1 Department of Microbiology
and Immunology, Asthma is frequently associated with abnormal
airway smooth muscle (ASM) growth that may contribute to airway
narrowing and hyperresponsiveness to contractile agents. Although
numerous hormones and cytokines have been shown to induce human ASM
(HASM) proliferation, the cellular and molecular mechanisms underlying
HASM hyperplasia are largely unknown. Here we characterize the roles of
the mitogen-activated protein kinase (MAPK) superfamily [p42/p44
MAPK, c-Jun amino-terminal kinase/stress-activated protein kinase
(JNK/SAPK), and p38] in mediating hormone- and cytokine-induced
HASM proliferation. Significant enhancement of
[3H]thymidine
incorporation in HASM cultures was observed only by treatment with
agents (epidermal growth factor, platelet-derived growth factor,
thrombin, and phorbol 12-myristate 13-acetate) that promoted a strong
and sustained activation of p42/p44 MAPK. Significant activation of the
JNK/SAPK and p38 pathways was only observed on stimulation with
interleukin (IL)-1
asthma; mitogen-activated protein kinase; inflammation; G
protein-coupled receptor; receptor tyrosine kinase
INCREASED AIRWAY SMOOTH MUSCLE (ASM) mass consequent to
cellular hypertrophy or hyperplasia has long been implicated in the pathophysiology and pathogenesis of airway diseases such as asthma (14,
20). Although numerous factors are known to contribute to excessive
airway narrowing, an increase in ASM mass has been asserted to be the
most important abnormality responsible for the increased airway
resistance observed in response to bronchoconstricting agents in both
asthma and chronic obstructive pulmonary disease (16). Thus
considerable interest lies in the characterization of stimuli and
associated intracellular mechanisms that regulate ASM proliferation.
The mitogen-activated protein kinases (MAPKs) constitute a family of
serine/threonine kinases that mediate the transduction of external
stimuli [typically via receptor tyrosine kinase (RTK) or G
protein-coupled receptor (GPCR) activation] into intracellular signals that regulate cell growth and differentiation. Among the best-characterized mammalian MAPKs are
1) the 42- and 44-kDa extracellular signal-regulated kinases (ERKs) ERK2 and ERK1, also collectively referred to as p42/p44 MAPK; 2) the
c-Jun amino-terminal kinase (JNK) or stress-activated protein kinase
(SAPK); and 3) p38 MAPK. Each is
activated by a signaling cascade composed of a series of sequentially
activated protein kinases acting downstream from small G proteins
representative of the Ras superfamily. These highly homologous MAPK
cascades, strongly conserved through evolution, are subject to
regulation at numerous junctures and exhibit significant cross talk
among themselves and other pathways for the purpose of integrating
various intercellular signals into discrete physiological responses
[for reviews of MAPK signaling, see van Biessen et al. (33),
Gutkind (9), and Hershenson et al. (11)].
To date, studies of ASM mitogenesis have focused primarily on the
identification of agents capable of inducing ASM proliferation. Unfortunately, little is known regarding the intracellular signaling pathways in ASM involved in transducing signals from external mitogenic
stimuli. Although a handful of studies have asserted the role of
p42/p44 MAPK in promoting nonhuman ASM proliferation, similar studies
in human ASM (HASM) are lacking. Moreover, the roles of other MAPK
homologs (JNK/SAPK, p38) and other signaling intermediates associated
with cell cycle regulation have remained largely uncharacterized in ASM.
In the present report, we characterize the relationship between HASM
proliferation and activation of the p42/44, JNK/SAPK, and p38 MAPKs by
numerous physiologically relevant agents. A fundamental role of p42/p44
activation in regulating HASM proliferation is revealed by data that
demonstrate its requirement for and correlation with mitogen-activated
growth. In addition, comparison of these data with those observed from
other species suggests possible explanations for the species-specific
differences among studies in ASM mitogenesis and offers future
directions for studies in this field.
Materials.
p20-5XGal4-Luc (35) and MLV.Gal4-Elk-1 (19) were provided by Channing
Der (Univ. of North Carolina, Chapel Hill, NC). HASM cell culture.
HASM cultures were established as described by Panettieri et al. (24)
from human tracheae obtained from lung transplant donors in accordance
with procedures approved by the University of Pennsylvania Committee on
Studies Involving Human Beings. Briefly, a segment of trachea superior
to the carina was dissected under sterile conditions, and the
trachealis muscle was isolated. Approximately 1 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, 10 mg of soybean trypsin inhibitor, and 10 U/ml of
elastase. Tissue was digested 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). Aliquots of cell suspension were plated at a
density of 1.0 × 104
cells/cm2 in Ham's F-12 medium
supplemented with 10% FBS, 100 U/ml of penicillin, 100 µg/ml of
streptomycin, and 100 µg/ml amphotericin B. Characterization of this
cell line with regard to immunofluorescence of smooth muscle actin and
agonist-induced changes in cytosolic calcium has been previously
reported (24).
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
and tumor necrosis factor-
,
agents that did not appreciably stimulate HASM proliferation. Two
different inhibitors of MAPK/extracellular signal-regulated kinase
kinase (MEK), PD-98059 and U-0126, inhibited mitogen-induced [3H]thymidine
incorporation in a manner consistent with their ability to inhibit
p42/p44 activation. Elk-1 and activator protein-1 reporter activation by mitogens was similarly inhibited by inhibition of MEK,
suggesting a linkage between p42/p44 activation, transcription factor
activation, and HASM proliferation. These findings establish a
fundamental role for p42/p44 activation in regulating HASM
proliferation and provide insight into species-specific differences
observed among studies in ASM mitogenesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
FosdE6AP-1-Luc (8)
was provided by Craig Hauser (The Burnham Institute, La Jolla, CA).
[Methyl-3H]thymidine
(1 µCi/ml) and enhanced chemiluminescence reagents were purchased
from Amersham (Arlington Heights, IL). Phosphorylation state-specific
and phosphorylation state-independent antibodies against p42/p44 MAPK,
JNK/SAPK, and p38 were purchased from New England Biolabs (Beverly,
MA). Luciferase assay reagent was purchased from Promega (Madison, WI).
PD-98059 was purchased from Calbiochem (La Jolla, CA). U-0126 was a
gift from DuPont Pharmaceuticals (Wilmington, DE). All other reagents
were purchased from Sigma (St. Louis, MO) or from previously identified
sources (21).
HASM cell proliferation assay.
After 48 h of treatment with IT medium, cells were stimulated with
various agents as indicated. In selected experiments, cells were first
pretreated for 30 min with either vehicle (0.1% dimethyl sulfoxide),
30 µM PD-98059, or 10 µM U-0126. Standard concentrations of agents,
unless otherwise noted, were as follows: epidermal growth factor (EGF),
0.01-10 ng/ml; platelet-derived growth factor (PDGF)-BB, 10 ng/ml;
thrombin, 1 U/ml; interleukin (IL)-1, 20 U/ml; tumor necrosis factor
(TNF)-
, 10 ng/ml; histamine, 10 µM; carbachol, 1 mM; and phorbol
12-myristate 13-acetate (PMA), 100 µM. After 16 h of stimulation,
cells were labeled with 3.0 µCi [methyl-3H]thymidine
(1 µCi/ml) and incubated at 37°C for 24 h. Cells were then washed
with PBS, harvested with 0.05% trypsin-0.53 mM EDTA, and lysed with
20% trichloroacetic acid. The precipitate was aspirated onto filter
paper and counted in scintillation vials. Data points from individual
proliferation experiments represent the mean values derived from six wells.
Analysis of MAPK phosphorylation. HASM cells were plated in six-well plates as described in HASM cell culture and stimulated with various agents for 0-12 h. At the indicated time points, cells were washed once with cold PBS and lysed by addition of 1% SDS sample buffer. Lysates were boiled for 5 min, and 20 µl of cell lysate were electrophoresed on a standard 10% SDS-polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membranes. To detect MAPK activation, blots were probed with antibodies that recognize the phosphorylated forms of p42/p44, p38, or JNK. These antibodies specifically recognize the phosphorylated amino acids Thr202/Tyr204 of p42/p44, Thr180/Tyr182 of p38, or Thr183/Tyr185 of JNK. Briefly, blots were incubated for 1 h in blocking buffer consisting of 20 mM Tris · HCl, pH 7.5, 150 mM NaCl [Tris-buffered saline (TBS)], 0.05% Tween 20 [TBS-Tween (TBS-T)] containing 5% (wt/vol) dried nonfat milk, followed by incubation overnight at 4°C in fresh blocking buffer (5% milk in TBS-T for p42/p44; 5% BSA in TBS-T for p38 and JNK) containing a 1:1,000 dilution of the phospho-specific MAPK antibody. The next day, blots were washed three times with TBS-T, followed by a 1-h room-temperature incubation with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody. Blots were then washed three times in TBS-T and visualized by enhanced chemiluminescence. To control for uniformity of gel loading, blots were first stained with 0.2% Ponceau S or parallel blots were run and probed with antibodies that recognize both phosphorylated and nonphosphorylated forms of the respective MAPKs. Signals were quantitated on a densitometer (Molecular Dynamics, Sunnyvale, CA) with the use of autoradiographs that depicted bands within a linear range of exposure.
Elk-1 and activator protein-1 reporter
assays.
HASM cells were transfected with the use of a replication-defective
adenovirus (Ad5-GPT) as previously described (28) with either
1) MLV.Gal4-Elk-1 (19) and
p20-5XGal4-Luc (35) or 2) FosdE6AP-1-Luc (8). MLV.Gal4-Elk-1 contains the Gal4 DNA binding domain linked to the Elk-1 transcription activation domain (19). Luciferase reporter activity in p20-5XGal4-Luc is under control of five
repeats of the Gal4 DNA binding domain (35). Although transfection by
the adenovirus method employed arrests HASM cell cycling by an
undetermined mechanism, this method has been determined useful for
examining acute signaling events and represents the only transfection
method identified to date that transfects HASM with reasonable
efficiency (~40-60%) (28). Twelve hours after transfection,
cells were passaged into 12-well dishes at a density of 2.0 × 104
cells/cm2 in Ham's F-12 medium
supplemented with 10% FBS. Eight hours later, the medium was switched
to IT medium, and cells were maintained for 48 h. Cells were pretreated
for 30 min with either vehicle or 10 µM U-0126 and then stimulated
for 15 h with various agonists; washed twice in
Ca2+- and
Mg2+-free PBS; lysed in 25 mM
Tris · HCl, pH 7.8, 2 mM dithiothreitol, 2 mM EGTA,
10% glycerol, and 1% Triton X-100; and harvested. Results were
qualitatively similar in experiments examining effects of 10 h of
stimulation (data not shown). Samples were briefly centrifuged to
pellet Triton-insoluble material, and the supernatants were transferred
to Eppendorf tubes and frozen at
70°C. Samples were subsequently assayed for luciferase activity with the use of firefly luciferase substrate as per the manufacturer's directions.
Data analysis. Data are presented as means ± SE. Statistically significant differences among groups were assessed by t-test for paired or independent samples, with P values < 0.05 sufficient to reject the null hypothesis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of p42/p44 MAPK and cell
proliferation.
HASM cells were cultured such that experiments examining the effects of
various agents on both cell proliferation and MAPK superfamily
activation could be performed simultaneously. Stimuli included
activators of RTKs (EGF and PDGF) and GPCRs (thrombin, histamine, and
carbachol), the cytokines IL-1 and TNF-
, and the protein kinase C
activator PMA. As shown in Fig.
1A,
EGF, PDGF, thrombin, and PMA strongly induced
[3H]thymidine
incorporation in HASM, whereas histamine, carbachol, IL-1
, and
TNF-
produced little or no effect. We have previously demonstrated
that EGF, thrombin, and PMA-induced increases in [3H]thymidine
incorporation in HASM are associated with significant increases in cell
number, whereas small increases in DNA synthesis elicited by numerous
other agents are not associated with proliferation (4, 22, 23, 25). In
parallel cultures, these agents were also tested for their capacity to
activate p42/p44 MAPK in a time-dependent manner. EGF, PDGF, thrombin,
and PMA induced a strong and sustained activation of p42/p44 MAPK for
up to 3 h after cell stimulation (Fig. 1,
B and
C), after which activation declined
toward basal levels by 12 h (see legend to Fig. 1 and below). In
contrast, the cytokines IL-1
and TNF-
each activated p42/p44 MAPK
more transiently, inducing maximal activation of p42/p44 at 30 min, with activated p42/p44 levels rapidly returning to basal levels thereafter. Histamine- and carbachol-stimulated p42/p44 activation was
also transient (peak activation at 10 min) and decidedly less robust
(approximately one-third of that induced by EGF at 10 min). When the
integrated p42/p44 response was calculated over the first 3 h of
stimulation, responses determined for EGF, thrombin, and PMA
stimulation were significantly greater (~2.5- to 10-fold) than those
determined for IL-1
, TNF-
, histamine, and carbachol (Fig.
1D). This disparity in
integrated activity was even greater (~5- to 20-fold) when
values were calculated from experiments examining p42/p44 activation
from 0 to 12 h (data not shown). These data suggest that the ability of
a given agonist to induce HASM cell proliferation is dependent on its
capacity to provoke a strong and sustained activation of p42/p44 MAPK.
|
Activation of JNK/SAPK and p38 kinases.
The activation of JNK and p38 kinases has been shown in several cell
types to influence cell proliferation and differentiation (27).
Examination of p38 (Fig. 2)
and JNK/SAPK (Fig. 3) activation in HASM
cultures revealed that IL-1 and TNF-
were the only agents capable
of significantly activating these kinases. With respect to p38, EGF,
thrombin, and histamine weakly and transiently activated p38 (up to
4-fold over basal levels over the first 30 min), whereas PMA and
carbachol had virtually no effect (Fig. 2A). In
separate experiments, PDGF also exhibited a weak and transient
activation of p38 (~3-fold of basal at 30 min; data not shown).
IL-1
and TNF-
activated p38 maximally at 10-30 min (both
~25-fold), with levels diminishing significantly by 3 h (Fig.
2B). As noted, however, IL-1
and TNF-
were not able
to induce significant [3H]thymidine
incorporation (Fig. 1A), suggesting that activation of
the p38 kinase pathway does not significantly stimulate HASM proliferation.
|
|
Effect of MEK1 and -2 inhibition on mitogenesis and
p42/p44 activation.
To further explore the relationship between p42/p44 MAPK activation and
HASM proliferation, we tested the effects of PD-98059 and U-0126, both
specific inhibitors of the p42/p44 activators MEK1 and MEK2 (1, 7).
Thirty minutes of pretreatment with 30 µM PD-98059 or 10 µM U-0126
inhibited PMA-, thrombin-, EGF-, and PDGF-mediated activation of
p42/p44 (Fig
4A).
Interestingly, U-0126 was significantly more effective than PD-98059 in
inhibiting mitogen-induced phosphorylation of p42/p44. We attribute
this effect largely to the disparity in apparent
IC50 values between the two
compounds (~1 µM and 10-50 µM for U-0126 and PD-98059, respectively) and to the limitation of solubility of PD-98059, which
renders it difficult to achieve effective concentrations in intact
cells. PD-98059 and U-0126 also had similar effects on mitogen-induced
HASM proliferation, demonstrating inhibition of
[3H]thymidine
incorporation commensurate with their respective abilities to inhibit
p42/p44 MAPK activation (Fig. 4B).
Interestingly, 90% of EGF-stimulated
[3H]thymidine
incorporation could still be inhibited when U-0126 was added up to 6 h
after stimulation with EGF (Fig.
4C). When added 12 h after
stimulation, a significant mitogenic effect of EGF was retained,
although U-0126 exposure throughout the remaining 28 h of the
experiment was still able to inhibit ~50% of EGF-stimulated [3H]thymidine
incorporation. These data suggest that late activation of p42/p44
influences the magnitude of EGF-stimulated DNA synthesis. Alternatively, the capacity of U-0126 to reduce p42/p44 activity significantly below basal levels may influence these results if a
minimal level of p42/p44 activity is required during the period of
[3H]thymidine
incorporation.
|
Dose-dependent effect of EGF on HASM proliferation
and p42/p44 activation.
To further examine the relationship between the magnitude and duration
of p42/p44 activation and HASM proliferation, we assessed agonist-induced
[3H]thymidine
incorporation and the time-dependent activation of p42/p44 in parallel
cultures stimulated by EGF in a dose-dependent manner. As shown in Fig.
5, A and
B, stimulation with increasing concentrations of EGF (0.01-10 ng/ml) resulted in a progressive and sustained activation of p42/p44
(EC50 of the integrated response ~0.5 ng/ml). A concentration of 0.01 ng/ml of EGF produced a very weak activation of p42/p44. A significantly greater and more sustained activation of p42/p44 was observed after stimulation with 0.1 ng/ml of
EGF. Progressively greater p42/p44 activation by 1.0 and 10 ng/ml of
EGF was observed over the first 3 h, whereas, at later time points,
activation of p42/p44 was attenuated but relatively sustained. The
dose-dependent effect of EGF on HASM [3H]thymidine
incorporation was similar, displaying an
EC50 of ~0.5 ng/ml, consistent
with previous observations (21). When values of
[3H]thymidine
incorporation are plotted as a function of the integrated p42/p44
activation, a relationship is revealed in which EGF-stimulated p42/p44
activation correlates (r = 0.96) with
proliferation (data not shown).
|
Transcriptional activation of Elk-1 and activator
protein-1.
HASM cultures were transfected with luciferase reporter constructs
responsive to intracellular phosphorylation/activation of the
transcription factors Elk-1 and activator protein (AP)-1. As shown in
Fig. 6, each of the mitogens,
EGF, PDGF, thrombin, and PMA, stimulated Elk-1 and AP-1 activation
approximately two- to fourfold that of basal activity. None of the
other agents tested (histamine, carbachol, IL-1, and TNF-
)
appreciably induced Elk-1 luciferase activity, although stimulation
with histamine and IL-1
did significantly increase AP-1 luciferase
activity to ~1.5-fold of basal levels (data not shown). Pretreatment
with 10 µM U-0126 before stimulation with mitogens essentially
eliminated the mitogen-induced increases in both Elk-1 and AP-1
luciferase activity. These findings suggest a requirement for p42/p44
activation for Elk-1 and AP-1 activation in HASM as well as linkage
between p42/p44 MAPK activation, Elk-1/AP-1 activation, and HASM
proliferation.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study we demonstrate that potent HASM mitogens such as EGF,
PDGF, thrombin, and phorbol esters produce a strong and sustained
activation of p42/p44 MAPK. In contrast, agents that do not
significantly promote HASM proliferation, such as histamine, carbachol,
IL-1, and TNF-
, produced only a transient increase in p42/p44
MAPK activation. PD-98059 and U-0126, both specific inhibitors of
p42/p44 MAPK activation, inhibited mitogen-induced [3H]thymidine
incorporation in a manner consistent with their ability to inhibit
p42/p44 activation. The apparent requirement of sustained p42/p44 MAPK
activation for promoting HASM mitogenesis is further suggested by
studies examining the dose-dependent effects of EGF. Low concentrations
of EGF resulted in weak, transient increases in p42/p44 activation that
were paralleled by minimal enhancements in
[3H]thymidine
incorporation. Increasing concentrations of EGF progressively increased
both the integrated (0-12 h) p42/p44 and mitogenic responses. These results are similar to those observed in studies of bovine (12,
13, 18), rat (32, 37), and guinea pig ASM (30) that suggest a
relationship between sustained p42/p44 activation and proliferation.
Our data also suggest that the two other members of the MAPK
superfamily, JNK/SAPK and p38, can be activated in HASM but do not
appear to stimulate mitogenesis (or cannot be sufficiently stimulated
to do so). Small numbers of studies examining JNK/SAPK and p38 in ASM
have been performed to date. Shapiro et al. (32) identified JNK
activation by endothelin, thrombin, and TNF- in rat ASM, and Pyne et
al. (31) observed JNK activation in guinea pig ASM treated with
sphingosine and ceramides. Larsen et al. (17) identified p38 activity
in canine ASM stimulated with carbachol, and Pyne and Pyne (29) found
evidence of PDGF-stimulated p38 activity in guinea pig ASM. However,
the sum of information to date has provided little insight into the
roles that JNK/SAPK or p38 might play in regulating ASM mitogenesis.
Studies in other cell types have attributed growth (2, 5, 15, 40), but more frequently anti-proliferative or apoptotic effects (3, 36, 38,
39), to JNK/SAPK and p38. Although our findings suggest
that activation of p38 or JNK/SAPK is not sufficient to stimulate
growth, an inhibitory or facilitatory role of these MAPKs in HASM
proliferation cannot be excluded. Of note, pretreatment of HASM cells
with the p38 inhibitor SB-203580 (6) did result in a small
(~20-30%) but significant increase in both basal and mitogen-induced
[3H]thymidine
incorporation (data not shown). However, this effect was partially
mimicked by the analogous control compound SB-202474, which lacks the
ability to inhibit p38. Thus any effect of p38 on growth in HASM would
appear to be minimal, and further insight into the roles of JNK/SAPK
and p38 in modulating HASM mitogenesis will likely depend on future
development of specific pharmacological inhibitors or
overexpression/dominant-negative strategies, the application of which
in ASM cultures to date has been problematic (28).
Although p42/p44 activation appears required for HASM proliferation and modulation of its activity by a given mitogen appears to correlate with growth, the level of p42/p44 activation observed among the different HASM mitogens is not a precise predictor of an agent's relative mitogenicity. For example, in the present study, thrombin exhibited one of the strongest effects in stimulating [3H]thymidine incorporation but was the least efficacious among the mitogens in activating p42/p44. Conversely, EGF exhibited the most robust activation of p42/p44 but stimulated [3H]thymidine incorporation to a lesser extent than did thrombin and PDGF. This disparity between the rank order of agents with respect to their induction of proliferation and p42/p44 activation suggests that other signaling pathways are important in modulating HASM growth.
Interestingly, the profile of agents that promote proliferation of HASM
differs significantly from that observed for nonhuman ASM. EGF, a
potent mitogen in HASM, is a weak mitogen in bovine ASM (13).
Conversely, insulin-derived growth factor I is mitogenic in bovine ASM
but is a relatively weak mitogen in HASM (Panettieri, unpublished
observations). IL-1 and IL-6 can be shown to induce guinea pig ASM
proliferation through secondary activation of the PDGF receptor (29),
although this does not appear to occur in HASM. Histamine appears to
induce a greater mitogenic response in canine ASM (26) than in HASM. In
addition, histamine has been shown to inhibit both GPCR- and
RTK-activated mitogenesis in bovine ASM (10) in contrast to preliminary
data from our laboratory that suggest that histamine potentiates
EGF-stimulated mitogenesis in HASM (Penn, unpublished data).
What accounts for the differences in the capacity of various agents to induce proliferation among different species of ASM? As mentioned, data from the present study suggest that sustained p42/p44 activation is required for significant enhancement of HASM proliferation, a finding consistent with previous studies of bovine (12, 13, 18), rat (32, 37), and guinea pig ASM (30). For example, in bovine ASM, activation of p42/p44 by PDGF and insulin-like growth factor I is strong and sustained (12, 13) compared with that of EGF, which is relatively weak and transient (13) (similar to that observed for histamine- and carbachol-stimulated p42/p44 in HASM). Thus the cumulative evidence leads us to suggest [as previously postulated by others (11)] that species-specific responses to potential mitogens may not reflect differences in the fundamental mitogenic-signaling pathway mechanisms. Instead, a more likely explanation may be that differences among species with respect to expression level, coupling, and/or regulation of various cell surface receptors dictate the relative mitogenic effect of a given agent. In HASM, high levels of EGF receptor expression are suggested by the low EC50 values (0.5 ng/ml) for both EGF-stimulated p42/p44 activation and proliferation and by the observation that EGF receptor autophosphorylation can be significantly inhibited without diminishing either p42/p44 activation or the mitogenic response to 10 ng/ml of EGF (i.e., evidence of spare receptors) (Krymskaya, Panettieri, and Penn, unpublished observations). Conversely, EGF receptors in bovine ASM may have significantly lower expression or be poorly coupled as suggested by the weak and transient p42/p44 response to EGF stimulation and the relatively higher EC50 (~1.0 ng/ml) for EGF-stimulated [3H]thymidine incorporation (13). The effect of histamine as an inhibitor of RTK-mediated mitogenesis in bovine ASM can be explained by differential expression of histamine receptors linked to different heterotrimeric G protein-signaling pathways. In bovine ASM, activation of H2-histamine receptors coupled to Gs protein, cAMP production, and protein kinase A activation mediates the inhibition of HASM proliferation via inhibition of raf-1 (10). In HASM, histamine does not appreciably stimulate cAMP production (Penn and Benovic, unpublished observations), suggesting that histamine effects in HASM are mediated primarily through the activation of the H1-histamine receptor coupled to Gq protein. Thus, in two specific instances, the expression or complement of receptors expressed in a given ASM culture appears important in mediating the mitogenic response to agonists. A more extensive characterization of RTK and GPCR signaling in ASM should help to elucidate the mechanisms underlying species-specific differences in the response to mitogens.
In summary, the present study establishes a fundamental role of p42/p44 MAPK in regulating HASM proliferation and suggests that activation of JNK/SAPK and p38 pathways has little effect on HASM growth. Small differences in the relationship between p42/p44 activation and HASM proliferation among mitogens suggest that input from other mitogenic pathways serves to modulate the proliferative response to different agents. Future studies examining the interplay between MAPK and other putative mitogenic pathways (34) will help to establish further the cellular and molecular bases for the relationship between asthma and increased ASM mass.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Kristin Brodbeck and Sybil Kane for technical assistance, Channing Der and Craig Hauser for providing reporter constructs, and James Trzaskos for advice concerning the use of U-0126.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-58506 and HL-55301 and National Institute of General Medical Sciences Grant GM-44944.
J. L. Benovic is a recipient of the American Heart Association Established Investigator Award. R. A. Panettieri, Jr., is a recipient of the Career Investigator Award from the American Lung Association.
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: R. B. Penn, Kimmel Cancer Institute, Thomas Jefferson Univ., Rm. 930 Bluemle Life Sciences Bldg., 233 S. 10th St., Philadelphia, PA 19107 (E-mail: rpenn{at}lac.jci.tju.edu).
Received 15 December 1998; accepted in final form 14 April 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alessi, D. R.,
A. Cuenda,
P. Cohen,
D. T. Dudley,
and
A. R. Saltiel.
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase in vitro and in vivo.
J. Biol. Chem.
270:
27489-27494,
1995
2.
Bogoyevitch, M. A.,
J. Gillespie-Brown,
A. J. Ketterman,
S. J. Fuller,
R. Ben-Levy,
A. Ashworth,
C. J. Marshall,
and
P. H. Sugden.
Stimulation of the stress-activated mitogen-activated protein kinase subfamilies in perfused heart. p38/RK mitogen-activated protein kinases and c-Jun N-terminal kinases are activated by ischemia/reperfusion.
Circ. Res.
79:
162-173,
1996
3.
Chen, Y.-R.,
C. F. Meyer,
and
T. H. Tan.
Persistent activation of c-Jun N-terminal kinase 1 (JNK1) in gamma radiation-induced apoptosis.
J. Biol. Chem.
271:
631-624,
1996
4.
Cohen, M. D.,
V. Ciocca,
and
R. A. Panettieri.
TGF-beta 1 modulates human airway smooth-muscle cell proliferation induced by mitogens.
Am. J. Respir. Cell Mol. Biol.
16:
85-90,
1997[Abstract].
5.
Coso, O. A.,
M. Chiariello,
J. C. Yu,
H. Teramoto,
P. Crespo,
N. Xu,
T. Miki,
and
J. S. Gutkind.
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
Cell
81:
1137-1146,
1995[Medline].
6.
Cuenda, A.,
J. Rouse,
Y. N. Doza,
R. Meier,
P. Cohen,
T. F. Gallager,
P. R. Young,
and
J. C. Lee.
SB203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stress and interleukin-1.
FEBS Lett.
364:
229-233,
1995[Medline].
7.
Favata, M. F.,
K. Y. Horiuchi,
E. J. Manos,
A. J. Daulerio,
D. A. Stradley,
W. S. Feeser,
D. E. Van Dyck,
W. J. Pitts,
R. A. Earl,
F. Hobbs,
R. A. Copeland,
R. L. Magolda,
P. A. Scherle,
and
J. M. Trzaskos.
Identification of a novel inhibitor of mitogen-activated protein kinase kinase.
J. Biol. Chem.
273:
18623-18632,
1998
8.
Galang, C. K.,
J. J. Garcia-Ramirez,
P. A. Solski,
J. K. Westwick,
C. J. Der,
N. N. Neznanov,
R. G. Oshima,
and
C. A. Hauser.
Oncogenic neu/erbB-2 increases Et-1, Ap-1 and NF-kb-dependent gene expression, and inhibiting Ets activation blocks neu-mediated cellular transformation.
J. Biol. Chem.
271:
7992-7998,
1996
9.
Gutkind, J. S.
The pathways connecting G-protein coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades.
J. Biol. Chem.
273:
1839-1842,
1998
10.
Hershenson, M. B.,
T. S. Chao,
M. K. Abe,
I. Gomes,
M. D. Kelleher,
J. Solway,
and
M. R. Rosner.
Histamine antagonizes serotonin and growth-factor-induced mitogen-activated protein kinase activation in bovine tracheal smooth muscle cells.
J. Biol. Chem.
270:
19908-19913,
1995
11.
Hershenson, M. B.,
E. T. Naurekas,
and
J. Li.
Mitogen-activated signaling in cultured airway smooth muscle cells.
Can. J. Physiol. Pharmacol.
75:
898-910,
1997[Medline].
12.
Karpova, A. Y.,
M. K. Abe,
J. Li,
P. Liu,
J. M. Rhee,
W. L. Kuo,
and
M. B. Hershenson.
MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L558-L565,
1997
13.
Kelleher, M. D.,
M. K. Abe,
T. S. Chao,
M. Jain,
J. M. Green,
J. Solway,
M. R. Rosner,
and
M. B. Hershenson.
Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L894-L901,
1995
14.
Knox, A. J.
Airway remodelling in asthma: role of airway smooth muscle.
Clin. Sci. (Colch.)
86:
647-652,
1994[Medline].
15.
Komuro, I.,
S. Kudo,
T. Yamzaki,
Y. Zou,
I. Shiojima,
and
Y. Yazaki.
Mechanical stretch activates the stress-activated protein kinases.
FASEB J.
10:
631-636,
1996
16.
Lambert, R. K.,
B. R. Wiggs,
K. Kuwano,
J. C. Hogg,
and
P. D. Pare.
Functional significance of increased airway smooth muscle in asthma and COPD.
J. Appl. Physiol.
74:
2771-2781,
1993[Abstract].
17.
Larsen, J. K.,
I. A. Yamboliev,
L. A. Weber,
and
W. T. Gerthoffer.
Phosphorylation of the 27-kDa heat shock protein via p38 MAP kinase and MAPKAP kinase in smooth muscle.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L930-L940,
1997
18.
Malarkey, K.,
E. R. Chilvers,
M. F. Lawson,
and
R. Plevin.
Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells.
Br. J. Pharmacol.
116:
2267-2273,
1995[Abstract].
19.
Marais, R.,
J. Wynne,
and
R. Triesman.
The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain.
Cell
73:
381-393,
1993[Medline].
20.
Panettieri, R. A.
Regulation of growth of airway smooth muscle by second messenger systems.
In: Airway Wall Remodeling in Asthma, edited by A. G. Stewart. Boca Raton, FL: CRC, 1996, p. 269-293.
21.
Panettieri, R. A.,
R. G. Goldie,
P. J. Rigby,
A. J. Eszterhas,
and
D. W. P. Hay.
Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.
Br. J. Pharmacol.
118:
191-197,
1996[Abstract].
22.
Panettieri, R. A.,
and
M. M. Grunstein.
Airway smooth hyperplasia and hypertrophy.
In: Asthma, edited by P. J. Barnes,
M. M. Grunstein,
A. R. Leff,
and A. J. Woolcock. New York: Raven, 1997, p. 823-842.
23.
Panettieri, R. A.,
I. P. Hall,
C. S. Maki,
and
R. K. Murray.
Alpha-thrombin increases cytoslic calcium and induces human airway smooth muscle cell proliferation.
Am. J. Respir. Cell Mol. Biol.
13:
205-216,
1995[Abstract].
24.
Panettieri, R. A.,
R. K. Murray,
L. R. DePalo,
P. A. Yadvish,
and
M. I. Kotlikoff.
A human smooth muscle cell line that retains physiological responsiveness.
Am. J. Physiol.
256 (Cell Physiol. 25):
C329-C335,
1989
25.
Panettieri, R. A.,
N. A. Rubenstein,
B. Feuerstein,
and
M. I. Kotlikoff.
Beta-adrenergic inhibition of airway smooth muscle inhibition (Abstract).
Am. Rev. Respir. Dis.
143:
A1608,
1991.
26.
Panettieri, R. A.,
P. A. Yadvish,
A. M. Kelly,
N. A. Rubinstein,
and
M. I. Kotlikoff.
Histamine stimulates proliferation of airway smooth muscle and induces c-fos expression.
Am. J. Physiol.
259 (Lung Cell. Mol. Physiol. 3):
L365-L371,
1990
27.
Paul, A.,
S. Wilson,
C. M. Belham,
C. J. M. Robinson,
P. H. Scott,
G. W. Gould,
and
R. Plevin.
Stress-activated protein kinases: activation, regulation and function.
Cell. Signal.
9:
403-410,
1997[Medline].
28.
Penn, R. B.,
R. A. Panettieri, Jr.,
and
J. L. Benovic.
Mechanisms of acute desensitization of the 2AR-adenylyl cyclase pathway in human airway smooth muscle.
Am. J. Respir. Cell Mol. Biol.
19:
338-348,
1998
29.
Pyne, N. J.,
and
S. Pyne.
Platelet-derived growth factor activates a mammalian Ste20 coupled mitogen-activated protein kinase in airway smooth muscle.
Cell. Signal.
9:
311-317,
1997[Medline].
30.
Pyne, N. J.,
P. A. Stevens,
N. Moughal,
and
S. Pyne.
PKC-dependent activation of the type II adenylate cyclase in airway smooth muscle limits the bradykinin-stimulated ERK-2 pathway.
Biochem. Soc. Trans.
23:
200S,
1995[Medline].
31.
Pyne, S.,
J. Chapman,
L. Steele,
and
N. Pyne.
Sphingomyelin-derived lipids differentially regulate the extracellular signal-regulated kinase 2 (ERK-2) and c-Jun N-terminal kinase (JNK) signal cascades in airway smooth muscle.
Eur. J. Biochem.
237:
819-826,
1996[Abstract].
32.
Shapiro, P. S.,
J. N. Evans,
R. J. Davis,
and
J. A. Posada.
The seven-transmembrane-spanning receptors for endothelin and thrombin cause proliferation of airway smooth muscle cells and activation of the extracellular regulated kinase and c-Jun NH2terminal kinase groups of mitogen-activated protein kinases.
J. Biol. Chem.
271:
5750-5754,
1996
33.
Van Biessen, T.,
L. M. Luttrell,
B. E. Hawes,
and
R. J. Lefkowitz.
Mitogenic signaling via G-protein coupled receptors.
Endocr. Rev.
17:
698-714,
1996[Medline].
34.
Walker, T. R.,
S. M. Moore,
M. F. Lawson,
R. A. Panettieri,
and
E. R. Chilvers.
Platelet-derived growth factor-BB and thrombin activate phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth muscle proliferation.
Mol. Pharmacol.
54:
1007-1015,
1998
35.
Westwick, J. K.,
A. D. Cox,
C. J. Der,
M. H. Cobb,
M. Hibi,
M. Karin,
and
D. A. Brenner.
Oncogenic Ras acitivates c-Jun via a separate pathway from the activation of extracellular signal-related kinases.
Proc. Natl. Acad. Sci. USA
91:
6030-6034,
1994[Abstract].
36.
Westwick, J. K.,
Q. T. Lambert,
G. J. Clark,
M. Symons,
L. van Aelst,
R. G. Pestell,
and
C. J. Der.
Rac regulation of transformation, gene expression and actin reorganization by multiple PAK-independent pathways.
Mol. Cell. Biol.
17:
1324-1335,
1997[Abstract].
37.
Whelchel, A.,
J. Evans,
and
J. Posada.
Inhibition of ERK activation attenuates endothelin-stimulated airway smooth muscle cell proliferation.
Am. J. Respir. Cell Mol. Biol.
16:
589-596,
1997[Abstract].
38.
Wilson, D. J.,
K. A. Fortner,
D. H. Lynch,
R. R. Mattingly,
I. G. Macara,
J. A. Posada,
and
R. C. Budd.
JNK, but not MAPK, activation is associated with Fas-mediated apoptosis in human T cells.
Eur. J. Immunol.
26:
989-994,
1996[Medline].
39.
Xia, Z.,
M. Dickens,
J. Raingeaud,
R. J. Davis,
and
M. E. Greenberg.
Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis.
Science
270:
1326-1331,
1995[Abstract].
40.
Zohn, I. E.,
H. Yu,
X. Li,
A. D. Cox,
and
H. S. Earp.
Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.
Mol. Cell. Biol.
15:
6160-6168,
1995[Abstract].