TNF-alpha inhibits SP-A gene expression in lung epithelial cells via p38 MAPK

Olga L. Miakotina and Jeanne M. Snyder

Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Surfactant protein A (SP-A), the major lung surfactant-associated protein, mediates local defense against pathogens and modulates inflammation in the alveolus. Tumor necrosis factor (TNF)-alpha , a proinflammatory cytokine, inhibits SP-A gene expression in lung epithelial cells. Inhibitors of the phosphatidylinositol 3-kinase pathway, i.e., wortmannin, LY-294002, and rapamycin, did not block the inhibitory effects of TNF-alpha on SP-A mRNA levels. An inhibitor of the p44/42 mitogen-activated protein kinase (MAPK) pathway, PD-98059, was also ineffective. PD-169316 and SB-203580, inhibitors of p38 MAPK, blocked the TNF-alpha -mediated inhibition of SP-A mRNA levels. TNF-alpha increased the phosphorylation of p38 MAPK within 15 min. Anisomycin, an activator of p38 MAPK, increased p38 MAPK phosphorylation and decreased SP-A mRNA levels in a dose-dependent manner. Finally, TNF-alpha increased the phosphorylation of ATF-2, a transcription factor that is a p38 MAPK substrate. We conclude that TNF-alpha downregulates SP-A gene expression in lung epithelial cells via the p38 MAPK signal transduction pathway.

tumor necrosis factor-alpha ; surfactant protein A; H441 cells; p38 mitogen-activated protein kinase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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SURFACTANT IS A LIPOPROTEIN that lowers the air-liquid surface tension in the alveolus and also functions as a component of local defense mechanisms in the lung (7, 19). Surfactant protein A (SP-A) is the most abundant surfactant-associated protein (19). SP-A has been shown to bind to pulmonary pathogens and increase their phagocytosis by macrophages (7). SP-A may also modulate inflammatory responses in the alveolus (15, 22). The time- and dose-dependent inhibitory effect of tumor necrosis factor (TNF)-alpha on SP-A protein and mRNA levels was first observed in H441 cells, a human lung epithelial cell line (40). In further studies, it was shown that TNF-alpha causes inhibition of SP-A gene transcription within 6 h (39). The physiological significance of the TNF-alpha -mediated decrease in SP-A gene expression is not known; however, SP-A may act as a general inhibitor of cytokine-mediated inflammation in the lung (4, 15, 22). In agreement with this hypothesis, lipopolysaccharide causes an increased inflammatory response in SP-A null mice compared with its effect in control, wild-type mice (4). Thus the TNF-alpha -mediated decrease in SP-A levels may permit a more robust inflammatory response to occur in the distal lung.

TNF-alpha is a homotrimer of 17-kDa peptide subunits and is produced by many different cell types in response to inflammation (1). TNF-alpha has a wide spectrum of functions, including promoting cell proliferation, differentiation, apoptosis or survival, and is implicated in the pathogenesis of many diseases (1). TNF-alpha has been shown to play an important role in several lung inflammatory conditions, including pulmonary fibrosis, acute respiratory distress syndrome, septic shock, and chronic lung diseases in infants (8, 11, 27, 37).

TNF-alpha transduces its cellular signal via a complex of receptors, adapter proteins, and kinases (1). TNF-alpha binds to two TNF receptors (TNFR), p55 and p75 (type I and type II TNFR, respectively), with equal affinity (1). H441 cells express primarily TNFR type I mRNA (30). Interactions between ligand-bound TNFR and intracellular adapter proteins lead to the activation of either c-Jun NH2-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), or nuclear factor (NF)-kappa B in lung epithelial cells (3, 5, 18, 25, 31, 32). Activation of the transcription factor NF-kappa B by TNF-alpha is probably not involved in the regulation of SP-A gene expression by TNF-alpha (31). TNF-alpha also activates protein kinase C in human and bovine lung epithelial cells (3, 41). In isolated rat type II cells and human lung epithelial cells, TNF-alpha increases p44/42 MAPK activity (5, 25). TNF-alpha has been shown to inhibit de novo phosphatidylcholine synthesis in H441 cells via an increase in ceramide production (35). A less common signaling pathway for TNF-alpha described in other cell types involves phosphatidylinositol 3-kinase (PI 3-kinase) (9, 16, 24). However, the signaling pathways utilized by TNF-alpha to inhibit SP-A gene expression in pulmonary epithelial cells remain unknown.

In the present study, we used several inhibitors and activators of key signaling kinases as tools to identify the mediators of TNF-alpha inhibition of SP-A gene expression in H441 cells. Our results suggest that the phosphorylation and activation of p38 MAPK in response to TNF-alpha results in decreased levels of SP-A mRNA.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell culture. Human lung epithelial cells, H441 cells, produce SP-A mRNA and protein (28). H441 cells were obtained from American Type Culture Collection (Rockville, MD) and maintained in monolayer culture at 37°C and in an atmosphere of 5% CO2 in RPMI 1640 media that contained 10% fetal bovine serum, 0.25 µg/ml amphotericin B, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were split 1:4 weekly, and media were changed every 3-4 days. For most experiments, subconfluent cultures of H441 cells were incubated in serum-free media for 24 h before the experiment, pretreated with signal transduction inhibitors or vehicle in fresh media for 2 h, and then incubated in the presence or absence of the inhibitors plus TNF-alpha (0.1-100 ng/ml) or vehicle (0.1% bovine serum albumin in PBS) for an additional 24 h. To evaluate the time dependence of protein kinase phosphorylation, serum-deprived cells were incubated in the presence of either TNF-alpha or vehicle for 15 min, 30 min, 2 h, and 16 h.

Reagents. PD-98059 was purchased from New England Biolabs (Beverly, MA). LY-294002, rapamycin, and wortmannin were obtained from Sigma Chemical (St. Louis, MO). Anisomycin and SB-203580 were obtained from BioMol (Plymouth Meeting, PA), PD-169316 was from Calbiochem (San Diego, CA), and human recombinant TNF-alpha was from either Calbiochem or Sigma. Stock solutions of PD-98059, wortmannin, LY-294002, rapamycin, anisomycin, SB-203580, and PD-169316 were prepared in DMSO as 10 mM, 1 mM, 50 mM, 50 µM, 10 mg/ml, 20 mM, and 10 mM stock solutions, respectively, aliquoted, and stored at -80°C. Antibodies against total p44/42 MAPK, p38 MAPK, and stress-activated protein kinase/JNK were obtained from Santa Cruz (Santa Cruz, CA). Antibodies against phosphospecific forms of p44/42 MAPK, p38 MAPK, JNK, activating transcription factor-2 (ATF-2), and c-Jun were purchased from Cell Signaling (Beverly, MA).

Northern blot analysis. Control and treated cells were harvested, and total RNA was isolated and then separated in formamide-containing agarose gels according to previously described methods (26). A photograph of the ethidium-stained RNA gel was taken to normalize SP-A mRNA levels to 18S ribosomal RNA levels to correct for loading errors, as previously described (26). The RNA was transferred to a nylon membrane (Nytran SuPerCharge; 0.45-µm pore, Schleicher and Schuell, Keene, NH) by capillary transfer. A human SP-A cDNA was labeled with [alpha -32P]dCTP using random primers and hybridized to the membrane that contained the immobilized RNAs. Membranes were then washed, dried, and exposed to X-ray film for 3-24 h at -70°C with an intensifier screen.

Immunoblot analysis. Control and treated cells were rinsed twice with ice-cold 1× PBS and then lysed in buffer (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl2, 50 mM NaF, 5 mM sodium pyrophosphate, 0.2 mM sodium orthovanadate, 2 µg/ml aprotinin, 2 µg/ml leupeptin, 10% glycerol, 1% Triton X-100, 0.5% Polydet P-40, and 1 mM phenylmethylsulfonyl fluoride) for 1 h with shaking at 4°C. The lysed cells were scraped and then centrifuged at 12,000 g for 10 min. The lysate supernatant was aliquoted, boiled for 5 min with 1× electrophoresis sample buffer (125 mM Tris · HCl, pH 6.8, 1% 2-mercaptoethanol, 2% SDS, 5% glycerol, and 0.003% bromphenol blue), and stored at -20°C. Equal amounts of protein (50 µg) were separated on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and then nonspecific binding was blocked by incubating the membrane in Tris-buffered saline with 5% nonfat dried milk and 0.1% Tween 20 (33). To detect total p44/42 MAPK, p38 MAPK, JNK1, or c-Jun proteins, rabbit polyclonal primary antibodies were utilized at 1:1,000, 1:500, 1:200, and 1:1,000 dilutions, respectively. Rabbit polyclonal phosphospecific antibodies were used to detect phosphorylated p44/42 MAPK (Thr202/Tyr204), p38 MAPK (Thr180/Tyr182), JNK (Thr183/Tyr185), ATF-2 (Thr71), and c-Jun (Ser73) (all used at a dilution of 1:1,000). Polyclonal anti-rabbit IgG horseradish peroxidase-conjugated antibodies were used as the secondary antibody (1:2,000). An enhanced chemiluminescence Western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to visualize the immunoreactive bands on X-ray film.

p38 MAPK activity assay. The activity of p38 MAPK was measured using a p38 MAPK assay kit (Cell Signaling) according to the manufacturer's instructions. Control and treated cells were rinsed with PBS and lysed. Phospho-p38 MAPK was then immunoprecipitated from the lysate with an immobilized monoclonal antibody, washed, and used in a kinase reaction with ATP and ATF-2 fusion protein. The products of the kinase reaction were separated on an SDS-polyacrylamide gel and immunoblotted with a phospho-ATF-2 antibody as the primary antibody. Immunoreactive bands were detected as described above.

Cell viability. To evaluate cell viability in the presence of the signal transduction inhibitors, cells were pretreated with anisomycin, SB-203580, or vehicle for 2 h and were then incubated for an additional 24 h in the absence of the reagent. H441 cells were also treated with PD-169316, LY-294002, wortmannin, rapamycin, PD-98059, TNF-alpha , or vehicle for 24 h. We then performed trypan blue staining, phase-contrast microscopy, and cell counting on trypsinized H441 cell cultures. In some experiments, we also measured the lactate dehydrogenase content of the media (26).

Quantitation and statistical analysis. Reactive bands detected on X-ray films were scanned using a GS-710 scanner (Bio-Rad, Hercules, CA) and quantitated using Quantity One image analysis software (Bio-Rad). Data from treated conditions were normalized to the control conditions, which were made equal to one. Data are presented as means ± SE or SD. All experiments were performed at least three times unless otherwise stated. One-way analysis of variance followed by Dunnett's test or unpaired Student's t-test were used to evaluate significant differences between the treated and control conditions (39).


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REFERENCES

The toxicities of all of the inhibitors and effectors used in this study were evaluated by either light microscopy with trypan blue staining or by lactate dehydrogenase activity measurements. We evaluated H441 cells treated with TNF-alpha (20 and 100 ng/ml), SB-203580 (20 µM), anisomycin (5 µg/ml), PD-169316 (1 and 3 µM), LY-294002 (5 µM), wortmannin (200 nM), rapamycin (20 nM), and PD-98059 (10 µM) and compared the cell viability with corresponding untreated controls. Cell viability was not different from controls for all effectors and signal transduction inhibitor conditions (data not shown).

TNF-alpha inhibits SP-A gene expression. In initial experiments, we treated serum-starved H441 cells with TNF-alpha (0.1-100 ng/ml) in serum-free media for 24 h. TNF-alpha significantly inhibited SP-A mRNA in a dose-dependent manner, with a maximum ~70% inhibition achieved at TNF-alpha concentrations of >= 10 ng/ml and a half-maximal inhibition (IC50) at ~4 ng/ml (Fig. 1). In all further experiments, TNF-alpha was used at concentrations of 10-100 ng/ml, i.e., at concentrations that resulted in consistently high levels of SP-A inhibition.


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Fig. 1.   Surfactant protein A (SP-A) mRNA levels in tumor necrosis factor (TNF)-alpha -treated H441 cells. H441 cells were incubated with TNF-alpha (0.1-100 ng/ml) for 24 h. Northern blot analysis was then performed to evaluate human SP-A mRNA levels. A: representative Northern blot of SP-A mRNA in the presence or absence of TNF-alpha . The bottom image is of ethidium bromide-stained ribosomal RNA bands (18S and 28S) of the total RNA samples used in the Northern blot shown (top). B: densitometric analysis of Northern blot data obtained from multiple independent experiments. Data are expressed as means ± SE. TNF-alpha significantly decreased SP-A mRNA levels in a dose-dependent manner. The TNF-alpha -induced decrease was significant at concentrations of 10, 20, and 100 ng/ml (ANOVA, Dunnett's test, * P < 0.05, n = 3-21).

TNF-alpha does not inhibit SP-A gene expression via the PI 3-kinase or p44/42 MAPK signal transduction pathways. Wortmannin and LY-294002 are structurally and mechanistically different inhibitors of PI 3-kinase with an IC50 of 1-5 nM and 1.4 µM, respectively (2, 36). Rapamycin inhibits the mammalian target of rapamycin, a protein kinase upstream from p70 S6 kinase, which is a terminal kinase in the PI 3-kinase pathway with an IC50 of ~0.04-0.4 nM (6). TNF-alpha -mediated inhibition of SP-A gene expression was not affected by any of the inhibitors of the PI 3-kinase signaling pathway, i.e., LY-294002, wortmannin, or rapamycin (Fig. 2A). In addition, LY-294002, wortmannin, and rapamycin had no significant effect on basal SP-A mRNA levels when added alone (Fig. 2A). We also examined the effects of an inhibitor of the p44/42 MAPK signaling pathway, PD-98059, on the TNF-alpha -mediated decrease in SP-A gene expression. PD-98059 blocks activation of MAPK/extracellular signal-regulated kinase kinase with an IC50 of 10 µM (10). PD-98059 did not reverse the TNF-alpha -mediated inhibition of SP-A mRNA levels (Fig. 2B). However, PD-98059 significantly decreased basal levels of SP-A mRNA when it was added alone (Fig. 2B).


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Fig. 2.   Effects of phosphatidylinositol (PI) 3-kinase and p44/42 mitogen-activated protein kinase (MAPK) pathway inhibitors on SP-A gene expression. H441 cells were treated with either control media or with various signal transduction inhibitors with or without TNF-alpha (100 ng/ml). Northern blot analysis was used to detect human SP-A mRNA. Densitometric data from 3 experiments are presented as means ± SE; * significant difference from control condition. A: effect of PI 3-kinase pathway inhibitors. H441 cells were incubated with wortmannin (Wort, 200 nM), LY-294002 (LY, 5 µM), or rapamycin (Rapa, 20 nM) in the presence (+) or absence (-) of TNF-alpha for 24 h. TNF-alpha inhibited SP-A gene expression by ~60% (ANOVA, Dunnett's test, P < 0.05, n = 3). None of the PI 3-kinase pathway inhibitors reversed the TNF-alpha -induced decrease in SP-A mRNA levels. B: effect of a p44/42 MAPK pathway inhibitor, PD-98059. H441 cells were treated with PD-98059 (10 µM) and/or TNF-alpha for 24 h. PD-98059 diminished SP-A mRNA levels when added alone. TNF-alpha inhibited SP-A gene expression by ~60%; however, PD-98059 did not prevent the TNF-alpha -mediated inhibition of SP-A gene expression (ANOVA, Dunnett's test, P < 0.05, n = 3).

Inhibitors of p38 MAPK partially block the TNF-alpha -induced inhibition of SP-A gene expression. We used two inhibitors of the stress-activated protein kinase p38 MAPK, i.e., SB-203580 and PD-169316, to evaluate the possible role of this signaling pathway in TNF-alpha action in H441 cells (42, 43). Both inhibitors are pyrydinylimidazole compounds and bind to the ATP pocket of p38 MAPK (42). SB-203580 and PD-169316 specifically inhibit p38 MAPK with an IC50 equal to 74 nM and 89 nM, respectively (12). Both inhibitors may block the activity of another stress-activated protein kinase, JNK, at high concentrations (23, 43). SB-203580 is known to inhibit the alpha - and beta -isoforms of p38 MAPK (38). Northern blot analysis demonstrated that SB-203580, added alone, had no effect on SP-A mRNA levels in control cells. However, SB-203580 partially blocked the TNF-alpha -induced inhibition of SP-A mRNA levels in a dose-dependent manner (Fig. 3, A and B). When added alone, PD-169316 had no effect on SP-A mRNA levels at any concentrations used (Fig. 3, C and D). However, PD-169316 restored SP-A mRNA levels from ~40% of control levels in the TNF-alpha alone condition to ~75% of control levels in the TNF-alpha plus PD-169316 condition. The effect of PD-169316 to block the inhibitory effect of TNF-alpha was dose dependent.


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Fig. 3.   Effect of SB-203580 and PD-169316, inhibitors of stress-activated protein kinases p38 MAPK and c-Jun NH2-terminal kinase (JNK), on SP-A mRNA levels in presence or absence of TNF-alpha . Cells were incubated with either SB-203580 (3 or 20 µM) or PD-169316 (0.02-3 µM) in presence or absence of TNF-alpha (10 ng/ml) for 24 h. * Significant difference from TNF-alpha added alone condition. A: effects of SB-203580. H441 cells were preincubated with SB-203580 for 2 h and then treated further with control media or media that contained TNF-alpha , without the inhibitor present, for an additional 24 h. Top: representative Northern blot shows SP-A mRNA levels. Bottom: ethidium bromide-stained ribosomal RNA bands of total RNA used in Northern blot shown above. B: densitometric data from 3 SB-203580 experiments are expressed as means ± SE. TNF-alpha inhibited SP-A mRNA levels by ~70%. SB-203580 added alone had no effect on SP-A mRNA levels. SB-203580 partially blocked inhibitory effect of TNF-alpha on SP-A gene expression in a dose-dependent manner (Student's t-test, P < 0.05, n = 3). C: effects of PD-169316. Cells were pretreated with PD-169316 for 2 h and then further incubated with inhibitor and/or TNF-alpha for an additional 24 h. Top: representative Northern blot for SP-A mRNA; bottom: ethidium bromide-stained RNA gel from same experiment. D: densitometric data from 3 PD-169316 experiments are expressed as means ± SE. PD-169316 added alone had no effect on SP-A mRNA levels. TNF-alpha inhibited SP-A gene expression by ~60%. PD-169316 partially blocked inhibitory effect of TNF-alpha in a dose-dependent manner (ANOVA, Dunnett's test, P < 0.05, n = 3).

Stress-activated protein kinase inhibitors do not affect TNF-alpha -induced phosphorylation of p38 MAPK and JNK. TNF-alpha , added alone, significantly increased the phosphorylation of p38 MAPK (Fig. 4, A and C). TNF-alpha also modestly increased the phosphorylation of JNK; however, the effect was not significant (Fig. 4, B and D). In additional experiments, TNF-alpha at a concentration of 10 ng/ml caused a 2.62 ± 0.77-fold increase (means ± SE, n = 6, P = 0.062, Student's t-test) in JNK phosphorylation after a 15-min exposure compared with a 14.24 ± 3.55-fold increase (means ± SE, n = 7, P < 0.05, Student's t-test) in p38 MAPK phosphorylation. SB-203580 or PD-169316, added alone, caused a small increase in the basal phosphorylation of p38 MAPK, but these effects were not significant (Fig. 4, A and C). Neither SB-203580 nor PD-169316 had a significant effect on the TNF-alpha -induced phosphorylation of p38 MAPK (Fig. 4, A and C). SB-203580, added alone, also tended to increase the phosphorylation of JNK at concentrations <20 µM (Fig. 4B). SB-203580 inhibited the TNF-alpha -induced phosphorylation of JNK only at the 20-µM concentration (Fig. 4B). PD-169316, added alone, tended to increase JNK phosphorylation in a dose-dependent manner (Fig. 4D). PD-169316 did not significantly affect TNF-alpha -induced JNK phosphorylation at any concentration tested (Fig. 4D).


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Fig. 4.   Effects of SB-203580 and PD-169316 on phosphorylation of p38 MAPK and JNK. H441 cells were pretreated with SB-203580 (0.02-20 µM) or PD-169316 (0.02-3 µM) for 2 h and then incubated in presence of TNF-alpha (10 ng/ml) and/or inhibitor for 15 min. A: effect of SB-203580 on p38 MAPK phosphorylation in presence or absence of TNF-alpha . Representative immunoblot for phosphorylated p38 (P-p38) MAPK (top) and densitometric data (bottom) show SB-203580 slightly increased p38 MAPK phosphorylation when added alone. TNF-alpha added alone dramatically increased p38 MAPK phosphorylation. SB-203580 had no effect on TNF-alpha -induced p38 MAPK phosphorylation. Data are means of 2 experiments ± SD. B: effect of SB-203580 on JNK phosphorylation in presence or absence of TNF-alpha . Representative immunoblot for phosphorylated JNK is shown (top) as is densitometry (bottom). TNF-alpha modestly increased JNK phosphorylation (P-JNK) after 15 min of incubation. SB-203580 added alone slightly increased JNK phosphorylation at concentrations of 0.5 and 3 µM. SB-203580 had no effect on TNF-alpha -mediated increase in JNK phosphorylation at 0.02-3 µM but blocked JNK phosphorylation at 20-µM concentration. Data are means of 2 experiments ± SD. C: top, representative immunoblot of phosphorylated p38 MAPK in presence of PD-169316 and/or TNF-alpha . Densitometric data (bottom) are means ± SE of 5 experiments. TNF-alpha caused a large increase in phosphorylation of p38 MAPK (ANOVA, Dunnett's test, P < 0.05). PD-169316 added alone slightly increased p38 MAPK phosphorylation. PD-169316 did not significantly inhibit TNF-alpha -induced p38 MAPK phosphorylation at any concentration. * Significant difference from controls. D: effects of PD-169316 and/or TNF-alpha on JNK phosphorylation. Representative immunoblot is shown (top) as is densitometric data (bottom). TNF-alpha added alone increased JNK phosphorylation modestly. PD-169316 at concentrations >0.1 µM increased phosphorylation of JNK when added alone. TNF-alpha -induced JNK phosphorylation was not affected by presence of PD-169316. Data are the mean of 3 experiments ± SE.

TNF-alpha increases the phosphorylation of p38 MAPK and JNK in a time-dependent manner. We next examined the time course of the TNF-alpha -mediated increase in the phosphorylation of MAPK family protein kinases in H441 cells. The phosphorylation state of the MAPKs was slightly increased by the media change before the experiment but declined within 30 min to 2 h (Fig. 5). TNF-alpha caused a synchronous increase in the phosphorylation of both p38 MAPK and JNK at the 15-min time point (Fig. 5, B and C). This increase in phosphorylation of the stress-activated protein kinases was significant for p38 MAPK (Student's t-test, P < 0.05, n = 3) but not for JNK (Student's t-test, P = 0.149, n = 3) compared with corresponding controls. An effect of TNF-alpha on the phosphorylation of these two enzymes was absent by the 30-min time point (Fig. 5, B and C). In further experiments, we found that 20 ng/ml of TNF-alpha caused a 3.66 ± 1.38-fold increase (means ± SE, P = 0.09, n = 5, Student's t-test) in JNK phosphorylation after a 15-min exposure compared with a 8.18 ± 1.59-fold increase (means ± SE, P < 0.05, n = 6, Student's t-test) in p38 MAPK phosphorylation. TNF-alpha did not increase the phosphorylation of p44/42 MAPK at any time point (Fig. 5A). TNF-alpha also did not alter the total amount of any of the three MAPKs present in the H441 cells at any time point evaluated (data not shown).


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Fig. 5.   Phosphorylation state of p44/42 MAPK, p38 MAPK, and JNK in H441 cells after various intervals in presence or absence of TNF-alpha . Cells were incubated with TNF-alpha (20 ng/ml) or vehicle for indicated time periods. Cells were harvested, and a protein lysate was used for immunoblots with rabbit polyclonal phosphospecific antibodies directed against either p44/42 MAPK (Thr202/Tyr204), p38 MAPK (Thr180/Tyr182), or JNK (Thr183/Tyr185). Protein molecular weight standards are indicated with each blot (left). Densitometric data are means of 3 experiments ± SE. * Significant difference from corresponding controls. Data were normalized to control value at 15-min time point that was made equal to one. A: representative immunoblot (top) and densitometric data concerning levels of phospho-p44/42 (P-p44/P-p42) MAPK in H441 cells incubated with or without TNF-alpha for 15 min, 30 min, 2 h, and 16 h. TNF-alpha did not affect phosphorylation of p44/42 MAPK at any time point examined. B: representative immunoblot and densitometric data concerning levels of phospho-p38 MAPK in presence or absence of TNF-alpha at various time points. TNF-alpha caused a rapid and dramatic increase in phosphorylation of p38 MAPK at 15 min that decreased to control levels thereafter (Student's t-test, P < 0.05, n = 3). C: representative immunoblot and densitometric data for phospho-JNK in H441 cells incubated with or without TNF-alpha for various intervals. TNF-alpha modestly increased phosphorylation of JNK within 15 min, but levels decreased to control values thereafter.

TNF-alpha increases p38 MAPK activity. Serum-starved H441 cells were treated with TNF-alpha (10 ng/ml) or vehicle for 15 min, harvested, and assayed for p38 MAPK activity. Densitometric data from three experiments showed that in the presence of TNF-alpha , p38 MAPK activity was significantly increased and equal to 5.81 ± 2.15 (arbitrary units, means ± SE, Student's t-test, P < 0.05, n = 3) compared with activity in the control conditions that were made equal to one in each experiment.

An activator of p38 MAPK inhibits SP-A gene expression. Anisomycin is a protein synthesis inhibitor that activates all four p38 MAPK isoforms (alpha , beta , gamma , and delta ) and JNK2alpha and their upstream kinases as well as p70/85 S6 kinase (14). Activation of stress-activated protein kinases by anisomycin has been shown to lead to the phosphorylation of several transcription factors, i.e., c-Jun, ATF-2, and ternary complex factor, and strongly induces the transcription of early responding genes (14). Pretreatment of H441 cells with anisomycin significantly increased p38 MAPK phosphorylation, in a dose-dependent manner, to approximately three times the control levels at the 5-µg/ml concentration (Fig. 6, A and B). Treatment with anisomycin did not affect the total amount of p38 MAPK at any concentration tested (data not shown, n = 3). Anisomycin had essentially no effect on JNK phosphorylation in H441 cells (Fig. 6, A and B). In a parallel experiment, anisomycin significantly inhibited SP-A gene expression after a 24-h incubation in a dose-dependent manner, with an IC50 of ~0.4 µg/ml (Fig. 6, C and D).


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Fig. 6.   Effects of anisomycin (Anis), an activator of p38 MAPK, on phosphorylation of p38 MAPK and JNK and on SP-A mRNA levels. H441 cells were treated with anisomycin (0.1-5 µg/ml) for 2 h and then incubated in control media for an additional 24 h. Cells were harvested, and protein lysates were used for immunoblotting using antibodies directed against phosphorylated p38 MAPK and JNK (A and B). In parallel experiments, cells were harvested, total RNA was isolated, and Northern blot analysis for SP-A mRNA was performed (C and D). * Significant difference from control cells. A: immunoblots showing effect of anisomycin on phosphorylated p38 MAPK and JNK. Top: anisomycin applied at concentrations from 0.1 to 5 µg/ml increased phosphorylation of p38 MAPK. Bottom: anisomycin did not affect phosphorylation of JNK in cells. B: densitometric analysis of phosphorylated p38 MAPK and JNK in presence or absence of anisomycin. Levels in control cells were made equal to one. Data are means ± SE for phospho-p38 MAPK (n = 3) and means ± SD for phospho-JNK (n = 2). Anisomycin increased p38 MAPK phosphorylation in a dose-dependent manner (ANOVA, Dunnett's test, P < 0.05, n = 3) but had no effect on JNK phosphorylation. C: effect of anisomycin on SP-A mRNA levels. Top: representative Northern blot of SP-A mRNA levels in presence of indicated concentrations of anisomycin. Bottom: ribosomal RNA (28S and 18S) in total RNA used for Northern blot shown above. D: densitometric analysis of SP-A mRNA levels in presence or absence of anisomycin. Data from 3 experiments are expressed as means ± SE. SP-A mRNA levels were diminished by anisomycin in a dose-dependent manner (ANOVA, Dunnett's test, P < 0.05, n = 3).

TNF-alpha increases phosphorylation of transcription factors ATF-2 and c-Jun. ATF-2 is a transcription factor phosphorylated by both p38 MAPK and JNK (21). The transcription factor c-Jun is a JNK substrate (21). Because TNF-alpha caused an increased phosphorylation of p38 MAPK after 15 min of incubation, we evaluated whether the increase in the phosphorylation of p38 MAPK resulted in ATF-2 phosphorylation. Because we observed a synchronous but smaller effect of TNF-alpha on JNK phosphorylation, we also examined the phosphorylation state of c-Jun in response to TNF-alpha . Some cells were incubated with anisomycin (10 µg/ml) for 2 h as a positive control. Figure 7A shows that ATF-2 phosphorylation on Thr71 was increased ~10-fold after 15 min of incubation with TNF-alpha and that the effect was gone by 30 min of incubation, a time course similar to the TNF-alpha -induced increase in p38 MAPK phosphorylation. As shown in Fig. 7B, the phosphorylation of c-Jun on Ser73 was increased only modestly (~1.7-fold) in response to TNF-alpha at 15 min. Anisomycin treatment of the cells greatly increased the phosphorylation of ATF-2 and modestly increased the phosphorylation of c-Jun (Ser73; Fig. 7).


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Fig. 7.   Effect of TNF-alpha on phosphorylation state of transcription factors, activating transcription factor -2 (ATF-2) and c-Jun. H441 cells were incubated in presence or absence of TNF-alpha (20 ng/ml) or vehicle for 15 min, 30 min, 2 h or 16 h. Some cells were treated with anisomycin (10 µg/ml) as a positive control. Protein molecular weight standards are shown with immunoblots (left). Data were normalized to control values at the 15-min time point that was made equal to one. A: representative immunoblot for phosphorylated ATF-2 (Thr71). Graph represents densitometric data from 4 experiments expressed as means ± SE. * Significant difference from corresponding control. TNF-alpha caused a dramatic phosphorylation of ATF-2 within 15 min (Student's t-test, P < 0.05, n = 4). B: representative immunoblot for phosphorylated c-Jun (Ser73). Immunoreactive (phosphorylated) bands were used to obtain densitometric data presented (bottom) as means of 2 experiments ± SD. TNF-alpha increased c-Jun phosphorylation modestly at 15 min but had no effect thereafter.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TNF-alpha inhibits SP-A gene transcription in H441 cells within 6 h (39). Some progress has been achieved in understanding the molecular events that accompany TNF-alpha -mediated suppression of the SP-A gene. For example, Pryhuber et al. (31) have shown that TNF-alpha probably does not inhibit SP-A gene expression via the activation of NF-kappa B. However, the signaling pathways utilized by TNF-alpha in modulating SP-A gene expression in pulmonary epithelial cells remain unknown.

To gain insight in the TNF-alpha signal transduction mechanisms in lung epithelial cells, we performed experiments based in part on the TNF-alpha signaling pathways previously described in lung epithelial cells and in other cell types. Other investigators have shown that TNF-alpha can activate the PI 3-kinase/Akt pathway in some cell types, for example, to reduce apoptosis of human endothelial cells, to stimulate survival of rat adult retinal ganglion cells, to promote protein synthesis in rat cardiac myocytes, and to interfere with insulin signaling at the insulin receptor substrate-1 (IRS-1) level in kidney cells (9, 16, 24, 29). We found that the TNF-alpha -induced inhibition of SP-A gene expression was not blocked by either LY-294002 or wortmannin, inhibitors of PI 3-kinase, or by rapamycin, an inhibitor of the mammalian target of rapamycin, a protein kinase that regulates p70 S6 kinase (34). Thus our results in H441 cells suggest that the PI 3-kinase pathway is not involved in TNF-alpha -mediated SP-A gene inhibition in lung epithelial cells.

TNF-alpha has been shown to activate p44/42 MAPK as well as p38 MAPK and JNK in rat and human lung epithelial cells (3, 5, 18, 25). In our study, in which we used human lung epithelial cell line H441, we found that TNF-alpha does not effect the phosphorylation/activation of p44/42 MAPK. In agreement with these results, inhibition of the p44/42 MAPK pathway using PD-98059 did not reverse TNF-alpha inhibition of SP-A gene expression. Interestingly, PD-98059 added alone decreased the basal levels of SP-A mRNA, a result that suggests the possible involvement of the p44/42 MAPK pathway in maintaining basal SP-A gene expression in H441 cells. Our observation of a lack of TNF-alpha -induced p44/42 MAPK phosphorylation in H441 cells differs from the sustained activation of this kinase in rat type II cells reported by Mallampalli and coworkers (25) and in H292 cells, a human alveolar epithelial carcinoma cell line, as shown by Chen and coworkers (5). This discrepancy may be the result of differences in the cell types studied and in the experimental conditions. However, based on our results, we conclude that the p44/42 MAPK pathway is probably not involved in TNF-alpha -mediated inhibition of SP-A gene expression in H441 cells.

TNF-alpha rapidly increased the phosphorylation and activity of p38 MAPK. The activation of p38 MAPK was observed at 15 min with a return to control levels within 30 min of incubation. When we inhibited p38 MAPK activity with either SB-203580 or PD-169316, SP-A mRNA levels in the presence of TNF-alpha were partially restored to the control levels. SB-203580 and PD-169316 are selective inhibitors of p38 MAPK alpha - and beta -isoforms and may also inhibit some isoforms of JNK at higher concentrations (12, 23, 38, 42, 43). It has been shown that SB-203580 binds to the ATP pocket of p38 MAPK and blocks its catalytic activity but does not affect p38 Thr180 and Tyr182 phosphorylation (20, 38, 42). In agreement with this, we found that SB-203580 and PD-169316 do not inhibit the TNF-alpha -induced phosphorylation of p38 MAPK in H441 cells. Furthermore, we showed that anisomycin, an activator of p38 MAPK, increased the phosphorylation of p38 MAPK and also caused a dramatic inhibition of SP-A gene expression. We also showed that the activation of p38 MAPK is accompanied by a synchronical increase in the phosphorylation of ATF-2, a transcription factor that is a p38 MAPK substrate (21). Thus we conclude that p38 MAPK is probably an important signaling pathway by which TNF-alpha acts to inhibit SP-A gene expression.

TNF-alpha modestly increased the phosphorylation of JNK simultaneously with p38 MAPK; therefore, we questioned whether TNF-alpha might also act via this kinase to inhibit SP-A gene expression. There is evidence of some overlap of targets of the p38 MAPK and JNK signaling pathways (21). For example, TNF-alpha -induced activation of p38 MAPK leads to phosphorylation of transcription factors such as ATF-2 and Elk-1, whereas activated JNK phosphorylates c-Jun, ATF-2, and Elk-1 (21). Our data suggest that the JNK pathway may be activated in parallel with the p38 MAPK pathway but that the effects of TNF-alpha on JNK phosphorylation are much smaller in magnitude than the effects on p38 MAPK. In agreement with these results, TNF-alpha caused only a small increase in the phosphorylation of c-Jun, a transcription factor phosphorylated solely by JNK, compared with a large increase in ATF-2 phosphorylation, which can potentially be mediated by either protein kinase, p38 MAPK or JNK (21). We conclude that TNF-alpha probably inhibits SP-A gene expression primarily via the p38 MAPK signaling pathway and not via the JNK pathway.

Our results agree with data reported by Awasthi and coworkers (3) that TNF-alpha causes a rapid phosphorylation of p38 MAPK that peaks within 15 min in H441 cells. The authors of this study suggested that p38 MAPK is involved in ceramide signaling. In another human alveolar epithelial cell line, H292 cells, TNF-alpha treatment resulted in the phosphorylation of both stress-activated protein kinases p38 MAPK and JNK as well as p44/42 MAPK (5). These authors suggest that MAPKs are activated by TNF-alpha via an increase in ceramide formation and that this may contribute to NF-kappa B-mediated induction of cyclooxygenase-2 gene expression by TNF-alpha (5). We found that in H441 cells, TNF-alpha caused synchronical phosphorylation of p38 MAPK and JNK within 15 min that ended by 30 min of exposure, a finding that suggests protein phosphatase-dependent dephosphorylation of these MAPKs may be involved in their regulation (13).

The phosphorylation of c-Jun, ATF-2, and Elk-1, along with an induction of early response gene transcription by MAPKs, contribute to activator protein-1 (AP-1) activation (21). TNF-alpha increases AP-1 binding to the gamma -glutamylcysteine synthetase heavy subunit promoter in human lung epithelial cells (32). The human SP-A gene promoter may contain DNA elements responsive to transcription factor complexes such as AP-1, which are controlled by the stress-activated protein kinase pathways. Hoover and coworkers (17) have suggested that binding of transcription factors to an AP-1 site in the first intron of the SP-A gene (+318/+324) may mediate the inhibitory effects of phorbol ester on SP-A gene expression. This binding may inhibit SP-A gene transcription by protein-protein interactions and effects on a basic transcription complex or by interaction with other cis-acting elements in the regulatory region of the SP-A gene (17).

In summary, our data suggest that a stress-activated protein kinase signal transduction pathway, primarily p38 MAPK, is important in the TNF-alpha regulation of SP-A gene expression in lung epithelial cells. Neither the p44/42 MAPK nor the PI 3-kinase signal transduction pathways mediate TNF-alpha inhibition of SP-A gene expression. Our data do not completely exclude a possible involvement of JNK in TNF-alpha action. Additional investigation will be needed to identify downstream effectors in the p38 MAPK signaling pathway that are activated by TNF-alpha in lung epithelial cells and to determine how their interaction with regulatory sequences in the SP-A gene or with other trans-acting factors results in an inhibition of SP-A gene transcription.


    ACKNOWLEDGEMENTS

The authors thank Jean Gardner for typing the manuscript.


    FOOTNOTES

The research was supported by National Institutes of Health Grants HL-50050 and DK-25295.

Address for reprint requests and other correspondence: J. M. Snyder, Dept. of Anatomy and Cell Biology, College of Medicine, Univ. of Iowa, 51 Newton Road, 1-550 BSB, Iowa City, IA 52242-1109 (E-mail: jeanne-snyder{at}uiowa.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00470.2001

Received 11 December 2001; accepted in final form 20 March 2002.


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