Protein kinase C is involved in enhanced airway smooth muscle cell growth in hyperresponsive rats

M. E. Zacour and J. G. Martin

Meakins-Christie Laboratories and the Heisler Laboratory of the Montreal Chest Institute Research Centre, McGill University, Montreal, Quebec, Canada H2X 2P2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta I and decreased activation of PKC-delta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta I and -delta but not for the other signaling molecules studied in the enhanced ASM cell growth of the highly inbred, hyperresponsive Fischer rat strain.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha , mouse anti-PKC-beta II, mouse anti-PKC-gamma , rabbit anti-PKC-delta , 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-beta 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Growth of Fischer airway smooth muscle (ASM) cells (open bar and open circle ) was enhanced compared with that in Lewis ASM cells (solid bar and ) in response to both 10% fetal bovine serum (FBS) (A; * P < 0.05) or epidermal growth factor (EGF) (B; P < 0.01 for Fischer vs. Lewis rats). Growth was assayed by [3H]thymidine incorporation. Value of 0 was assigned to control cells receiving no EGF; doses of EGF ([EGF]) from 1.6-1,600 pM are expressed as logs of these values. Error markers are SE. CPM, counts/min.

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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Fig. 2.   Growth response to 10% FBS was inhibited by the tyrosine kinase inhibitors genistein (A) or herbimycin A (B). Values are normalized to growth effect of 10% FBS in each preparation. open circle , Mean of 5 Fischer ASM cells; , mean of 5 Lewis ASM cells. There were no significant differences between ASM cells from Fischer and Lewis rats. Error markers are SE.

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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Fig. 3.   Representative Western blot showing tyrosine phosphorylation without (-) or with (+) 30 min of stimulation with 10% FBS. Each F is cell preparation from separate Fischer rat and each L is cell preparation from separate Lewis rat. Lane 3 contains molecular-mass markers.



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Fig. 4.   Growth stimulation caused increase in tyrosine phosphorylation of 70-kDa (solid bars) and 115-kDa (open bars) bands, which was abolished by genistein. There were no differences between ASM cells from Fischer and Lewis rats. Western blotting followed either no stimulation (Control, 3 Fischer and 4 Lewis rats, no significant difference, are represented in 1 set of bars), 30 min of stimulation with 10% FBS (5 Fischer and 5 Lewis rats), or 30 min of stimulation with 10% FBS in the presence of 40 µM genistein (Genistein, 4 Fischer and 4 Lewis rats, no significant difference, are represented in 1 set of bars). Ordinate shows density of bands expressed as percent of internal standard. Inset, kinetics of tyrosine phosphorylation following stimulation, with time in minutes on abscissa and optical density of band (in arbitrary units) on ordinate. Only 1 band (70 kDa) from 1 rat strain (Lewis) is shown; similar results were obtained for 115-kDa band and in Fischer cells.

There were no differences in either the baseline or the FBS-induced levels of tyrosine phosphorylation of the 70- or 115-kDa proteins, comparing cells from Fischer and Lewis rats following either 30 min (Fig. 4) or 2 min of stimulation. Optical densities following 2 min of stimulation for p70 were Fischer 20.5 ± 6.5 and Lewis 24.0 ± 4.4 and for p115 Fischer 8.3 ± 2.3 and Lewis 5.8 ± 0.6 (means ± SE, n = 6 each strain, no significant difference). This was verified three times for each of five different cellular preparations for each rat strain. Results were virtually identical using a second anti-phosphotyrosine antibody obtained from a different commercial source. Tyrosine-phosphorylated proteins of 200, 180, 55, 44, and 42 kDa were also present in FBS-stimulated cells from both strains of rat. These proteins were less abundant than the 70- or 115-kDa proteins but were nonetheless evident on longer exposures. Densitometric analysis revealed no differences in tyrosine phosphorylation of these proteins, comparing Fischer and Lewis ASM cells (data shown below for 42- and 44-kDa proteins).

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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Fig. 5.   There was no difference between Fischer (open circle ) and Lewis () rat ASM cells in level of tyrosine phosphorylation of 42- or 44-kDa band following 30 min of mitogenic stimulation with 10% FBS. Two preparations from separate animals in each rat strain are shown; these were representative of 4 experiments. Inset, anti-phosphotyrosine immunoblot: lane A is a 45-kDa molecular-mass marker, lanes B and C are unstimulated Lewis and Fischer ASM cells, respectively, and lanes D-G are cell preparations following 30 min of stimulation (D and F are Lewis ASM cells and E and G are Fischer ASM cells). AU, arbitrary units.



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Fig. 6.   Bar graph shows densitometric analysis of mitogen-activated protein kinase-immunoreactive band, which shifted from 42 kDa before stimulation (-) to 43 kDa following stimulation for 30 min with 10% FBS (+) in Fischer (open bars) and Lewis (solid bars) ASM cells. There were no significant differences between rat strains. A second band, which shifted from 44 to 45 kDa, demonstrated same pattern (data not shown). Inset, representative Western blot: lanes 1 and 3 are Fischer ASM cells, lanes 2 and 5 are Lewis ASM cells, and lane 4 is a 45-kDa molecular-mass marker.

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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Fig. 7.   Serine/threonine phosphorylation of 115-kDa protein following growth stimulation for 30 min with 10% FBS was significantly greater in Fischer (open bars) than in Lewis (closed bars) ASM cells (* P < 0.05, n = 5 in each rat strain). A 70-kDa protein was also phosphorylated but with no difference between strains. Error markers indicate SE. Inset, representative Western blot of 2 separate cell preparations from each strain.

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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Fig. 8.   Inhibition of ASM cell growth response to 10% FBS by the broad protein kinase inhibitor staurosporine (A) or specific protein kinase C (PKC) inhibitor calphostin C (B). open circle , Fischer ASM cells. , Lewis ASM cells. n, 5 or 6 for each data point, with no significant difference between strains.



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Fig. 9.   Effect of depletion of phorbol ester-sensitive PKC isoforms on growth stimulation by 10% FBS. Fischer or Lewis ASM cells were stimulated with 10% FBS with (+) or without (-) 48-h preincubation with phorbol 12-myristate 13-acetate (PMA 1.6 µM). Incubation with PMA decreased growth response of Fischer cells to 10% FBS (* P < 0.05, n = 3) but not that of Lewis cells (n = 5). Error markers are SE.

Expression of phorbol ester-sensitive PKC isoforms. Of the phorbol ester-sensitive PKC isoforms examined, Fischer and Lewis whole cell lysates expressed immunoreactive PKC-alpha (a doublet of approximately 79 and 80 kDa), -beta I (a doublet of approximately 81 and 82 kDa), and -delta (a doublet of approximately 76 and 78 kDa), whereas the isoforms PKC-beta II and -gamma 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-alpha , -beta I, and -delta (data not shown).

We fractionated lysates into their membrane and cytosolic components to reveal PKC translocation from the cytosol to the membrane with growth stimulation (Fig. 10). Successful separation of the two fractions was confirmed by immunoblotting for a known membrane-bound protein, the PDGF receptor, which was clearly detectable in the membrane fraction of these lysates but absent in the cytosolic fraction (data not shown). After 30 min of stimulation with 10% FBS, the expression of PKC-beta I was enhanced in Fischer compared with Lewis cell membranes, primarily due to an increase in the lower (81-kDa) band of the doublet. This suggests greater activation of PKC-beta I in growth-stimulated Fischer cells (Fig. 10, middle and inset; note the bar represents densitometry of both doublet bands together). No differences between rat strains in membrane expression of PKC-alpha were detected (Fig. 10, left and inset). The lower band of the PKC-delta doublet (76 kDa) was decreased in stimulated Fischer compared with Lewis ASM cell membranes, consistent with a decreased activation of this isoform in growth-stimulated Fischer relative to Lewis ASM cells (Fig. 10, right and inset; note bars represent densitometry of 76-kDa band only; the 80-kDa band of PKC-delta doublet did not differ between strains). The results shown were obtained by pooling cell lysates from five separate animals from each rat strain.


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Fig. 10.   Expression of cytosolic (C)- and membrane (M)-localized PKC-alpha (left), PKC-beta I (middle), and PKC-delta (right) in Fischer (open bars) and Lewis (solid bars) ASM cells following stimulation for 30 min with 10% FBS. Bottom bar graphs demonstrate densitometry of 1 experiment, whereas bar graphs in insets show means ± SE of 3-4 experiments (membrane fractions only; * P < 0.05). Insets, representative Western blot of 5 pooled Fischer (left) and 5 pooled Lewis (right) membrane fractions.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta I, beta II, and gamma ) and novel (delta , epsilon , eta , theta , and µ) isoforms of PKC are activated in vivo by DAG (20), whereas the atypical isoforms (zeta  and lambda ) 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-beta 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-beta I promotes proliferation of rat fibroblasts (4) and smooth muscle cells via an acceleration of S phase entry (37). The observed enhancement in PKC-beta 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-delta in stimulated Fischer compared with Lewis cells. This too is consistent with the enhanced Fischer proliferative response because PKC-delta 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.


    ACKNOWLEDGEMENTS

We are grateful to Dr. B. Tolloczko for performing measurements of intracellular calcium and to Dr. Elizabeth Fixman for valuable comments on the manuscript.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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