1 Pulmonary and Critical Care
Division, Hypertrophy and hyperplasia of airway smooth
muscle (ASM) are important pathological features that contribute to
airflow obstruction in chronic severe asthma. Despite considerable
research effort, the cellular mechanisms that modulate ASM growth
remain unknown. Recent evidence suggests that mitogen-induced
activation of phosphoinositide (PI)-specific phospholipase C (PLC) and
PI-dependent calcium mobilization are neither sufficient nor necessary
to stimulate human ASM proliferation. In this study, we identify
phosphatidylinositol (PtdIns) 3-kinase as a key regulator of human ASM
proliferation. Pretreatment of human ASM with the PtdIns 3-kinase
inhibitors wortmannin and LY-294002 significantly reduced thrombin- and
epidermal growth factor (EGF)-induced DNA synthesis
(IC50 ~10 nM and ~3 µM,
respectively). In separate experiments, wortmannin and LY-294002
markedly inhibited PtdIns 3-kinase and 70-kDa S6 protein kinase
(pp70S6k) activation induced by
stimulation of human ASM cells with EGF and thrombin but had no effect
on EGF- and thrombin-induced p42/p44 mitogen-activated protein kinase
(MAPK) activation. The specificity of wortmannin and LY-294002
was further suggested by the demonstrated inability of
these compounds to alter thrombin-induced calcium transients,
total PI hydrolysis, or basal cAMP levels. Transient expression of
constitutively active PtdIns 3-kinase (p110*) activated pp70S6k, whereas a
dominant-negative PtdIns 3-kinase (
asthma; thrombin; airway remodeling; epidermal growth factor; p42/p44 mitogen-activated protein kinase; 70-kDa S6 protein kinase
NUMEROUS CONTRACTILE AGONISTS and growth factors have
been shown to induce proliferation of airway smooth muscle (ASM) cells in culture and thus may play an important role in stimulating the
smooth muscle hypertrophy and hyperplasia associated with several
disease states (32, 34). Although numerous studies have
identified agonists associated with acute bronchoconstriction in
asthmatic patients as potent effectors of human ASM (HASM) cell
proliferation, the mechanism by which spasmogens induce human ASM
growth is not known (reviewed in Ref. 32). Because contractile responses are mediated primarily by increases in intracellular phosphoinositide (PI) turnover and calcium, these elements represent potentially important regulators of HASM growth. However, the inability
of some contractile agonists to induce HASM growth suggests that
increased PI turnover and calcium flux per se are not sufficient to
induce HASM proliferation and that activation of other signaling pathways is likely required (34).
PIs play a key role in signal transduction. To date, two PI pathways
have been characterized. In the canonical PI pathway, activation of one
of a family of PI-specific phospholipase C (PLC; PI-PLC) enzymes
hydrolyzes phosphatidylinositol (PtdIns) 4,5-bisphosphate [PtdIns(4,5)P2]
to inositol 1,4,5-trisphosphate
[Ins(1,4,5)P3] and diacylglycerol. In the 3-PI pathway, activation of PtdIns 3-kinase
phosphorylates PIs at the D3 position of the inositol ring, leading to
the formation of PtdIns 3-phosphate
(PtdIns3P), PtdIns 3,4-bisphosphate
[PtdIns(3,4)P2],
and PtdIns 3,4,5-trisphosphate [PtdIns(3,4,5)P3].
PtdIns(3,4,5)P3
plays a significant role as a second messenger in signaling induced by
a wide array of extracellular stimuli and appears to be the immediate
and dominant PtdIns 3-kinase product in vivo (43, 46).
PtdIns 3-kinases are a family of structurally diverse enzymes divided
into three main classes (48). Each class exhibits distinct substrate
specificity. Class IA PtdIns 3-kinase is a cytosolic heterodimer
composed of a 110-kDa (p110 D3-PIs are not substrates for any known PLC (43, 46) and are not
components of the classic or conventional PI turnover pathway. Auger et
al. (3) were the first to report that platelet-derived growth factor
(PDGF) activated PtdIns 3-kinase in human vascular smooth muscle (VSM)
cells; however, the role of PtdIns 3-kinase in modulating cell growth
was not clarified. Several studies have since demonstrated that PtdIns
3-kinase and its lipid product(s) can regulate mitogenesis in a
mitogen- and cell-specific manner (e.g., Ref. 8). In human umbilical
vein VSM cells, PtdIns 3-kinase appears to be required for PDGF-induced
focal adhesion kinase activation (40). Focal adhesion kinase activation
is an important integration site for a number of extracellular signals
including integrin-receptor family members, G protein-coupled
receptors, and both receptor and non-receptor tyrosine kinases (40).
Conversely, PtdIns 3-kinase is not required for the mitogenic signaling
of thrombin in bovine VSM (52). These studies suggested that the role
of PtdIns 3-kinase in regulating smooth muscle growth remains largely
unknown, and no study to date has examined the role of PtdIns 3-kinase
in mediating HASM cell proliferation.
An essential step in the pathway by which growth factors trigger
cellular proliferation is the biogenesis of translational components
regulating protein synthesis required for cell cycle progression in
G1 phase. The 70/85-kDa S6 protein
kinases, referred to collectively as
pp70S6k, are the principal kinases
that exert translational control of mRNA transcripts and are required
for growth factor-induced G1 progression (19, 24, 28). The
pp70S6k signaling pathway appears
to be distinct from those that activate c-Ras, c-Raf, mitogen-activated
protein kinase (MAPK)/extracellular signal-regulated kinase kinase,
MAPK, and ribosomal S6 kinase (5). In some cell types,
pp70S6k appears to be an essential
downstream effector of PtdIns 3-kinase (9). In response to mitogen
stimuli, pp70S6k is phosphorylated
and activated by PI-dependent kinase 1, a multifunctional effector of
PtdIns 3-kinase (2, 38). Mitogen-induced activation of
pp70S6k is also necessary for
expression of early-immediate genes such as
c-fos, and
pp70S6k activation is required for
the duration of the G1 phase of
the cell cycle in rat embryo fibroblasts (24). Whether activation of
pp70S6k is necessary to stimulate
growth factor- or spasmogen-induced smooth muscle cell proliferation
remains unexplored.
In this study, we examined the relationship between mitogen-induced
activation of PtdIns 3-kinase and ASM cell mitogenesis. We show that
epidermal growth factor (EGF) and thrombin induce PtdIns 3-kinase
activation and that inhibition of PtdIns 3-kinase abolishes
mitogen-stimulated ASM cell growth. In addition, we demonstrate that
transient expression of constitutively active PtdIns 3-kinase (p110*)
activates pp70S6k. Moreover,
expression of a dominant-negative PtdIns 3-kinase ( ASM cell culture. Human tracheae were
obtained from lung transplant donors in accordance with procedures
approved by the University of Pennsylvania (Philadelphia, PA) Committee
on Studies Involving Human Beings. A segment of trachea just proximal
to the carina was removed under sterile conditions, and the trachealis
muscle was isolated. With this technique, 0.5 g of wet tissue was
obtained, minced, centrifuged, and resuspended in 10 ml of buffer
containing 0.2 mM CaCl2, 640 U/ml
of collagenase, 1 mg/ml of soybean trypsin inhibitor, and 10 U/ml of
elastase. Enzymatic dissociation of the tissue was performed for 90 min
in a shaking water bath at 37°C. The cell suspension was filtered
through 105-µm Nytex mesh, and the filtrate was washed with equal
volumes of cold Ham's F-12 medium supplemented with 10% fetal bovine
serum (FBS; HyClone, Logan, UT). Aliquots of the cell suspension were
plated at a density of 1.0 104
cells/cm2. The cells were cultured
in Ham's F-12 medium supplemented with 10% FBS, 100 U/ml of
penicillin, 0.1 mg/ml of streptomycin, and 2.5 µg/ml of amphotericin
B, and this was replaced every 72 h. Cell counts were obtained from
triplicate wells with 0.5% trypsin in a 1 mM EDTA solution.
HASM cells in subculture during the second through fifth cell passages
were used because during these cell passages, the cells retain native
contractile protein expression as demonstrated by immunocytochemical
staining for smooth muscle actin and myosin (33). These cells retain
functional cell-excitation coupling systems as determined by fura 2 measurements of agonist-induced changes in cytosolic calcium (29, 33).
All experiments were performed with a minimum of three different cell
lines. Each ASM cell line was established with tracheal tissue from a
single human donor.
Cytosolic calcium measurements.
Cultured HASM cells were grown to near confluence on 22-mm glass
coverslips and loaded with fura 2-AM (2.5 µM in medium 199 supplemented with 1 mg/ml of BSA for 30 min at 37°C). After being
loaded with fura 2, the cells were washed with a HEPES-buffered saline
solution as previously described (13) and placed in a thermostatically
controlled cell chamber on a Nikon inverted microscope (Diaphot). The
cells were imaged with a ×40 (oil) fluorescence objective lens.
Excitation energy was switched between 340- and 380-nm wavelengths from
a 75-W xenon lamp source via a fura 2 dichroic mirror (Chroma
Technology, Brattleboro, VT). Emitted light was passed through a 510-nm
filter and diverted to an image-intensified charge-coupled device
camera (Hamamatsu, Hamamatsu City, Japan) attached to the video side port of the microscope. Images were digitized with a video
analog-to-digital board (Maatrox). Image analysis of individual
cursor-defined regions corresponding to individual cells was
accomplished with the Image-1 AT/fluor program (Universal Imaging, West
Chester, PA). The 340- to 380-nm ratio was converted to an estimate of
cytosolic calcium with previously described calibration methods (13,
29, 30). Calibration measurements were made with ionomycin (10 µM)
and added calcium (total calcium = 12 mM) to measure the ratio of the
340-nm signal to the 380-nm signal under saturating conditions of
calcium (Rmax) or with the
addition of a stoichiometric excess of EGTA to achieve the ratio of the
340-nm signal to the 380-nm signal in the absence of calcium
(Rmin). Values used for the
calibration equation were Rmin = 0.3, Rmax = 5.0, dissociation
constant = 224 nM, and the ratio of the 380-nm fluorescence at high
calcium to that at low calcium = 5. Additions of plasminogen-free
bovine Accumulation of total
[3H]inositol phosphates.
[3H]inositol phosphate
formation in cultured cells was determined as previously reported (6,
53), with minor modifications. The medium was aspirated from
near-confluent cell monolayers in 24-well plates and replaced with 300 µl of inositol-free Dulbecco's modified Eagle's medium (DMEM)
containing
[myo-3H]inositol
(10 20 Ci/mmol; DuPont-New England Nuclear, Boston, MA) at a
concentration of 2 µCi/ml. The cells were then incubated for 24 h,
the medium was removed, and the cells were washed twice with
inositol-free DMEM containing 10 mM LiCl and then incubated with this
medium for 30 min at 37°C. Agonists were added in a volume of 10 µl, and the final incubation volume was 300 µl. Using this labeling
protocol, Daykin et al. (10) have shown that membrane inositol phospholipids achieve isotopic equilibrium and that over the
time course of these experiments, no significant depletion of labeled
membrane inositol phospholipids occurs. The reactions were stopped by
removing the medium from each well and adding 1 ml of methanol-0.12 M
HCl (1:1 vol/vol) previously stored at 20°C. The samples were then
stored at
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
p85) blocked EGF- and
thrombin-stimulated pp70S6k
activity. Collectively, these data suggest that activation of PtdIns
3-kinase is required for the mitogenic effect of EGF and thrombin in
human ASM cells. Further investigation of the role of PtdIns 3-kinase
may offer new therapeutic approaches in the treatment of diseases
characterized by smooth muscle cell hyperplasia such as asthma
and chronic bronchitis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
, -
, or -
) catalytic subunit and an
85-kDa (p85) adaptor protein. In response to growth factor stimulation,
class IA PtdIns 3-kinases complex with activated receptor protein
tyrosine kinases (through SH2 domains of the p85 subunit) or
tyrosine-phosphorylated proteins. The class IB PtdIns 3-kinase-
does
not have an adaptor protein but instead is activated by G
subunits of G proteins (44). Class II PtdIns 3-kinases contain a
COOH-terminal C2 domain that appears involved in the regulation of this
class by phospholipid and calcium (11). The recently identified class
III PtdIns 3-kinases are structurally related to the product of the
Saccharomyces cerevisiae gene Vps34 (51).
p85) blocks EGF-
and thrombin-induced pp70S6k
activity, suggesting that PtdIns 3-kinase is required for
pp70S6k activation by EGF and
thrombin. These data suggest that mitogenic signals elicited by
contractile agonists and growth factors share common signaling pathways
that converge to activate PtdIns 3-kinase.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-thrombin (0.01 1 U/ml) were made by direct bath additions.
Fura 2, ionomycin, bradykinin, and
-thrombin (1,000 U/mg protein) were obtained from Calbiochem (La Jolla, CA). All other reagents were
obtained from Sigma (St. Louis, MO).
20°C overnight. An 800-µl aliquot of each sample
was then neutralized to pH 7 with 4.2 ml of buffer containing 25 mM
Tris-0.5 M NaOH-H2O
(0.238:0.025:0.737 vol/vol/vol). Total
[3H]inositol
phosphates were finally separated from free
[myo-3H]inositol
by anion-exchange chromatography (53).
[3H]thymidine and cell
proliferation assays.
Proliferation studies were performed on near-confluent, growth-arrested
HASM cells. The cells were growth arrested by incubating the cultures
on day 10 in serum-free medium consisting of Ham's F-12 medium with 5 ng/ml of insulin and 5 ng/ml of transferrin (36). Near-confluent,
growth-arrested cells were used because cells can be synchronized in
the
G0/G1
phase of the cell cycle and, at this baseline, minimally incorporate
[35S]methionine and
[3H]thymidine (33,
36). After 24 h in serum-free medium, the cells were then stimulated
with either 10 ng/ml of EGF (Sigma), 10% FBS, or plasminogen-free
bovine -thrombin (1,000 U/mg protein; Calbiochem). In some
experiments, monolayers were treated with wortmannin or LY-294002 at
varying doses for 20 min and then stimulated with mitogens. After
16-18 h of mitogen stimulation, HASM cells were labeled with 1 µCi/ml of
[methyl-3H]thymidine
(40-60 Ci/mmol; Amersham, Arlington Heights, IL) for 12-18 h.
The cells were then scraped and lysed, and the protein and DNA were
precipitated with 12% TCA. The precipitant was aspirated onto glass
filters, extensively washed, dried, and counted (33, 36).
[3H]thymidine
incorporation experiments were performed in parallel with cell
proliferation assays. Cell number was quantified by removing cells from
the 12-well plates with a 0.5% trypsin-1 mM EDTA solution. Cell counts
were then obtained from each well with a Coulter Counter (Coulter
Electronics, Hialeah, FL).
Assay of p70S6k activity.
Cells stimulated and washed as described in
Preparation of cell lysates and
immunoprecipitation were solubilized in a
50 mM Tris buffer (pH 8.0) containing 120 mM NaCl, 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 60 min at 4°C
(lysis buffer) (28). The lysates were centrifuged at 1,000 g for 2 min, and equal quantities of
the supernatant (500 µl, 150-200 µg of protein) incubated with
2 µg of a polyclonal anti-p70/85rsk antibody (Upstate
Biotechnology). After 90 min, the lysates were further incubated with
50 µl of protein A-Sepharose (60 µl; Pharmacia Biotech). The
immunoprecipitates were washed twice in lysis buffer and twice in the
same buffer without detergents. For in vitro kinase assay, the
immunoprecipitates were washed twice in 25 mM HEPES (pH 7.4) containing
20 mM -glycerophosphate, 20 mM
MgCl2, 3 mM EGTA, and 2 mM
dithiothreitol and then incubated in the same buffer containing 100 µM S40 substrate peptide (28), 10 µM protein kinase (PK) A
inhibitor peptide, and 10 µM
[
-32P]ATP (2.5 µCi) in a volume of 30 µl for 20 min at 30°C. The reaction was
terminated by the addition of 10 µl of 300 mM
H3PO4.
The phosphorylated peptide was separated from the other products by
ion-exchange chromatography on P81 ion-exchange paper with 75 mM
H3PO4
and quantified by liquid scintillation counting.
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RESULTS |
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PtdIns 3-kinase inhibition abolishes EGF- and
thrombin-induced ASM cell growth. Because PtdIns
3-kinase activation is required for the induction of cell proliferation
in some cell lines, we investigated whether PtdIns 3-kinase mediated
ASM cell growth induced by EGF or thrombin. Confluent ASM cells were
growth arrested for 24 h, pretreated with the PtdIns 3-kinase
inhibitors wortmannin or LY-294002, and then stimulated with 1 U/ml of
thrombin or 10 ng/ml of EGF. DNA synthesis was subsequently measured by
assessment of
[3H]thymidine
incorporation. Wortmannin and LY-294002 abrogated thrombin- and
EGF-induced DNA synthesis in a concentration-dependent manner
(IC50 ~10 nM and ~3 µM, respectively;
Fig. 1, A and
B, respectively). Wortmannin (100 nM) inhibited thrombin-
and EGF-induced DNA synthesis by 93 ± 9.2 and 67 ± 7.8%,
respectively (Fig. 1A). This effect of
wortmannin was not observed if wortmannin was added 17 h after the
cells were stimulated with mitogens (Fig. 1,
C and D). Pretreatent of
HASM cells with 30 µM LY-294002 before stimulation with thrombin or
EGF completely inhibited DNA synthesis (Fig.
1B). The effects of LY-294002 (30 µM) and wortmannin (100 nM) on the basal levels of
[3H]thymidine
incorporation were not different from those of diluent alone. These
data suggest that PtdIns 3-kinase modulates early mitogenic signaling
events involved in agonist-induced ASM cell growth.
|
Because incorporation of [3H]thymidine correlates with DNA synthesis and may occur without cell division, we investigated whether wortmannin inhibited thrombin- and EGF-stimulated increases in cell number. After 36 h of agonist stimulation, thrombin stimulated a 17.6 ± 2.0% increase in cell number compared with that induced by diluent alone (Fig. 1E). EGF and 10% serum increased cell number by 22.3 ± 2.1 and 27 ± 2.5%, respectively. Pretreatment of cells with 100 nM wortmannin inhibited thrombin- and EGF-induced cell proliferation by 85 ± 5.2 and 61 ± 6%, respectively (Fig. 1D). Cell counts from monolayers treated with wortmannin alone were comparable to those of diluent-treated cells. Trypan blue staining of cells treated with 100 nM wortmannin alone revealed 99.9% viability, suggesting that wortmannin is not toxic to ASM cells. Collectively, these data suggest that PtdIns 3-kinase activation is important in modulating ASM cell proliferation induced by growth factors and contractile agonists.
Mitogens activate PtdIns 3-kinase in ASM
cells. Although wortmannin and LY-294002 inhibited EGF-
and thrombin-induced myocyte proliferation, studies were also performed
to confirm that mitogens directly activate PtdIns 3-kinase in HASM
cells. Confluent, growth-arrested cells were stimulated with 10 ng/ml
of EGF for 1, 3, 5, and 10 min or 1 U/ml of thrombin for 1, 5, 15, and
30 min. PtdIns 3-kinase activities were detected in anti-PTyr
immunoprecipitates from cell lysates. Immunoprecipitates were incubated
with PtdIns and [-32P]ATP, and in
vitro phosphorylation of PtdIns was examined by TLC and autoradiography
as described in METHODS. EGF maximally increased PtdIns 3-kinase activity at 1 min (Fig.
2A).
Interestingly, maximal PtdIns 3-kinase activation by thrombin was
observed at 15 min (Fig. 2B).
Bradykinin, a contractile agonist that is not a HASM cell mitogen (34),
did not activate PtdIns 3-kinase (data not shown).
|
In other studies, experiments were performed to address whether EGF also increased the amount of PtdIns 3-kinase associated with tyrosine-phosphorylated proteins in EGF-stimulated ASM cells. Immunoblot analysis with an anti-p85 subunit of PtdIns 3-kinase antibody was performed on anti-PTyr immunoprecipitates from EGF- and diluent-treated cells. EGF treatment markedly increased association of PtdIns 3-kinase with tyrosine-phosphorylated proteins when compared with that obtained from cells treated with diluent alone (Fig. 2C). Immunoblot testing of whole cell lysate confirmed the presence of PtdIns 3-kinase in HASM cells. Cell lysates from Jurkat T cells, which contain high levels of PtdIns 3-kinase, were used as positive controls. Negative controls included isotype-matched nonimmune IgG for immunoprecipitations in the PtdIns 3-kinase activity assays and immunoblots to support the specificity of protein-protein interactions (data not shown).
To establish the effectiveness of PtdIns 3-kinase inhibitors in HASM cells, the cells were pretreated for wortmannin or LY-294002 and then stimulated with EGF or thrombin. Wortmannin (100 nM) pretreatment inhibited EGF-induced activation of PtdIns 3-kinase (Fig. 2D). Similarly, LY-294002 also decreased activity of the enzyme and, in a concentration-dependent manner, abolished PtdIns 3-kinase activation by EGF at 30 µM (Fig. 2E). Pretreatment of cells with wortmannin (1 100 nM) before thrombin stimulation also induced concentration-dependent inhibition of PtdIns 3-kinase activation (Fig. 2F). In separate experiments, pretreatment of cells with U-73122, a PLC inhibitor, had no effect on EGF- or thrombin-induced PtdIns 3-kinase activation (data not shown).
PtdIns 3-kinase inhibition has no effect on
mitogen-induced calcium transients or PI turnover. To
address the specificity of wortmannin for inhibition of PtdIns 3-kinase
and to determine whether mitogen-induced calcium mobilization is
necessary to induce ASM cell growth, we examined the effects of
wortmannin on mitogen-evoked calcium transients and PI turnover.
Monolayers were pretreated with wortmannin (100 nM) and then stimulated
with 1 U/ml of thrombin. Cytosolic calcium measurements in fura
2-loaded ASM cells were performed as described in
METHODS. As shown in Fig.
3A,
pretreatment with wortmannin did not affect thrombin-induced calcium
transients. In parallel experiments, the effects of wortmannin and
LY-294002 on total PI turnover were studied. ASM cell monolayers were
pretreated with 100 nM wortmannin for 10 min and stimulated with either
1 U/ml of thrombin, 10 ng/ml of EGF, or diluent. PI turnover was measured after 30 min of agonist stimulation as described in
METHODS. As shown in Fig.
3B, thrombin and EGF increased
[3H]InsP1-4
formation in ASM cells by 2.59 ± 0.06- and 3.09 ± 0.07-fold,
respectively. Wortmannin pretreatment did not alter either thrombin- or
EGF-induced PI turnover. Similarly, pretreatment of HASM cells with 30 µM LY-294002 for 30 min had no effect on thrombin-induced PI turnover
(data not shown). In addition, pretreatment of cells overnight with 100 nM wortmannin had no effect on thrombin- and EGF-evoked calcium
transients (data not shown). These data suggest that the effects of
wortmannin or LY-294002 on cell growth are not due to nonspecific
cytotoxicity and that mitogen-induced calcium mobilization and PI
turnover are not sufficient to stimulate ASM cell proliferation.
|
PtdIns 3-kinase inhibition does not significantly
alter p42/p44 MAPK activation. Because a previous study
(15) in non-HASM cells has asserted a requirement for p42/p44 MAPK
activation in mitogen-induced proliferation, we examined the effects of
LY-294002 and wortmannin on EGF- and thrombin-induced p42/p44 MAPK
activation. Growth-arrested HASM cells were pretreated with 30 µM
LY-294002 or 100 nM wortmannin before EGF (10 ng/ml) or thrombin (1 U/ml) stimulation for 0-180 min, and immunoblot analysis of p42/p44 MAPK phosphorylation was subsequently performed. As depicted in Fig.
4, no significant effects of either
LY-294002 or wortmannin pretreatment were observed in basal and
agent-stimulated p42/p44 MAPK phosphorylation. These data suggest that
the effects of PtdIns 3-kinase, LY-294002, and wortmannin on
mitogenesis are primarily mediated through pathways distinct from that
of p42/p44 MAPK.
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Treatment of HASM with wortmannin or LY-294002 does not stimulate cAMP accumulation. Numerous studies (see Ref. 32) have demonstrated that stimulation of the cAMP-dependent PKA can inhibit mitogenic signaling that includes the Raf-dependent p42/p44 MAPK and PI 3-kinase pathways in multiple cell types. We therefore investigated whether treatment with wortmannin or LY-294002 could induce cAMP accumulation (possibly either directly or secondarily through exocytotic release of an activating agent) in HASM and thereby activate PKA. Treatment of HASM with 100 nM wortmannin or 30 µM LY-294002 for 0-60 min produced cAMP accumulation identical to that of (vehicle-stimulated) basal levels (data not shown). Thus the antimitogenic effects of wortmannin and LY-294002 do not appear to be mediated by nonspecific activation of PKA.
Activation of pp70S6k in ASM cells is
mediated by stimulation of PtdIns 3-kinase.
To further assess mechanisms by which mitogens induce HASM cell
proliferation, we investigated the relationship between PtdIns 3-kinase
and pp70S6k activation in
modulating HASM mitogenesis. Growth-arrested ASM cells were treated
with fetal calf serum, thrombin, or bradykinin for 15 min and lysed,
pp70S6k was immunoprecipitated,
and activity was assessed as described in
METHODS. As shown in Fig.
5A,
thrombin and serum stimulated pp70S6k activity in ASM cells by
5.4 ± 1.1- and 5.9 ± 0.9-fold, respectively. Conversely,
bradykinin, a contractile agonist that evokes levels of intracellular
calcium concentration and PI turnover comparable to those induced by
thrombin in HASM cells (34) did not activate pp70S6k. In other experiments, the
specificity of the in vitro activity assay for
pp70S6k was studied. ASM cell
monolayers were treated with EGF and rapamycin, a
pp70S6k inhibitor, EGF alone, or
diluent alone for 15 min. EGF induced a 9.6 ± 0.5-fold increase in
pp70S6k activity as shown in Fig.
5B. Pretreatment of cells with
rapamycin completely inhibited EGF-induced
pp70S6k activation. The effect of
rapamycin on EGF-induced pp70S6k
activation was shown to be both time and concentration dependent (data
not shown). Rapamycin also inhibited EGF- and thrombin-induced DNA
synthesis in a concentration-dependent manner
(IC50 ~0.2 µM for EGF and
~0.4 µM for thrombin), and 5 µM rapamycin blocked EGF- and
thrombin-induced DNA synthesis by 99.1 ± 0.2 and 98.6 ± 2.5%,
respectively (Fig. 5C).
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DISCUSSION |
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The principal finding of this study is that PtdIns 3-kinase activation
is necessary for mitogen-induced HASM proliferation. -Thrombin and
EGF, both potent ASM cell mitogens, evoked comparable increases in
cytosolic calcium, PI hydrolysis, and DNA synthesis and activated
PtdIns 3-kinase, p42/p44 MAPK, and
pp70S6k. The PtdIns 3-kinase
inhibitors wortmannin and LY-294002 both abrogated mitogen-induced
stimulation of PtdIns 3-kinase and ASM proliferation in a time- and
dose-dependent manner. The specificity of these compounds was suggested
by experiments demonstrating their failure to alter PI turnover and
calcium-, cAMP-, and MAPK-regulated signaling. Last, a role for
pp70S6k in PtdIns
3-kinase-mediated mitogenesis was suggested by effective inhibition of
mitogen-activated pp70S6k activity
by wortmannin and LY-294002 and by expression of
p85.
PtdIns 3-kinase and its lipid products are pivotal signaling events in
regulating various cellular functions including mitogenesis (8, 20,
22). PtdIns 3-kinase is required for DNA synthesis induced by some, but
not all, growth factors. Microinjection of 3T3 fibroblasts with a
neutralizing antibody to the p110 catalytic subunit of PtdIns 3-kinase
completely inhibited PDGF- and EGF-induced mitogenesis (39).
Stimulation of a PDGF-receptor mutant incapable of binding PtdIns
3-kinase could not induce DNA synthesis in a human hepatome cell line
HepG2, and mitogenic signaling was restored when PtdIns 3-kinase
binding sites were added back to the mutant (47). Microinjection of a
monoclonal antibody against the
NH2-terminal SH2 domain of the p85
regulatory subunit of PtdIns 3-kinase induced stimulation of DNA
synthesis in Chinese hamster ovary cells, an effect blocked by
coinjection of antibodies against
pp70S6k or Ras or inhibition of
MAPK (26). Expression of an inducible form of PtdIns 3-kinase in rat
embryo fibroblasts demonstrated that activation of PtdIns 3-kinase is
sufficient for cell cycle progression into the S phase (21). Other
studies (4, 41, 42, 52) suggested a role for PtdIns 3-kinase in ASM and
VSM cell proliferation. Wortmannin- and LY-294002-mediated inhibition of DNA synthesis was demonstrated in bovine ASM and porcine and rat VSM
cells stimulated with PDGF, basic fibroblast growth factor, angiotensin
II, and serum. Stimulation of
1-adrenergic receptors with
norepinephrine activated mitogenesis, Ras, MAPK, and PtdIns 3-kinase in
human VSM cells in a wortmannin-sensitive manner (17). In rat thoracic
aorta VSM cells, wortmannin completely blocked angiotensin II-induced
Ras activation but had no effect on MAPK activation and protein
synthesis (45). These data suggest that in both ASM and VAM cells
PtdIns 3-kinase is involved in mitogenic signaling induced by numerous agents.
The role of PtdIns 3-kinase activation in modulating cell proliferation is cell-type specific. In some cell types, bombesin and lysophosphatidic acid, which induce cell proliferation by activating receptors coupled to G proteins, stimulate cell growth in the absence of PtdIns 3-kinase activity (8, 39). Our data, however, suggest that thrombin, which induces HASM cell growth by activating a receptor presumably coupled to both Gi and Gq proteins (34), requires PtdIns 3-kinase activation to mediate its growth effects (35). Bradykinin, a contractile agonist that potently mediates bronchoconstriction in asthmatic patients, stimulates similar increases in PI turnover and cytosolic calcium comparable to those stimulated by either thrombin or EGF (34). However, bradykinin is not a HASM cell mitogen (34) and does not activate PtdIns 3-kinase. In bovine ASM, the mitogenic effects of PDGF or endothelin-1 have been attributed to their ability to stimulate PtdIns 3-kinase or pp70S6k (42). Taken together, these studies suggest that mitogens may activate different intracellular signaling pathways in a cell-specific manner. Our data demonstrate a role for PtdIns 3-kinase in mediating human smooth muscle cell proliferation induced by both growth factors and contractile agonists.
PIs are generated from two separate and distinct pathways. Activation of PI-PLC hydrolyzes PtdIns(4,5)P2 to Ins(1,4,5)P3 and diacylglycerol. Activation of PtdIns 3-kinase, which involves protein tyrosine kinase-mediated recruitment, phosphorylates PIs at the D3 position of the inositol ring and leads to the formation of PtdIns3P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 (43, 46). Recently, these phospholipids have been recognized as a new class of second messengers. Based on a number of studies (43, 46), PtdIns(3,4,5)P3 appears to be the critical signaling 3-phospholipid. This assumption is supported by the time course of accumulation and the subsequent metabolism of the individual 3-PIs after agonist stimulation (12, 18). The 3-PIs are not substrates for any known PLC (8, 43, 49) and are not components of the canonical PI turnover pathway. Moreover, their rapid accumulation on growth factor stimulation suggests that the lipids themselves act as second messengers mediating PtdIns 3-kinase mitogenic signals (8, 46). The recent finding that PtdIns 3-kinase, through its products (3-PIs), exerts control of the novel and atypical isoforms of PKC may have important implications in understanding the mechanisms that induce smooth muscle cell proliferation, insomuch as myocyte growth is thought to be PKC dependent (1, 25, 27, 31).
In our studies, numerous approaches were used to investigate the role of PtdIns 3-kinase in mediating ASM cell proliferation. We demonstrate that EGF and thrombin, which transduce their signals by activating distinct pathways associated with receptor tyrosine kinase and heterotrimeric G protein activation, respectively, both induce PtdIns 3-kinase activity in HASM cells. Ligand-induced PtdIns 3-kinase activity was similar to that observed when p110* was expressed in ASM. Wortmannin, a fungal metabolite that binds covalently to the 110-kDa subunit of PtdIns 3-kinase, and LY-294002, another PtdIns 3-kinase inhibitor, the site of action of which differs from that of wortmannin (54), both inhibited EGF-stimulated PtdIns 3-kinase activity. Nanomolar concentrations of wortmannin have been shown to inhibit PtdIns 3-kinase in numerous mammalian cell types (7, 20, 50). At these concentrations, wortmannin does not affect other PKs and lipid kinases, which include PtdIns 4-kinase (IC50 value of >100 µM), PI-PLC (IC50 value of 100 µM), or myosin light chain kinase (IC50 value of 1 µM) (7, 54). Our data suggest that wortmannin inhibited ASM cell proliferation with an IC50 value of 10 ± 0.3 nM. At this concentration, it is likely that the effects of wortmannin on ASM growth are specific for PtdIns 3-kinase. In addition, because wortmannin had no effect on agonist-induced calcium transients, this suggests that wortmannin does not inhibit PI-PLC activation in ASM cells at these concentrations. To further characterize the specificity of these inhibitors in ASM cells, we examined whether LY-294002 modulated EGF- or thrombin-induced ASM cell growth. LY-294002 also inhibited mitogen-induced DNA synthesis in ASM cells at concentrations that abolished PtdIns 3-kinase activation.
The specificity of wortmannin and LY-294002 for PtdIns 3-kinase in HASM cells was further supported by experiments examining their effects on p42/p44 MAPK activation and cAMP accumulation. p42/p44 MAPK activation, shown to be required for proliferation in numerous cell types including non-HASM cells, was not affected by maximal concentrations of either wortmannin or LY-294002. This finding also suggests that PtdIns 3-kinase activation by mitogens in HASM cells is of little consequence to p42/p44 MAPK activation (i.e., PtdIns 3-kinase does not regulate p42/p44 MAPK activity). cAMP accumulation and consequent activation of PKA has the potential to inhibit several promitogenic pathways in numerous cell types. We observed no significant stimulation of cAMP production by either wortmannin or LY-294002, suggesting that the inhibition of PtdIns 3-kinase activity or HASM cell proliferation by wortmannin or LY-294002 is not mediated by nonspecific activation of PKA.
In numerous cell types, PtdIns 3-kinase has been shown to be an
important mediator of pp70S6k
activation in response to serum and growth factors (9).
pp70S6k, a critical enzyme for
mitogen-induced cell cycle progression through the
G1 phase and translational control
of mRNA transcripts that contain a polypyrimidine tract at their
transcriptional start site (19, 24, 28), is phosphorylated and
activated by PI-dependent kinase 1, a multifunctional effector of
PtdIns 3-kinase (2, 38). In T cells, interleukin-2 appears to induce
cell proliferation by stimulating PtdIns 3-kinase, which, in turn,
activates pp70S6k. Inhibition of
pp70S6k with rapamycin, an
immunosuppressant, did not alter interleukin-2 activation of PtdIns
3-kinase but inhibited T-cell growth (9, 28). In another study
(24), microinjection of neutralizing antibodies to
pp70S6k completely inhibited
serum-induced DNA synthesis in an embryonic fibroblast cell line. These
data suggest that in some cell types activation of
pp70S6k is necessary for cell
progression from the G1 to the S
phase of the cell cycle and that
pp70S6k appears to be an essential
downstream effector of PtdIns 3-kinase. The kinetics of
pp70S6k activation suggest that
this kinase may also be an attractive target for therapeutic
interventions. Mitogen-induced activation of
pp70S6k is essential for
expression of early-immediate genes such as c-fos, and
pp70S6k expression is required for
the duration of the G1 phase of
the cell cycle (24). Whether activation of
pp70S6k is necessary to stimulate
growth factor- or contractile agonist-induced smooth muscle cell
proliferation remains unknown. Our data, however, suggest that EGF and
thrombin significantly stimulate
pp70S6k, and wortmannin,
LY-294002, and rapamycin completely block this activation. Bradykinin,
a contractile agonist that does not induce ASM cell growth, had no
effect on pp70S6k activation.
Moreover, overexpression of p85 abolished EGF and thrombin
pp70S6k activation. Transient
expression of p110* activates
pp70S6k in the absence of
stimulation with mitogens. Thus EGF and thrombin induce activation of
pp70S6k in HASM cells and
mitogen-induced activation of
pp70S6k appears PtdIns 3-kinase dependent.
In summary, results from the present study demonstrate a requirement for PtdIns 3-kinase and pp70S6k activation in both EGF- and thrombin-stimulated HASM cell proliferation. Future studies are needed to identify features by which those signaling intermediates are regulated as well as the effect of PtdIns 3-kinase and pp70S6k activity on downstream signaling elements involved in HASM mitogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. A. Klippel for the constitutively active
phosphatidylinositol 3-kinase (p110*) expression vector and Dr. M. Kasuga for the dominant-negative phosphatidylinositol 3-kinase (p85) expression vector. We also thank Mary McNichol for expert assistance in
preparing this manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-02647, HL-55301, and HL-58506; a Career Investigator Award from the American Lung Association; the National Aeronautics and Space Administration; The National Asthma Campaign (United Kingdom); and The Wellcome Trust (United Kingdom).
E. R. Chilvers is a Wellcome Trust Senior Research Fellow in Clinical Science.
Address for reprints requests and other correspondence: R. A. Panettieri, Jr., Pulmonary and Critical Care Division, Room 805 East Gates Building, Hospital of the University of Pennsylvania, 3400 Spruce St., Philadelphia, PA 19104-4283 (E-mail: rap{at}mail.med.upenn.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 23 December 1998; accepted in final form 19 March 1999.
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