Insulin utilizes the PI 3-kinase pathway to inhibit SP-A gene expression in lung epithelial cells
© The Author(s) 2002
Received: 17 January 2002
Accepted: 17 July 2002
Published: 23 October 2002
It has been proposed that high insulin levels may cause delayed lung development in the fetuses of diabetic mothers. A key event in lung development is the production of adequate amounts of pulmonary surfactant. Insulin inhibits the expression of surfactant protein A (SP-A), the major surfactant-associated protein, in lung epithelial cells. In the present study, we investigated the signal transduction pathways involved in insulin inhibition of SP-A gene expression.
H441 cells, a human lung adenocarcinoma cell line, or human fetal lung explants were incubated with or without insulin. Transcription run-on assays were used to determine SP-A gene transcription rates. Northern blot analysis was used to examine the effect of various signal transduction inhibitors on SP-A gene expression. Immunoblot analysis was used to evaluate the levels and phosphorylation states of signal transduction protein kinases.
Insulin decreased SP-A gene transcription in human lung epithelial cells within 1 hour. Insulin did not affect p44/42 mitogen-activated protein kinase (MAPK) phosphorylation and the insulin inhibition of SP-A mRNA levels was not affected by PD98059, an inhibitor of the p44/42 MAPK pathway. In contrast, insulin increased p70 S6 kinase Thr389 phosphorylation within 15 minutes. Wortmannin or LY294002, both inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase), or rapamycin, an inhibitor of the activation of p70 S6 kinase, a downstream effector in the PI 3-kinase pathway, abolished or attenuated the insulin-induced inhibition of SP-A mRNA levels.
Insulin inhibition of SP-A gene expression in lung epithelial cells probably occurs via the rapamycin-sensitive PI 3-kinase signaling pathway.
Fetuses of diabetic mothers with uncontrolled blood glucose levels tend to be hyperglycemic and hyperinsulinemic . An increased incidence of neonatal respiratory distress syndrome (RDS) has been observed in infants of diabetic mothers . RDS is caused by inadequate amounts of pulmonary surfactant due to delayed lung development . It has been proposed that high insulin levels can delay lung development in the fetus of the diabetic mother .
Surfactant, a lipoprotein comprised of phospholipids (~80%), cholesterol (~10%), and proteins (~10%), functions to reduce surface tension and prevents alveolar collapse at end expiration . The surfactant-associated proteins (SP) A, B, C and D, which are required for proper surfactant function, are developmentally and hormonally regulated . We, and others, have shown that the SP-A levels in the amniotic fluid of diabetic mothers are significantly decreased . Low SP-A levels in amniotic fluid have been correlated with an increased incidence of neonatal RDS . Our previous studies have shown that insulin down-regulates SP-A mRNA and protein levels in human lung epithelial cells in a concentration and time-dependent manner via an inhibition of gene transcription [6, 7].
Insulin transduces its cellular signal in most cell types via two signaling pathways, i.e., the p44/42 mitogen-activated protein kinase (MAPK) pathway and the phosphatidylinositol 3-kinase (PI 3-kinase) pathway . Insulin initially binds to the insulin receptor causing autophosphorylation of the β-subunit of the receptor . The protein kinase domain of the β-subunit then phosphorylates tyrosine residues in insulin receptor substrate (IRS)-1, IRS-2, IRS-3, or Shc, which then activates the p44/42 MAPK signaling cascade . PD98059 is a cell permeable, non-competitive, reversible inhibitor of MAPK/extracellular signal-regulated kinase kinase (MEK), a protein kinase upstream of p44/42 MAPK . Tyrosine phosphorylated IRS also activates the PI 3-kinase pathway [8, 10]. There are at least three, well-characterized inhibitors of the PI 3-kinase pathway, i.e., wortmannin, LY294002, and rapamycin [11–13]. Wortmannin and LY294002 block the activation of PI 3-kinase itself [11, 12], while rapamycin prevents the phosphorylation of a downstream effector in the pathway, p70 S6 kinase .
In the present study, we used two experimental models, a human lung adenocarcinoma cell line, NCI-H441, and human fetal lung explants. H441 cells resemble bronchiolar epithelial Clara cells in phenotype and produce both SP-A and SP-B mRNA and protein . The hormonal regulation of SP-A and SP-B gene expression in H441 cells is similar to that observed in differentiated type II cells in human fetal lung explants [6, 7, 15, 16]. We treated H441 cells with the four inhibitors of signal transduction: wortmannin, LY294002, rapamycin and PD98059, to elucidate which signaling pathways are activated when insulin inhibits SP-A gene expression. To confirm these results, we repeated key experiments with insulin and the signal transduction inhibitors using human fetal lung explants. We then examined the levels and phosphorylation state of key enzymes in the p44/42 MAPK and PI 3-kinase signaling pathways. We found that insulin probably inhibits SP-A gene expression in lung epithelial cells via the rapamycin-sensitive PI 3-kinase pathway.
Materials and methods
Cell and explant cultures
A human lung adenocarcinoma cell line, NCI-H441, was maintained in vitro in monolayer culture in 10% fetal bovine serum in the presence of penicillin (100 U/ml), streptomycin (100 μg/ml) and fungizone (0.25 μg/ml) at 37°C in a 5% CO2 atmosphere . Culture media were changed every 3 days and cells were passed (1:4) weekly. For the inhibitor experiments, the H441 cells were grown until ~80% confluent, then incubated in serum-free media for 24 hours prior to an experiment. The cultured cells were subsequently exposed to fresh serum-free media and pretreated for 30–60 minutes with either control media that contained the vehicle for the inhibitors (dimethyl sulphoxide) or with media that contained wortmannin (5–200 nM), LY294002 (2–50 μM), rapamycin (1–100 nM), or PD98059 (2.5–25 μM). After pretreatment, either insulin (2.5 μg/ml) or vehicle (dilute HCl) was added for an additional 12–16 hours. Experiments were repeated three to five times unless otherwise noted.
Human lung tissue was obtained from mid-tremester abortuses (15–21 weeks old), dissected free from blood vessels and conducting airways, then minced with a sterile razor blade into ~1 mm3 explants . The explants were maintained on lens paper-covered stainless steel grids at the air-media interface in Waymouth's media at 37°C and 5% CO2 for 6 days. The media were changed daily. On the last day of culture, explants were pretreated with signal transduction inhibitors, either wortmannin, rapamycin or PD98059, for 30–60 min and then further treated with insulin (2.5 μg/ml) for 12–24 hours. Experiments were performed in duplicate and were repeated twice.
Porcine insulin was purchased from Calbiochem (San Diego, CA, USA), wortmannin, LY294002, and rapamycin were purchased from Sigma-Aldrich Company (St. Louis, MO, USA), and PD98059 was purchased from New England Biolabs (Beverly, MA, USA). Insulin was prepared as 2.5 mg/ml stock solution in ~0.01 N HCl, aliquoted and stored at -80°C. Wortmannin, LY294002, rapamycin and PD98059 were reconstituted in dimethyl sulphoxide as 1 mM, 50 mM, 50 μM and 10 mM stock solutions, respectively, and stored at -80°C in aliquots. Insulin causes a time- and dose-dependent inhibition of SP-A gene expression with maximum effect at 0.25–2.5 μg/ml (~40 to 400 nM) . In order to achieve a maximal inhibitory effect of insulin, we used a concentration of 2.5 μg/ml in the present study.
Transcription run-on assay
Nuclear isolation, transcription elongation reactions and hybridizations were performed as described previously with minor modifications . Subconfluent H441 cells were incubated in serum-free media for 24 hours and then exposed to media plus either vehicle or insulin (2.5 μg/ml) for an additional 1, 4, 8 and 24 hours. The cells were then rinsed and trypsinized, and nuclei from control and treated cells were harvested. The transcription elongation reaction was performed with 20 × 106 nuclei. Labeled, newly synthesized RNA was then isolated and purified from the nuclei. Nytran membranes with immobilized cDNAs for the BlueScript vector, human SP-A and human α-actin were prehybridized in 1 ml of buffer (50% formamide, 5 X SSC, 5 X Denhardt's solution, 100 μg/ml denatured herring sperm, 0.1% SDS) for 4 hours at 45°C and then hybridized to the labeled RNAs (6 × 106 cpm in the presence of 500 units of RNasin) in duplicate for an additional 60 hours. Afterwards, the hybridized membranes were washed twice in 0.2 X SSC with 0.1% SDS at 55°C for 1 hour, once in 2 X SSC at 55°C for 15 min, once in 2 X SSC with 10 μg/ml RNase A at 37°C for 30 min and then rinsed twice in 2 X SSC at room temperature for 15 min. Membranes were exposed to a Storage Phosphor Screen (Molecular Dynamics, San Francisco, CA, USA) for 3–5 days, scanned using a PhosphorImager (Molecular Dynamics) and data quantitated using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
Northern blot analysis
Northern blot analysis was used to semi-quantitate SP-A mRNA levels. H441 cells were harvested and total RNA isolated as described previously . Equal amounts of total RNA from each condition were separated on agarose gels, transferred to Nytran-plus membranes (Schleicher and Schuell, Keene, NH, USA), and subsequently hybridized with a radiolabeled human SP-A cDNA. Radioactive bands were detected using X-ray film after a 1–4 hour exposure with an intensifier screen at -70°C. The relative intensity of the reactive bands was estimated by densitometry and corrected for RNA loading as described previously .
To perform a northern blot analysis of the human fetal lung explant samples, 4 μg of total RNA from each condition were immobilized on Nytran-plus membranes (0.2 μm, Schleicher and Schuell) in triplicate in denaturing solution (66 % formamide, 7.9 % formaldehyde, 26 mM MOPS buffer, pH 7.0, 1.3 mM EDTA, 0.066 M sodium acetate) using a slot blot apparatus. The membranes were then processed for northern blot analysis as described above. In addition, 10 μg of total RNA from each explant sample were separated on agarose gels to ensure the quality of the RNA samples and to correct for RNA loading.
The total amount and phosphorylation states of p44/42 MAPK and p70 S6 kinase were assessed using immunoblot analysis according to the methods described by Sharma and coworkers . H441 cells were incubated in serum-free media for 24 hours then exposed to fresh control media plus either insulin (2.5 μg/ml) or vehicle for 15 min, 30 min, 2 hours or 16 hours. Control and treated cells were rinsed twice with PBS and then incubated with 1 ml of lysis 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% NP-40, 1 mM phenylmethylsulfonyl fluoride) for 1 hour with shaking at 4°C. Equal amounts of protein lysates from each condition in 1X sample buffer (125 mM Tris-HCl, pH 6.8, 1% β-mercaptoethanol, 2% SDS, 5% glycerol, 0.003% bromphenol blue) were separated on 7.5 or 10% polyacrylamide gels and subsequently transferred to nitrocellulose membranes. Immunoblotting of membranes for phospho-MAPK (extracellular signal-regulated kinase [Erk]1/2) was performed using a Phospho-MAPK Antibody Sampler kit (Cell Signaling, Beverly, MA, USA) according to the manufacturer's instructions. The phospho-MAPK antibody recognizes dual phosphorylated Erk1 and Erk2 at Thr202/Tyr204. Total p44/42 MAPK was detected with rabbit polyclonal anti-MAPK (Erk1) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), according to the manufacturer's instructions. Membranes were then incubated with a secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG (1:4000, ICN Biomedical Research Products, Costa Mesa, CA, USA). The reactive bands were detected using chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA). Immunoblotting experiments for the detection of total and phosphorylated p70 S6 kinase were performed using a PhosphoPlus p70 S6 Kinase (Thr389, Thr421/Ser424) Antibody kit according to the manufacturer's instructions (Cell Signaling).
Lactate dehydrogenase assay
Lactate dehydrogenase (LDH) activity was determined in cell culture media. Pyruvate conversion into lactate was measured in 0.2 M Tris-HCl (pH 7.2) buffer in the presence of sodium pyruvate (0.037 mg/ml, Fisher Scientific, Fair Lawn, NJ, USA) and the reduced form of β-nicotinamide adenine dinucleotide (0.057 mg/ml, Sigma) at 22°C by measuring absorbance at 340 nm. LDH activity was determined as the rate of decrease in absorbency per min using a standard curve for lactate hydrogenase (2–40 mU, L-lactic dehydrogenase, porcine muscle, Sigma). LDH activity in the media samples was normalized to controls, which were made equal to one.
All experiments were repeated at least three times unless otherwise stated. Student's t-test or one-way analysis of variance (ANOVA) followed by Dunnett's test was used to estimate the statistical significance of the results .
Lactate dehydrogenase assay
LDH activity was measured in media obtained from control H441 cells and cells treated with the various signal transduction inhibitors. LDH activity was not different from controls in cells treated for 12 to 16 hours with either 200 nM wortmannin, 5 μM LY294002, 20 nM rapamycin, or 10 μM PD98059 (data not shown).
Effects of signal transduction inhibitors and insulin on SP-A mRNA levels
To confirm the inhibitory effect of wortmannin on insulin action, a structurally different PI 3-kinase inhibitor, LY294002, was used. LY294002 is a competitive inhibitor of the PI 3-kinase ATP-binding site, with an IC50 = 1.4 μM in vitro. LY294002, added alone, decreased SP-A mRNA levels at concentrations greater than 10 μM (data not shown). However, 2 μM LY294002 abolished the inhibitory effects of insulin, but did not affect basal SP-A mRNA levels. In the control group, insulin significantly decreased SP-A mRNA levels to 57 ± 11% of control levels, however, in presence of 2 μM LY294002, insulin only decreased SP-A mRNA levels to 87 ± 15% of control levels versus 99 ± 3% of control levels in samples treated with LY294002 alone (mean ± SE, n = 4, n indicates the number of independent experiments).
Rapamycin inhibits activation of p70 S6 kinase, a downstream effector in the PI 3-kinase signaling pathway . Rapamycin forms a complex with FK506-binding protein 12 and this complex blocks the kinase activity of mTOR (mammalian target of rapamycin), a kinase upstream of p70 S6 kinase . The IC50 for p70 S6 kinase inhibition by rapamycin is ~0.04–0.4 nM . When added alone at concentrations of 1–100 nM, rapamycin did not affect SP-A mRNA levels (Fig. 1B). However, rapamycin abolished the insulin-mediated inhibition of SP-A gene expression in a dose-dependent manner.
To examine whether the p44/42 MAPK pathway mediates insulin signaling in the H441 cells, PD98059, an inhibitor of the p44/42 MAPK pathway, was used . PD98059 binds to the dephosphorylated form of MEK and prevents its activation in vivo with an IC50 of 10 μM . PD98059, added alone, significantly decreased SP-A mRNA levels in a dose-dependent manner (Fig. 1C). PD98059 had no effect on the insulin inhibition of SP-A mRNA levels.
Transcription rate of human SP-A
Phosphorylation-activation of protein kinases
Immunoblot analysis of control and insulin-treated H441 cells using phosphospecific antibodies for p44/42 MAPK and p70 S6 kinase revealed that both kinases were in a partially activated state at the 15 min time point. The phosphorylation of the protein kinases in the controls declined over time and remained at minimum levels after 30 min to 2 hours of incubation. We attribute this partial activation of the signaling kinases to the media change that occurred immediately prior to insulin or vehicle addition .
Since it is possible that the insulin-mediated stimulation of p44/42 MAPK phosphorylation had ended within a 15 min exposure to insulin, we performed additional experiments in order to evaluate the possibility of a shorter time course of insulin action. Serum-starved cells were incubated with fresh serum-free media for 1 hour at 37°C (in order to return the phosphorylation levels of p44/42 MAPK to the basal state) and then treated with insulin (2.5 μg/ml) or vehicle for 5 min. Insulin did not activate p44/42 MAPK within this time frame. The intensity of the immunoreactive bands of phospho-p44/42 MAPK from insulin-treated cells was 0.92 ± 0.10 relative to controls, which were made equal to one (data are the mean of two experiments performed in duplicate plus or minus the standard deviation of the mean). In order to prove that p44/42 MAPK can be stimulated in H441 cells by an activator of this kinase, some cells were also treated with 10 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong stimulator of p44/42 MAPK in H441 cells, for 5 min . TPA caused an approximately twofold increase in p44/42 MAPK phosphorylation over control levels.
p70 S6 kinase
Inhibitors of p44/42 MAPK and p70 S6 kinase
Rapamycin completely abolished the phosphorylation of Thr389 in p70 S6 kinase in insulin-treated cells and also abolished the basal phosphorylation of p70 S6 kinase in control cells, at both time points examined (Fig. 6C). Rapamycin also down regulated the Thr421/Ser424 phosphorylation in p70 S6 kinase in control and insulin-treated cells at both time-points (Fig. 6E). PD98059 did not affect Thr389 or Thr421/Ser424 phosphorylation in p70 S6 kinase in insulin-treated cells at either time point, but slightly decreased their phosphorylation when added alone (Figs. 6D and 6F).
Nothing is known about the relationship between signaling pathways and the regulation of SP-A gene expression by insulin. In contrast, insulin signal transduction pathways are well-characterized in a wide variety of cell types [8, 10]. Insulin is known to primarily transduce its cellular actions via two signaling pathways, the p44/42 MAPK pathway and the PI 3-kinase pathway [8, 10]. In the present study, we investigated the signaling pathways that mediate the inhibitory effects of insulin on the expression of the SP-A genes in human lung epithelial cells.
Two inhibitors of PI 3-kinase, wortmannin and LY294002, and an inhibitor of p70 S6 kinase activation, rapamycin, blocked the inhibitory effect of insulin on SP-A gene expression. We confirmed these observations by showing that insulin increased the phosphorylation of a specific rapamycin-sensitive residue (threonine 389), present in the linker region of p70 S6 kinase, a phosphorylation event that is known to activate this enzyme . In contrast, PD98059, an inhibitor of p44/42 MAPK activation, did not block the inhibitory effects of insulin on SP-A gene expression. In addition, insulin had no effect on p44/42 MAPK phosphorylation during a 2-hour incubation. The effects of the PI 3-kinase and the MAPK pathway inhibitors on SP-A gene expression were evaluated using two experimental models, a human immortalized epithelial cell line (H441 cells) and human fetal lung explants. We also demonstrated that transcriptional inhibition of SP-A gene commences within 1 hour of exposure to the hormone. Together, our results are suggestive that insulin activates the rapamycin-sensitive PI 3-kinase pathway in lung epithelial cells and that this event leads to an inhibition of SP-A gene transcription.
We found that wortmannin and rapamycin had no effect on basal SP-A mRNA levels, at any concentration tested. However, PD98059, added alone, abolished p44/42 MAPK phosphorylation in control cells and also inhibited basal SP-A mRNA levels in a dose-dependent manner. We hypothesize that a basal level of p44/42 MAPK activity may be required for SP-A gene expression. Chess and coworkers have shown that transforming growth factor-α and hepatocyte growth factor increase the phosphorylation of p44/p42 MAPK in H441 cells . Epidermal growth factor, which binds to the same receptor as transforming growth factor-α, increases SP-A content in human fetal lung . Hepatocyte growth factor has also been shown to upregulate the synthesis of SP-A in rat type II cells . Thus, phosphorylation of p44/42 MAPK may be involved in the up-regulation of SP-A gene expression, results consistent with our observation that inhibition of basal levels of p44/42 MAPK phosphorylation may decrease SP-A gene expression.
In summary, our studies are suggestive that in lung epithelial cells, SP-A gene transcription is inhibited by insulin via the rapamycin-sensitive PI 3-kinase pathway, but not the p44/42 MAPK pathway. In cultured human muscle cells, only the PI 3-kinase – p70 S6 kinase pathway, and not the MAPK pathway, mediates insulin up-regulation of p85α PI 3-kinase gene expression . Similarly, in L6 myotubes, insulin induces hexokinase II gene transcription via the rapamycin-sensitive PI 3-kinase pathway, but not via the MAPK pathway . The PI 3-kinase – p70 S6 kinase pathway, but not the MAPK pathway, mediates insulin induction of glucose-6-phosphate dehydrogenase gene expression in primary rat hepatocytes . In H411E liver cells, insulin down-regulates cAMP-induced phosphoenolpyruvate carboxykinase gene transcription via the PI 3-kinase pathway, but not via the MAPK pathway . Phosphorylated p70 S6 kinase has been shown to be present in the nucleus in insulin-treated cells . Moreover, it has been shown that p70 S6 kinase phosphorylates and activates members of the cAMP response element modulator family of transcription factors . These data raise the possibility that transcriptional regulation of SP-A gene expression occurs via the PI 3-kinase – p70 S6 kinase pathway. Further studies will be required to elucidate the function of p70 S6 kinase in the nuclear events involved in SP-A gene transcription.
Our studies are suggestive that insulin inhibits SP-A gene transcription via the PI 3-kinase pathway in lung epithelial cells, specifically via the rapamycin-sensitive activation of p70 S6 kinase. Insulin does not activate p44/42 MAPK and does not inhibit SP-A gene transcription via the MAPK pathway. Our findings further characterize the signal transduction pathways that control surfactant protein gene expression. This information could lead to the possible modification of these pathways by pharmacological agents in order to modulate the composition of pulmonary surfactant, which is critical for normal respiratory function and for host defense mechanisms in the lung.
analysis of variance
extracellular signal-regulated kinase
- IC50 :
insulin receptor substrate
mitogen-activated protein kinase
MAPK/extracellular signal-regulated kinase kinase
mammalian target of rapamycin
- PI 3-kinase:
respiratory distress syndrome
standard deviation of the mean
standard error of the mean
The authors thank Jean Gardner for typing this manuscript. This research was supported by grants from the National Institutes of Health NIH-HL-50050 and DK-25295.
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