL-1beta was a strong activator of JNK and ERK in rat pulmonary myofibroblasts.

Both staurosporine and IL-1beta activated p38 MAP kinase in rat pulmonary myofibroblasts, and upregulation of 125I-PDGF-AA binding by either of these agents was significantly inhibited by SB-203580. This observation suggests that staurosporine and IL-1beta act through a similar mechanism to induce PDGFR-alpha gene expression. IL-1beta activates a diversity of signaling intermediates including NF-kappa B, PKC isozymes, and all three classes of MAP kinases (ERKs, JNKs, and p38 MAP kinase). The broad spectrum of intracellular mediators activated by IL-1beta has complicated the search for signaling pathways that control PDGFR-alpha expression. To our knowledge, p38 MAP kinase is the only signaling intermediate that is strongly activated by staurosporine in rat pulmonary myofibroblasts, and all other IL-1-activated pathways (NF-kappa B, ERK, JNK, and PKC) are not affected by staurosporine. IL-1beta -induced upregulation of the PDGFR-alpha also requires activation of p38 MAP kinase, which serves to stabilize the mRNA that encodes the PDGFR-alpha (38). In contrast to staurosporine, IL-1beta activates ERK, which leads to suppression of PDGFR-alpha expression (23). Thus the suppression or induction of PDGFR-alpha by IL-1beta appears to involve activation of both ERK and p38 MAP kinases, respectively. In future studies, staurosporine will serve as a useful tool to better understand the mechanisms through which p38 MAP kinase regulates PDGFR-alpha expression.

Other investigators have shown that staurosporine upregulates the epidermal growth factor (EGF) receptor in PC-12 cells (28), the tumor necrosis factor-alpha (TNF-alpha ) receptor in myeloid and epithelial cells (40), and the human serotonin receptor in choriocarcinoma cells (29). In all of these cases, receptor upregulation was associated with enhanced biological responses. Raffioni and Bradshaw (28) showed that staurosporine increased EGF receptor expression in PC-12 cells and that staurosporine enhanced EGF-induced receptor tyrosine phosphorylation. In contrast, we found that staurosporine inhibited PDGF-induced receptor tyrosine phosphorylation, even though it upregulated PDGFR-alpha . These results are in agreement with those of Secrist et al. (31) wherein staurosporine was reported as a potent inhibitor of PDGFR phosphorylation. We demonstrated in this report that inhibition of PDGFR tyrosine phosphorylation was not a mechanism that contributes to PDGFR-alpha upregulation because genistein and tyrphostin both blocked PDGFR phosphorylation but did not induce PDGFR-alpha expression. Similar results were obtained by Zhang et al. (40) who found that upregulation of the TNF-alpha receptor by staurosporine in a human erythroblastoid leukemic cell line was not due to inhibition of tyrosine kinases.

In contrast to IL-1beta - or lipopolysaccharide-induced upregulation of PDGFR-alpha (10, 22), staurosporine-induced upregulation of PDGFR-alpha did not result in an enhanced mitogenic response to PDGF isoforms but, instead, inhibited PDGF-stimulated [3H]thymidine uptake in myofibroblasts (Fig. 7). This is likely due to the fact that staurosporine inhibited PDGFR tyrosine kinase activity (Fig. 4). Basal [3H]thymidine uptake in the presence of 0.5% FBS-DMEM with no PDGF was also suppressed in a concentration-dependent manner by staurosporine, and this is consistent with the well-known activity of staurosporine in the nanomolar range to cause growth arrest via the inhibition of cell cycle kinases (14, 41). Higher concentrations of staurosporine (i.e., 100-1,000 µM) have been reported to cause apoptosis in a variety of cell types (4, 11). However, we did not observe significant cytotoxicity in rat pulmonary myofibroblasts within the concentration range used in this study (1-100 nM).

We investigated the possibility that the signal transduction pathways mediating PDGFR-alpha upregulation by IL-1beta and staurosporine could converge at the level of transcriptional activation by NF-kappa B. Activation of NF-kappa B is necessary for the induction of gene expression by IL-1beta in a number of cells (1, 2, 13, 17, 18), and a kappa B site exists in the PDGFR-alpha promoter region (19). Furthermore, activation of NF-kappa B by staurosporine and IL-1beta in EL4 thymoma cells has been reported as a common mechanism that leads to increased IL-2 production (24), and Chabot and Breton (8) reported that staurosporine activated NF-kappa B in human keratinocytes. However, we clearly demonstrated that staurosporine did not activate NF-kappa B in rat pulmonary myofibroblasts, nor did staurosporine cause degradation of cytosolic Ikappa B-alpha in these cells. These findings are in agreement with some other investigators (3, 15, 16) who reported no effect of staurosporine on NF-kappa B activation. Also, a report by Warshamana et al. (37) demonstrated that dexamethasone upregulated PDGFR-alpha mRNA and protein, although dexamethasone suppresses NF-kappa B activity. In further support of this idea, we have reported that TNF-alpha strongly activates NF-kappa B in pulmonary myofibroblasts but does not upregulate PDGFR-alpha (23). Thus it appears that NF-kappa B activation is not related to upregulation of the PDGFR-alpha , even though some inducers of PDGFR-alpha expression (IL-1beta and lipopolysaccharide) are NF-kappa B activators.

In summary, we report that the PK inhibitor staurosporine upregulates PDGFR-alpha in rat pulmonary myofibroblasts via a p38 MAP kinase-dependent pathway. Induction of PDGFR-alpha by staurosporine was not due to inhibition of PKC, PKA, or receptor tyrosine kinase activity. Moreover, the effect of staurosporine on PDGFR-alpha expression was not due to the production of IL-1beta , the major endogenous inducer of PDGFR-alpha in the lung. Although staurosporine upregulated PDGFR-alpha expression, it also caused growth arrest of rat pulmonary myofibroblasts and inhibited PDGF-stimulated mitogenesis. Collectively, these data support the idea that the PDGFR-alpha is a growth arrest-specific gene and demonstrate that staurosporine is a useful tool for elucidating the signaling mechanisms that regulate PDGFR expression in lung connective tissue cells.


    FOOTNOTES

Address for reprint requests and other correspondence: J. C. Bonnner, NIEHS, PO Box 12233, Research Triangle Park, NC 27709 (E-mail: bonnerj{at}niehs.nih.gov).

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.

Received 2 February 2000; accepted in final form 14 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anisowicz, A, Messineo M, Lee SW, and Sager R. An NF-kappa B transcription factor mediates IL-1/TNF-alpha induction of gro in human fibroblasts. J Immunol 147: 520-527, 1991[Abstract/Free Full Text].

2.   Beg, AA, Finco TS, Nantermet PV, and Baldwin AS. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of Ikappa Balpha : a mechanism for NF-kappa B activation. Mol Cell Biol 13: 3301-3310, 1993[Abstract].

3.   Beyaert, R, Vanhaesebroeck B, Heyninck K, Boone E, De Valck D, Schulze-Osthoff K, Haegeman G, Roy FV, and Fiers W. Sensitization of tumor cells to tumor necrosis factor action by the protein kinase inhibitor staurosporine. Cancer Res 53: 2623-2630, 1993[Abstract].

4.   Bijur, GN, De Sarno P, and Jope RS. Glycogen synthase kinase-3beta facilitates staurosporine- and heat shock-induced apoptosis. J Biol Chem 275: 7583-7590, 2000[Abstract/Free Full Text].

5.   Bonner, JC, Lindroos PM, Rice AB, Moomaw CR, and Morgan DL. Induction of PDGFR-alpha in rat myofibroblasts during pulmonary fibrogenesis in vivo. Am J Physiol Lung Cell Mol Physiol 274: L72-L80, 1998[Abstract/Free Full Text].

6.   Bostrom, H, Willetts K, Pekny M, Leveen P, Lindahl P, Hedstrand H, Pekna M, Hellstrom M, Gebre-Medhin S, Schalling M, Nilsson M, Kurland S, Tornell J, Heath JK, and Betsholtz C. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85: 863-873, 1996[ISI][Medline].

7.   Centrella, M, McCarthy TL, Kusmik WF, and Canalis E. Isoform-specific regulation of platelet-derived growth factor activity and binding in osteoblast-enriched cultures from fetal rat bone. J Clin Invest 98: 1076-1084, 1992[Abstract/Free Full Text].

8.   Chabot, FM, and Breton JJ. Effect of staurosporine on transcription factor NF-kappaB in human keratinocytes. Biochem Pharmacol 56: 71-78, 1998[ISI][Medline].

9.   Claesson-Welsh, L. Platelet-derived growth factor receptor signals. J Biol Chem 269: 32023-32026, 1994[Free Full Text].

10.   Coin, PG, Lindroos PM, Bird GSJ, Osornio-Vargas AR, Roggli VL, and Bonner JC. Lipopolysaccharide up-regulates platelet-derived growth factor (PDGF) alpha -receptor expression in rat lung myofibroblasts and enhances response to all PDGF isoforms. J Immunol 156: 4797-4806, 1996[Abstract/Free Full Text].

11.   Deng, X, Ruvolo P, Carr B, and May WS. Survival function of ERK1/2 as IL-3-activated, staurosporine-resistant Bcl2 kinases. Proc Natl Acad Sci USA 97: 1578-1583, 2000[Abstract/Free Full Text].

12.   Fitzer-Attas, C, Eldar H, Eisenbach L, and Livneh E. The expression of PDGF-alpha but not PDGF-beta receptors is suppressed in Swiss/3T3 fibroblasts over-expressing protein kinase C-alpha . FEBS Lett 342: 165-170, 1994[ISI][Medline].

13.   Friedman, WJ, Thakur S, Seidman L, and Rabson AR. Regulation of nerve growth factor mRNA by interleukin-1 in rat hippocampal astrocytes is mediated by NFkappa B. J Biol Chem 271: 31115-31120, 1996[Abstract/Free Full Text].

14.   Gadbois, DM, Peterson S, Bradbury EM, and Lehnert BE. CDK4/cyclin D1/PCNA complexes during staurosporine-induced G1 arrest and G0 arrest of human fibroblasts. Exp Cell Res 220: 220-225, 1995[ISI][Medline].

15.   Hecker, M, Preib C, and Schini-Kerth VB. Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: role of NF-kappa B, CREB and C/EBPbeta . Br J Pharmacol 120: 1067-1074, 1997[Abstract].

16.   Hohmann, HP, Remy R, Aigner L, Brockhaus M, and van Loon APGM Protein kinases negatively affect nuclear factor-kappa B activation by tumor necrosis factor-alpha at two different stages in promyelocytic HL60 cells. J Biol Chem 267: 2065-2072, 1992[Abstract/Free Full Text].

17.   Joshi-Barve, SS, Rangnekar VV, Sells SF, and Rangnekar VM. Interleukin-1-inducible expression of gro-beta via NF-kappa B activation is dependent upon tyrosine kinase signaling. J Biol Chem 268: 18018-18029, 1993[Abstract/Free Full Text].

18.   Kessler, DJ, Duyao MP, Spicer DB, and Sonenshein GE. NF-kappa B-like factors mediate interleukin 1 induction of c-myc gene transcription in fibroblasts. J Exp Med 176: 787-792, 1992[Abstract].

19.   Kitami, Y, Inui H, Uno S, and Inagami T. Molecular structure and transcriptional regulation of the gene for the platelet-derived growth factor alpha  receptor in cultured vascular smooth muscle cells. J Clin Invest 96: 558-567, 1995[ISI][Medline].

20.   Lasky, JA, Tonthat B, Liu JY, Friedman M, and Brody AR. Upregulation of the PDGF-alpha receptor precedes asbestos-induced lung fibrosis in rats. Am J Respir Crit Care Med 157: 1652-1657, 1998[Abstract/Free Full Text].

21.   Lih, C-J, Cohen SN, Wang C, and Lin-Chao S. The platelet-derived growth factor alpha -receptor is encoded by a growth-arrest-specific (gas) gene. Proc Natl Acad Sci USA 93: 4617-4622, 1996[Abstract/Free Full Text].

22.   Lindroos, PM, Coin PG, Osornio-Vargas AR, and Bonner JC. Interleukin 1 beta (IL-1 beta) and the IL-1 beta-alpha 2-macroglobulin complex upregulate the platelet-derived growth factor alpha-receptor on rat pulmonary fibroblasts. Am J Respir Cell Mol Biol 13: 455-465, 1995[Abstract].

23.   Lindroos, PM, Rice AB, Wang YZ, and Bonner JC. Role of nuclear factor-kappa B and mitogen-activated protein kinase signaling pathways in IL-1beta -mediated induction of alpha -PDGFR expression in rat pulmonary myofibroblasts. J Immunol 161: 3464-3468, 1998[Abstract/Free Full Text].

24.   Mahon, TM, Matthews JS, and O'Neill LAJ Staurosporine, but not Ro 31-8220, induces interleukin 2 production and synergizes with interleukin-1alpha in EL4 thymoma cells. Biochem J 325: 39-45, 1997[ISI][Medline].

25.   Martinet, Y, Rom WN, Grotendorst GR, Martin GR, and Crystal RG. Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis. N Engl J Med 317: 202-209, 1987[Abstract].

26.   Nagaoka, I, Trapnell BC, and Crystal RG. Upregulation of platelet-derived growth factor-A and -B gene expression in human alveolar macrophages of individuals with idiopathic pulmonary fibrosis. J Clin Invest 85: 2023-2027, 1990[ISI][Medline].

27.   Osornio-Vargas, AR, Lindroos PM, Coin PG, Badgett A, Hernandez-Rodriguez NA, and Bonner JC. Maximal PDGF-induced lung myofibroblast chemotaxis requires the PDGFR-alpha . Am J Physiol Lung Cell Mol Physiol 271: L93-L99, 1996[Abstract/Free Full Text].

28.   Raffioni, S, and Bradshaw RA. Staurosporine causes epidermal growth factor to induce differentiation in PC-12 cells via receptor upregulation. J Biol Chem 270: 7568-7572, 1995[Abstract/Free Full Text].

29.   Ramamoorthy, JD, Ramamoorthy S, Papapetropoulos A, Catravas JD, Leibach FH, and Ganapathy V. Cyclic AMP-independent upregulation of the human serotonin transporter by staurosporine in choriocarcinoma cells. J Biol Chem 270: 17189-17195, 1995[Abstract/Free Full Text].

30.   Rupp, E, Seigbahn A, Ronnstrand L, Wernstedt C, Claesson-Welsh L, and Heldin CH. A unique autophosphorylation site in the platelet-derived growth factor alpha  receptor from a heterodimeric receptor complex. Eur J Biochem 225: 29-41, 1994[Abstract].

31.   Secrist, JP, Sehgal I, Powis G, and Abraham RT. Preferential inhibition of the platelet-derived growth factor receptor tyrosine kinase by staurosporine. J Biol Chem 265: 20394-20400, 1990[Abstract/Free Full Text].

32.   Seifert, RA, Hart CE, Phillips PE, Forstrom JW, Ross R, Murray MJ, and Bowen-Pope DF. Two different subunits associate to create isoform-specific platelet-derived growth factor receptors. J Biol Chem 264: 8771-8778, 1989[Abstract/Free Full Text].

33.   Seifert, RA, van Koppen A, and Bowen-Pope DF. PDGF-AB requires PDGFR alpha-subunits for high-affinity, but not for low-affinity, binding and signal transduction. J Biol Chem 268: 4473-4480, 1993[Abstract/Free Full Text].

34.   Souza, P, Kuliszewski M, Wang JX, Tseu I, Tanswell AK, and Post M. PDGF-AA and its receptor influence early lung branching via an epithelial-mesenchymal interaction. Development 121: 2559-2567, 1995[Abstract/Free Full Text].

35.   Tsukamoto, T, Matsui T, Nakata H, Ito M, Natazuka T, Fukase M, and Fujita T. Interleukin-1 enhances the response of osteoblasts to platelet-derived growth factor through the alpha receptor-specific upregulation. J Biol Chem 266: 10143-10147, 1991[Abstract/Free Full Text].

36.   Wang, Y-Z, Zhang P, Rice AB, and Bonner JC. Regulation of interleukin-1beta -induced platelet-derived growth factor receptor-alpha expression in rat pulmonary myofibroblasts by p38 mitogen-activated protein kinase. J Biol Chem 275: 22550-22557, 2000[Abstract/Free Full Text].

37.   Warshamana, GS, Martinez S, Lasky JA, Corti M, and Brody AR. Dexamethasone activates expression of the PDGF-alpha receptor and induces lung fibroblast proliferation. Am J Physiol Lung Cell Mol Physiol 274: L499-L507, 1998[Abstract/Free Full Text].

38.   Xiao, YQ, Someya K, Morita H, Takahashhi K, and Ohuchi K. Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal macrophages. Biochim Biophys Acta 1450: 155-163, 1999[ISI][Medline].

39.   Yao, R, Yoshihara M, and Osada H. Specific activation of a c-Jun NH2-terminal kinase isoform and induction of neurite outgrowth in PC-12 cells by staurosporine. J Biol Chem 272: 18261-18266, 1997[Abstract/Free Full Text].

40.   Zhang, L, Higuchi M, Totpal K, Chaturvedi MM, and Aggarwal BB. Staurosporine induces the cell surface expression of both forms of human tumor necrosis factor receptors on myeloid and epithelial cells and modulates ligand-induced cellular response. J Biol Chem 269: 10270-10279, 1994[Abstract/Free Full Text].

41.   Zong, ZP, Fujikawa-Yamamoto K, Li AL, Yamaguchi N, Chang YG, Murakami M, Odashima S, and Ishikawa Y. Both low and high concentrations of staurosporine induce G1 arrest through downregulation of cyclin E and cdk2 expression. Cell Struct Funct 24: 457-463, 1999[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 280(2):L354-L362