Vol. 276, Issue 4, L564-L570, April 1999
Gi
but not
Gq is linked to activation of
p21ras
in human airway smooth muscle cells
Charles W.
Emala,
Feng
Liu, and
Carol A.
Hirshman
Department of Anesthesiology, Columbia University College of
Physicians and Surgeons, New York, New York 10032
 |
ABSTRACT |
Airway smooth
muscle hypertrophy contributes to the narrowing of asthmatic airways.
Activation of the mitogen-activated protein kinases is an important
event in mediating cell proliferation. Because the monomeric G protein
p21ras is an important
intermediate leading to activation of mitogen-activated protein
kinases, we questioned which heterotrimeric G protein-coupled receptors
were linked to the activation of
p21ras in cultured human airway
smooth muscle and which of the heterotrimeric G protein subunits (
or 
) transmitted the activation signal. Carbachol and
endothelin-1 increased GTP-bound
p21ras in a pertussis
toxin-sensitive manner [ratio of
[32P]GTP to
([32P]GTP + [32P]GDP): control, 30 ± 1.7; 3 min of 1 µM carbachol, 39 ± 1.1; 3 min of 1 µM
endothelin-1, 40 ± 1.2], whereas histamine, bradykinin, and
KCl were without effect. Transfection of an inhibitor of the G protein
-subunit [the carboxy terminus
(Gly495-Leu689)
of the -adrenoceptor kinase 1] failed to
inhibit the carbachol-induced activation of
p21ras. These data suggest that
Gi- but not
Gq-coupled receptors activate p21ras in human airway smooth
muscle cells, and this effect most likely involves the -subunit.
G proteins; carbachol; endothelin; epidermal growth factor; bradykinin; histamine; pertussis toxin
| |
INTRODUCTION |
INCREASED MASS of the smooth muscle lining the airway
(5, 11) and airway hyperresponsiveness are characteristic
features of human asthma, and increased airway smooth muscle DNA
synthesis is seen in a number of animal models of airway
hyperresponsiveness (12, 14, 21). The association between increased
airway smooth muscle mass and airway responsiveness suggests that
enhanced airway smooth muscle proliferation may be involved in the
pathogenesis of the disease. However, the signaling pathways that
regulate the growth and development of airway smooth muscle cells to
various stimuli have not been well characterized.
A number of signal transduction pathways leading to the activation of
mitogen-activated protein (MAP) kinases or extracellular signal-regulated kinases (ERKs) have been implicated in the control of
cell proliferation in airway smooth muscle cells. These include growth
factors that stimulate receptor tyrosine kinases (15) and contractile
agonists that signal through G protein-coupled receptors (13, 19). The
major pathway involved in MAP kinase or ERK activation by growth
factors requires the activation of p21ras (4), a membrane-bound
21-kDa monomeric G protein. Heterogeneity exists in the mechanisms by
which G protein-coupled receptors activate MAP kinases. Depending on
the receptor being activated and on the cell type, MAP kinase
activation may be mediated by pertussis toxin-sensitive (1, 27) or
pertussis toxin-insensitive G proteins (10) and be either protein
kinase C (PKC) (10, 13) or p21ras
dependent (1, 25, 27). In airway smooth muscle cells, a major pathway
by which agonists activate MAP kinases is pertussis toxin insensitive
and is mediated by PKC without the involvement of
p21ras (15).
In native airway smooth muscle cells, carbachol activates
M2 and
M3 muscarinic receptors to
activate Gi and
Gq pathways, respectively. However, the cultured airway smooth muscle cells used in this study
predominantly express M2
muscarinic receptors (26). These M2 muscarinic receptors and
endothelin receptors are coupled to the pertussis toxin-sensitive G
protein Gi, whereas histamine H1, bradykinin, and endothelin
receptors are coupled to Gq (9). Agonists that activate these receptors are present in the airway of
humans in both the presence and absence of disease. Acetylcholine is
released from parasympathetic postganglionic nerves, histamine and
bradykinin are potent inflammatory mediators thought to be important in
asthma and allergy, and endothelin-1 is found in increased
concentrations in the airways of humans with asthma (22).
Because contractile agonists that elicit proliferative responses in
airway smooth muscle cells in culture can be coupled to either
Gq or
Gi, because MAP kinases are
important regulators of cell proliferation, and because
p21ras is an important
intermediate in MAP kinase activation, we questioned whether
stimulation of receptors coupled to
Gi and/or
Gq induced p21ras activation in human airway
smooth muscle cells, and if so, which G protein subunits were involved.
We therefore tested the effects of contractile agonists coupled to
Gi (carbachol and endothelin) and
Gq (histamine, endothelin, and
bradykinin) on p21ras activation
in human airway smooth muscle cells in the presence and absence of
pertussis toxin and a G protein -subunit inhibitor.
| |
METHODS |
Cell culture and 32P loading.
Primary cultures of previously characterized human
tracheal smooth muscle cells (26) were maintained in
M-199 medium containing antibiotics (100 U/ml of
penicillin G, 100 µg/ml of streptomycin, and 0.25 µg/ml of
amphotericin B) and 10% fetal bovine serum (FBS), unless otherwise
stated, at 37°C in an atmosphere of 5%
O2-95% air. Cells were grown to
confluence in six-well culture plates and used between
passages 6 and
12. Confluent cells were rinsed once
and cultured overnight in serum-free and phosphate-free DMEM (2 ml
medium/well). 32P loading of cells was performed the
following day for 4 h by adding 30 µl of phosphorus-32 (9,000 Ci/mmol, 10 mCi/ml) to each well. Medium was then removed, and wells
were rinsed once with serum-free and phosphate-free DMEM. One
milliliter of serum-free and phosphate-free DMEM was then added to each
well, and the indicated effectors were added to wells for the indicated
times. Cells were untreated (control) or treated with epidermal growth
factor (EGF; 100 ng/ml) for 5 min (positive control) or 1 µM
carbachol, 1 µM endothelin-1, 1 µM histamine, or 1 µM bradykinin
for 3 min. KCl (40 mM) was used in some experiments to determine
whether elevation of cellular calcium alone was sufficient to activate
Ras. In some experiments, cells were pretreated with
pertussis toxin (100 ng/ml for 4 h) before treatment with carbachol or
endothelin-1. Reactions were terminated by the removal of medium and
the immediate addition of 500 µl of cold lysis buffer (25 mM Tris, pH
7.5, 150 mM NaCl, 16 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, and 10 µg/ml of
aprotinin) containing 10 µg/ml of
p21ras primary antibody
[anti-v-H-ras (Ab-1)]. Plates were incubated on ice
for 30 min. Cell lysates were scraped from wells into microcentrifuge tubes and centrifuged in a microcentrifuge for 10 min at 16,000 g at 4°C. Supernatants were
transferred to a clean microcentrifuge tube, and an additional 5 µg
of p21ras primary antibody were
added to each tube and allowed to incubate for 1 h at 4°C. Protein
G Sepharose beads were diluted 1:1 with lysis buffer without antibody, and 100 µl of these diluted beads were
added to each tube and allowed to incubate for 1 h with gentle mixing
at 4°C. Tubes were centrifuged at 82 g for 1 min at 4°C to collect the
beads, which were then washed three times for 1 min in 1 ml of washing
buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 16 mM
MgCl2, and 1% Nonidet P-40).
Beads were transferred to a clean tube and washed once more, removing
the final supernatant completely. Twenty microliters of elution buffer
(2 mM EDTA, 0.2% SDS, and 2 mM 1,4-dithiothreitol) were added to each
tube, and tubes were then boiled for 3 min. Beads were pelleted by
centrifugation at 16,000 g for 10 min
at room temperature. Supernatants were transferred to a new
microcentrifuge tube and stored at 
20°C until
immunoprecipitated
[32P]GDP and
[32P]GTP were
separated and quantitated by thin-layer chromatography (TLC).
TLC was performed on 20 × 20-cm cellulose PEI-F
Baker-flex sheets that had been prerun in deionized
distilled water and allowed to completely air-dry.
Two-microliter aliquots of each sample were counted in a scintillation
counter so that equal amounts of radioactivity for each sample could be
loaded on TLC plates. Typically, 500 counts/min of each sample in a
final volume of 10 µl of elution buffer were spotted 3 cm from the
bottom of the TLC plates. Plates were placed in 100 ml (~0.5 cm deep)
of tank buffer (0.75 M
H2KPO4,
pH 3.4) in a glass tank (25 × 27 × 7 cm) with a glass lid.
Migration of the tank buffer up the plate was allowed to continue until
it reached the top of the plate (~2 h). TLC plates were removed from
the tanks, allowed to air-dry, wrapped in plastic wrap, and exposed to
Fuji phosphorimager plates overnight. Exposed
phosphorimager plates were read in a Fujix BAS 1000 phosphorimager
(Fuji Medical Systems, Stanford, CT). The relative
intensities of separated
[32P]GDP and
[32P]GTP were
quantitated from these phosphorimages with Mac BAS 2.0 software (Fuji
Medical Systems). Data are expressed as the ratio of
[32P]GTP to
([32P]GTP + [32P]GDP).
Transfection with pRK--adrenoceptor
kinase 1 fragment. Transient expression of the carboxy
terminus
(Gly495-Leu689)
of the -adrenoceptor kinase (-ARK) 1, also
known as the G protein-coupled receptor kinase
(GRK) 2, functions as a cellular antagonist for
liberated G protein -subunits and allows one to distinguish
between G protein - and -mediated processes (16, 27). The day
before transfection, human airway smooth muscle cells were seeded at
70% confluency in six-well plates containing M-199 medium and 10% FBS
without antibiotics. Two micrograms of plasmid DNA
[pRK--ARK1 or the control plasmid
pRK5 lacking an insert (16, 17)] were diluted in
50 µl of M-199 medium. In a separate tube, 10 µg (5 µl) of
lipofectamine (GIBCO BRL, Gaithersburg, MD) were
diluted in 50 µl of M-199 medium. We slowly pipetted the diluted
lipofectamine into the diluted DNA, avoiding excessive agitation. We
added 1 µl (1.1 × 109
plaque-forming units) of the replication-deficient adenovirus (Ad5CMVntlacZ) to each tube, gently tapping the tube to
mix. The DNA-lipofectamine-virus mixture was allowed to
incubate at room temperature for 45 min. Just before transfection, the
cells were washed twice with serum-free and antibiotic-free M-199
medium. Five milliliters of serum-free and antibiotic-free M-199 medium were added to each well of the six-well plate. The
DNA-lipofectamine-virus mixture was added to the well and incubated at
37°C in an atmosphere of 5%
CO2-95% air for 4 h. The
transfection mixture was removed, and the cells were rinsed once and
then incubated in 5 ml of M-199 medium and 10% FBS. The medium was
replaced the following day with M-199 medium with 10% FBS and
antibiotics. For immunoblot analysis, cells were incubated for 72 h
before being harvested. For
p21ras assays, the medium was
replaced at 48 h with DMEM without phosphate or serum for assays to be
performed at 72 h posttransfection.
Immunoblot analysis of transfected
cells. To confirm that transfection was successful,
immunoblot analysis was performed on lysed cells that had been
transfected with the -ARK1 minigene or sham transfected
with the control plasmid (pRK5) or were untransfected (control).
Seventy-two hours posttransfection, cells in representative wells of
six-well plates were rinsed with M-199 medium and then lysed in 0.5 ml
of Laemmli buffer (62.5 mM Tris, pH 6.8, 2% SDS, 10% glycerol, and
5% -mercaptoethanol). Aliquots (100 µl) were boiled, subjected to
electrophoresis through discontinuous SDS-12% polyacrylamide gels,
electrophoretically transferred to polyvinylidene difluoride membranes,
and immunoblotted as previously described (6) with a rabbit polyclonal
primary antibody raised against the carboxy terminus (residues
675-689) of -ARK1 (or GRK2) and a secondary
antibody coupled to alkaline phosphatase. Immunoreactive bands were
identified by chemiluminescence according to the manufacturer's protocol (ImmunoLite II, Bio-Rad, Hercules, CA) and exposed to autoradiographic film.
Materials. Cell culture media, serum,
and antibiotics were purchased from GIBCO BRL. Phosphorus-32 (9,000 Ci/mmol, 10 mCi/ml) was purchased from NEN (Boston, MA). Pertussis
toxin was purchased from List Biological Laboratories (Campbell, CA).
The primary antibody to p21ras
[anti-v-H-ras (Ab-1)] was purchased
from Calbiochem (La Jolla, CA), the primary antibody specific for the
-ARK1 fragment (GRK2) was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA), and protein G Sepharose 4 Fast Flow beads were
purchased from Pharmacia Biotech (Piscataway, NJ). TLC plates
(20 × 20-cm cellulose PEI-F Baker-flex) were
purchased from VWR Scientific Products (Baltimore, MD). pRK--ARK1
minigene and the pRK5 control plasmid were generous gifts from Walter
J. Koch (Duke University, Durham, NC) (16, 17). The
replication-deficient adenovirus
Ad5CMVntlacZ was purchased from the Gene Transfer
Vector Core at the University of Iowa (Iowa City, IA). Cultured human
airway smooth muscle cells were a kind gift from Dr. Ian P. Hall
(Queens Medical Centre, Nottingham, UK).
Statistical analysis. All
data are presented as means ± SE;
n indicates number of separate
experiments. Control versus treated groups were analyzed
for an increase in the
[32P]GTP-to-([32P]GTP + [32P]GDP) ratio by
ANOVA, with Bonferroni posttest comparisons between groups. A
P value < 0.05 was considered significant.
| |
RESULTS |
Cultured human tracheal smooth muscle cells exposed to 1 µM carbachol
or endothelin-1 for 3 min had a significant increase in the amount of
[32P]GTP
immunoprecipitated by v-H-ras-specific
antibodies compared with untreated control cells (Fig.
1). In the control cells, the [32P]GTP-to-([32P]GTP + [32P]GDP) ratio
averaged 30 ± 1.7, whereas in carbachol-treated cells, the ratio
increased to 39 ± 1.1 (P < 0.01 compared with control value; n = 7)
and in endothelin-1-treated cells, the ratio increased to 40 ± 1.2 (P < 0.001 compared with control
value; n = 11). In cells treated with
the positive control EGF, which activates
p21ras independent of a
heterotrimeric G protein (3, 27), the
[32P]GTP-to-([32P]GTP + [32P]GDP) ratio
increased to 59 ± 1.8 (P < 0.001 compared with control value; n = 5;
Fig. 1). Pretreatment of cells for 4 h with pertussis toxin blocked the
ability of carbachol (Fig. 2) or
endothelin-1 (Fig. 3) to activate
p21ras [ratio of
[32P]GTP to
([32P]GTP + [32P]GDP): carbachol
alone, 39 ± 1.1; carbachol + pertussis toxin, 31 ± 1.0 (n = 7); endothelin-1 alone, 40 ± 1.2; endothelin-1 + pertussis toxin, 29 ± 1.1 (n = 10)]. Pertussis toxin
pretreatment had no significant effect on either basal levels of
p21ras activation or on the
activation of p21ras by EGF
[ratio of
[32P]GTP to
([32P]GTP + [32P]GDP): basal, 30 ± 1.7; pertussis alone, 29 ± 1.7 (n = 12); EGF alone, 59 ± 1.8 (n = 9); EGF + pertussis toxin, 56 ± 0.9 (n = 6); Fig.
4].
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Fig. 1.
A: Ras activation in
human airway smooth muscle cells. Cells were exposed for 3 min to 1 µM carbachol, 1 µM endothelin-1, 1 µM bradykinin, 1 µM
histamine, or 40 mM KCl or for 5 min to 100 ng/ml of epidermal growth
factor (EGF; positive control). Ras-bound
[32P]GTP and
[32P]GDP were
immunoprecipitated, separated by TLC, and quantitated by
phosphorimaging. Activation of
Gi-coupled receptors by carbachol
and endothelin-1, but not of
Gq-coupled receptors by bradykinin
or histamine, resulted in a significant increase in amount of
immunoprecipitated
[32P]GTP. Elevation of
cellular calcium alone with KCl was insufficient to activate Ras,
whereas positive control EGF, which does not couple to heterotrimeric G
proteins, was a potent activator of Ras. GTP/GTP+GDP, ratio of
[32P]GTP to
([32P]GTP + [32P]GDP).
* P < 0.05 compared with
control group. B: representative
phosphorimage of TLC separation of immunoprecipitated
[32P]GDP and
[32P]GTP bound to Ras
after indicated agonist exposure.
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Fig. 2.
Pertussis toxin inhibition of carbachol-induced Ras activation in human
airway smooth muscle cells. Cells in parallel 6-well plates were
preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h
during 32P loading. Cells were exposed to 1 µM carbachol
for 3 min, and Ras-bound
[32P]GTP and
[32P]GDP were
immunoprecipitated, separated by TLC, and quantitated by
phosphorimaging. Pretreatment with pertussis toxin
blocked carbachol-induced activation of Ras, indicating that carbachol
was activating Ras via a pertussis toxin-sensitive heterotrimeric G
protein. * P < 0.05 compared
with carbachol alone. B:
representative phosphorimage of TLC separation of immunoprecipitated
[32P]GTP and
[32P]GDP bound to Ras
after carbachol exposure with and without pertussis toxin
pretreatment.
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Fig. 3.
Pertussis toxin inhibition of endothelin-1-induced Ras activation in
human airway smooth muscle cells. Cells in parallel 6-well plates were
preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h
during 32P loading.
Cells were exposed to 1 µM endothelin-1 for 3 min, and Ras-bound
[32P]GTP and
[32P]GDP were
immunoprecipitated, separated by TLC, and quantitated by
phosphorimaging. Pretreatment with pertussis toxin blocked
endothelin-1-induced activation of Ras, indicating that endothelin-1
was activating Ras via a pertussis toxin-sensitive heterotrimeric G
protein. * P < 0.05 compared
with endothelin-1 alone. B:
representative phosphorimage of TLC separation of immunoprecipitated
[32P]GTP and
[32P]GDP bound to Ras
after endothelin-1 exposure with and without pertussis toxin
pretreatment.
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Fig. 4.
Pertussis toxin inhibition of basal or EGF-induced Ras activation in
human airway smooth muscle cells. Cells in parallel 6-well plates were
preincubated with 100 ng/ml of pertussis toxin or medium alone for 4 h
during 32P loading. Cells were exposed to medium alone or
EGF for 5 min, and Ras-bound
[32P]GTP and
[32P]GDP were
immunoprecipitated, separated by TLC, and quantitated by
phosphorimaging. Pretreatment with pertussis toxin had no effect on
basal levels or EGF-induced activation of Ras.
B: representative phosphorimage of TLC
separation of immunoprecipitated
[32P]GTP and
[32P]GDP bound to Ras
after no stimulation (control) or EGF exposure with and without
pertussis toxin pretreatment.
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In contrast to the activation of
p21ras by carbachol or
endothelin-1, 1 µM histamine or 1 µM bradykinin for 3 min had no
effect on p21ras activation. The
[32P]GTP-to-([32P]GTP + [32P]GDP) ratio
after histamine was 32 ± 1.8 (n = 5) and after bradykinin was 32 ± 1.3 (n = 7) compared with the control
value of 30 ± 1.7 (n = 20).
Elevation of cellular calcium alone with 40 mM KCl did not
significantly activate p21ras (30 ± 3.3; n = 4) compared with
control (Fig. 1).
In an attempt to determine whether the carbachol-mediated activation of
p21ras in airway smooth muscle
cells was mediated through the - or -subunit of heterotrimeric
G proteins, carbachol-induced
p21ras activation was measured in
cells transiently transfected with the -ARK1 minigene product
[a known intracellular antagonist of liberated G protein
-subunits (17)]. Carbachol-induced p21ras activation was compared
between untransfected cells, cells transfected with the -ARK1
minigene, and cells sham transfected with the control pRK5 plasmid
lacking an insert. Transfection with the -ARK1 minigene did not
effect carbachol-induced activation of p21ras (Fig.
5A),
suggesting that the activation of
p21ras by carbachol is not
mediated via G protein -subunits in human airway smooth muscle
cells. Transfection with pRK--ARK1 also had no effect on basal or
EGF-induced p21ras activation
(data not shown). Sham transfection with the empty pRK5 plasmid had no
effect on basal or carbachol- or EGF-mediated p21ras activation (data not
shown).
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Fig. 5.
A: carbachol-induced Ras activation in
transfected human airway smooth muscle cells. To determine whether
carbachol activation of Ras was mediated via G protein - or
-subunit in human airway smooth muscle cells, cells were either
not transfected (None), transiently transfected with an empty control
plasmid (pRK5; Sham), or transiently transfected with a plasmid
encoding carboxy-terminal fragment of -adrenoceptor kinase 1 (pRK--ARK1; ARK1), which functions as a
cellular G protein antagonist before measurement of
carbachol-induced Ras activation. Transient transfection with -ARK1
fragment had no effect on carbachol-induced Ras activity.
B: representative immunoblot analysis
of human airway smooth muscle cell lysates after no transfection, sham
transfection, or -ARK1 transfection. An expected immunoreactive
protein of ~24 kDa (arrowhead) was detected with a primary antibody
directed against -ARK1 only in cells transfected with -ARK1
minigene. A larger band of ~80 kDa, likely representing native
full-length -ARK-related proteins, was seen in all cells.
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To confirm that transfection with the -ARK1 minigene results in
expression of the -ARK1 protein fragment, immunoblot analysis was
performed with an antibody that recognized the transfected shorter-length carboxy-terminal fragment of -ARK1. In
pRK--ARK1-transfected cells, an immunoreactive band was detected at
~24 kDa (Fig. 5B) consistent with
the expected size of the -ARK1 minigene protein product (17). No
immunoreactive band within this molecular-mass range was
detected in nontransfected control cells or in control cells sham
transfected with the empty pRK5 plasmid. Preliminary experiments in which transfection was performed with a plasmid encoding
-galactosidase showed that ~70% of cells were successfully transfected. These results suggest that abundant expression of the
carboxy terminus
(Gly495-Leu689)
of -ARK1, the G protein -subunit antagonist, occurred in the
transfected cells.
| |
DISCUSSION |
In the current study, we found that carbachol and endothelin-1 but not
histamine, bradykinin, or KCl activated the monomeric G protein
p21ras in cultured human airway
smooth muscle cells. Activation of
p21ras by carbachol or
endothelin-1 was blocked by pertussis toxin but not by overexpression
of a G protein -subunit scavenger, suggesting that muscarinic and
endothelin receptors couple to a heterotrimeric Gi protein that activates the
monomeric G protein p21ras via
the G protein -subunit. The current study is the first study in
airway smooth muscle cells to directly measure
p21ras activation and to link
this activation to a G protein-coupled pathway.
In native airway smooth muscle cells, carbachol activates
M2 and
M3 muscarinic receptors to
activate Gi and
Gq pathways, respectively. However, the cultured airway smooth muscle cells used in this study
predominantly express M2
muscarinic receptors (26). The very low levels of
M3 muscarinic receptors are
demonstrated by minimal stimulation by carbachol of inositol phosphate
synthesis in these cells (9). Endothelin also couples to both the
Gi and
Gq pathways, and thus pertussis
toxin was used with both carbachol and endothelin to implicate a
Gi-mediated pathway. Histamine and bradykinin, which couple to the Gq
pathway in these airway smooth cells (9), did not activate
p21ras. The effect of pertussis
toxin in inhibiting the carbachol and endothelin activation of
p21ras appeared to be specific
for Gi activation because
pertussis toxin had no effect on basal or EGF-induced levels of
activated p21ras.
Activation of Gq by histamine or
bradykinin or elevation of cellular calcium alone with KCl was
insufficient to activate p21ras.
These data are not in conflict with a previously published
study on signaling in airway smooth muscle cells (15)
because that study of mitogenic signaling measured the activation or
phosphorylation of the distal MAP kinases, not
p21ras activation directly as in
the current study. Directly measuring p21ras activation is necessary to
determine whether p21ras is a
signaling intermediate leading to activation of MAP kinases because
receptors coupled to Gq can
activate MAP kinases by both p21ras-dependent and
p21ras-independent pathways (10,
23). Our results are therefore consistent with those of Kelleher et al.
(15) in cultured bovine airway smooth muscle cells in
which the Gq-coupled receptor
5-hydroxytryptamine type 2 activated MAP kinases via a pathway proposed
to be independent of p21ras. Thus
the findings of Kelleher et al. together with those of the present
study suggest that in airway smooth muscle cells MAP kinase activation
via the Gq pathway occurs through
PKC and is p21ras independent,
whereas MAP kinase activation via the
Gi pathway is
p21ras dependent.
Although initial studies in a variety of cell types suggested that
Gi-coupled receptors
(M2 muscarinic,
2-adrenergic, lysophosphatidic acid, and thrombin) activate MAP kinases through
p21ras activation (1, 24, 27) and
Gq-coupled receptors activate MAP
kinases via a PKC-dependent,
p21ras-independent pathway (10),
exceptions to these suppositions are emerging. In a recent study (18)
in transfected COS-7 cells, stimulation of MAP kinase
by thyrotropin-releasing hormone was shown to be pertussis toxin
insensitive and partially impaired by scavenging of G protein
-subunits. Additionally, a pertussis toxin-insensitive,
p21ras-dependent pathway that
activated MAP kinase has been shown for both thrombin and angiotensin
receptors in fibroblasts and myocytes, respectively (2, 20), and a
constitutively active Gq mutant activates p21ras in NIH/3T3 cells
(25). The emerging picture is that the responsible heterotrimeric G
protein, the transmission of the signal by G protein - or
-subunit, and the involvement of PKC and/or
p21ras in the activation of MAP
kinases are cell-type dependent.
Our results also agree with the findings of Koch and colleagues (16,
17) in fibroblasts and COS-7 cells in which the
Gi protein mediated the activation
of p21ras. Having incriminated
the Gi pathway in the activation
of p21ras in these cells, we next
sought to determine whether the G protein - or -subunit of
Gi transmitted the signal to
downstream proteins. We utilized a strategy of transiently transfecting
a carboxy-terminal fragment of the -ARK1 enzyme. This protein
fragment functions as an intracellular scavenger of liberated G protein
-subunits and has been used extensively to distinguish between G
protein - and -mediated processes (16, 17). Unlike standard
liposome-mediated transfection protocols that have been successful with
bovine airway smooth muscle (13), successful transient transfection in
the human airway smooth muscle cells used in this study was only
obtained when a liposome formulation was combined with a
replication-deficient adenovirus that together mediated entry of the
pRK--ARK1 plasmid construct (7). Immunoblot analysis revealed
abundant expression of an immunoreactive protein at an expected
molecular mass of ~24 kDa, indicative of successful expression of
this G protein antagonist.
However, in airway smooth muscle cells transfected with this G protein
antagonist, carbachol activated
p21ras to a similar level to that
seen in untransfected cells or cells transfected with a control plasmid
without an insert. These results suggest that the
Gi-mediated activation of
p21ras in these cells is not
transmitted via the G protein -subunits but rather is likely
transmitted by the G protein -subunit. This is in contrast to the
findings of two studies (8, 10) in which the
Gi -subunit
was responsible for the activation of MAP kinase pathways in
fibroblasts or COS-7 cells. This difference may be explained by unique
signaling pathways among different cell types. It is also possible that
the efficiency of transfection was too low in the current study to
block liberated G protein -subunits in the majority of cells.
However, this is unlikely considering the abundant expression of the
-ARK1 minigene product identified by immunoblotting in transfected
cells and the ~70% transfection efficiency demonstrated by
-galactosidase staining. Confirming that expression
levels of the -ARK1 minigene product were sufficiently high to block
a G protein -mediated event is difficult because the G protein
-subunit has not been shown to activate a specific cellular
signal in human airway smooth muscle cells. Other cells have pathways
known to utilize the G protein -subunit; however, successful
transfection and signal blockade in another cell type does not confirm
successful transfection and sufficient expression in an airway smooth
muscle cell.
In summary, this study shows that activation of
Gi- but not of
Gq-coupled receptors in airway
smooth muscle leads to the activation of the monomeric G protein
p21ras via liberated G protein
-subunits. Activation of
p21ras by
M2 muscarinic and endothelin
receptors by chronic release of acetylcholine by parasympathetic nerves
and endothelin by inflammatory cells, respectively, likely contributes
to mitogenesis of airway smooth muscle cells and may contribute to the
asthmatic process.
| |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Ian P. Hall (Queens Medical Centre,
Nottingham, UK) for providing the human airway smooth muscle cells used
in this study.
| |
FOOTNOTES |
This worked was supported by National Heart, Lung, and Blood Institute
Grants HL-58519 and HL-62340.
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: C. W. Emala,
Dept. of Anesthesiology, Columbia-Presbyterian Medical Center, 630 W. 168th St., PH 525, New York, NY 10032 (E-mail:
cwe5{at}columbia.edu).
Received 27 August 1998; accepted in final form 21 December 1998.
| |
REFERENCES |
1.
Alblas, J.,
E. J. Van Corven,
P. L. Hordijk,
G. Milligan,
and
W. H. Moolenaar.
Gi-mediated activation of the p21ras-mitogen-activated protein kinase pathway by 2-adrenergic receptors expressed in fibroblasts.
J. Biol. Chem.
268:
22235-22238,
1993[Abstract/Free Full Text].
2.
Chen, Y. H.,
D. Grall,
A. E. Salcini,
P. G. Pelicci,
J. Pouysségur,
and
E. V. Obberghen-Schilling.
Shc adapter proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor.
EMBO J.
15:
1037-1044,
1996[Medline].
3.
Cobb, M. H.,
T. G. Boulton,
and
D. J. Robbins.
Extracellular signal-regulated kinases: ERKs in progress.
Cell Regul.
2:
965-978,
1991[Medline].
4.
DeVries-Smits, A. M. M.,
B. M. T. Burgering,
S. J. Leevers,
C. J. Marshall,
and
J. L. Bos.
Involvement of p21ras in activation of extracellular signal-regulated kinase 2.
Nature
357:
602-604,
1992[Medline].
5.
Ebina, M.,
H. Yaegashi,
R. Chiba,
T. Takahashi,
M. Motomiya,
and
M. Tanemura.
Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles.
Am. Rev. Respir. Dis.
141:
1327-1332,
1990[Medline].
6.
Emala, C. W.,
C. A. Hirshman,
and
M. A. Levine.
G protein subunits in lung cells.
Life Sci.
55:
593-602,
1994[Medline].
7.
Fasbender, A.,
J. Zabner,
M. Chillon,
T. O. Moninger,
A. P. Puga,
B. L. Davidson,
and
M. J. Welsh.
Complexes of adenovirus with polycationic polymers and cationic lipids increase the efficiency of gene transfer in vitro and in vivo.
J. Biol. Chem.
272:
6479-6489,
1997[Abstract/Free Full Text].
8.
Garnovskaya, M. N.,
T. van Biesen,
B. E. Hawes,
S. C. Ramos,
R. J. Lefkowitz,
and
J. R. Raymond.
Ras-dependent activation of fibroblast mitogen-activated protein kinase by 5-HT1A receptor via a G protein -subunit-initiated pathway.
Biochemistry
35:
13716-13722,
1996[Medline].
9.
Hall, I. P.,
and
M. Kotlikoff.
Use of cultured airway myocytes for study of airway smooth muscle.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L1-L11,
1995[Abstract/Free Full Text].
10.
Hawes, B. E.,
T. van Biesen,
W. J. Koch,
L. M. Luttrell,
and
R. J. Lefkowitz.
Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation.
J. Biol. Chem.
270:
17148-17153,
1995[Abstract/Free Full Text].
11.
Hossain, S.,
and
B. E. Heard.
Hyperplasia of bronchial muscle in chronic bronchitis.
J. Pathol.
101:
171-184,
1970[Medline].
12.
Hershenson, M. B.,
S. Aghili,
N. Punjabi,
C. Hernandez,
D. W. Ray,
A. Garland,
S. Glagov,
and
J. Solway.
Hyperoxia-induced airway hyperresponsiveness and remodeling in immature rats.
Am. J. Physiol.
262 (Lung Cell. Mol. Physiol. 6):
L263-L269,
1992[Abstract/Free Full Text].
13.
Hershenson, M. B.,
T. O. 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[Abstract/Free Full Text].
14.
Hershenson, M. B.,
A. Garland,
M. D. Kelleher,
A. Zimmerman,
C. Hernandez,
and
J. Solway.
Hyperoxia-induced airway remodeling in immature rats. Correlation with airway responsiveness.
Am. Rev. Respir. Dis.
146:
1294-1300,
1992[Medline].
15.
Kelleher, M. D.,
M. K. Abe,
T. O. 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[Abstract/Free Full Text].
16.
Koch, W. J.,
B. E. Hawes,
L. F. Allen,
and
R. J. Lefkowitz.
Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G
activation of p21ras.
Proc. Natl. Acad. Sci. USA
91:
12706-12710,
1994[Abstract/Free Full Text].
17.
Koch, W. J.,
B. E. Hawes,
J. Inglese,
L. M. Luttrell,
and
R. J. Lefkowitz.
Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attentuates G
-mediated signaling.
J. Biol. Chem.
269:
6193-6197,
1994[Abstract/Free Full Text].
18.
Palomero, T.,
F. Barros,
D. del Camino,
C. G. Viloria,
and
P. Pena.
A G protein dimer-mediated pathway contributes to mitogen-activated protein kinase activation by thyrotropin-releasing hormone receptors in transfected cos-7 cells.
Mol. Pharmacol.
53:
613-622,
1998[Abstract/Free Full Text].
19.
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[Abstract/Free Full Text].
20.
Sadoshima, J. I.,
and
S. Izumo.
The heterotrimeric Gq protein-coupled angiotensin II receptor activates p21ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.
EMBO J.
15:
775-787,
1996[Medline].
21.
Sapienza, S.,
T. Du,
D. H. Eidelman,
N. S. Wang,
and
J. G. Martin.
Structural changes in the airways of sensitized Brown Norway rats after antigen challenge.
Am. Rev. Respir. Dis.
144:
423-427,
1991[Medline].
22.
Sofia, M.,
M. Mormile,
S. Faraone,
M. Alifano,
S. Zofra,
L. Romano,
and
L. Carratu.
Increased endothelin-like immunoreactive material on bronchoalveolar lavage fluid from patients with bronchial asthma and patients with interstitial lung disease.
Respiration
60:
89-95,
1993[Medline].
23.
Van Biesen, 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].
24.
Van Corven, E. J.,
P. L. Hordijk,
R. H. Medema,
J. L. Bos,
and
W. H. Moolenaar.
Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts.
Proc. Natl. Acad. Sci. USA
90:
1257-1261,
1993[Abstract/Free Full Text].
25.
Watanabe, T.,
I. Waga,
Z. Honda,
K. Kurokawa,
and
T. Shimizu.
Prostaglandin F2
stimulates formation of p21ras-GTP complex and mitogen-activated protein kinase in NIH-3T3 cells via Gq-protein-coupled pathway.
J. Biol. Chem.
270:
8984-8990,
1995[Abstract/Free Full Text].
26.
Widdop, S.,
K. Daykin,
and
I. P. Hall.
Expression of muscarinic M2 receptors in cultured human airway smooth muscle cells.
Am. J. Respir. Cell Mol. Biol.
9:
541-546,
1993.
27.
Winitz, S.,
M. Russell,
N. X. Qian,
A. Gardner,
L. Dwyer,
and
G. L. Johnson.
Involvement of ras and raf in the Gi-coupled acetylcholine muscarinic M2 receptor activation of mitogen-activated protein (MAP) kinase kinase and MAP kinase.
J. Biol. Chem.
268:
19196-19199,
1993[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 276(4):L564-L570
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