Signal transduction events involved in TPA downregulation of SP-A gene expression
Olga L. Miakotina and
Jeanne M. Snyder
Department of Anatomy and Cell Biology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242-1109
Submitted 1 December 2003
; accepted in final form 26 January 2004
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ABSTRACT
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Surfactant protein A (SP-A), the most abundant pulmonary surfactant protein, plays a role in innate host defense and blocks the inhibitory effects of serum proteins on surfactant surface tension-lowering properties. SP-A mRNA and protein are downregulated by phorbol esters (TPA) via inhibition of gene transcription. We evaluated the TPA signaling pathways involved in SP-A inhibition in a lung cell line, H441 cells. TPA caused sustained phosphorylation of p44/42 mitogen-activated protein kinase (MAPK), p38 MAPK, and c-Jun-NH2-terminal kinase. An inhibitor of conventional and novel isoforms of protein kinase C (PKC) and two inhibitors of p44/42 MAPK kinase partially or completely blocked the inhibitory effects of TPA on SP-A mRNA levels. In contrast, inhibitors of conventional PKC-
and -
, stress-activated protein kinases, protein phosphatases, protein kinase A, and the phosphatidylinositol 3-kinase pathway had no effect on the TPA-mediated inhibition of SP-A mRNA. TPA also stimulated the synthesis of c-Jun mRNA and protein in a time-dependent manner. Inhibitors of the p44/42 MAPK signaling pathway and PKC blocked the TPA-mediated phosphorylation of p44/42 MAPK and the increase in c-Jun mRNA. We conclude that TPA inhibits SP-A gene expression via novel isoforms of PKC, the p44/42 MAPK pathway, and the activator protein-1 complex.
12-O-tetradecanoylphorbol-13-acetate; surfactant protein A; signal transduction; protein kinase C; mitogen-activated protein kinases; H441 cells; activator protein 1
SURFACTANT IS A LIPOPROTEIN COMPLEX that forms a monolayer at the air-aqueous interface in the pulmonary alveolus and serves as a surface tension-reducing agent that facilitates the re-expansion of alveoli during inspiration (50). Surfactant consists primarily of phospholipids (
80%), cholesterol (
10%), and the surfactant-associated proteins (SP-A, -B, -C, and -D) (
10%), which contribute to surfactant biophysical properties and also function in innate host defense mechanisms in the lung (7, 48). The surfactant proteins are regulated during development and by cytokines, hormones, and growth factors (48). TPA is a structural homolog of diacylglycerol (DAG) that mimics its activation of protein kinase C (PKC). This kinase influences cell growth and differentiation and is part of the signal transduction pathways of many growth factors and cytokines involved in regulating gene expression in the lung, including SP-A. Treatment of rabbits with TPA in vivo causes lung pathology similar to that observed in acute respiratory distress syndrome (ARDS) (28). Phorbol ester (TPA) has been shown to inhibit SP-A and SP-B gene expression in lung epithelial cells at the level of gene transcription within a few hours (34, 49). TPA treatment decreases the nuclear content of thyroid transcription factor-1 and hepatocyte nuclear factor-3 transcription factors, and this in turn inhibits SP-B gene transcription in H441 cells (25). TPA has been shown to downregulate the SP-A promoter via an activator protein (AP)-1 response element in the first intron of the SP-A genes (18). There are no data available concerning the cellular signaling mechanisms by which phorbol esters inhibit SP-A in epithelial cells.
PKC is a family of lipid-regulated serine/threonine protein kinases that is divided into three classes: conventional or classical, novel, and atypical PKCs. Conventional PKC isoforms are activated by Ca2+ and diacylglycerol (DAG); novel isoforms are only dependent on DAG, whereas atypical PKC isoforms are not stimulated by either Ca2+ or DAG. Phorbol esters and DAG bind and activate conventional and novel PKC isoforms with high affinity. Phorbol esters also bind nonkinase DAG/phorbol ester receptors (37). Activation of PKC impairs the barrier function of epithelia and, in the lung, increases endothelial permeability and can lead to pulmonary edema and respiratory distress syndrome (RDS) (5, 43). Several studies have documented the involvement of certain PKC isoforms, i.e., PKC-µ and Ca-/phosphatidylserine (PS)-dependent PKC, in surfactant secretion in primary cultures of alveolar type II cells (14, 39).
Different isoforms of PKC act via different signal transduction pathways to regulate gene expression, cell survival, etc. (21). Activated PKC isoforms generally transduce their intracellular signal via either mitogen-activated protein kinases (MAPK) pathways, usually p44/42 MAPK and c-Jun NH2-terminal kinase (JNK), or via MAPK-independent pathways, for example the phosphatidylinositol 3-kinase (PI 3-kinase) pathway (11, 23, 36, 44, 46). Downstream targets of phorbol esters that are involved in gene regulation include transcription factors such as the AP-1 complex, cyclic AMP binding protein (CREB), nuclear factor (NF)-
B, or Sp1 (9, 23, 46, 52).
In the present study, we characterized the signaling pathways involved in TPA-mediated inhibition of SP-A mRNA levels in lung epithelial cells (H441 cells). SP-A is regulated by hormonal effectors such as insulin and retinoic acid in a similar manner in human fetal lung explants and the H441 cell line (10, 13, 29, 30). Thus H441 cells are a good in vitro model for studying the effects of regulatory agents on SP-A mRNA levels in lung epithelial cells. In the present study, we used several specific inhibitors of signal transduction pathways. We confirmed the effects of these inhibitors by evaluating changes in the phosphorylation state and/or total amount of the various signal transduction protein kinases and their substrates. Our results indicate that TPA activates PKC isoforms, which in turn activate p44/42 MAPK and stimulate the synthesis of c-Jun mRNA and protein. We found that inhibitors of PKC and p44/42 MAPK blocked both c-Jun gene expression and the TPA-induced inhibition of SP-A mRNA levels. Thus TPA probably decreases SP-A mRNA levels by activating novel PKCs and p44/42 MAPK, followed by an increase in c-Jun levels that inhibits SP-A gene transcription via an interaction with AP-1 site(s) in the SP-A genes.
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MATERIALS AND METHODS
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Cell culture and reagents.
Human lung epithelial cells, NCI-H441 cells, were maintained in monolayer culture in RPMI 1640 medium that contained 10% fetal bovine serum, 0.25 µg/ml amphotericin B, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO2. Cells were passed weekly, and medium was changed every 34 days.
For most experiments, subconfluent cells (
70% confluence) were incubated in serum-free medium for 24 h before the experiment, pretreated with either signal transduction inhibitors or vehicle for 1 h, and then incubated with TPA, 510 nM, in the presence or absence of the signal transduction inhibitors for an additional 24 h. To evaluate the phosphorylation state of the MAPK family protein kinases, we incubated serum-deprived cells in the presence or absence of TPA for 15 min, 30 min, 2 h, and 16 h.
PD-98059 and U-0126 were purchased from New England Biolabs (Beverly, MA). SB-203580, SP-600125, GF-109203X, myristoylated protein kinase C [2028] (PKCI), okadaic acid, and H-89 were purchased from BioMol (Plymouth Meeting, PA). TPA, wortmannin, LY-294002, and rapamycin were obtained from Sigma Chemical (St. Louis, MO). Stock solutions of PD-98059, U-0126, SB-203580, SP-600125, GF-109203X, okadaic acid, wortmannin, LY-294002, rapamycin, TPA, and H-89 were prepared in DMSO as 10 mM, 1 mM, 5 mM, 20 mM, 5 mM, 1 mM, 100 µM, 50 mM, 50 µM, 1 mg/ml, and 40 mM stocks, respectively, and stored at 80°C in aliquots. These inhibitors have been shown previously to be relatively reliable and specific inhibitors of signal transduction protein kinases (8, 33, 47). Antibodies directed against phosphorylated protein kinases, p44/42 MAPK, p38 MAPK, and SAPK/JNK, as well as against phospho-activating transcription factor (ATF)-2, phospho-c-Jun, and total c-Jun were purchased from Cell Signaling (Beverly, MA). Antibodies against total p44/42 MAPK (ERK1), p38 MAPK, and JNK1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).
Immunoblot analysis.
Immunoblotting was performed as described previously (31). In short, control and treated cells were rinsed twice with ice-cold phosphate-buffered saline and then lysed in a 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% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride) for 30 min at 4°C followed by centrifugation at 12,000 g for 10 min. The supernatant, which contained the protein lysate, was boiled with an equal volume of 2x electrophoresis sample buffer (250 mM Tris·HCl, pH 6.8, 2% 2-mercaptoethanol, 4% SDS, 10% glycerol, and 0.006% bromphenol blue) for 5 min, and then equal amounts of protein from each condition were separated on 10% SDS polyacrylamide gels and immunoblotted according to the instructions of the antibody supplier. Phosphospecific antibodies directed against p44/42 MAPK, p38 MAPK, and SAPK/JNK, ATF-2, c-Jun, and an antibody directed against total c-Jun were used at dilutions of 1:1,000, whereas ERK 1, p38 MAPK, and JNK1 antibodies were diluted 1:1,000, 1:500, and 1:200, respectively. Primary antibodies were visualized with polyclonal anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibodies at a 1:2,000 dilution. Immunoblots were subsequently treated with chemiluminescence reagent (ECL Western blotting detection system; Amersham Pharmacia Biotech, Piscataway, NJ) and exposed to X-ray film.
Northern blot analysis.
Control and treated cells were harvested, and total RNA was isolated, separated on formaldehyde-containing agarose gel, and transferred to a nitrocellulose membrane according to previously described methods (31). Human SP-A cDNA, human c-Jun, or c-Fos cDNAs were labeled with [
-32P]dCTP and hybridized to membranes that contained the immobilized total RNA. Membranes were then washed, dried, and exposed to X-ray film. Radioactive bands were scanned and quantitated with Quantity One image analysis software (Bio-Rad Laboratories, Hercules, CA). Intensities of reactive bands were corrected to intensities of correspondent 18S rRNA bands (31). Each experiment was performed at least three times unless otherwise stated.
Quantitation of results and statistical analysis.
Results were quantitated relative to the densitometric data in the control condition, which was made equal to one. To estimate the statistical significance of the results, one-way analysis of variance (ANOVA) followed by Dunnett's test or unpaired t-test was used (51).
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RESULTS
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To evaluate the viability of the H441 cells in the presence of the various signal transduction inhibitors, we treated cells with the highest concentration used of U-0126, SB-203580, SP-600125, GF-109203X, PKCI, PD-98059, wortmannin, LY-294002, rapamycin, DMSO, or TPA (10 nM) for 24 h. Other plates were preincubated with okadaic acid or H-89 for 1 h, then media were discarded, and the cells were exposed to fresh media for an additional 23 h. Examination of the cells under phase-contrast microscopy and trypan blue exclusion, as well as by measurement of the lactate dehydrogenase level in the media for some agents, revealed no difference in cell viability in the treated cells vs. controls.
TPA inhibits SP-A mRNA levels.
To choose an optimal TPA concentration that would inhibit SP-A mRNA levels in the H441 cells, we applied 0.130 nM TPA to the cells for 24 h and then measured the amount of human SP-A mRNA present in the treated vs. control cells. Figure 1 shows that TPA caused a dose-dependent decrease in SP-A mRNA levels in H441 cells with maximum inhibition of
90% by 30 nM TPA and a half-maximal effect (IC50) at
35 nM.

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Fig. 1. The effect of TPA on surfactant protein (SP)-A mRNA levels in H441 cells. H441 cells were exposed to TPA (0.130 nM) for 24 h. Cells were harvested, total RNA was isolated, and Northern blot analysis for human SP-A mRNA was performed. Reactive bands were detected by exposure to X-ray film and quantitated by densitometry. Data are normalized to values in untreated control cells and presented as the means ± SE. A, top: representative Northern blot for SP-A mRNA in the presence of the indicated concentrations of TPA. Bottom: ethidium bromide-stained rRNA bands, 28S and 18S, in the samples shown in the Northern blot above. B: quantitative data from 3 experiments. TPA caused a dose-dependent decrease in SP-A mRNA levels. *Significant difference from untreated controls (ANOVA, Dunnett's test, P < 0.05, n = 3).
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Inhibitors of PKC partially block the TPA inhibition of SP-A mRNA levels.
Phorbol esters are well-characterized activators of PKC (21). To determine whether PKC activation is involved in the inhibition of SP-A mRNA levels by TPA, two well-characterized PKC inhibitors, i.e., PKCI and GF-109203X, were used. PKCI is a selective inhibitor of two conventional PKC isoforms,
and
, whereas GF-109203X blocks a wide spectrum of PKCs, including
-,
I-,
II-,
-, and
-isoforms, i.e., it inhibits both conventional and novel PKC isoforms. As shown in Fig. 2A, PKCI had no effect on the TPA-induced inhibition of SP-A mRNA levels. In contrast, GF-109203X partially blocked the TPA inhibition of SP-A in a dose-dependent manner at concentrations of 0.050.5 µM (Fig. 2B). Thus TPA probably inhibits SP-A mRNA levels via the activation of novel PKC isoforms but not via activation of conventional PKC-
or -
.

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Fig. 2. The effects of protein kinase (PK) CI and GF-109203X (GF), both inhibitors of PKC, on SP-A mRNA levels in the presence (+) or absence () of TPA. H441 cells were pretreated with 0.110.0 µM PKCI or 0.055.0 µM GF-109203X for 1 h and then treated with 10 nM TPA for an additional 24 h. The treated and untreated (control) cells were harvested, and Northern blot analysis was performed for human SP-A mRNA. Data from 34 independent experiments are expressed as means ± SE. *Statistically significant difference from untreated controls, which were made equal to 1 (ANOVA, Dunnett's test, P < 0.05). A, top: representative Northern blot and the ethidium bromide-stained rRNA of samples shown in the Northern blot. Bottom: graph of densitometric data from 4 independent experiments showing that there was no effect of PKCI on the TPA-mediated inhibition of SP-A mRNA levels. B, top: representative Northern blot and respective ethidium bromide-stained rRNA in the samples used in the blot. Bottom: densitometric data from 3 independent experiments demonstrate a partial and dose-dependent restoration of TPA-inhibited SP-A mRNA levels in presence of GF-109203X. #Statistically significant difference from the TPA added alone condition (ANOVA, Dunnett's test, P < 0.05, n = 3).
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Phorbol esters activate all three protein kinases of the MAPK family.
To further evaluate the signaling pathways activated by TPA in the H441 cells, we examined the effects of TPA on the phosphorylation of three key enzymes in the MAPK family of protein kinases. H441 cells were incubated in the presence of TPA for 15 min, 30 min, 2 h, and 16 h, and the protein lysates were used for immunoblotting with phosphospecific antibodies. Figure 3 demonstrates that TPA upregulated the phosphorylation of all three MAPK family protein kinases in a time-dependent manner. TPA increased the phosphorylation of p44/42 MAPK as early as after 15 min, and this 1.5- to 4-fold increase in phosphorylation over control levels lasted for at least 16 h (Fig. 3A). TPA also increased the phosphorylation of p38 MAPK by a maximum of
10-fold over controls at the 30-min time point (Fig. 3B). This increased level of phosphorylation also lasted for at least 16 h. TPA induced a 1.5- to 3-fold increase in the phosphorylation of SAPK/JNK with a temporal pattern that was similar to the increase in p44/42 MAPK phosphorylation (Fig. 3C). The effect was significant at 15 and 30 min. The addition of fresh media to the cells at the start of the experiment may have caused increased phosphorylation of p44/42 MAPK and SAPK/JNK during the initial 15-min incubation period. The level of phosphorylated enzyme in controls declined to baseline levels thereafter. This pattern has been observed in previous studies (2, 31). TPA had no effect on the total amount of p44/42 MAPK, p38 MAPK, or JNK1 proteins in the cells (data not shown).

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Fig. 3. The effects of TPA on the phosphorylation (P) of p44/42 MAPK (A), p38 MAPK (B), and SAPK/JNK (C). Subconfluent cells were treated with or without TPA (10 nM) for 15 min, 30 min, 2 h, and 16 h. The cells were harvested, and then protein lysates were used for immunoblotting with phosphospecific antibodies directed against the MAPK family protein kinases. Densitometric data from 3 independent experiments are expressed as means ± SE relative to untreated controls at 15-min time point, which were made equal to 1. *Significant difference from respective untreated controls (unpaired t-test, P < 0.05). A: representative immunoblot of phospho-p44/42 MAPK (top) and a graph of densitometric data (bottom) show that TPA significantly increased the phosphorylation of p44/42 MAPK at all time points compared with corresponding untreated controls. B: representative immunoblot of phospho-p38 MAPK (top) and a graph of densitometric data (bottom) illustrate that TPA significantly upregulated the phosphorylation of p38 MAPK at all time points with a maximal increase at the 30-min time point. C: representative immunoblot of phospho-SAPK/JNK (top) and a graph with densitometric results (bottom) demonstrate that TPA significantly increased JNK/SAPK phosphorylation at the 15- and 30-min time points in a temporal pattern similar to the TPA-mediated increase in p44/42 MAPK phosphorylation.
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Inhibition of p44/42 MAPK blocks TPA-induced decrease in SP-A mRNA synthesis.
TPA signaling frequently involves the activation of the PKC-Ras-p44/42 MAPK pathway (12, 21, 40). The results of our initial experiments were suggestive that the classical MAPK pathway (p44/42 MAPK) is activated by TPA in H441 cells (Fig. 3A). To further test this hypothesis, we used U-0126, an inhibitor of MAPK kinase (MEK) 1/2, a dual protein kinase upstream from p44/42 MAPK. Cells were treated with U-0126 (0.013 µM) in the presence or absence of TPA (5 nM) for 24 h. Figure 4A shows that U-0126 dramatically decreased the TPA-induced phosphorylation of p44/42 MAPK in a dose-dependent manner, reaching control levels at the 3 µM concentration. U-0126 also decreased basal levels of p44/42 MAPK phosphorylation in the control cells at high concentrations. Finally, U-0126 blocked the TPA-induced inhibition of SP-A mRNA levels in a dose-dependent manner (Fig. 4B). U-0126 also inhibited basal SP-A mRNA levels at high concentrations. To confirm these results, we repeated the experiment using another well-characterized inhibitor of MEK1, PD-98059. Whereas 24-h treatment with TPA added alone significantly inhibited SP-A mRNA levels to 35 ± 2% of controls, PD-98059 (10 µM) in the presence of TPA (10 nM) restored SP-A mRNA levels to 68 ± 7% of control levels compared with 64 ± 7% of SP-A mRNA in the presence of PD-98059 alone (ANOVA, Dunnett's test, P < 0.05, n = 3). Together, these results indicate an important role for p44/42 MAPK in mediating both basal SP-A mRNA levels and the inhibitory effect of TPA on SP-A mRNA levels.

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Fig. 4. Treatment with U-0126, an inhibitor of p44/42 MAPK phosphorylation, blocks the inhibitory effect of TPA on SP-A mRNA levels. Subconfluent cells were pretreated with U-0126 (0.013 µM) for 1 h and then incubated with TPA (5 nM) for an additional 24 h. The cells were harvested, and total RNA was isolated to perform Northern blot analysis for human SP-A mRNA. In another series of experiments, cells were pretreated with U-0126, stimulated with TPA for 30 min, and harvested, and protein lysates were used for an analysis of phospho-p44/42 MAPK levels. Control cells were not treated with either TPA or the inhibitor. A: representative immunoblot of 2 experiments showing that U-0126 had a dose-dependent inhibitory effect on basal and TPA-stimulated p44/42 MAPK phosphorylation with a maximal inhibition achieved at the 3 µM concentration. B: representative Northern blot for SP-A mRNA (top) and the ethidium bromide-stained rRNA (28S and 18S) in the same samples (bottom). A graph of densitometric data at the bottom demonstrates that U-0126 blocked the TPA-induced inhibition of SP-A mRNA levels in a dose-dependent manner. U-0126 also decreased basal levels of SP-A mRNA in a dose-dependent manner. Data from 3 experiments are presented as means ± SE. *Significant difference from untreated controls, which were made equal to 1; #statistically significant difference from the TPA added alone condition (ANOVA, Dunnett's test, P < 0.05, n = 3).
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Only the classical MAPK pathway, not p38 MAPK or JNK, is involved in TPA signaling.
We have shown that TPA causes an increased and sustained phosphorylation of the SAPKs, p38 MAPK and JNK (Fig. 3, B and C). To determine whether the SAPKs are also involved in TPA signal transduction in the H441 cells, we pretreated the cells with SB-203580 (0.0210 µM), an inhibitor of p38 MAPK
- and
-isoforms, or SP-600125 (0.520 µM), an inhibitor of JNK, for 1 h, and then exposed the cells to TPA (10 nM) for 24 h and measured the relative amount of human SP-A mRNA. Figure 5, A and B, shows that neither SB-203580 nor SP-600125 had any effect on the TPA-induced inhibition of SP-A mRNA levels. SB-203580 also had no effect on basal SP-A mRNA levels, whereas SP-600125 decreased SP-A mRNA levels in a dose-dependent manner. These results are suggestive that neither SB-203580-sensitive p38 MAPK nor JNK mediates the TPA inhibition of SP-A mRNA levels. JNK may be involved in the regulation of basal SP-A mRNA levels, however.

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Fig. 5. Inhibitors of SAPKs do not modulate the TPA inhibition of SP-A mRNA levels. H441 cells were incubated with SB-203580 (SB, 0.0210 µM) or SP-600125 (SP, 0.520.0 µM) for 1 h and then further treated with TPA (10 nM) for an additional 24 h. Controls were not exposed to either TPA or the inhibitors. The cells were harvested, and isolated total RNA was used to perform Northern blot analysis for human SP-A mRNA. A representative Northern SP-A blot along with a photograph of the ethidium bromide-stained rRNA of the respective samples used in the same experiment is shown at the top of each panel. Graphs of densitometric data from several independent experiments are shown at bottom (n = 26). The data are expressed as means ± SE. *Significant difference from untreated controls, which were made equal to 1 (ANOVA, Dunnett's test, P < 0.05). A: effect of SB-203580, an inhibitor of p38 MAPK, on the TPA-induced inhibition of SP-A mRNA. SB-203580 had no effect on basal SP-A levels or on the TPA-inhibition of SP-A mRNA (n = 36). B: effect of SP-600125, an inhibitor of JNK, on the TPA inhibition of SP-A mRNA levels. SP-600125 had a dose-dependent, inhibitory effect on basal SP-A mRNA levels; however, it had no effect on the TPA-mediated inhibition of SP-A mRNA (n = 23).
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The PI 3-kinase pathway is not involved in SP-A inhibition by TPA.
The PI 3-kinase signaling pathway may also be involved in TPA mechanisms of action (11). To examine whether this signaling pathway plays a role in TPA inhibition of SP-A mRNA levels, we evaluated the effects of three inhibitors of the PI 3-kinase pathway on the TPA-mediated decrease in SP-A mRNA levels. Wortmannin and LY-294002 are two structurally and mechanistically different inhibitors of PI 3-kinase with IC50 of
5 nM and 1.4 µM, respectively (42). Rapamycin inhibits the mammalian target of rapamycin, a downstream PI 3-kinase effector that activates p70 S6 kinase with IC50 of
0.040.4 nM (42). None of these PI 3-kinase pathway inhibitors modified the TPA-induced decrease in SP-A mRNA levels (Table 1). In addition, none of them had an effect on basal SP-A mRNA levels. We conclude that the PI 3-kinase pathway probably does not mediate the inhibitory effect of TPA on SP-A mRNA levels.
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Table 1. Effects of inhibitors of the phosphatidylinositol 3-kinase signal transduction pathway on SP-A mRNA levels in the presence or absence of TPA in H441 cells
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PKA or protein phosphatase does not affect TPA inhibition of SP-A.
Previous studies have indicated that either protein phosphatases or PKA may modulate TPA signaling in cells (16, 20, 26, 40). To evaluate this possibility, we treated H441 cells with TPA plus or minus okadaic acid (1200 nM), an inhibitor of protein phosphatase 2A, or H-89 (0.15 µM), a PKA inhibitor, and measured the levels of human SP-A mRNA. Neither okadaic acid nor H-89 had an effect on the TPA-mediated inhibition of SP-A mRNA levels (Fig. 6, A and B). In addition, neither inhibitor affected basal SP-A mRNA levels.

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Fig. 6. The effects of a protein phosphatase inhibitor, okadaic acid (Ok), and a PKA inhibitor, H-89, on the TPA-mediated decrease in SP-A mRNA levels. H441 cells were preincubated with okadaic acid (1200 nM) or H-89 (0.15 µM) for 1 h, then the media were changed, and the cells were incubated with TPA (10 nM) for an additional 24 h. Untreated cells (controls, no inhibitors or TPA) and treated cells were harvested and then RNA was isolated and used in Northern blots for human SP-A mRNA. Top: representative Northern blots and the ethidium bromide-stained rRNA of the corresponding samples. Bottom: densitometric data from several independent experiments presented as means ± SE (n = 25). *Significant difference from untreated controls, which were made equal to 1 (ANOVA, Dunnett's test, P < 0.05). A: Okadaic acid had no significant effect on either basal or TPA-inhibited SP-A mRNA levels at any concentration tested. B: H-89 had no effect on the TPA-induced decrease in SP-A mRNA levels. H-89 also had no significant effect on basal SP-A mRNA levels.
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TPA stimulates the synthesis of c-Jun mRNA and protein.
TPA can modify gene expression via AP-1, a transcription factor that consists of homo- or heterodimers of several proteins, i.e., c-Jun, JunB, JunD, c-Fos, or ATF-2 (22). Hoover and coworkers (18) have reported that TPA inhibits SP-A gene expression in H441 cells via an AP-1 response element localized in first intron of the human SP-A genes. We measured the levels of c-Jun and c-Fos mRNA in H441 cells at various times after TPA application and observed a significant increase in c-Jun mRNA levels after a 1-h incubation with TPA (Fig. 7A). In contrast, c-Fos mRNA was not detectable in the H441 cells in either the control or TPA-treated cells (data not shown). We also measured the total amount of c-Jun protein in TPA-treated and control cells at various time points. The maximal amount of c-Jun protein was detected after 2 h of incubation with TPA, whereas in control cells, c-Jun protein levels tended to decline over time (Fig. 7B). TPA inhibited SP-A mRNA levels during the 6- to 24-h time period (Fig. 7C). These results show that c-Jun mRNA and protein synthesis precede the inhibition of SP-A mRNA by TPA.

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Fig. 7. TPA stimulates synthesis of c-Jun mRNA and protein in time-dependent manner. H441 cells were exposed to TPA (10 nM) for 5 min ('), 15 min, 1 h, 2 h, 6 h, and 24 h. The cells were harvested, and RNA was isolated and used in Northern blot analysis for human c-Jun mRNA (A) and SP-A mRNA (C). In another experiment, H441 cells were incubated with TPA (10 nM) for 15 min, 30 min, 2 h, and 16 h and harvested, and immunoblotting was performed for total c-Jun protein (B). Representative blots are shown on the left, and densitometric data from several independent experiments are depicted on the right. Data were normalized to values in untreated controls at the zero time point (A and C) or the 15-min time point (B). A: TPA increased c-Jun mRNA levels with maximum levels achieved after a 1-h incubation. *Significant difference from untreated controls at the zero time point (means ± SE, ANOVA, Dunnett's test, P < 0.05, n = 4). B: TPA increased the amount of c-Jun protein in time-dependent manner. The maximal amount of c-Jun protein was detected after a 2-h incubation with TPA. In the untreated control cells, the amount of c-Jun protein tended to decrease over time. *Significant difference from respective untreated controls (unpaired t-test, P < 0.05, n = 3). C: TPA inhibited SP-A mRNA levels with maximal effect at the 24-h time point. Data from 23 experiments are expressed as means ± SE.
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Inhibition of phospho-p44/42 MAPK correlates with inhibition of c-Jun gene expression.
In further experiments, we evaluated the phosphorylation state of MAPK family protein kinases and their substrates in the presence of PKC and MAPK inhibitors and, in parallel experiments, examined the effect of these inhibitors on the TPA-mediated increase in c-Jun gene expression. TPA, when added alone, increased the phosphorylation of p44/42 MAPK, p38 MAPK, and JNK (Fig. 8A). TPA also increased the phosphorylation of ATF-2 and c-Jun, two proteins that can form AP-1 complexes and are targets of p38 MAPK and JNK (Fig. 8A). TPA increased the levels of c-Jun mRNA (Fig. 8B). Inhibition of p44/42 MAPK phosphorylation by U-0126 was accompanied by decreased JNK phosphorylation, as well as decreased phosphorylation of ATF-2 and c-Jun, both substrates of JNK. These effects correlated well with the complete inhibition of TPA-stimulated c-Jun gene expression by U-0126. An inhibitor of PKC, GF-109203X, partially blocked the TPA-induced increase in the phosphorylation of p44/42 MAPK and inhibited the TPA-mediated increase in c-Jun gene expression. Unexpectedly, GF-109203X completely blocked the TPA-induced phosphorylation of p38 MAPK. An
- and
-p38 MAPK inhibitor, SB-203580, did not affect the TPA-mediated increase in phosphorylated p38 MAPK or ATF-2 and partially inhibited the increased phosphorylation of c-Jun. An inhibitor of JNK, SP-600125, completely blocked the increased phosphorylation of JNK and its substrate, c-Jun. However, neither of the SAPK inhibitors, SB-203580 or SP-600125, had an effect on TPA-stimulated c-Jun gene expression (Fig. 8B). The total amounts of p44/42 MAPK, p38 MAPK, or JNK1 were not affected by any of the inhibitors (data not shown). Together these findings show that the TPA-stimulated c-Jun gene expression is probably mediated via the activation of PKC upstream of p44/42 MAPK activation. In contrast, activation of the two SAPKs, p38 MAPK and JNK, and subsequent phosphorylation of their substrates, ATF-2 and c-Jun, are probably not involved in the TPA-induced c-Jun gene expression.

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Fig. 8. Effect of signal transduction inhibitors on the phosphorylation of signal transduction protein kinases and their substrates and on the levels of c-Jun mRNA. H441 cells were pretreated with U-0126 (U0, 1 µM), GF-109203X (1 µM), SB-203580 (5 µM), SP-600125 (20 µM), or vehicle (controls) for 1 h and then exposed to TPA (10 nM) for an additional 30 min. Cells were harvested, and protein lysates used for immunoblotting using phosphospecific antibodies directed against p44/42 MAPK, p38 MAPK, JNK, activating transcription factor (ATF)-2, and c-Jun. In other experiments, cells pretreated with the indicated inhibitors or vehicle (controls) were incubated with TPA (10 nM) for an additional 1 h and used to perform Northern blot analysis for human c-Jun mRNA. A: representative immunoblots from 2 experiments are shown for phospho-p44/42 MAPK, phospho-p38 MAPK, phospho-JNK, phospho-ATF-2, and phospho-c-Jun. The TPA-mediated increase in p44/42 MAPK was inhibited by U-0126 and GF-109203X; the increase in phospho-p38 MAPK was inhibited by GF-109203X; the increase in phospho-JNK was inhibited by U-0126 and SP-600125. The TPA-mediated increase in phospho-ATF-2 was slightly decreased by U-0126, GF-109203X, and SP-600125. The TPA-mediated increase in phospho-c-Jun was decreased by U-0126, SB-203580, and SP-600125. B: densitometric data from 3 experiments showing the modulation of c-Jun gene expression by the various signal transduction inhibitors and TPA. Data are presented as means ± SE; *significant difference from untreated controls, which were made equal to 1 (ANOVA, Dunnett's test, P < 0.05, n = 3). TPA-induced c-Jun gene expression was inhibited by U-0126 and GF-109203X but was not inhibited by SB-203580 or SP-600125.
|
|
 |
DISCUSSION
|
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PKC is a family of lipid-regulated serine/threonine protein kinases that is divided into three classes: conventional (
-,
I-,
II-, and
-isoforms), novel (
,
,
,
, and µ) and atypical (
and
/
). Conventional PKC isoforms are activated by Ca2+ and DAG, novel isoforms are only DAG dependent, whereas atypical isoforms are stimulated by neither Ca2+ nor DAG. The activity of PKC isoforms is regulated by phosphorylation, localization, and substrate specificity. Phorbol esters and DAGs bind to cysteine-rich domains with high affinity and activate conventional and novel PKC isoforms in the presence of PS. Phorbol esters are also known to bind to nonkinase, DAG/phorbol ester receptors (37, 38).
Rat type II alveolar epithelial cells, both adult and newborn, contain PKC-
, -
I, -
II, -
, -
, -
, -
, and -µ (15). PKC isoforms
,
II,
, and
have been shown to be present in H441 cells (2). In rat type II cells, the phorbol ester TPA activates PKC-
, -
I, -
II, -
, and -
and has been shown to target PKC-µ to a membrane fraction (14). In fetal rabbit type II cells, TPA causes the translocation of PKC activity to a lamellar body fraction (39). TPA stimulates PKC activity associated with a membrane fraction rather than the cytosolic fraction of lung epithelial cell homogenates (2).
Activation of PKC-
by TPA increases the permeability of cellular tight junctions, which impairs the barrier function of epithelia and results in the movement of growth factors from luminal/apical fluids into lateral, intracellular spaces in epithelial cell lines and to interstitial fluid spaces in colon cancers (5). In acute lung injury, the activation of PKC, mainly PKC-
, can increase endothelial permeability, which can in turn lead to pulmonary edema and RDS. TPA, which is a potent activator of PKC, is used experimentally to simulate characteristics of acute lung injury, edema or RDS (43).
PKC isoforms are characterized by different signaling pathways that modulate gene expression and lead to terminal events such as neuronal differentiation, monocytic differentiation, and the induction of apoptosis (19, 27, 45). In two previous studies, it was shown that phorbol esters inhibit the gene expression of SP-A and -B in a dose- and time-dependent manner (34, 49). In H441 cells and human fetal lung explants, TPA profoundly decreases the SP-A transcription rate within 68 h of exposure; this effect lasts for 24 h (34, 49). However, there is no information available concerning the intracellular signaling pathways that are activated by TPA to inhibit surfactant protein gene expression.
To verify the involvement of certain PKC isoforms in TPA-mediated SP-A inhibition, we used two inhibitors of PKC, i.e., GF-109203X and PKCI. PKCI inhibits only conventional PKC-
and -
, whereas GF-109203X inhibits conventional and novel PKC isoforms, i.e.,
,
I,
II,
, and
. Although PKCI failed to modify the TPA inhibition of SP-A, GF-109203X was able to partially restore SP-A mRNA levels decreased by exposure to TPA. We conclude that novel PKCs rather than the conventional
- and
-isoforms mediate the TPA inhibition of SP-A mRNA levels.
Several studies are suggestive that TPA activates protein kinases of the MAPK family (1, 2, 19, 41). Awasthi and King (2) have previously reported TPA-mediated activation of p44/42 MAPK and p38 MAPK in H441 cells. In the present study, we confirmed that the phosphorylation/activation of p44/42 MAPK, p38 MAPK, and JNK are increased by TPA in a time-dependent manner, reaching maximum after 1530 min of application. To determine whether these protein kinases are involved in the TPA-mediated inhibition of SP-A mRNA levels, we used various inhibitors of the MAPKs. We found that neither an
- and
-p38 MAPK inhibitor, SB-203580, nor a JNK inhibitor, SP-600125, modified the TPA-mediated inhibition of SP-A mRNA levels. Because SB-203580 only inhibits the
- and
-isoforms of p38 MAPK, and SB-203580 did not inhibit the TPA-induced phosphorylation of ATF-2, it is possible that the H441 cells express the
- or
-forms of p38 MAPK. Further investigation will be necessary to evaluate whether the SB-203580-insensitive isoforms of p38 MAPK, i.e., the
- or
-isoforms, contribute to TPA-inhibition of SP-A. In contrast, when we used two inhibitors of the p44/42 MAPK pathway, i.e., PD-98059 and U-0126, which block MEK1 or MEK1/2, respectively, the TPA-mediated inhibition of SP-A mRNA levels was abolished. We conclude that p44/42 MAPK pathway plays a major role in transducing the TPA inhibitory signal to the SP-A gene.
Many cell types use the classical MAPK/ERK pathway to transmit signals induced by phorbol esters. For example, in MCF7 breast cancer cells, TPA induces a prolonged activation of ERK2 (1). In addition, PD-98059, an inhibitor of MAPK, blocks the inhibitory effect of TPA on cell cycle entry (1). TPA increases serine phosphorylation of Shc and activates the Ras/ERK signaling pathway in NIH 3T3 fibroblasts (12). TPA inhibits the induction of apoptosis in HL-60 cells in an ERK-dependent manner (44). Phorbol ester inhibits glucose-6-phosphatase gene expression via the activation of MEK and ERK in H4IIE hepatoma cells (40). In a gastric cancer cell line (AGS-B), TPA treatment increases tyrosine phosphorylation of ERK1 and 2, Elk-1- and c-Myc-dependent transactivation and stimulates the transcription of human histidine decarboxylase in a Raf-dependent, Ras-independent fashion (17).
In the present study, we demonstrated that GF-109203X, a PKC inhibitor, partially inhibited the increased phosphorylation of p44/42 MAPK induced by TPA treatment. This result is consistent with the concept that the novel PKC isoforms activated by TPA are probably coupled to the MAPK pathway in H441 cells.
Several other signal transduction pathways have been reported to be involved in TPA-mediated regulation of gene expression. These include the PI 3-kinase pathway, protein phosphatases, and the PKA pathway (3, 11, 16, 20, 26, 40). Our experimental results using multiple inhibitors of each of these signaling pathways rule out the possibility that any of them plays an important role in the TPA-mediated inhibition of SP-A mRNA levels.
In our study, TPA affected SP-A gene expression after prolonged exposure, for 24 h. TPA causes permanent activation of PKC and could potentially cause a depletion in PKC as a result of dephosphorylation and proteolysis (32). However, the low levels of TPA, 10 nM, used in our study are much less than the TPA levels required to deplete PKC in other studies, for instance, 200 nM in H441 cells (2), 1 µM in rat pituitary cells (6), or 2.5 µM in bovine luteal cells (4). In addition, the PKC isoforms that are most sensitive to depletion are the conventional isoforms, PKC-
and -
, which are probably not involved in inhibition of SP-A gene expression by TPA (24). Okadaic acid, which prevents dephosphorylation of PKC, failed to reverse the TPA inhibition of SP-A in the present study (16). Inhibitors of PKC usually cause the same effect as a depletion of PKC. In our study, both PKC inhibitors we used, GF-109203X and PKCI, added alone, did not affect SP-A mRNA levels. Finally, in the study of Awasthi and King (2), in H441 cells, PKC depletion resulted in fully inactivated p44/42 MAPK and partially inactivated p38 MAPK. In contrast, in our study, prolonged exposure of the H441 cells to low levels of TPA resulted in the activation of the three MAPKs to levels three- to fourfold above the controls. We conclude that PKC depletion is probably not a significant factor in the prolonged TPA-mediated inhibition of SP-A mRNA levels at low levels of the stimulus.
Downstream nuclear events in TPA signaling pathways leading to the regulation of gene expression include the activation of AP-1 complex, NF-
B, the CREB family of proteins, or Sp1 (9, 23, 46, 52). TPA causes organ-specific stimulation of AP-1 activity and phosphorylation of p44/42 and p38 MAPKs in transgenic mice that express an AP-1 luciferase reporter gene (53). Pryhuber and coworkers (35) showed that induction of NF-
B binding activity was not involved in the TPA-mediated inhibition of SP-A gene expression in H441 cells. Hoover and coworkers (18) have characterized a TPA-sensitive AP-1 binding element in the first intron of human SP-A genes and speculated that this response element binds an AP-1 complex consisting of a c-Jun homodimer. We found that TPA increases the expression of an early responding gene, c-Jun, with maximum effect at the 1-h time point followed by increase in the synthesis of c-Jun protein at the 2-h time point. c-Fos mRNA was not detectable in the cells. SP-A mRNA levels declined only after a 6-h exposure to TPA. In agreement with the hypothesis that TPA acts via c-Jun to decrease SP-A mRNA levels, we found that exposure of the H441 cells to U-0126, an MEK1/2 inhibitor, and GF-109203X, a PKC inhibitor, both of which block the inhibitory effects of TPA, decreased c-Jun mRNA levels. We conclude that TPA probably inhibits SP-A gene expression via a specific PKC isoform(s) that activates the p44/42 MAPK signaling pathway and increases c-Jun synthesis.
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GRANTS
|
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The research was supported by National Institutes of Health Grants HL-50050 and DK-25295.
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ACKNOWLEDGMENTS
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The authors thank Jean Gardner for typing this manuscript.
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FOOTNOTES
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Address for reprint requests and other correspondence: J. M. Snyder, Dept. of Anatomy and Cell Biology, Roy J. and Lucille A. Carver College of Medicine, Univ. of Iowa, 51 Newton Rd., 1-550 BSB, Iowa City, IA 52242-1109.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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