TGF-beta 1 modulates EGF-stimulated phosphatidylinositol 3-kinase activity in human airway smooth muscle cells

Vera P. Krymskaya1, Rebecca Hoffman2, Andrew Eszterhas1, Vincenzo Ciocca1, and Reynold A. Panettieri Jr.1

1 Pulmonary and Critical Care Division, Department of Medicine, and 2 Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulation of phosphatidylinositol (PI) 3-kinase plays an important role in modulating cellular function. We have previously shown that transforming growth factor (TGF)-beta 1 inhibited epidermal growth factor (EGF)-induced human airway smooth muscle (hASM) cell proliferation and that PI 3-kinase activation is a necessary signaling event in mitogen-induced hASM cell growth. In this study, we postulated that TGF-beta 1 may modulate EGF-induced PI 3-kinase activation. To date, no study has examined the effects of TGF-beta 1 on PI 3-kinase activity. In cultured hASM cells, EGF induced a 5.7 ± 1.2-fold activation of PI 3-kinase compared with diluent-treated cells. Although TGF-beta 1 alone did not alter PI 3-kinase activation, TGF-beta 1 markedly enhanced EGF-induced PI 3-kinase activity, with a 16.6 ± 1.9-fold increase over control cells treated with diluent alone. EGF significantly increased the association of PI 3-kinase with tyrosine phosphorylated proteins, and TGF-beta 1 pretreatment before EGF stimulation apparently did not alter this association. Interestingly, TGF-beta 1 did not modulate EGF-induced p70 S6 kinase activity, which is important for the progression of cells from the G0 to the G1 phase of the cell cycle. Immunoprecipitation of type I and type II TGF-beta receptors showed that PI 3-kinase was associated with both type I and type II TGF-beta receptors. TGF-beta 1, however, enhanced PI 3-kinase activity associated with the type I TGF-beta receptor. Although in some cell types inhibition of PI 3-kinase and treatment of cells with TGF-beta 1 mediate apoptosis, cell cycle analysis and DNA ladder studies show that PI 3-kinase inhibition or stimulation of hASM cells with TGF-beta 1 did not induce myocyte apoptosis. Although the inhibitory effects of TGF-beta 1 on hASM cell growth are not mediated at the level of PI 3-kinase and p70 S6 kinase, we now show that activation of the TGF-beta 1 receptor modulates PI 3-kinase activity stimulated by growth factors in hASM cells.

hyperplasia; signal transduction; cytokines; p70 S6 kinase; apoptosis ; transforming growth factor-beta 1; epidermal growth factor

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RECENT EVIDENCE SUGGESTS that patients who have chronic severe asthma develop an irreversible airflow obstruction that is refractory to bronchodilators and anti-inflammatory medications (19). Such an airflow obstruction may be a consequence of persistent structural changes in the airway wall due to frequent stimulation of airway smooth muscle (ASM) by contractile agonists, growth factors, and cytokines. Increased smooth muscle mass, which has been attributed to increases in myocyte number, is a well-documented pathological finding in the airways of chronic severe asthmatic patients. Little information is available, however, with respect to the factors that promote or inhibit human ASM (hASM) cell growth.

Studies suggest a role for phosphatidylinositol (PI) 3-kinase in regulating cell growth, differentiation, transformation, and apoptosis (3, 11). PI 3-kinase, a cytosolic heterodimer, is composed of an 85-kDa (p85) regulatory subunit and a 110-kDa (p110) catalytic subunit (11). The catalytic p110 subunit contains phosphoinositide kinase activity and also functions as a serine-threonine kinase (3). Activated PI 3-kinase specifically phosphorylates PI and other phosphoinositides (PI 4-monophosphate and PI 4,5-diphosphate) on the D-3 position of the inositol ring. In response to growth factor stimulation, PI 3-kinase, which contains SH3 and two SH2 domains on the p85 regulatory subunit, binds directly to the activated receptor or can associate with intermediary cytosolic proteins (11). Studies have shown that epidermal growth factor (EGF) stimulates PI 3-kinase activation (20, 23). In some cell types, PI 3-kinase regulates cell proliferation (3).

In most cell types, transforming growth factor (TGF)-beta 1 binds to transmembrane type I and type II TGF-beta receptors, which contain serine-threonine kinase activity, and forms a heteromeric complex (30). Little, however, is known about the downstream intracellular signaling events that follow TGF-beta receptor binding. In epithelial cells, TGF-beta 1 activates Ras (16) and increases mitogen-activated protein kinase activity (9). This cytokine also induces protein phosphatase 1 activity in keratinocytes (8) and phosphorylation of the adenosine 3',5'-cyclic monophosphate responsive element binding protein in Mv1Lu cells (14). In the carcinoma cell line PC3, TGF-beta 1 decreases protein kinase activity of the Src family tyrosine kinases pp60Src and pp53/56Lyn (1). These studies suggest that the downstream signaling events mediated by TGF-beta 1 are cell-type specific.

Recent data suggest that PI 3-kinase may be involved in TGF-beta 1 signaling. TGF-beta 1 reduces PI 3-kinase activity in polyoma middle T-transformed cell lines (6), and wortmannin, a PI 3-kinase inhibitor, inhibits TGF-beta 1-stimulated chemotaxis in human neutrophil leukocytes (26). Because TGF-beta 1 inhibited EGF-induced hASM cell growth (5) and because PI 3-kinase seems to be involved in growth factor-regulated hASM cell signaling (22), we postulated that TGF-beta 1 modulates mitogen-induced PI 3-kinase activity in hASM cells.

We now show that EGF stimulates PI 3-kinase activity and that TGF-beta 1 markedly potentiates EGF-induced PI 3-kinase activation. Together, our studies suggest that the TGF-beta 1 inhibition of hASM cell mitogenesis likely occurs downstream from PI 3-kinase activation. Importantly, for the first time, our data suggest that activation of the TGF-beta receptor may modulate PI 3-kinase activation induced by mitogens. Although the antiproliferative effects of TGF-beta 1 are not likely mediated by TGF-beta 1 modulation of PI 3-kinase activity, other cellular effects of TGF-beta 1 on matrix secretion or on cell cycle progression may be regulated by PI 3-kinase.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Ham's F-12 medium and trypsin were obtained from GIBCO BRL (Grand Island, NY). [32P]ATP (specific activity, 5,000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL). TGF-beta 1, EGF, PI, PI 4-monophosphate, and wortmannin were purchased from Sigma Chemical (St. Louis, MO). Anti-phosphotyrosine (PTyr) and anti-PI 3-kinase antibodies and the S6 kinase assay kit were obtained from Upstate Biotechnology (Lake Placid, NY). Anti-TGF-beta RI (T-19), anti-TGF-beta RI (V-22), anti-TGF-beta RII (L-21), and p70 S6 kinase (C-18) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC)-conjugated antibody was purchased from Becton-Dickinson (San Jose, CA). The apoptotic DNA ladder kit was obtained from Boehringer Mannheim (Indianapolis, IN). Normal rabbit immunoglobulin (Ig) G control and mouse IgG2B isotype control were obtained from R&D Systems (Minneapolis, MN). All other reagents were purchased from Sigma Chemical.

Cell culture. hASM cells were grown in 100-mm-diameter dishes and were maintained in Ham's F-12 medium supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. Details regarding the characterization of this cell line by indirect immunofluorescence of smooth muscle-specific actin have been previously reported by our laboratory (17). Confluent hASM cells were growth arrested in serum-free F-12 medium supplemented with 0.1% bovine serum albumin (BSA) for 48 h before experiments. In our studies, third and fourth passage hASM cells were used.

Preparation of cell lysates and immunoprecipitation. Growth factors and wortmannin were added to the cells for the indicated times at 37°C. The cells were washed two times with ice-cold wash buffer [137 mM NaCl, 20 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM MgCl2, 1 mM CaCl2, and 0.2 mM vanadate (pH 7.5)] and were lysed in lysis buffer [wash buffer plus 10% (vol/vol) glycerol, 1% (vol/vol) Nonidet P-40 (NP-40), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml of aprotinin, and 10 µg/ml of leupeptin] (23). The lysates were centrifuged at 13,200 g for 10 min. Supernatants, which were normalized for protein content with a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), were incubated with anti-PTyr (5 µg/ml), anti-PI 3-kinase (3 µg/ml), anti-TGF-beta receptor type I (ALK-1; T-19), anti-TGF-beta receptor type I (ALK-5; V-22), and anti-TGF-beta receptor type II (L-21) antibodies (10 µg/ml) for 16 h. Protein A-Sepharose (60 µl; Pharmacia Biotech, Uppsala, Sweden) was then added to the lysates for 2 h at 4°C. The immunoprecipitates were washed three times in phosphate-buffered saline (PBS)-1% NP-40, three times in 0.1 M Tris (pH 7.5)-0.5 M LiCl, and two times in 10 mM Tris-100 mM NaCl-1 mM EDTA, pH 7.5. All solutions contained 0.2 mM vanadate.

PI 3-kinase activity assay. PI 3-kinase activity assays were performed as described (23). Briefly, the sonicated PI in tris(hydroxymethyl)aminomethane (Tris)-ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (0.2 mg/ml final concentration) was added to the immunoprecipitates. The phosphorylation reactions were started by addition of MgCl2, ATP, and [gamma -32P]ATP (30 µCi/sample) to a final concentration of 4 mM MgCl2 and 50 mM ATP for 10 min at room temperature. Reactions were stopped by the addition of 20 µl of 6 N HCl and extracted with 160 µl of chloroform-methanol (1:1). Lipids were separated on oxalate-coated thin-layer chromatography plates (Silica Gel 60, Merck, Darmstadt, Germany) by using a chloroform-methanol-water-ammonium hydroxide (60:40:11.3:2) solvent system. The lipids were then visualized by autoradiography and quantitatively analyzed with a laser densitometry system and the public domain National Institutes of Health Image program (available on the Internet at http://rsb.info.nih.gov/ nih-image/).

Identification of proteins by Western blot assay. Immunoprecipitated proteins were subjected to 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot assays as described previously (23). The blots were exposed to anti-p85 PI 3-kinase antibody (1 µg/ml) in Tris-buffered saline (TBS)-0.5% Tween 20 (TBS-T) overnight at 4°C. After three washes in TBS-T, the nitrocellulose filters were exposed to an anti-rabbit horseradish peroxide secondary antibody (Boehringer Mannheim) at a 1:3,000 dilution. Filters were washed five times in TBS-T and were visualized with a chemiluminescence system (ECL, Amersham).

p70 S6 kinase activity assay. After stimulation with growth factors at the indicated times, the cells were washed two times in ice-cold TBS with 0.2 mM vanadate (pH 8.0) and lysed in TBS (pH 8.0) containing 20 mM NaF, 5 mM EGTA, 1 mM EDTA, 10 mM sodium pyrophosphate, 10 mM p-nitrophenyl phosphate, 1 mM benzamidine, 0.1 mM PMSF, and 1% (vol/vol) NP-40 for 30 min at 4°C (lysis buffer) (24). The lysates were centrifuged at 1,000 g for 2 min, and 3 µg of anti-p70 S6 kinase antibody were added to cell lysates that were normalized for protein content (Bio-Rad Protein Assay, Bio-Rad Laboratories). After incubation for 2 h, 50 µl of protein A-Sepharose were added, and the mixture was rocked for 1 h. The immunoprecipitates were washed two times in lysis buffer, two times in the same buffer without detergents, and two times in assay buffer [20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 25 mM beta -glycerol phosphate, 5 mM EGTA, 1 mM vanadate, and 1 mM dithiothreitol]. The S6 kinase activity of the immunoprecipitates was measured with S6 kinase assay kit components (Upstate Biotechnology).

Cell cycle analysis and DNA ladder assay. The effects of TGF-beta 1 on hASM cell cycle progression and apoptosis were determined by flow cytometric analysis of anti-BrdU-labeled cells or DNA ladder analysis, respectively.

Flow cytometric analysis was performed as previously described, with some modifications (18). Confluent monolayers of hASM cells were growth arrested in serum-free medium supplemented with 0.1% BSA for 48 h; samples were then treated with 10 ng/ml of EGF, 1 ng/ml of TGF-beta 1, or both agents in the presence of 20 µM BrdU for 40 h. The cells were washed two times with PBS containing 1% BSA before being harvested with trypsin-EDTA. After that, the cells were fixed in 70% ethanol at -20°C for 30 min and incubated with monoclonal anti-BrdU FITC-conjugated antibody for 30 min. Excess antibody was removed by washing in PBS. Finally, the samples were resuspended in PBS containing 5 µg/ml of propidium iodine. Dual-wavelength flow cytometric analysis was performed with Coulter Epics-XL (Coulter, Hialeah, FL). Laser excitation for FITC was at 488 nm, and emission was collected at 525 nm. Propidium iodine was detected at 610 nm.

Apoptosis was characterized by DNA fragmentation assay (DNA ladder kit, Boehringer Mannheim). Briefly, the cells were growth arrested for 24 h and then stimulated overnight with 10 ng/ml of EGF and 1 ng/ml of TGF-beta 1, pretreated with 1 ng/ml of TGF-beta 1 for 10 min and then stimulated with 10 ng/ml of EGF, or pretreated with 100 nM wortmannin for 10 min and then stimulated with 10 ng/ml of EGF. Extracted DNA was analyzed with agarose gel electrophoresis with ethidium bromide DNA staining. Experiments were performed in triplicate, and representative experiments are reported.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of TGF-beta 1 on EGF-induced PI 3-kinase activation. To examine whether TGF-beta 1 modulates PI 3-kinase activation, we measured PI 3-kinase activity in hASM cells treated with EGF and TGF-beta 1 and those pretreated with TGF-beta 1 and then stimulated with EGF (Fig. 1). EGF (10 ng/ml) markedly induced PI 3-kinase activity at 1 min that was sustained for 10 min (Fig. 1A). To determine whether EGF activates PI 3-kinase activity, cells were pretreated for 10 min with 100 nM wortmannin, an inhibitor of PI 3-kinase (31), and then stimulated with EGF for 1 min. Wortmannin completely abrogated EGF-stimulated PI 3-kinase activity compared with cells treated with EGF alone (Fig. 1A). These studies suggest that PI 3-kinase is activated by EGF in a time-dependent and specific manner in hASM cells.


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Fig. 1.   Effects of transforming growth factor (TGF)-beta 1 on phosphatidylinositol (PI) 3-kinase in epidermal growth factor (EGF)-stimulated human airway smooth muscle (hASM) cells. A: time course of EGF on PI 3-kinase activity in hASM cells. Growth-arrested cells were stimulated with EGF (10 ng/ml) for various times (n = 3) and compared with those treated with diluent alone. hASM cells were incubated with wortmannin (100 nM) for 10 min and then activated with EGF (10 ng/ml, 1 min). Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine (PTyr) antibody, and PI 3-kinase activity was measured as described in MATERIALS AND METHODS. These data are representative of 3 separate experiments. +, Presence; -, absence. PIP, [32P]phosphatidylinositol monophosphate. B: TGF-beta 1 augments EGF-stimulated PI 3-kinase activity. hASM cells were stimulated with EGF (10 ng/ml, 1 min) or with TGF-beta 1 (1 or 10 ng/ml, 10 min) + EGF (10 ng/ml, 1 min). PI 3-kinase activity was measured as described in MATERIALS AND METHODS. C: TGF-beta 1 augments EGF-stimulated PI 3-kinase activity as shown by densitometric analysis of data presented in B. Data are means ± SE from n separate experiments: EGF-stimulated hASM cells (n = 7); cells stimulated with TGF-beta 1 alone, 1 ng/ml, 10 min (n = 3); and cells treated with TGF-beta 1 (1 ng/ml, 10 min, n = 11; or 10 ng/ml, 10 min, n = 9) and then stimulated with EGF (10 ng/ml, 1 min). D: effects of TGF-beta 1 and EGF on PI 3-kinase association with tyrosine phosphorylated proteins. hASM cells were stimulated with EGF alone (10 ng/ml, 1 min) or were treated with TGF-beta 1 (10 ng/ml, 10 min) and then stimulated with EGF (10 ng/ml, 1 min). Cell lysates were immunoprecipitated with anti-PTyr antibody. Immunoprecipitated proteins were separated on an 8% SDS-polyacrylamide gel electrophoresis, and immunoblot assays were performed with an anti-85-kDa (p85) PI 3-kinase antibody (1 µg/ml). WCL, whole hASM cells lysate; JCL, Jurkat cells lysate. This is a representative immunoblot analysis of 3 separate experiments.

In cells pretreated with TGF-beta 1 and then stimulated with 10 ng/ml of EGF, PI 3-kinase activity was markedly enhanced by TGF-beta 1 compared with cells treated with EGF or diluent alone (Fig. 1B). Quantitative analysis of these experiments revealed that PI 3-kinase activity was increased 5.7 ± 1.2 times in cells stimulated with EGF (seven separate experiments; Fig. 1C). Interestingly, TGF-beta 1 alone did not affect PI 3-kinase activity (four separate experiments) compared with cells treated with diluent alone. In cells treated with TGF-beta 1 before stimulation with EGF, PI 3-kinase activity was augmented markedly by 16.6 ± 1.9-fold (1 ng/ml of TGF-beta 1; six separate experiments) and 17.8 ± 1.8-fold (10 ng/ml of TGF-beta 1; three separate experiments), respectively, compared with diluent-treated control cells. These studies suggest that although TGF-beta 1 alone does not alter PI 3-kinase activity, pretreatment of hASM cells with TGF-beta 1 significantly augments EGF-induced activation of PI 3-kinase.

Growth factor-stimulated PI 3-kinase associates with tyrosine phosphorylated proteins. To determine the effects of EGF, TGF-beta 1, and TGF-beta 1 + EGF on PI 3-kinase association with tyrosine phosphorylated proteins, immunoblot analysis of anti-PTyr immunoprecipitates from cell lysates normalized for total protein was performed (Fig. 1D). After EGF stimulation (10 ng/ml, 1 min), the association of the p85 regulatory subunit of PI 3-kinase with tyrosine phosphorylated proteins was markedly increased compared with control cells. TGF-beta 1 stimulation (1 ng/ml, 10 min) did not alter the amount of p85 compared with cells treated with diluent alone (data not shown). There was no difference in the amount of p85 from cells pretreated with TGF-beta 1 and then stimulated with EGF compared with those stimulated with EGF alone. Identical experiments performed with an isotype-matched nonimmune mouse IgG2B did not immunoprecipitate any p85 protein, suggesting specificity of the anti-PTyr antibody (data not shown). These data suggest that EGF significantly enhances PI 3-kinase association with tyrosine phosphorylated proteins. TGF-beta 1 pretreatment before EGF stimulation apparently does not alter this association. Together, these data suggest that TGF-beta 1 may directly modulate the activation of PI 3-kinase rather than the association of the enzyme with tyrosine phosphorylated proteins.

PI 3-kinase associates with TGF-beta receptors and activation of the type I TGF-beta receptor increases PI 3-kinase activity. To characterize the TGF-beta receptor subtypes that may mediate EGF-induced PI 3-kinase activation, PI 3-kinase association with type I and type II TGF-beta receptors was determined. hASM cell lysates were immunoprecipitated with specific polyclonal antibodies to these receptors [anti-type I (ALK-1, ALK-5) and anti-type II antibodies], and immunoblot analysis was then performed (Fig. 2A). The p85 subunit of PI 3-kinase was associated with both type I and type II TGF-beta receptors. Jurkat cell lysate and whole hASM cell lysate demonstrated considerable quantities of p85 subunit and served as a positive control. Immunoprecipitates, with a nonimmune rabbit IgG, did not precipitate the p85 subunit. In cells treated with mitogen or with a combination of mitogen and TGF-beta 1, PI 3-kinase was not associated exclusively with either the type I or type II receptor. PI 3-kinase activity associated with type II TGF-beta receptor was similar in cells stimulated with TGF-beta 1, EGF, and TGF-beta 1 + EGF compared with those treated with diluent alone (Fig. 2B). TGF-beta 1, however, induced activation of PI 3-kinase associated with the type I TGF-beta receptor (Fig. 2, C and D). These studies revealed that PI 3-kinase is associated in vivo with both TGF-beta receptor subtypes and that TGF-beta 1 stimulation enhances PI 3-kinase activity associated with type I TGF-beta receptor in hASM cells.


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Fig. 2.   PI 3-kinase associates with both type I (RI) and type II (RII) TGF-beta receptors. A: PI 3-kinase immunoprecipitates with type I and type II TGF-beta receptors. hASM cells were stimulated with TGF-beta 1 (1 ng/ml, 10 min) or pretreated with TGF-beta 1 (1 ng/ml, 10 min) and then stimulated with EGF (10 ng/ml, 1 min). hASM cell lysates were immunoprecipitated with polyclonal anti-TGF-beta receptor type I (ALK-1, ALK-5) and anti-TGF-beta receptor type II antibodies (10 µg/ml), and immunoblot analysis was performed with specific antibodies to p85 PI 3-kinase (1 µg/ml). IgG, immunoglobulin G. This is a representative immunoblot analysis of 4 separate experiments. B: PI 3-kinase activity associated with type II TGF-beta receptor. PI 3-kinase activity was measured in anti-TGF-beta receptor type II antibody immunoprecipitates as described in MATERIALS AND METHODS. These data are representative of 4 separate experiments. C: PI 3-kinase activity is associated with type I TGF-beta receptor. hASM cells were treated with diluent (lane 2) or 1 ng/ml of TGF-beta 1 for 30 (lane 3), 45 (lane 4), and 60 min (lane 5). Lane 1, nonimmune rabbit IgG. D: PI 3-kinase activity measured in anti-TGF-beta receptor type I (ALK-5) antibody immunoprecipitates as described in MATERIALS AND METHODS and quantitated. These data are representative of 3 separate experiments.

TGF-beta 1 does not alter p70 S6 kinase activity in hASM cells. p70 S6 kinase activation is necessary for cell progression through the G0 and G1 phases of the cell cycle (15). In addition, PI 3-kinase appears to be the upstream signaling protein that modulates p70 S6 kinase activation (29). To dissect mechanisms by which TGF-beta 1 may inhibit EGF-induced hASM cell proliferation, we investigated whether TGF-beta 1 affects p70 S6 kinase activity. Growth-arrested hASM cells were treated with either EGF for 15 min or TGF-beta 1 for 25 min or were pretreated with TGF-beta 1 for 10 min and then stimulated with EGF for 15 min. After stimulation, cell lysates were prepared, and p70 S6 kinase was immunoprecipitated from the cell lysates that were normalized for total protein. Activation of p70 S6 kinase was measured with an in vitro activity assay as described in MATERIALS AND METHODS. As shown in Fig. 3, EGF stimulated p70 S6 kinase activation in hASM cells by 3.13 ± 0.52-fold compared with cells treated with diluent alone. TGF-beta 1 alone did not alter p70 S6 kinase activity, and pretreatment of hASM cells with TGF-beta 1 did not affect EGF-induced p70 S6 kinase activation. These data suggest that EGF induces activation of p70 S6 kinase in hASM cells and that TGF-beta 1 growth inhibitory effects do not involve p70 S6 kinase.


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Fig. 3.   TGF-beta 1 does not alter p70 S6 kinase activity in hASM cells. Growth-arrested hASM cells were stimulated with EGF for 15 min or TGF-beta 1 for 25 min or were pretreated with TGF-beta 1 for 10 min and stimulated with EGF for 15 min. Activation of p70 S6 kinase was measured in an in vitro activity assay as described in MATERIALS AND METHODS. Cont, control. Data are means ± SE from 3 separate experiments. * P < 0.005 by analysis of variance.

TGF-beta 1 does not induce apoptosis of hASM cells. To determine whether the growth inhibitory effects of TGF-beta 1 were due to induction of apoptosis, we studied whether this cytokine induces DNA fragmentation as determined by DNA ladder analysis. In parallel experiments, cell cycle analysis was performed to confirm that TGF-beta 1 inhibits EGF-induced DNA synthesis. Growth-arrested hASM cells were incubated with 1 ng/ml of TGF-beta 1 overnight, the cells were lysed, and DNA was extracted. As shown in Fig. 4A, DNA fragmentation was not observed in hASM cells treated with TGF-beta 1, TGF-beta 1 + EGF, or wortmannin. As shown in Fig. 4B, cell cycle analysis revealed that TGF-beta 1 inhibited EGF-induced hASM cell progression from the Go/G1 to S phase of the cell cycle and that TGF-beta 1 alone or in combination with EGF did not induce apoptosis (Fig. 4B). These data suggest that growth inhibitory effects of TGF-beta 1 are not mediated by stimulation of apoptosis of hASM cells. Because TGF-beta 1 did not inhibit mitogen-induced p70 S6 kinase activation, an event that occurs early in the G1 phase of the cell cycle, it is likely that inhibitory effects of TGF-beta 1 on hASM cell growth occur late in the G1 phase of the cell cycle.


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Fig. 4.   TGF-beta 1 does not induce apoptosis of hASM cells. A: TGF-beta 1 does not induce DNA fragmentation in hASM cells. Growth-arrested hASM cells were incubated with 1 ng/ml of TGF-beta 1 for 16 h, and DNA was extracted and separated with agarose gel electrophoresis with ethidium bromide DNA staining. Lane 1, base pair markers; lane 2, control; lane 3, EGF; lane 4, TGF-beta 1; lane 5, TGF-beta 1-EGF; lane 6, wortmannin-EGF; lane 7, DNA harvested from an apoptotic monocyte cell line (U-937 cells). B: TGF-beta 1 inhibits EGF-induced hASM cell cycle progression but does not induce apoptosis. Growth-arrested hASM cells were treated in presence of 20 µM bromodeoxyuridine (BrdU) with diluent alone (1), stimulated with 10 ng/ml of EGF (2), treated with 1 ng/ml of TGF-beta 1 (3), and pretreated with 1 ng/ml of TGF-beta 1 for 10 min and stimulated with 10 ng/ml of EGF (4). Cell cycle progression was measured with dual-wavelength staining with anti-BrdU fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody and propidium iodine (PI). Staining with flow cytometric analysis was performed as described in MATERIALS AND METHODS. x-Axis, DNA content measured by PI staining; y-axis, DNA synthesis measured by anti-BrdU FITC staining. PI Int, PI fluorescence intensity; log Grn, log of anti-BrdU FITC fluorescence intensity. a, Go/G1 phase of cell cycle; b, S phase of cell cycle; c, G2/M phase of cell cycle.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Recent studies (3, 11) showed that PI 3-kinase and its products are critical in generating mitogenic signals. Previously, it was determined that EGF is a potent and effective hASM cell mitogen (5) and that wortmannin, a PI 3-kinase inhibitor, abrogates EGF-induced hASM cell growth (19) and bovine ASM cell proliferation (22). In another study, our laboratory (5) has shown that TGF-beta 1 inhibited EGF-induced hASM cell proliferation via a pathway that is independent of mitogen-activated protein kinase activation. In the present study, we postulated that TGF-beta 1 may inhibit hASM cell growth by modulating mitogen-induced activation of PI 3-kinase. TGF-beta 1 alone, however, had no effect on PI 3-kinase activation and, interestingly, augmented PI 3-kinase activity induced by growth factors. In addition, TGF-beta 1 modulation of EGF-induced PI 3-kinase activity did not modulate p70 S6 kinase activity, which is downstream from signaling events stimulated by PI 3-kinase (29). To the best of our knowledge, this is the first report that describes the coupling of TGF-beta receptors to PI 3-kinase activation. Our data also suggest that the growth inhibitory effects of TGF-beta 1 on hASM cells are likely mediated by signaling events downstream from PI 3-kinase or potentially from a parallel signaling pathway. PI 3-kinase activation, therefore, may be necessary but not sufficient for EGF-induced hASM cell growth.

TGF-beta 1, which mediates its effects through type I and type II receptors, is a multifunctional cytokine that stimulates or inhibits cell proliferation depending on the cell type (30). To date, we are aware of no studies that have examined the effects of TGF-beta 1 on PI 3-kinase activation. In epithelial cells, Mulder and Morris (16) and Hartsough and Mulder (9) showed that TGF-beta 1 activated p21ras and p44mapk, signaling events thought to be promitogenic. TGF-beta 1, however, inhibited epithelial cell growth (9, 16). In most cells, TGF-beta 1 binds to constitutively active type II receptors that, in turn, transduce signals by recruitment and phosphorylation of type I receptors (13, 30). To date, no intracellular protein or serine-threonine kinase target has been described that directly associates with TGF-beta receptors. Recent studies using a yeast two-hybrid system showed that, in vitro, the type II receptor associates with and phosphorylates the WD domain-containing protein TRIP-1 (4); the type I receptor then interacts with immunophilin FKBP-12 (28), farnesyl-protein transferase-alpha (12), and p21ras farnesyltransferase-alpha subunit (27). The role of these downstream signaling events in modulating eukaryotic cell growth remains unknown. In our study, the finding that PI 3-kinase coimmunoprecipitates with both types of TGF-beta receptor suggests that PI 3-kinase is a potential candidate in mediating TGF-beta 1 signals. PI 3-kinase isotypes are involved in a number of signaling systems and may act as a lipid kinase, a protein kinase, or an adapter protein (3).

Why is it important to study the effects of TGF-beta 1 on PI 3-kinase activation induced by EGF? Evidence suggests that, in a variety of diseases and in normal reparative processes, the integrated signals imparted by EGF and TGF-beta 1 may profoundly affect cell function (2, 21). In cultured Leydig cells, TGF-beta 1 and EGF act synergistically to regulate androgen formation (24). TGF-beta 1, in combination with EGF, markedly increased secretion of parathyroid hormone-related protein in squamous carcinoma cells to a greater extent than either molecule alone (25). In regulating growth responses, the roles of TGF-beta 1 and EGF are complex. In fetal rat hepatocytes, TGF-beta 1 acts synergistically with EGF to maintain the differentiated state of fetal hepatocytes. Interestingly, at high doses of EGF (>= 20 ng/ml), TGF-beta 1-induced apoptosis is abrogated in these cells (7). In hASM cells, TGF-beta 1 did not induce apoptosis and did not inhibit PI 3-kinase activity. In cervical epithelial cells, although TGF-beta 1 increases EGF-receptor expression, these receptors are unable to undergo autophosphorylation, which suggests that the growth inhibitory effects of TGF-beta 1 on EGF-induced mitogenesis may, in part, be modulated by the upregulation of a low-affinity and less active EGF receptor (10). Together, these studies show that in some cell types TGF-beta 1 can modulate EGF-induced mitogenesis, apoptosis, and secretion of hormones or cytokines. No study, however, has examined the signaling pathways that mediate these effects. Given our present findings, we speculate that the synergistic effects of EGF and TGF-beta 1 on cell function may, in part, be mediated by activation of PI 3-kinase (5).

The identification that phosphoinositides, which are phosphorylated at the D-3 position of the inositol ring by specific PI 3-kinases, could serve as a source of novel second messengers has stimulated considerable research interest in understanding the role of these molecules in regulating cell function. Although PI 3-kinase was initially thought to be a single enzyme, recent studies determined that PI 3-kinases are encoded by a family of genes (32). To date, there exist three specific human p110 catalytic subunits, termed alpha , beta , and gamma . Although all these isoenzymes have the ability to phosphorylate the D-3 position of the inositol ring, the specific function of the isoenzymes remains unknown. Although the results of the present study suggest that TGF-beta 1 augments EGF-induced activation of PI 3-kinase, the functional consequences of this augmentation remain speculative. It is plausible that TGF-beta 1 may activate a distinct PI 3-kinase isoenzyme that differs from that which is activated by EGF. Although an interesting hypothesis, further studies are needed to address this issue.

In the present study, we have determined that 1) TGF-beta 1 modulates EGF-stimulated PI 3-kinase activation; 2) EGF increases PI 3-kinase association with tyrosine phosphorylated proteins, and TGF-beta 1 pretreatment before EGF stimulation does not apparently affect this association; 3) PI 3-kinase associates with both TGF-beta receptors, and TGF-beta 1 enhances PI 3-kinase activity associated with the type I receptor; 4) TGF-beta 1 does not alter basal p70 S6 kinase activity and its stimulation by EGF; and 5) growth inhibitory effects of TGF-beta 1 are not mediated by apoptosis of hASM cells. Our data suggest, for the first time, that TGF-beta 1 may modulate PI 3-kinase activation and that the TGF-beta 1 signaling pathway may modulate the growth factor signaling pathways. The precise mechanism by which TGF-beta 1 modulates mitogen-induced PI 3-kinase remains unknown. Further studies are needed to address whether TGF-beta 1 potentiation of mitogen-induced activation of PI 3-kinase modulates myocyte function, which may include extracellular matrix secretion, cytokine production, or cell cycle arrest. Identification of the critical cellular signaling pathways that modulate hASM cell growth and function will be necessary before the role of ASM hyperplasia in the pathogenesis of asthma can be addressed and therapeutic measures to prevent or abrogate these alterations can be developed.

    ACKNOWLEDGEMENTS

We thank Dr. K. Baron for help in laser densitometry analysis, Jaehyuk Choi for help in carrying out experiments, and Mary McNichol for assistance in preparing the manuscript.

    FOOTNOTES

These studies were supported by National Heart, Lung, and Blood Institute Grant R01-HL-55301; National Aeronautics and Space Administration Grant NRA-94-OLMSA-02; and a Career Investigator Award from the American Lung Association (all to R. A. Panettieri, Jr.).

Address for reprint requests: V. P. Krymskaya, Pulmonary and Critical Care Division, Rm. 815 East Gates Bldg., Hospital of the Univ. of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104-4283.

Received 20 February 1997; accepted in final form 4 September 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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