Activation of class IA PI3K stimulates DNA synthesis in human
airway smooth muscle cells
Vera P.
Krymskaya,
Alaina
J.
Ammit,
Rebecca K.
Hoffman,
Andrew J.
Eszterhas, and
Reynold A.
Panettieri Jr.
Pulmonary, Allergy, and Critical Care Division, Department of
Medicine, University of Pennsylvania Medical Center, Philadelphia,
Pennsylvania 19104
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ABSTRACT |
The precise mechanisms that regulate
increases in airway smooth muscle (ASM) mass in asthma are unknown.
This study determined whether class IA phosphatidylinositol 3-kinase
(PI3K) is sufficient to stimulate DNA synthesis and characterized the
PI3K isoforms expressed in human ASM cells. ASM cells express class IA,
II, and III PI3K but not class IB. Because thrombin induces ASM cell proliferation, we investigated whether thrombin can stimulate class IA
PI3K. Transient transfection of ASM cells with hemagglutinin-tagged p85
PI3K followed by immunostaining revealed that in quiescent cells, p85
was expressed diffusely in the cytoplasm and after stimulation with
thrombin p85 translocated to the cell membrane. Microinjection of ASM
cells with a dominant negative class IA PI3K inhibited thrombin-induced
DNA synthesis by 30% and epidermal growth factor (EGF)- or
serum-induced DNA synthesis by 13 and 28%, respectively
(P < 0.05 by
2 analysis). In parallel
experiments, transfection or microinjection of cells with
constitutively active PI3K markedly increased DNA synthesis in
transfected cells 10.5-fold and in microinjected cells 12.7-fold
(P < 0.05 by
2 analysis) compared with
cells transfected or microinjected with control plasmid. Interestingly,
constitutively active PI3K augmented EGF-induced DNA synthesis but had
little effect on that induced by serum or thrombin in ASM cells.
Collectively, these data suggest that class IA PI3K is activated by
thrombin and is sufficient to induce ASM cell growth.
phosphatidylinositol 3-kinase; airway remodeling; asthma; signaling
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INTRODUCTION |
IN MANY CELL
TYPES, activation of class IA phosphatidylinositol 3-kinase
(PI3K) regulates myriad cellular functions including cell
proliferation, differentiation, transformation, motility, and
apoptosis. Evidence suggests that PI3K exists as a family of
enzymes that phosphorylates membrane phosphoinositides on the D3
position of the inositol ring and serves as a protein kinase, phosphorylating serine residues of target proteins. The D3
phosphoinositides function as second messengers and activate downstream
effector molecules such as p70S6k, protein kinase C-
,
and Akt (27). The ability of PI3K to regulate diverse
functions may be due to the existence of multiple isoforms that have
specific substrate specificities and reside in unique cytoplasmic
locations within the cell (32).
PI3K isoforms are divided into three classes based on their structure
and substrate specificity. Class IA PI3Ks are cytoplasmic heterodimers
composed of a 110-kDa (p110
, -
, or -
) catalytic subunit and an
85-kDa (p85, p55, or p50) adaptor protein. The catalytic subunits
p110
and p110
are ubiquitously expressed in mammalian cells.
Catalytic subunit p110
is expressed predominantly in lymphocytes and
lymphoid tissues and therefore may play a role in PI3K-mediated
signaling of immune responses (4). Class IA isoforms are
mainly activated by receptor and non-receptor tyrosine kinases (RTKs),
whereas class IB p110
is activated by G
subunits of G
protein-coupled receptors (GPCRs) (31). Class II isoforms are mainly associated with the phospholipid membranes and are present
in the endoplasmic reticulum and Golgi apparatus (8). Class III isoforms, structurally related to the yeast vesicular sorting
protein Vps34p (33), are mammalian homologs that use only
membrane phosphatidylinositol as a substrate and generate phosphatidylinositol 3-monophosphate. Cellular levels of
phosphatidylinositol 3-monophosphate are usually maintained at constant
levels, suggesting that class III isoforms do not respond to
extracellular stimuli. Despite PI3K playing an essential role in
modulating mitogen-induced smooth muscle proliferation (15,
17), the repertoire of PI3K isoforms expressed and their
attendant functions have not been well studied in airway or vascular
smooth muscle cells.
In asthma, chronic bronchitis, and atherosclerosis, smooth muscle mass
is increased, in part, because of myocyte hyperplasia. Smooth muscle
hyperplasia may be a normal response to injury and repair or,
alternatively, myocyte growth may be a critical component of the
pathobiology of these diseases. Despite considerable effort, the
precise signaling mechanisms that regulate smooth muscle cell proliferation in these diseases remain unknown. Evidence clearly suggests that smooth muscle mitogens may activate at least two types of
receptors: GPCRs and those with intrinsic tyrosine kinases (RTKs). On
ligand binding, these disparate receptors activate a variety of
downstream signaling pathways that appear, in part, to converge to
stimulate PI3K (32). In a variety of cell types (7,
18, 31), GPCR activation preferentially stimulates the class IB
p110
isoform of PI3K, whereas RTK activation favors activation of
either class IA or IB isoforms. Our studies, which used pharmacological
inhibitors, suggest that Ras/mitogen-activated protein kinase and PI3K
pathways are required for mitogen-induced airway smooth muscle (ASM)
cell proliferation and that these pathways may act in parallel
(1, 15-17, 29). Epidermal growth factor (EGF) and
thrombin, which are human ASM cell mitogens and are present in
increased quantities in bronchoalveolar lavage fluid from asthmatic
patients after allergen challenge, robustly stimulate PI3K via
activation of the RTK and GPCR pathways, respectively (17). In these cells, PI3K then stimulates
p70S6k, the principal kinase that regulates translation of
the mRNA transcripts necessary for G1 cell cycle
progression. Although such studies suggest that PI3K plays a central
role in mediating mitogen-induced ASM cell proliferation, most
investigators have relied on the specificity of pharmacological
inhibitors to dissect such pathways; thus whether PI3K is sufficient
and/or necessary to stimulate ASM cell growth remains unclear.
In this study, we characterized the specific isoforms expressed in
human ASM cells. Using newly developed transient transfection techniques, we also investigated whether PI3K is necessary to thrombin-
and EGF-induced DNA synthesis and whether PI3K activation alone is
sufficient to induce DNA synthesis in these cells. Such studies
are critically important in improving our understanding of the basic
mechanisms that regulate smooth muscle cell proliferation and,
hopefully, will foster the development of new therapeutic approaches to prevent or abrogate myocyte hyperplasia.
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METHODS |
Cell culture.
Human tracheal tissue was obtained from lung transplant donors in
accordance with procedures approved by the University of Pennsylvania
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, and 0.1 mg/ml of streptomycin; this was replaced every
72 h.
Human ASM 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 (23). These cells also retain functional signaling
pathways that are important in mediating ASM contraction as determined by fura 2 measurements of agonist-induced changes in cytosolic calcium
(21, 23). All experiments were performed with a minimum of
three different cell lines. Each human ASM cell line was established with tracheal tissue from a single human donor. Jurkat and U937 cells
were purchased from the American Type Culture Collection (Manassas, VA).
Transfection of human ASM cells.
Cultures of human ASM cells were transfected with the calcium phosphate
transfection system (Life Technologies, Grand Island, NY) according to
the manufacturer's protocol. The plasmid DNAs used in these studies
were purified with the QIAGEN EndoFree Plasmid Maxi Kit (QIAGEN,
Valencia, CA) and were endotoxin free. Cells were incubated with 10 µg of either pEGFP-N1 (Stratagene, La Jolla, CA), pCG, pCG-p110*-Myc,
pSR
, or pSR
-p85-HA plasmids (11) for 16 h. For
experiments designed to examine whether transfection affects
mitogen-induced DNA synthesis in ASM cells, the cells were washed with
PBS and incubated for 48 h in Ham's F-12 medium supplemented with
10% FBS. Cultures were then trypsinized, resuspended in PBS, and
sorted with the use of dual-wavelength flow cytometric analysis on a
Coulter Epics-XL (Coulter, Hialeah, FL). Laser excitation for green
fluorescent protein (GFP) was at 488 nm, with an emission wavelength of 525 nm. Sorted cells were plated at a density of 80,000 cells/well on two-well plastic chamber slides (Nunc, Naperville, IL)
and incubated for 24 h in Ham's F-12 medium supplemented with 10% FBS. Monolayers were growth arrested by incubation in Ham's F-12
medium with 0.1% BSA for 48 h. Subsequently, the cells were treated with either 10 ng/ml of EGF (R&D Systems, Minneapolis, MN),
10% FBS, 1 U/ml of thrombin (Calbiochem, San Diego, CA), or diluent.
After 16-18 h of mitogen stimulation, human ASM cells were labeled
with 10 µM 5-bromo-2'-deoxyuridine (BrdU) for 24 h. Detection of
BrdU incorporation was performed as described in Microinjection
and measurement of DNA synthesis.
For the cloning of stable transfectants of human ASM cells, the cells
were transfected with the aforementioned calcium phosphate method with
a pEGFP-N1 plasmid that encoded the neomycin resistance gene. After
48 h, the cells were trypsinized and replated on 100-mm plates at
a 1:20 dilution and were cultured in 10% FBS-containing Ham's F-12
medium supplemented with 300 µg/ml of Geneticin (G418; Life
Technologies, Grand Island, NY). The colonies were then tested for
expression of GFP with a Nikon microscope equipped with appropriate GFP
filters. Clones containing 100% GFP-positive cells were then plated on
24-well plates at a density of 18,000 cells/well for [3H]thymidine incorporation studies.
Microinjection and measurement of DNA synthesis.
Near-confluent human ASM cells grown on two-well plastic chamber slides
(Nunc, Naperville, IL) were growth arrested. Microinjection pipettes
(Femtotip I, Eppendorf, Hamburg, Germany) were filled with either
Tris-EDTA buffer with 5 mg/ml of rabbit IgG, 0.375 mg/ml of pCG-p110*
with 5 mg/ml of rabbit IgG, or 0.375 mg/ml of pCG-p110DN with 5 mg/ml
of rabbit IgG. Rabbit IgG served as a marker for the microinjected cells.
Microinjection was performed as described previously (1).
Briefly, ~2 h after microinjection, human ASM cells were treated with
10 ng/ml of EGF, 1 U/ml thrombin, 10% FBS, or diluent. Fifteen hours
later, the thymidine analog BrdU (10 µM) was added to all wells.
Twenty-four hours after the addition of BrdU, the cell monolayers were
fixed with 3.7% paraformaldehyde (Polysciences, Warrington, PA) and
then permeabilized with 0.1% Triton X-100. After denaturation of the
DNA with 4 N HCl (3 min at room temperature), the monolayers were
incubated for 1 h at 37°C with 2 µg/ml of murine anti-BrdU
antibody (Becton Dickinson, San Jose, CA) and then with 10 µg/ml of
Texas Red-conjugated anti-mouse antibody for 1 h at 37°C
(Jackson ImmunoResearch Laboratories, West Grove, PA) to detect
BrdU-positive cells.
The slides were examined with a fluorescence microscope (Aristoplan,
Leica, Wetzlar, Germany) with the appropriate fluorescence filters at
×200 magnification. Results are presented as mitotic index, defined as
the percentage of BrdU-positive nuclei per number of cells microinjected.
[3H]thymidine incorporation assays.
DNA synthesis was measured with a [3H]thymidine
incorporation assay. Cells were growth arrested by incubating the
cultures in serum-free medium consisting of Ham's F-12 medium with 5 ng/ml of insulin and 5 ng/ml of transferrin (24) on
day 10. Near-confluent, growth-arrested cells were then used
because cells can be synchronized in the G0/G1
phase of the cell cycle, and at this baseline, they minimally
incorporate [35S]methionine and
[3H]thymidine (23, 24). After 24 h in
serum-free medium, the cells were stimulated with either 10 ng/ml of
EGF, 10% FBS, or 1 U/ml of thrombin. After 16-18 h of
stimulation, human ASM cells were labeled with 3 µCi/ml of
[methyl-3H]thymidine (40-60 Ci/mmol;
Amersham Pharmacia, Arlington Heights, IL) for 24 h. The cells
were then scraped and lysed, and the protein or DNA was precipitated
with 10% trichloroacetic acid. The precipitant was aspirated onto
glass filters, extensively washed, dried, and counted (23,
24).
RT-PCR analysis.
RT-PCR analysis was performed as described (5). Briefly,
total RNA was extracted from growth-arrested ASM cells with the acid-phenol method (5). cDNA was produced from this RNA by reverse transcription with oligo(dT) primers (Promega, Madison, WI) and
Superscript II reverse transcriptase (Life Technologies, Grand Island,
NY). Specific cDNAs for the PI3K isoforms were amplified with primer
pairs (Table 1) by PCR for 30 cycles:
denaturing at 94°C for 30 s, annealing at 56°C for 30 s,
and extension at 72°C for 30 s. The primer pairs yielded the
appropriate sizes of RT-PCR products as determined by electrophoresis
in 1% agarose gels (5). The RT-PCR products were
subcloned into Bluescript SK+ (Stratagene, La Jolla, CA), and their
nucleotide sequences were confirmed by dideoxy sequencing with
Sequenase (USB/Amersham).
Immunolocalization of hemagglutinin-tagged p85.
ASM cells transiently transfected with pSR
-p85-HA (11)
or control pSR
cDNA were growth arrested for 48 h in Ham's
F-12 medium supplemented with 0.1% BSA. The cells were then stimulated with 1 U/ml of thrombin for 5 or 15 min or treated with diluent alone.
Cells were washed with ice-cold PBS, fixed with 2% paraformaldehyde, and blocked with 2% FBS in PBS. Immunostaining was performed with 0.5 µg/ml of a primary anti-hemagglutinin (HA) tag mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and a 1:200 dilution of secondary Alexa 594 antibody (Molecular Probes, Eugene, OR). Immunofluorescence was viewed at ×630 magnification with a Zeiss
Axiovert LSM150 confocal microscope.
Data analysis.
Data points from individual assays represent the mean values of
triplicate measurements. Significant differences among groups were
assessed with ANOVA (Bonferroni-Dunn test) or by
2
analysis, with values of P < 0.05 sufficient to reject
the null hypothesis for all analyses. All experiments were designed
such that matched control conditions existed within each experiment to
enable statistical comparison of paired samples. Each set of experiments was performed with a minimum of three different human ASM
cell lines.
 |
RESULTS |
PI3K isoforms in human ASM cells.
In most cell types, a variety of PI3K isoforms are expressed.
Activation of PI3K isoforms appears to modulate specific cellular functions. To date, the repertoire of PI3K isoforms expressed in human
ASM remains unknown. With the use of specific primers (Table 1) and
RT-PCR, the PI3K isoforms expressed in human ASM were characterized. As
shown in Fig. 1, analysis of the RT-PCR products from human ASM cells revealed expression of class IA p110
and p110
, class II, and class III PI3K isoforms. Class IB p110
PI3K was not detected by RT-PCR. To confirm the specificity of the
primers designed for class IB p110
PI3K, RT-PCR was performed with
total RNA from Jurkat and U937 cells, which are known to express this
isoform. In Jurkat and U937 cells, but not human ASM cells, p110
was
present. Protein expression of class IA PI3K was also examined by
immunoprecipitation and immunoblotting with specific antibodies to the
p85, p110
, and p110
isoforms as shown in Fig.
2. Immunoprecipitation and immunoblotting
with an anti-p110
antibody revealed no p110
isoforms in human ASM
cell lysates (data not shown). These data suggest that human ASM cells
express class IA, II, and III PI3K but not the class IB p110
isoform. The absence of class IB p110
is interesting because this
isoform has been implicated in GPCR activation, which mediates
agonist-induced cytosolic calcium mobilization in a variety of cells.

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Fig. 1.
Expression of class IA phosphatidylinositol 3-kinase
(PI3K) isoforms in human airway smooth muscle (HASM) cells. Total RNA
from cultured HASM, Jurkat, or U937 cells was reverse transcribed, and
the resulting cDNA was amplified with primers specific for the
catalytic and regulatory subunits of PI3K isoforms. PCR products were
size fractionated on a 1% agarose gel. Top: catalytic
subunit of PI3Ks expressed in HASM cells. PCR products are 452 bp from
p110 , 432 bp from p110 , 381 bp from PI3K-C2, and 353 bp from
Vps34p. Middle: detection of p110 expression in HASM,
Jurkat, and U937 cells. The PCR product from p110 was 346 bp.
Bottom: regulatory subunit of PI3Ks expressed in HASM cells.
Products are 425 bp from p85, 336 bp from p55, and 271 bp from p50.
This is a representative experiment from 3 replicate experiments with
different HASM cell lines.
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Fig. 2.
Class IA catalytic subunits of PI3K p110 and p110
form heterodimers with p85 regulatory subunits. HASM cells were
immunoprecipitated with antibodies specific to either the p85
regulatory subunit or the p110 or - catalytic subunits. Each
immunoprecipitate (IP) was split into 3 samples, subjected to denatured
SDS-PAGE, and immunoblotted (IB) with either anti-p85, anti-p110 , or
anti-p110 antibodies. These are representative immunoblots from 3 separate experiments with the same results.
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The HA-p85 regulatory subunit of class IA PI3K translocates to the
cell membrane after thrombin stimulation of human ASM cells.
Evidence suggests that GPCRs may activate class IA or class IB PI3K and
that this activation appears to be cell and tissue specific. In most
cell types, the class IB p110
isoform appeared to be important in
mediating GPCR effects. Because our laboratory (17) previously
described the fact that thrombin stimulates human ASM cell DNA
synthesis by activation of a pertussis toxin-sensitive GPCR and because
human ASM cells do not express class IB p110
but activate PI3K, we
were interested in characterizing the PI3K isoform that mediates the
effects of thrombin in these cells. To determine whether class IA PI3K
modulates thrombin-induced signaling in human ASM cells, monolayers
were transiently transfected with the pSR
-HA-p85 encoding HA-tagged
p85 regulatory subunit of class IA PI3K. Transfected cells were plated
on chamber slides, growth arrested for 24 h, and then stimulated
with thrombin for 5 and 15 min or treated with diluent alone. Thrombin
(1 U/ml) was used because in a previous study (17), this
concentration was optimal for activation of PI3K and DNA synthesis in
human ASM cells. The translocation of the HA-tagged p85 was
characterized by immunofluorescence microscopy. In control cells,
HA-p85 was diffusely present throughout the cytoplasm as shown in Fig.
3. After 5 min of thrombin stimulation,
HA-p85 was no longer diffusely distributed in the cytosol but clustered
near the cell membrane. Relocation of HA-p85 was observed 5 and 15 min
after stimulation with thrombin. These data suggest that thrombin
activates a receptor that induces translocation from the cytosol to the
plasmalemma of the transiently expressed HA-p85 regulatory subunit of
class IA PI3K.

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Fig. 3.
Thrombin-induced translocation
of hemagglutinin (HA)-p85 PI3K. Growth-arrested HASM cells were treated
for 5 and 15 min with 1 U/ml of thrombin or diluent alone (Control).
Monolayers were washed, fixed, and immunostained with primary HA tag
and Alexa 594 secondary antibodies as described in METHODS.
Immunofluorescence was visualized on an LSM150 confocal microscope
under ×630 magnification. The number of cells analyzed was >600.
These are representative images from 4 separate experiments with
similar results.
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Transfected human ASM cells retain proliferative responses to
mitogens.
In a variety of cell types, PI3K appears to be necessary for the
mediation of cell growth and proliferation. In human ASM cells, it has
been shown (6, 12, 17) that both RTK- and GPCR-dependent human ASM cell proliferation require PI3K activation. To
date, however, there are no studies to suggest that PI3K activation in
smooth muscle is sufficient to induce cell proliferation. Such studies
require the ability to transfect cells with high-efficiency plasmids
that would constitutively activate PI3K and not alter the ability of
the cells to undergo DNA synthesis. Despite a number of laboratories
having successfully transiently transfected ASM cells (22,
25), to date no laboratory has demonstrated that the transiently
transfected cells retain mitogen-induced proliferative responses.
To overcome these experimental obstacles, we developed a technique to
transiently transfect human ASM cells with a modified calcium phosphate
technique that did not interfere with the proliferative capacity of the
cells. Monolayers transfected with pEGFP-N1, which expresses a red
shift variant of wild-type GFP, enabled us to sort transfected cells
with flow cytometry. After 24 h, human ASM cells expressing GFP
were sorted from untransfected cells. Flow cytometric analysis of cells
transfected with pEGFP-N1 demonstrated a significant (up to 70%)
percentage of cells expressing GFP compared with cells treated with
carrier DNA as shown in Fig. 4. After cell sorting, the population of human ASM cells expressing GFP was
enriched to >99% as shown in Fig. 5. To
determine whether the transfected cells were capable of undergoing
mitogen-induced DNA synthesis, cells expressing and not expressing GFP
were plated on chamber slides, growth arrested, and then stimulated
with 10% FBS, EGF, thrombin, or diluent alone. After 40 h, BrdU
incorporation, a measure of DNA synthesis, was determined with an
anti-BrdU immunocytochemical analysis. Mitotic indexes were
calculated for each of the growth stimuli. As shown in Fig.
6, mitogen-induced DNA synthesis in cells
transfected with pEGFP-N1 was no different from that in mock-transfected cells stimulated by mitogens. These data demonstrate that human ASM cells transfected with pEGFP-N1 reporter plasmid retained the ability to undergo mitogen-induced DNA synthesis.

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Fig. 4.
Flow cytometric analysis of HASM cells transiently
transfected with pEGFP-N1. HASM cells were trypsinized, resuspended in
PBS, and sorted with dual-wavelength flow fluorescence-activated cell
sorting analysis (FACS). Top: green fluorescent protein
(GFP) profile of control cells and pEGFP-N1-transfected cells.
Bottom: quantitative analysis of sorted cells. Area
C (from GFP profile at top) presents total number of
cells in sample; area B (from GFP profile at top)
presents number of cells identified by FACS analysis as expressing GFP.
This is a representative experiment of 7 with similar results.
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Fig. 5.
HASM cells expressing GFP. Cells sorted by flow cytometry for GFP
expression were visualized on a confocal microscope (original
magnification, ×400). Left: cells under visible light.
Right: FITC (narrow band) filter. This is a representative
image of 7 separate experiments.
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Fig. 6.
Transiently transfected HASM cells retain proliferative
responsiveness to mitogens. HASM cells transiently transfected with
pEGFP-N1 were growth arrested for 48 h and then stimulated with
either 10% fetal bovine serum (FBS), 10 ng/ml of epidermal growth
factor (EGF), 1 U/ml of thrombin (Thr), or diluent. DNA synthesis was
assessed by 5-bromo-2'deoxyuridine (BrdU) incorporation as described in
METHODS. Mitotic index is the percentage of BrdU-positive
cells to the total number of cells expressed as a ratio (>600
cells/condition). Data are means ± SE from 3 separate
experiments, with each condition performed in triplicate.
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To further investigate whether human ASM cells are capable of stable
transfection and whether stably transfected human ASM cells retain
mitogenic responses, cells were transfected with a pEGFP-N1 reporter
plasmid containing a Geneticin-resistant gene that enabled the
selection of cells that were stably transfected. After transfection,
Geneticin was added, and >99% of the viable cells expressed GFP.
These confluent cells were then growth arrested and stimulated
with 10% FBS, thrombin, or EGF. As shown in Fig. 7, FBS, thrombin, and EGF stimulated
[3H]thymidine incorporation to 3,146 ± 377, 3,867 ± 142, and 1,362 ± 195 counts · minute
1
(cpm) · well
1, respectively, compared with that
in diluent-treated cells (101 ± 29 cpm/well). Similar responses
were seen in cells transfected and subsequently grown through the
eighth passage. Collectively, these data show that stable transfectants
of human ASM cells retained proliferative responses to mitogens.

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Fig. 7.
Mitogen-induced DNA synthesis of HASM cell clones
expressing GFP. Confluent, growth-arrested HASM cell clones were
stimulated with either 10% FBS, 1 U/ml of thrombin, 10 ng/ml of EGF,
or diluent. [3H]thymidine incorporation was subsequently
assessed as described in METHODS. Data are means ± SE
from 3 experiments. *P < 0.01 by one-way ANOVA
(Bonferroni-Dunn).
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PI3K is sufficient to induce DNA synthesis in human ASM cells.
Because human ASM cells could be stably transfected and because
transfected cells retained proliferative responses, experiments were
performed to address whether PI3K is sufficient to induce mitogenesis.
Monolayers were transfected with p110* cDNA expressing the Myc-tagged
constitutively active form of class IA PI3K, which can activate
signaling pathways independent from receptor stimulation (13,
14). To characterize whether p110* was present in transfected cells, immunoprecipitation of cell lysates with Myc-tagged specific antibodies followed by immunoblotting was performed. As shown in Fig.
8A, human ASM cells
transfected with pCG-p110*-Myc markedly expressed p110*-Myc compared
with cells transfected with empty pCG plasmid. To address whether
constitutively active PI3K is sufficient to induce DNA synthesis,
monolayers were transfected with constitutively active p110* PI3K, and
BrdU incorporation was then measured. As shown in Fig. 8B
and Table 2, expression of constitutively
active p110* alone was sufficient to induce BrdU incorporation compared
with cells transfected with empty plasmid. Mitotic index of
pCG-p110*-transfected cells was 8.196% compared with 0.780% in
control cells (P < 0.05 by
2 test).
Interestingly, expression of p110* also significantly augmented
EGF-stimulated DNA synthesis but had little effect on thrombin- or
FBS-induced DNA synthesis. In parallel experiments, cells were
transfected with a dominant negative PI3K, and mitogen-induced DNA
synthesis was determined. Unfortunately, transient transfection of ASM
cells with dominant negative p110DN PI3K caused monolayers to detach
from the culture plates (data not shown). We speculated that during the
prolonged exposure to the p110DN PI3K, cells may have experienced
toxicity. To address these concerns, we performed single-cell
microinjection techniques utilizing p110DN PI3K and investigated the
effect of transfecting this plasmid on mitogen-induced DNA synthesis.
This technique decreased the exposure of the cells to the dominant
negative plasmid from 7 days to 40 h. As shown in Fig.
9 and Table
3, cells microinjected with the p110DN
PI3K had significantly decreased EGF-, thrombin-, and FBS-induced DNA synthesis compared with those cells microinjected with empty plasmid. Interestingly, the dominant negative p110DN PI3K did not completely abrogate mitogen-induced DNA synthesis. Importantly, cells injected with constitutively active p110* but not stimulated with mitogens had
markedly enhanced BrdU incorporation compared with cells microinjected with control vector. Furthermore, EGF- and thrombin-induced DNA synthesis was enhanced in the presence of microinjected pCG-p110* compared with cells microinjected with vector alone. Interestingly, serum-induced DNA synthesis was unaffected in cells microinjected with
the constitutively active PI3K. Collectively, these data show that
constitutively active p110* is sufficient to induce DNA synthesis in
human ASM cells and that EGF- but not thrombin- or serum-induced DNA
synthesis can be augmented in cells transfected with constitutively
active p110* PI3K.

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Fig. 8.
Constitutively active p110* PI3K is sufficient for DNA
synthesis in HASM cells. A: HASM cells were transiently
transfected with empty pCG plasmid or pCG-p110*-Myc (p110*) plasmid
expressing Myc-tagged constitutively active p110* PI3K.
Immunoprecipitation and immunoblot analysis with specific anti-Myc tag
antibody were performed to detect p110*-Myc protein expression.
B: HASM cells transfected with constitutively active p110*
PI3K or control DNA were growth arrested and then treated with 10 ng/ml
of EGF, 1 U/ml of thrombin, or 10% FBS for 16 h or with diluent
alone (control). After BrdU treatment for 24 h, DNA synthesis was
then measured with anti-BrdU immunofluorescence. Data represent
proportions of >734 cells/condition from pooled experiments; results
are expressed as the mitotic index. Statistical analysis was performed
with the 2 test *Significantly augmented compared with
control cells, P < 0.05.
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Fig. 9.
Microinjection of HASM cells with p110*-PI3K augmented
basal and EGF- and thrombin-induced cell proliferation, whereas EGF-,
thrombin-, and FBS-induced cell proliferation was inhibited by
p110DN-PI3K. Near-confluent, growth-arrested HASM cells were
microinjected with p110*CA-PI3K or p110DN-PI3K and compared with cells
microinjected with vehicle alone. Cells were then treated with 10 ng/ml
of EGF, 1 U/ml of thrombin, 10% FBS, or diluent alone (control) for
16 h. After BrdU treatment for another 24 h, DNA synthesis
was measured with indirect anti-BrdU immunofluorescence. TE, Tris-EDTA.
Data represent proportions of >150 microinjected cells/condition from
pooled experiments; results are expressed as the mitotic index.
Statistical analysis was performed with the 2 test
*Significantly augmented compared with vehicle alone, P < 0.05. **Significantly inhibited compared with vehicle alone,
P < 0.05.
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View this table:
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Table 3.
Effect of microinjection of constitutively active p110* and dominant
negative p110DN PI3K on BrdU incorporation
|
|
 |
DISCUSSION |
In a variety of diseases, smooth muscle cell growth and
proliferation represent pathogenetic mechanisms or responses to injury and repair that markedly alter myocyte function. In asthma, ASM hyperplasia characteristically defines airway remodeling, a complex lesion observed in the bronchi of patients with chronic severe asthma.
Despite a considerable research effort, the precise mechanisms that
regulate ASM cell proliferation remain unclear. New evidence, however,
suggests that mitogens activate receptors whose downstream signaling
events activate PI3K. In vitro studies with pharmacological inhibitors
suggest that PI3K is essential for myocyte proliferation. PI3K,
however, is a family of kinases whose members modulate differential functions in the cell. To date, the repertoire of PI3K isoforms expressed in ASM and their function remain unclear. In this study, we
demonstrated that human ASM cells express class IA, II, and III PI3K
but not the class IB isoform. We showed that activation of a GPCR by
thrombin induced translocation of class IA PI3K and that this isoform
not only mediated thrombin-induced DNA synthesis but also that of EGF-
and serum-induced responses. We now show that constitutively active
p110* class IA PI3K is sufficient to induce DNA synthesis in human ASM cells.
Current evidence suggests that PI3K activation modulates a variety of
physiological functions that appear to be cell specific. With regard to
cell proliferation, PI3K activation appears to be required for cell
growth induced by some but not all mitogens (28).
Homozygous deletion of the p110
catalytic subunit of PI3K in
transgenic mice induces early embryonic lethality that may be due to
profound alterations in the proliferative capacities of a variety of
cell types (3). In another study (3),
fibroblasts isolated from p110
knockout mice failed to replicate in
serum supplemented with growth factors (3). The
p85
-p55
-p50
-deficient mice also succumbed a few days after
birth and had markedly impaired development and proliferation of B
cells (9). Interestingly, transgenic mice expressing
constitutively active class IA PI3K, which is driven by the cardiac
-myosin heavy chain promoter, underwent marked cardiac hypertrophy,
whereas mice expressing the dominant negative PI3K mutant controlled by
the same promoter had markedly attenuated heart sizes
(30). Taken together, these studies suggest that class IA
PI3K, not only in vitro but also in vivo, plays a critical role in
modulating cell growth in mammalian development. In our study, we
showed that human ASM cells express most PI3K isoforms but appear to be
lacking the class IB p110
isoform.
In some cell types, activation of GPCR stimulates class IB PI3K-
activation (10, 19, 20, 26, 31, 34). Despite these data,
it is known that some cells do not express PI3K-
(2).
The precise mechanisms by which GPCR activation stimulates PI3K remain
unknown. Our laboratory (17) previously found that thrombin, a
mitogen that activates a pertussis toxin-sensitive GPCR, stimulates
PI3K activation and that the p85 regulatory subunit of class IA PI3K
associates with tyrosine-phosphorylated proteins in response to
thrombin. With wortmannin or LY-294002, we (17) showed
that thrombin-induced ASM mitogenesis was completely abrogated. Collectively, these data suggest that in human ASM cells, GPCR activation stimulates class IA PI3K and that this isoform may modulate
proliferative responses. In the current study, we demonstrated that
after thrombin stimulation, HA-tagged p85 PI3K translocated from the
cytosol to the cell membrane. These data suggest that a GPCR is capable
of translocating class IA PI3K in human ASM cells.
To determine whether PI3K activation is necessary or sufficient to
stimulate ASM cell DNA synthesis, we used a newly developed transfection technique that did not alter the proliferative capacity of
the cells to respond to mitogens. The cells were transfected with a
chimeric model of class IA PI3K in which the inter-SH2 region of the
p85 regulatory subunit was covalently linked to its binding site at the
p110 NH2-terminal region of the catalytic subunit. Klippel
et al. (14), using this form of constitutively active p110* in COS-7 cells, demonstrated that PI3K was sufficient for
stimulation of Akt/Rac, c-Jun amino-terminal kinase, and
p70S6k. Previously, our laboratory also showed
(17) that constitutively active p110* was sufficient for
activation of p70S6k in human ASM cells. We now show that
transient expression of constitutively active p110* is sufficient to
induce DNA synthesis in ASM cells in the absence of mitogens. This
finding is in agreement with the work of Klippel et al.
(13), who showed that activation of PI3K was sufficient to
stimulate rat embryonic fibroblasts and COS-7 cells to enter the cell
cycle. Interestingly, in human ASM cells, the level of p110*-induced
DNA synthesis was markedly lower than that induced by EGF, thrombin, or
serum, and thus although PI3K activation is sufficient to induce DNA
synthesis in ASM cells, other signaling pathways that act in parallel
or that are more effective inducers of PI3K may play a role in
modulating mitogen-induced DNA synthesis in these cells. This notion is
also supported by the experiments with the dominant negative PI3K
construct. EGF-, thrombin-, or FBS-induced BrdU incorporation was
decreased in cells microinjected with the dominant negative class IA
p110 but was not completely inhibited.
Our study yields novel information on the regulation of human ASM cell
mitogenesis. Several limitations regarding our study need to be
addressed, however. A major limitation concerns the variability of
transfection efficiency and the difficulty of interpretation of data
obtained from a mixed population of transfected and untransfected cells. To overcome these difficulties, we performed
fluorescence-activated cell sorting that allowed us to enrich the
population of transfected cells to ~99% as shown in Fig. 5. Although
effective, these studies required a large number of cells and
sophisticated cell-sorting techniques. In addition, the experimental
approach in this study required cotransfection of cells with two
plasmids, one containing GFP and the other the target gene. The cell
sorting recognizes only GFP and assumes that the plasmid with the
target gene is coinfected. Further studies need to be performed with
the target gene cloned into the GFP-containing plasmids and thus
eliminate this concern. The physiological relevance of overexpression
of target proteins can also be called into question. It was evident that prolonged overexpression of p110DN PI3K induced the cells to
detach from the culture plates, which induced toxicity. Fortunately, our microinjection experiments decreased cell exposure time to p110DN
and yielded useful data. A high level of p110* expression, as shown in
Fig. 8A (17), may also affect other cell
functions apart from proliferation. We attempted to overcome these
obstacles by using both transient transfection and microinjection
techniques. As investigators, however, we recognize the limitations of
such studies.
Together, our data show that in human ASM cells, most PI3K isoforms are
expressed. More importantly, we showed that thrombin activates class IA
PI3K and that this isoform is sufficient to induce DNA synthesis in
human ASM cells without mitogen stimulation. Interestingly, inhibition
of class IA PI3K, in part, inhibits mitogen-induced DNA synthesis. The
lack of complete abrogation of mitogen-induced proliferative responses
in human ASM cells suggests that other pathways parallel to PI3K
activation likely modulate myocyte growth. Further studies are
necessary to identify such pathways to define the mechanisms that
regulate smooth muscle cell growth and that lead to the pathological
findings of smooth muscle hyperplasia in such diseases as asthma,
chronic bronchitis, and atherosclerosis.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Klippel for the pCG and pCG-p110*-Myc; Dr. M. Kasuga for the pSR
and pSR
-p85-HA expression vectors; and Dr. R. Penn, Dr. S. Harada, and Dr. J. Zhang for helpful discussion about the
transfection protocols. 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-55301 and HL-64063 (to R. A. Panettieri), American Heart
Association and Lymphangioleiomyomatosis Foundation grants (to V. P. Krymskaya), and a grant from the National Health and Medical Research Council of Australia (C. J. Martin Fellowship 977301 to A. J. Ammit).
Address for reprint requests and other correspondence: V. P. Krymskaya, Pulmonary, Allergy and Critical Care Division, Univ. of
Pennsylvania, Rm. 847 BRB II/III, 421 Curie Blvd., Philadelphia, PA
19104-6160 (E-mail: krymskay{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. Section 1734 solely to indicate this fact.
Received 14 July 2000; accepted in final form 6 November 2000.
 |
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