1 Pulmonary and Critical Care
Division, Regulation of phosphatidylinositol (PI)
3-kinase plays an important role in modulating cellular function. We
have previously shown that transforming growth factor (TGF)-
hyperplasia; signal transduction; cytokines; p70 S6 kinase; apoptosis ; transforming growth factor- 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)- Recent data suggest that PI 3-kinase may be involved in TGF- We now show that EGF stimulates PI 3-kinase activity and that TGF- 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- 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- 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( 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
Cell cycle analysis and DNA ladder
assay. The effects of TGF- 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- 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- Effects of TGF-
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-
1 may modulate EGF-induced PI 3-kinase activation. To date, no study has examined the effects of TGF-
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-
1 alone did not alter PI
3-kinase activation, TGF-
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-
1
pretreatment before EGF stimulation apparently did not alter this
association. Interestingly, TGF-
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-
receptors showed that
PI 3-kinase was associated with both type I and type II TGF-
receptors. TGF-
1, however, enhanced PI 3-kinase activity associated
with the type I TGF-
receptor. Although in some cell types
inhibition of PI 3-kinase and treatment of cells with TGF-
1 mediate
apoptosis, cell cycle analysis and DNA ladder studies show that PI
3-kinase inhibition or stimulation of hASM cells with TGF-
1
did not induce myocyte apoptosis. Although the inhibitory effects
of TGF-
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-
1
receptor modulates PI 3-kinase activity stimulated by growth factors in hASM cells.
1; epidermal growth
factor
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 binds to
transmembrane type I and type II TGF-
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-
receptor binding. In epithelial cells,
TGF-
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-
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-
1 are
cell-type specific.
1
signaling. TGF-
1 reduces PI 3-kinase activity in polyoma middle T-transformed cell lines (6), and wortmannin, a PI 3-kinase inhibitor,
inhibits TGF-
1-stimulated chemotaxis in human neutrophil leukocytes
(26). Because TGF-
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-
1 modulates
mitogen-induced PI 3-kinase activity in hASM cells.
1
markedly potentiates EGF-induced PI 3-kinase activation. Together, our
studies suggest that the TGF-
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-
receptor may modulate PI 3-kinase activation induced by mitogens.
Although the antiproliferative effects of TGF-
1 are not likely
mediated by TGF-
1 modulation of PI 3-kinase activity, other cellular
effects of TGF-
1 on matrix secretion or on cell cycle progression
may be regulated by PI 3-kinase.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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-
RI (T-19), anti-TGF-
RI (V-22), anti-TGF-
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.
receptor type I
(ALK-1; T-19), anti-TGF-
receptor type I (ALK-5; V-22), and
anti-TGF-
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.
-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
[
-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/).
-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).
1 on hASM cell cycle
progression and apoptosis were determined by flow cytometric analysis
of anti-BrdU-labeled cells or DNA ladder analysis, respectively.
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.
1, pretreated with 1 ng/ml of TGF-
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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
1 on EGF-induced PI
3-kinase activation. To examine whether TGF-
1
modulates PI 3-kinase activation, we measured PI 3-kinase activity in
hASM cells treated with EGF and TGF-
1 and those pretreated with
TGF-
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)- 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-
1 augments
EGF-stimulated PI 3-kinase activity. hASM cells were stimulated with
EGF (10 ng/ml, 1 min) or with TGF-
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-
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-
1 alone, 1 ng/ml, 10 min
(n = 3); and cells treated with
TGF-
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-
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-
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-1 and then stimulated with 10 ng/ml of
EGF, PI 3-kinase activity was markedly enhanced by TGF-
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-
1 alone did not affect PI 3-kinase activity (four separate experiments) compared with cells treated with diluent alone. In cells treated with
TGF-
1 before stimulation with EGF, PI 3-kinase activity was
augmented markedly by 16.6 ± 1.9-fold (1 ng/ml of TGF-
1; six
separate experiments) and 17.8 ± 1.8-fold (10 ng/ml of TGF-
1; three separate experiments), respectively, compared with
diluent-treated control cells. These studies suggest that although
TGF-
1 alone does not alter PI 3-kinase activity, pretreatment of
hASM cells with TGF-
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-1, and TGF-
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-
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-
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-
1
pretreatment before EGF stimulation apparently does not alter this
association. Together, these data suggest that TGF-
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-
receptors and activation of the type I TGF-
receptor
increases PI 3-kinase activity. To characterize the
TGF-
receptor subtypes that may mediate EGF-induced PI 3-kinase
activation, PI 3-kinase association with type I and type II TGF-
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-
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-
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-
receptor was similar in cells
stimulated with TGF-
1, EGF, and TGF-
1 + EGF compared with those
treated with diluent alone (Fig. 2B). TGF-
1, however, induced
activation of PI 3-kinase associated with the type I TGF-
receptor
(Fig. 2, C and
D). These studies revealed that PI
3-kinase is associated in vivo with both TGF-
receptor subtypes and
that TGF-
1 stimulation enhances PI 3-kinase activity associated with
type I TGF-
receptor in hASM cells.
|
TGF-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-
1 may inhibit EGF-induced hASM cell proliferation, we
investigated whether TGF-
1 affects p70 S6 kinase activity.
Growth-arrested hASM cells were treated with either EGF for 15 min or
TGF-
1 for 25 min or were pretreated with TGF-
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-
1 alone did not alter p70 S6 kinase
activity, and pretreatment of hASM cells with TGF-
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-
1
growth inhibitory effects do not involve p70 S6 kinase.
|
TGF-1 does not induce apoptosis of
hASM cells. To determine whether the growth inhibitory
effects of TGF-
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-
1 inhibits EGF-induced DNA synthesis.
Growth-arrested hASM cells were incubated with 1 ng/ml of
TGF-
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-
1,
TGF-
1 + EGF, or wortmannin. As shown in Fig.
4B, cell cycle analysis revealed that
TGF-
1 inhibited EGF-induced hASM cell progression from the
Go/G1
to S phase of the cell cycle and that TGF-
1 alone or in combination with EGF did not induce apoptosis (Fig.
4B). These data suggest that growth
inhibitory effects of TGF-
1 are not mediated by stimulation of
apoptosis of hASM cells. Because TGF-
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-
1 on
hASM cell growth occur late in the
G1 phase of the cell cycle.
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DISCUSSION |
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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-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-
1 may inhibit hASM cell growth by modulating
mitogen-induced activation of PI 3-kinase. TGF-
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-
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-
receptors to PI 3-kinase activation. Our data also suggest that the growth inhibitory effects of
TGF-
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-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-
1 on PI
3-kinase activation. In epithelial cells, Mulder and Morris (16) and
Hartsough and Mulder (9) showed that TGF-
1 activated
p21ras and
p44mapk, signaling events thought
to be promitogenic. TGF-
1, however, inhibited epithelial cell growth
(9, 16). In most cells, TGF-
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-
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-
(12), and p21ras
farnesyltransferase-
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-
receptor suggests that PI 3-kinase is a potential
candidate in mediating TGF-
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-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-
1 may profoundly affect cell function (2,
21). In cultured Leydig cells, TGF-
1 and EGF act synergistically to
regulate androgen formation (24). TGF-
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-
1 and EGF are
complex. In fetal rat hepatocytes, TGF-
1 acts synergistically with
EGF to maintain the differentiated state of fetal hepatocytes.
Interestingly, at high doses of EGF (
20 ng/ml), TGF-
1-induced
apoptosis is abrogated in these cells (7). In hASM cells, TGF-
1 did
not induce apoptosis and did not inhibit PI 3-kinase activity. In cervical epithelial cells, although TGF-
1 increases EGF-receptor expression, these receptors are unable to undergo autophosphorylation, which suggests that the growth inhibitory effects of TGF-
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-
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-
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 ,
,
and
. 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-
1 augments EGF-induced activation of
PI 3-kinase, the functional consequences of this augmentation remain
speculative. It is plausible that TGF-
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-1 modulates EGF-stimulated
PI 3-kinase activation; 2) EGF
increases PI 3-kinase association with tyrosine phosphorylated
proteins, and TGF-
1 pretreatment before EGF stimulation does not
apparently affect this association;
3) PI 3-kinase associates with both
TGF-
receptors, and TGF-
1 enhances PI 3-kinase activity
associated with the type I receptor;
4) TGF-
1 does not alter basal p70
S6 kinase activity and its stimulation by EGF; and
5) growth inhibitory effects of
TGF-
1 are not mediated by apoptosis of hASM cells. Our data suggest,
for the first time, that TGF-
1 may modulate PI 3-kinase activation
and that the TGF-
1 signaling pathway may modulate the growth factor
signaling pathways. The precise mechanism by which TGF-
1 modulates
mitogen-induced PI 3-kinase remains unknown. Further studies are needed
to address whether TGF-
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.
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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.
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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.
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REFERENCES |
---|
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---|
1.
Atfi, A.,
E. Drobetsky,
M. Boissonneault,
A. Chapdelaine,
and
S. Chevalier.
Transforming growth factor beta down-regulates Src family protein tyrosine kinase signaling pathways.
J. Biol. Chem.
269:
30688-30693,
1994
2.
Border, W. A.,
and
E. Ruoslahti.
Transforming growth factor- in disease: the dark side of tissue repair.
J. Clin. Invest.
90:
1-7,
1992[Medline].
3.
Carpenter, C. L.,
and
L. C. Cantley.
Phosphoinositide 3-kinase and the regulation of cell growth.
Biochim. Biophys. Acta
1288:
M11-M16,
1996[Medline].
4.
Chen, R. H.,
P. J. Miettinen,
E. M. Maruoka,
L. Choy,
and
R. Derynck.
A WD-domain protein that is associated with and phosphorylated by the type II TGF- receptor.
Nature
377:
548-552,
1995[Medline].
5.
Cohen, M. D.,
V. Ciocca,
and
R. A. Panettieri.
TGF-1 modulates human airway smooth muscle cell proliferation induced by mitogens.
Am. J. Respir. Cell Mol. Biol.
16:
85-90,
1997[Abstract].
6.
Dong, Q. G.,
A. Graziani,
C. Garlanda,
R. W. De Calmanovici,
M. Arese,
R. Soldi,
A. Vecchi,
A. Mantovani,
and
F. Bussolino.
Anti-tumor activity of cytokines against opportunistic vascular tumors in mice.
Int. J. Cancer
65:
700-708,
1996[Medline].
7.
Fabregat, I.,
A. Sanchez,
A. M. Alvarez,
T. Nakamura,
and
M. Benito.
Epidermal growth factor, but not hepatocyte growth factor, suppresses the apoptosis induced by transforming growth factor-beta in fetal hepatocytes in primary culture.
FEBS Lett.
384:
14-18,
1996[Medline].
8.
Gruppuso, P. A.,
R. Mikumo,
D. L. Brautigan,
and
L. Braun.
Growth arrest induced by transforming growth factor 1 is accompanied by protein phosphatase activation in human keratinocytes.
J. Biol. Chem.
266:
3444-3448,
1991
9.
Hartsough, M. T.,
and
K. M. Mulder.
Transforming growth factor activation of p44mapk in proliferating cultures of epithelial cells.
J. Biol. Chem.
270:
7117-7124,
1995
10.
Jacobberger, J. W.,
N. Sizemore,
G. Gorodeski,
and
E. A. Rorke.
Transforming growth factor regulation of epidermal growth factor receptor in ectocervical epithelial cells.
Exp. Cell Res.
220:
390-396,
1995[Medline].
11.
Kapeller, R.,
and
L. C. Cantley.
Phosphatidylinositol 3-kinase.
Bioessays
16:
565-576,
1994[Medline].
12.
Kawabata, M.,
T. Imamura,
K. Miyazono,
M. E. Engel,
and
H. L. Moses.
Interaction of the transforming growth factor-beta type I receptor with farnesyl-protein transferase-alpha.
J. Biol. Chem.
270:
29628-29631,
1995
13.
Kingsley, D. M.
The TGF- superfamily: new members, new receptors, and new genetic tests of function in different organisms.
Genes Dev.
8:
133-146,
1994[Medline].
14.
Kramer, I. M.,
I. Koornneef,
S. W. de Laat,
and
A. J. M. van den Eijnden-van Raaij.
TGF-1 induces phosphorylation of the cyclic AMP responsive element binding protein in ML-CC164 cells.
EMBO J.
10:
1083-1089,
1991[Abstract].
15.
Lane, H. A.,
A. Fernandez,
N. J. C. Lamb,
and
G. Thomas.
p70s6k Function is essential for G1 progression.
Nature
363:
170-172,
1993[Medline].
16.
Mulder, K. M.,
and
S. L. Morris.
Activation of p21ras by transforming growth factor in epithelial cells.
J. Biol. Chem.
267:
5029-5031,
1992
17.
Panettieri, R. A.,
L. R. DePalo,
R. K. Murray,
P. A. Yadvich,
and
M. I. Kotlikoff.
A human airway smooth muscle cell line that retains physiological responsiveness.
Am. J. Physiol.
256 (Cell Physiol. 25):
C329-C335,
1989
18.
Panettieri, R. A.,
A. L. Lazaar,
E. Puré,
and
S. M. Albelda.
Activation of cAMP-dependent pathways in human airway smooth muscle cells inhibits TNF--induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion.
J. Immunol.
154:
2358-2365,
1995
19.
Panettieri, R. A.,
V. Krymskaya,
P. Scott,
R. Plevin,
J. Al-Hafidh,
A. Eszterhas,
and
E. R. Chilvers.
Thrombin induces human airway smooth muscle (ASM) cell proliferation by activation of a novel signaling pathway (Abstract).
Am. J. Respir. Crit. Care Med.
153:
A742,
1996.
20.
Raffioni, S.,
and
R. A. Bradshaw.
Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells.
Proc. Natl. Acad. Sci. USA
89:
9121-9125,
1992[Abstract].
21.
Roberts, A. B.,
M. A. Anzano,
L. C. Lamb,
J. M. Smith,
and
M. B. Sporn.
New class of transforming growth factors potentiated by epidermal growth factor: isolation from neoplastic tissues.
Proc. Natl. Acad. Sci. USA
78:
5339-5343,
1981[Abstract].
22.
Scott, P. H.,
C. M. Belham,
J. Al-Hafidh,
E. R. Chilvers,
A. J. Peacock,
G. W. Gould,
and
R. Plevin.
A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells.
Biochem. J.
318:
965-971,
1996[Medline].
23.
Soltoff, S. P.,
K. L. Carraway,
S. A. Prigent,
W. G. Gullick,
and
L. C. Cantley.
ErbB3 is involved in activation of phosphatidylinositol 3-kinase by epidermal growth factor.
Mol. Cell. Biol.
14:
3550-3558,
1994[Abstract].
24.
Sordoillet, C.,
M. A. Chauvin,
J. C. Hendrick,
P. Franchimont,
A. M. Morera,
and
M. Benahmed.
Sites of interaction between epidermal growth factor and transforming growth factor-1 in the control of steroidogenesis in cultured porcine Leydig cells.
Endocrinology
130:
1352-1358,
1992[Abstract].
25.
Tait, D. L.,
P. C. MacDonald,
and
M. L. Casey.
Parathyroid hormone-related protein expression in gynecic squamous carcinoma cells.
Cancer
73:
1515-1521,
1994[Medline].
26.
Thelen, M.,
M. Uguccioni,
and
J. Bosiger.
PI 3-kinase-dependent and -independent chemotaxis of human neutrophil leukocytes.
Biochem. Biophys. Res. Commun.
217:
1255-1262,
1995[Medline].
27.
Wang, T.,
P. D. Danielson,
B.-Y. Li,
P. C. Shah,
S. D. Kim,
and
P. K. Donahue.
The p21ras farnesyltransferase subunit in TGF-
and activin signaling.
Science
271:
1120-1122,
1996[Abstract].
28.
Wang, T.,
P. K. Donahoe,
and
A. S. Zervos.
Specific interaction of type I receptors of the TGF- family with the immunophilin FKBP-12.
Science
265:
674-676,
1994[Medline].
29.
Weng, Q.-P.,
K. Andrabi,
A. Klippel,
M. T. Kozlowski,
L. T. Williams,
and
J. Avruch.
Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation.
Proc. Natl. Acad. Sci. USA
92:
5744-5748,
1995[Abstract].
30.
Wrana, J. L.,
L. Attisano,
R. Weuser,
F. Ventura,
and
J. Massague.
Mechanism of activation of the TGF- receptor.
Nature
370:
341-347,
1994[Medline].
31.
Yano, H.,
S. Nakanashi,
K. Kimura,
N. Hanai,
Y. Saitoh,
Y. Fukui,
Y. Nonomura,
and
Y. Matsuda.
Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells.
J. Biol. Chem.
268:
25846-25856,
1993
32.
Zvelebil, M. J.,
L. MacDougall,
S. Leevers,
S. Volinia,
B. Vanhaesebroeck,
I. Gout,
G. Panayotou,
J. Domin,
R. Stein,
F. Pages,
H. Koga,
K. Salim,
J. Linacre,
P. Das,
C. Panaretou,
R. Wetzker,
and
M. Waterfield.
Structural and functional diversity of phosphoinosidtide 3-kinases.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
351:
217-223,
1996[Medline].