Meakins-Christie Laboratories and the Heisler Laboratory of the Montreal Chest Institute Research Centre, McGill University, Montreal, Quebec, Canada H2X 2P2
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ABSTRACT |
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Fischer rat airway smooth muscle (ASM) models two
potential risk factors for asthma: hyperresponsiveness to contractile
agonists and to growth stimuli. The aim of this study was to identify
the mechanisms responsible for enhanced ASM mitogenic response in Fischer rats compared with the control Lewis strain. The enhanced Fischer ASM cell growth response to fetal bovine serum (FBS) could not
be accounted for by phospholipase C, mitogen-activated protein kinases,
or tyrosine kinase activities as assessed by pharmacological inhibition
and Western blotting. In contrast, depletion of phorbol ester-sensitive
isoforms of the serine/threonine kinase protein kinase C (PKC) removed
the difference in growth response between the rat strains.
Additionally, FBS selectively induced serine/threonine phosphorylation
of a 115-kDa protein in Fischer ASM cells. Enhanced activation of
PKC-I and decreased activation of PKC-
in Fischer compared with
Lewis cells following FBS stimulation were suggested by Western
blotting of membrane and cytosolic fractions. The data are consistent
with a role for PKC in the enhanced ASM cell growth of hyperresponsive rats.
proliferation; epidermal growth factor; tyrosine phosphorylation; serine/threonine phosphorylation; U-73122
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INTRODUCTION |
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ONE OF THE CONSEQUENCES of chronic airway inflammation in asthma is remodeling of the airways. Increased airway smooth muscle (ASM) mass has been documented both in asthmatics (9-11, 17, 19, 21) and in animal models of asthma (18, 31, 34) and may affect airway responses to spasmogens by both increasing contractile responses and increasing luminal closure for a given contractile response (28). Compared with other structural abnormalities in the airways of asthmatics, increased ASM mass is the most important determinant of excessive airway lumen narrowing (25).
Airway hyperresponsiveness is influenced by both environmental and genetic factors, and the growth of ASM may be similarly influenced. Current research on mechanisms of ASM growth focuses on proliferation as a secondary event, i.e., in response to inflammatory mediators. We have published evidence for an alternative (but not exclusive) hypothesis that smooth muscle growth in hyperresponsive airways reflects a genetic predisposition of ASM cells for enhanced growth during normal development (39). Enhanced mitogenic responsiveness of smooth muscle cells in the vasculature has likewise been implicated in the etiology of hypertension (13-16, 32, 33). Inasmuch as enhanced responsiveness of ASM cells to mitogenic stimuli may be important in the development of asthma, identification of the relevant growth mechanisms may lead to new therapeutic approaches for the treatment of asthma.
We used an inbred rat model of airway hyperresponsiveness and enhanced ASM growth; the highly inbred Fischer rat strain is hyperresponsive to inhaled agonists and has more ASM than the control Lewis strain (12). Fischer rat ASM also has a greater contractile response in vitro than Lewis rat ASM (22), and ASM cells from these rat strains show differences in calcium transients in response to contractile agonists (36). ASM cells from Fischer rats have an enhanced proliferative response compared with ASM cells from Lewis rats, which persists over many passages in culture and is determined by postreceptor mechanisms operative during G0 or G1 of the cell cycle (39). There are several pathways known to operate in G0 or G1 of the cell cycle, which could potentially produce enhanced signals in Fischer compared with Lewis ASM cells. Important members of these pathways include various tyrosine kinases, serine/threonine kinases [notably mitogen-activated protein (MAP) kinases and protein kinase C (PKC)], and phospholipase C (PLC).
In this study we have investigated differences between Fischer and
Lewis ASM cells in growth response, in inhibition of that response by
inhibitors of PLC, tyrosine kinases and PKC, and in phosphorylation on
tyrosine and serine/threonine residues following growth stimulation. In
addition, we have characterized the expression profile and membrane
translocation of PKC isozymes following growth stimulation in this
model. The data suggest a role for PKC-I and -
but not for the
other signaling molecules studied in the enhanced ASM cell growth of
the highly inbred, hyperresponsive Fischer rat strain.
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MATERIALS AND METHODS |
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Materials.
Standard chemicals were purchased from Sigma Immunochemicals (St.
Louis, MO), and cell culture solutions were obtained from GIBCO
Laboratories (Grand Island, NY). Electrophoresis reagents were from
Bio-Rad (Mississauga, ON), and enhanced chemiluminescence reagents and
film were from Amersham (Oakville, ON).
[3H]thymidine was
purchased from New England Nuclear (Lachine, PQ). Epidermal growth
factor (EGF) was obtained from ICN Biochemicals (Costa Mesa, CA).
U-73122 and U-73343 were purchased from Biomol Research Laboratories
(Plymouth Meeting, PA). Antibodies obtained from Upstate Biotechnology
(Lake Placid, NY) were mouse anti-phosphotyrosine clone 4G10 and rabbit
anti-rat MAP kinase R2. From Sigma Immunochemicals, we obtained
anti-phosphotyrosine mouse IgG1 clone PT-66, anti-phosphothreonine mouse IgG2b clone PTR-8, anti-phosphoserine mouse IgG1 clone PSR-45, rabbit anti-PKC-, mouse anti-PKC-
II, mouse anti-PKC-
, rabbit anti-PKC-
, rabbit anti-human platelet-derived growth factor (PDGF) receptor, secondary goat anti-mouse horseradish peroxidase (HRP), preadsorbed against rat proteins, and secondary biotinylated goat anti-rabbit IgG. Rabbit anti-PKC-
I was from Santa Cruz
Biotechnology (Santa Cruz, CA), and secondary donkey anti-rabbit HRP
was from Amersham.
Animals. Male Fischer and Lewis rats (7-9 wk old) were obtained from a commercial source (Harlan Sprague Dawley, Indianapolis, IN) and maintained on standard rat chow in a conventional animal care facility at McGill University. All studies were performed using protocols in accordance with standards set by the Canadian Council on Animal Care.
Cell culture. ASM cells were prepared according to a previously described enzymatic dispersion method, which was verified to produce cells of smooth muscle origin (8, 39). Acutely dissociated cells were seeded on 25-cm2 Falcon flasks (Becton Dickinson, Mississauga, ON), allowed to grow to confluence, and were then passaged at 5,000 cells/cm2 onto 24-well (2-cm2) Linbro plates (Flow Lab, Mississauga, ON). All experiments were done on passage 1-5 cells. Cells were maintained in 1:1 DMEM-Ham's F-12 culture medium supplemented with 10% fetal bovine serum (FBS) and changed every 3 days unless otherwise stated.
Growth assay. Cell growth was assessed by tritiated thymidine uptake, which was previously demonstrated to give similar results to direct cell counting in our system (39). [3H]thymidine incorporation was assayed as previously described (39). Mitogens with and without inhibitors were incubated for 24 h with cells that had been growth arrested for 3 days in the log phase of cell growth. Growth arrest was with 1% FBS, previously demonstrated by cell cycle analysis to produce effective arrest of these cells (39). Mitogenic stimuli were 10% FBS or 1% FBS with 1.6 pM to 16 nM EGF. Growth inhibitors included herbimycin A (0.01-5 µM), genistein (2.5-40 µM), staurosporine (1-100 nM), calphostin C (10 nM to 1 µM), phorbol 12-myristate 13-acetate (PMA; 1.6 µM incubated for 24 h before and throughout FBS stimulation), and the PLC inhibitor U-73122 or its inactive analog U-73343 (50 µM). Samples were assayed in triplicate, and each sample was prepared from cells originating from different animals.
Extraction and fractionation of cell lysates.
First to fifth passage ASM cells from four to six separate animals of
each strain were grown on 150 × 25-mm tissue culture dishes
(Becton Dickinson), matched for confluence between strains, and growth
arrested with 1% FBS for 3 days before stimulation with 10% FBS for
2-30 min. Confluence at the time of stimulation was approximately
80%. Cells were rinsed twice with ice-cold PBS, scraped with a rubber
policeman in lysis buffer (1% Nonidet P-40 detergent, 20 mM Tris, pH
8.0, 137 mM NaCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride,
0.15 U aprotinin/ml, and 1 mM sodium orthovanadate) and extracted at
4°C for 40 min with agitation. Lysates were
centrifuged at 4°C for 10 min at 14,000 rpm (Eppendorf centrifuge
5402), and supernatants were stored at 40°C for Western blots on whole cell lysates. For blots on specific subcellular fractions, lysates (supernatant and low-speed pellet) were sonicated and then recentrifuged at low speed as above, and then low-speed supernatant was ultracentrifuged for 1 h at 100,000 g (Beckman L-70 ultracentrifuge). High-speed supernatants (cytosolic fraction) were separated from high-speed pellets (membrane fraction). Pellets were rinsed with PBS and then resuspended in 1 ml of lysis buffer prior
to storage at
40°C. Successful fractionation was verified by
Western blotting for the known membrane-bound PDGF receptor.
Separation of proteins and Western blotting. Protein was quantified by the method of Bradford (5). Protein loads varied between 5 and 40 µg depending on the protein studied but were always equal between Fischer and Lewis samples; samples from each rat strain were always loaded together on the same gel. Proteins were resolved by 7.5% SDS-PAGE (Bio-Rad minigels) or 4-12% gradient SDS-PAGE (Novex minigels). The separated proteins were electroblotted onto 0.22-µm-pore nitrocellulose membranes. Posttransfer staining of the gel with Coomassie blue confirmed the efficacy of the transfer. Any variations from lane to lane in protein load or efficiency of electrophoretic transfer were corrected following densitometry of Ponceau S-stained nitrocellulose membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline with 0.05% Tween 20 before antibody incubation. For assessment of protein phosphorylation, primary antibodies were biotinylated, and for MAP kinases, the secondary antibody was biotinylated. Streptavidin-HRP incubation followed labeling with biotinylated antibodies. Secondary antibodies used for detection of PKC isoforms were themselves conjugated to HRP. All secondary antibodies were verified to produce no nonspecific binding in the absence of primary antibodies under the conditions of these experiments (data not shown). For studies of serine/threonine phosphorylation, the anti-phosphoserine and anti-phosphothreonine antibodies were mixed 1:1. Blots were developed by enhanced chemiluminescence and were routinely within the linear range of intensity for densitometric analysis. Signals were digitized on a Scanjet IIcx scanner (Hewlett-Packard, Palo Alto, CA), and the density of the bands was analyzed with a commercially available software package (Scanplot; Peter Cunningham, Calgary, Alberta).
Statistical analysis. Statistical analysis was done using Student's t-test or a paired Student's t-test where appropriate. Differences were not considered significant unless the two-tailed P value was <0.05. For dose-response curves, differences were tested by two-way ANOVA with dose and strain as independent variables. Differences between strains were not considered significant unless the P value for the strains was <0.05, and the P value for the dose times strain interaction was >0.15.
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RESULTS |
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Growth stimulation by FBS and EGF.
Fischer rat ASM cells stimulated with FBS have an enhanced mitogenic
response compared with Lewis rat ASM cells (Fig.
1A). This finding was extended to another mitogen, EGF (Fig.
1B). EGF was mitogenic to both
strains, but Fischer ASM cells had a greater maximal response to EGF.
The EC50 was 14.5 pM in Fischer
ASM cells and 9.6 pM in Lewis ASM cells (geometric means;
P = 0.61).
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Effect of PLC inhibition on growth. Inhibition of PLC-dependent processes by the inhibitor U-73122 did not inhibit ASM cell growth induced by 10% FBS in either rat strain. In fact a slight potentiation of growth was observed: 129,290 ± 22,223 counts/min with U-73122 vs. 108,510 ± 14,780 counts/min with vehicle in Fischer and 83,468 ± 7,254 counts/min with U-73122 vs. 59,666 ± 5,831 counts/min with vehicle in Lewis rats (means ± SE for 5 rats each). The difference was not attributable to the vehicle DMSO, which of itself had no effect (data not shown). Importantly, under the same conditions, this compound abolished PLC-mediated intracellular calcium release. As expected, U-73343, an inactive, close structural analog of U-73122 (3), did not prevent PLC-mediated calcium release. Growth in response to FBS was slightly inhibited in both Fischer and Lewis ASM cells in the presence of U-73343 (data not shown). The mechanisms by which U-73122 enhanced proliferation and U-73343 inhibited proliferation of both Fischer and Lewis ASM cells are unclear. The salient point with respect to the questions addressed in this study remains that enhanced growth of Fischer ASM cells with respect to Lewis ASM cells was not affected by PLC inhibition.
Growth inhibition with tyrosine kinase inhibitors.
Genistein dose dependently inhibited growth (Fig.
2A),
with an IC50 of 12.4 µM in
Fischer ASM cells and 11.3 µM in Lewis ASM cells (geometric means). A
second inhibitor, herbimycin A, showed similar results (Fig.
2B), inhibiting growth dose
dependently with an IC50 of 49.9 nM in Fischer cells and 59.9 nM in Lewis cells. Differences were not
statistically significant. There was no evidence of toxicity following
treatment with either compound.
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Tyrosine-phosphorylated proteins in ASM cell lysates.
Tyrosine-phosphorylated proteins of approximately 70 and 115 kDa were
present in unstimulated ASM cells from both Fischer and Lewis rats
(Fig. 3). After stimulation
with 10% FBS, tyrosine phosphorylation of these bands increased in a
time-dependent fashion, peaking at 30 min of stimulation (Fig.
4,
inset). The increased phosphorylation was inhibited by 40 µM genistein, the same
concentration of genistein that abolished the response to FBS in growth
assays (Fig. 4).
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MAP kinase activation following FBS stimulation.
Two anti-phosphotyrosine immunoreactive proteins had the same molecular
mass as the MAP kinases extracellular signal-regulated kinase (ERK 1)
and ERK2 (42 and 44 kDa). Tyrosine phosphorylation of these two
proteins, indicative of their activation by MAP kinase kinase (MEK),
was undetectable in unstimulated cells but was induced equally in
Fischer and Lewis ASM cells by 30 min of stimulation with FBS (Fig.
5). Similarly, an antibody specific to MAP
kinases revealed similar expression of p42 and p44 in unstimulated
Fischer and Lewis rat cells. After FBS stimulation for 30 min, a
portion of each of these proteins migrated more slowly (Fig.
6), reflecting increased MAP kinase
phosphorylation of tyrosine and threonine residues by MEK (2). This
change in mobility occurred in every stimulated preparation, with no
differences between Fischer and Lewis ASM, consistent with the
similarity between the strains of anti-phosphotyrosine immunoreactivity
of the 42- and 44-kDa proteins.
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Serine/threonine phosphorylation in ASM cell lysates.
There was no difference between Fischer and Lewis ASM cell lysates in
the expression of serine/threonine-phosphorylated proteins prior to
stimulation with 10% FBS (data not shown). However, stimulation with
10% FBS for 30 min induced significantly more phosphorylation of an
approximately 115-kDa protein in Fischer ASM cells compared with that
in Lewis ASM cells (Fig. 7).
Serine/threonine phosphorylation of a 70-kDa protein was also noted
following stimulation but did not differ between Fischer and Lewis ASM
cells.
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Growth inhibition with PKC inhibitors.
Growth of ASM cells from both strains of rat was dose dependently and
completely inhibited by the relatively nonspecific kinase inhibitor
staurosporine and by the specific PKC inhibitor calphostin C (Fig.
8). The
IC50 values in Fischer and Lewis
ASM cells, respectively, were 16.1 and 13.9 nM for staurosporine and
130.4 and 116.4 nM for calphostin C (geometric means). These were not
statistically significantly different. However, when phorbol
ester-sensitive isoforms of PKC were selectively depleted by prolonged
exposure to PMA, only the Fischer response to 10% FBS was
significantly inhibited, and the difference between Fischer and Lewis
growth responses was abolished (Fig. 9),
suggesting that enhanced growth of Fischer ASM cells is
mediated by one or more of the phorbol ester-sensitive PKC isoforms.
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Expression of phorbol ester-sensitive PKC isoforms.
Of the phorbol ester-sensitive PKC isoforms examined, Fischer and Lewis
whole cell lysates expressed immunoreactive PKC- (a doublet of
approximately 79 and 80 kDa), -
I (a doublet of approximately 81 and
82 kDa), and -
(a doublet of approximately 76 and 78 kDa), whereas
the isoforms PKC-
II and -
were undetectable even with fivefold
higher protein load (40 µg/lane). Preincubation for 48 h with PMA,
which abolished the growth difference between rat strains, also
abolished the expression of PKC-
, -
I, and -
(data not shown).
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DISCUSSION |
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We have characterized growth signaling pathways in ASM cells from Fischer and Lewis rats, a model for investigating the genetic component of airway hyperresponsiveness. This model holds promise as a tool for the identification of mechanisms that increase airway hyperresponsiveness and induce ASM growth in vivo, but because the enhanced growth responsiveness of Fischer ASM cells has only recently been described (39), mechanisms of growth signaling in these cells are as yet uncharacterized. We have examined several signaling molecules of potential importance to growth, and our data suggest that a number of these are unlikely to contribute to enhanced Fischer ASM cell growth. These include tyrosine kinases, PLC-mediated events, and MAP kinases. Instead, our data are consistent with a role for diacylglycerol (DAG)-dependent PKC isoforms in enhanced Fischer ASM growth.
In a previous study, Fischer rat ASM cells demonstrated a greater mitogenic response than Lewis cells to both FBS and PDGF (39). We have now shown that the Fischer growth response to another mitogen, EGF, is also enhanced. A greater response of Fischer cells to a third mitogen supports previous evidence that the mechanisms of enhanced Fischer growth are postreceptor (39) and suggests that the mechanisms responsible lie in a pathway shared by these mitogens.
EGF and PDGF both bind to specific receptors with intrinsic tyrosine kinase activity and induce tyrosine phosphorylation of a number of intracellular signaling molecules via both receptor and nonreceptor tyrosine kinases (6). We therefore examined the effect of tyrosine kinase inhibitors on proliferative responses of Fischer and Lewis ASM cells. Both genistein and herbimycin A inhibited growth dose dependently, with no difference between the rat strains. Additionally, direct examination of FBS-induced protein tyrosine phosphorylation revealed no differences between the rat strains. These data argue against a putative role for enhanced tyrosine kinase activity as a mechanism for enhanced Fischer ASM cell growth.
After initial tyrosine phosphorylations, growth signaling pathways continue through various serine/threonine phosphorylations. We therefore immunoblotted cell lysates with antibodies to phosphorylated serine and threonine residues. After 30 min of stimulation with FBS, Fischer cells had enhanced serine/threonine phosphorylation of a band of approximately 115 kDa, suggesting that a serine/threonine kinase, for which this protein is a substrate, is more active in Fischer than in Lewis ASM cells.
The MAP kinases ERK1 and ERK2 are serine/threonine kinases that are important for cell proliferation [reviewed by Johnson and Vaillancourt (23)]. ASM cells from both Fischer and Lewis rats expressed ERK1 and ERK2; however, no differences were apparent between Fischer and Lewis cells in either expression of these kinases or their activation [as reflected by phosphorylation and reduced mobility on SDS-PAGE (1, 2, 7, 35)]. These observations were made following 30 min of stimulation, corresponding to the stimulation time for enhanced p115 phosphorylation and clearly surpassing 2-10 min, which is the time frame in smooth muscle cells for disappearance of transient MAP kinase activation (24, 27). Prolonged, rather than transient, MAP kinase activation appears relevant to proliferative responses (26). The data therefore argue against a role for MAP kinases in both enhanced Fischer cell growth and enhanced serine/threonine phosphorylation of p115.
Other potential mediators of the enhanced growth response and enhanced
serine/threonine phosphorylation of Fischer rat ASM cells are members
of the PKC family. Members of this family of serine/threonine kinases
are involved in growth signaling by many mitogens, including PDGF, EGF,
and FBS (30). The conventional (,
I,
II, and
) and novel
(
,
,
,
, and µ) isoforms of PKC are activated in vivo by
DAG (20), whereas the atypical isoforms (
and
) are not.
Inhibition of all isoforms of PKC by staurosporine and calphostin C
inhibited FBS-induced growth dose dependently and completely, with no
difference between rat strains. However, selective depletion of
DAG-dependent isoforms of PKC by prolonged exposure to the phorbol
ester PMA abolished the enhancement in Fischer cell growth, indicating
that this enhanced growth response is dependent on DAG-sensitive PKC
isoforms. Consistent with this, we observed more membrane-localized
PKC-
I in Fischer than in Lewis ASM cells following FBS stimulation.
Translocation of DAG-sensitive PKC from the cytosol to the membrane is
commonly used as an indication of its activation (20). PKC-
I
promotes proliferation of rat fibroblasts (4) and smooth muscle cells via an acceleration of S phase entry (37). The observed enhancement in
PKC-
I activation in growth-stimulated Fischer cells may then be
related to the accelerated S phase entry we previously observed in
these cells (39). We also noted an apparent decrease in activation of
PKC-
in stimulated Fischer compared with Lewis cells. This too is
consistent with the enhanced Fischer proliferative response because
PKC-
promotes differentiation in other cell types (a phenotype that
is inversely related to proliferation) and it is commonly inactivated
in the neoplastic phenotype (29, 38). A number of other novel PKC
isoforms have not been investigated here and may also prove to be
associated with enhanced Fischer ASM cell growth responses; the
observed abolition of enhanced growth by PMA represents the net effect
of abolishing potentially opposing growth effects of the various
DAG-sensitive isoforms.
In conclusion, our data support a role for DAG-dependent isoforms of PKC in a genetically determined susceptibility to enhanced ASM growth. Although studies of the role of ASM in asthma have traditionally concentrated on airway hyperresponsiveness to contractile stimuli, it may prove to be that hyperresponsiveness of ASM to growth stimuli constitutes another crucial susceptibility to development of asthma.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. B. Tolloczko for performing measurements of intracellular calcium and to Dr. Elizabeth Fixman for valuable comments on the manuscript.
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FOOTNOTES |
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This study was supported by Medical Research Council Grant 7852.
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.
Address for reprint requests and other correspondence: J. G. Martin, Meakins-Christie Laboratories, McGill Univ., 3626 St. Urbain St., Montreal, Quebec, Canada H2X 2P2. (E-mail: jmartin{at}meakins.lan.mcgill.ca).
Received 26 June 1998; accepted in final form 6 August 1999.
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