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(Received for publication, June 5, 1995; and in revised form, June 26, 1995) From the
We examined the effects of the bronchoconstrictor agonists
serotonin (5-hydroxytryptamine; 5-HT) and histamine on
mitogen-activated protein (MAP) kinase activation in cultured bovine
tracheal myocytes. Kinase renaturation assays demonstrated activation
of the 42- and 44-kDa MAP kinases within 2 min of 5-HT exposure. MAP
kinase activation was mimicked by
Abnormal growth of airway smooth muscle may play a significant
role in the pathogenesis of two important human airways diseases,
asthma (1) and bronchopulmonary dysplasia(2) . Little
is known, however, about the signaling pathways responsible for such
proliferation. We have examined the role of mitogen-activated protein
(MAP) ( In the human airway diseases asthma and
bronchopulmonary dysplasia, excess airway smooth muscle mass coexists
with airway constrictor
hyperresponsiveness(1, 2, 5, 6) . In
addition, abnormal airway smooth muscle DNA synthesis and airway
hyperreactivity have been correlated in two animal models of airway
disease: hyperoxia-exposed, immature Sprague-Dawley rats (7) and ovalbumin-challenged brown Norway rats(8) . The
association of airway smooth muscle proliferation and
bronchoconstriction suggests that bronchoconstrictor agonists may
regulate not only airway smooth muscle tone but cell proliferation.
Thus, signaling events subsequent to airway cell stimulation with
bronchoconstrictor agonists are of particular interest. The biogenic
amines serotonin (5-HT) and histamine are potent bronchoconstrictors (9, 10) that have recently been implicated in the
regulation of cell growth. 5-HT, which is primarily synthesized by and
released from airway neuroendocrine cells in response to alterations in
airway gas chemical composition(11) , has been demonstrated to
stimulate vascular smooth muscle (12) and
fibroblast(13, 14) proliferation in vitro.
5-HT stimulates at least two different G protein-dependent signaling
pathways through distinct receptors. Stimulation of the 5-HT Histamine is released into the airways by mast cells following
allergen exposure(26) . Histamine has been shown to induce
cytosolic Ca In the current study, we tested the
effects of the 5-HT and histamine on MAP kinase activation. Our data
suggest a model whereby 5-HT activates MAP kinase via a protein kinase
C/Raf-1 pathway, and histamine inhibits MAP kinase activation via
stimulation of cAMP-dependent protein kinase A and inhibition of Raf-1.
Figure 1:
Panel A, typical kinase renaturation
assay demonstrating the effects of 5-HT and histamine (HIST)
on the phosphorylation of MBP by MAP kinase. After electrophoretic
resolution on a MBP-impregnated polyacrylamide gel, MAP kinases were
renatured to active form and detected by phosphorylation of the
substrate MBP. In the experiment depicted here, 5-HT induced
substantial MAP kinase activation, whereas histamine-treated cells
demonstrated a slight increase in MAP kinase activity 2 min after
exposure followed by a reduction to normal or subnormal levels
5-10 min after stimulation. Panel B, Western blotting of
MAP kinases. A slight shift in the MAP kinase bands toward a slower
mobility reflects the phosphorylation of MAP kinases at threonine and
tyrosine residues, which is required for enzyme activity. Responses to
PDGF, EGF, 5-HT, and histamine are shown (C, control sample).
Similar results were obtained in three separate
experiments.
Figure 2:
Panel A, kinase renaturation
assay demonstrating the effects of
It has been demonstrated that
cAMP inhibits Ras-dependent activation of Raf-1 via the activation of
protein kinase
A(20, 21, 22, 23, 24, 25) .
We therefore examined the effect of forskolin, which augments
intracellular cAMP concentration, on MAP kinase activation following
5-HT exposure. Pretreatment with forskolin (50 µM for 15
min) abolished MAP kinase activity (Fig. 2A),
suggesting that 5-HT-induced MAP kinase activation involves activation
of Raf-1. The activation of Raf-1 by 5-HT was confirmed by measurement
of Raf-1 kinase activity using a kinase-inactive MEK-1 as
substrate(24) . Administration of 5-HT increased Raf-1
activation 4-fold (Fig. 2C). The requirement of
Raf-1 for 5-HT-induced MAP kinase activation was tested by examining
ERK2 activation in bovine tracheal smooth muscle cells transiently
transfected with an epitope-tagged murine ERK2 and the p301-1
dominant-negative Raf-1. Treatment with 5-HT increased
MBP-phosphorylating activity 2-3-fold; expression of the mutant
Raf-1 returned 5-HT-induced ERK2 activity to base line (Fig. 2D).
Figure 3:
Panel A, kinase renaturation assay
demonstrating the effects of forskolin and histamine on MAP kinase
activation following stimulation with either 5-HT or IGF-1. Cells were
incubated with either forskolin (FSK) or histamine (HIST) and stimulated with either 5-HT (2 min) or IGF-1 (5
min). Kinase renaturation assays were performed as described under
``Experimental Procedures.'' C, control sample. Panel B, quantitation of MBP phosphorylation by MAP kinases
was measured by optical scanning. For each group, the results are
expressed as mean ± S.E. of three different experiments; *, p < 0.05, paired t test. Panel C, kinase
renaturation assay demonstrating the effects of forskolin and histamine
on MAP kinase activation following stimulation with either PDGF or
EGF.
Figure 4:
Panel
A, kinase renaturation examining the effects of H
We have demonstrated that the bronchoconstrictors 5-HT and
histamine each influence MAP kinase activation in cultured bovine
tracheal smooth muscle cells. 5-HT activates MAP kinase, likely via a
protein kinase C/Raf-1 pathway, whereas histamine attenuates MAP kinase
activation, apparently via stimulation of cAMP-dependent protein kinase
A and inhibition of Raf-1. The modulation of airway smooth muscle MAP
kinase activation by the physiologic effectors 5-HT and histamine may
hold significance for two important human airway diseases,
bronchopulmonary dysplasia and asthma, both of which have been
associated with an abnormal increase in airway smooth muscle mass (1, 2) . The observed activation of MAP kinase by
5-HT is consistent with the data of Meloche etal.(13) , who found that 5-HT induced MAP kinase
activation in CCL39 hamster fibroblasts. The precise pathways
responsible for stimulation of MAP kinase activity by 5-HT have yet to
be completely clarified, however. 5-HT stimulates at least two
different G protein-dependent signaling pathways through distinct
receptors. Stimulation of the 5-HT Stimulation of the
5-HT It has also been suggested that signals that stimulate G
protein-linked receptors activate MEK and MAP kinase via another
cytosolic serine/threonine kinase, MEK kinase(37) . The
presence of Raf-1 activation and inhibition of 5-HT-induced MAP kinase
activation by both forskolin pretreatment and the dominant-negative
p301 Raf-1 suggest that MEK kinase plays little if any role in the
activation of MAP kinase by 5-HT in bovine tracheal myocytes. We have
observed similar, cAMP-sensitive activation of Raf-1 by thrombin,
another extracellular signal requiring G protein-linked receptor
activation, in these cells. ( In contrast, histamine inhibited MAP kinase activity
following stimulation of bovine tracheal smooth muscle cells with
either 5-HT or IGF-1. A likely explanation for this effect of histamine
relates to its effects on adenyl cyclase. As noted above, histamine has
been shown to stimulate cAMP synthesis in cultured guinea pig tracheal
smooth muscle cells via the H We found that histamine markedly inhibited
Raf-1 kinase activity following treatment with 5-HT, IGF-1, PDGF, or
EGF. Despite this reduction in Raf-1 kinase activation, PDGF- and
EGF-induced MAP kinase activation were unaffected by histamine
pretreatment. Further, transfection of bovine tracheal smooth muscle
cells with the plasmid vector p301-1, which overexpresses a
dominant-negative Raf-1 mutant that interferes with Raf-1-mediated
intracellular signals, failed to abolish PDGF-induced ERK2 activation.
These data indicate that in bovine tracheal smooth muscle cells, MAP
kinase activation may not require the activation of Raf-1. We have
found similar examples of Raf-1-independent MAP kinase activation in
other systems; expression of the p301-1 dominant-negative Raf-1
mutant failed to reduce EGF-induced ERK2 activation in rat hippocampal
neurons stably transfected with a temperature-sensitive SV40 large T
antigen. ( It should be noted that although transient transfection with
the dominant-negative Raf-1 p301 plasmid attenuated PDGF-induced ERK 2
activation, pretreatment with forskolin, an inhibitor of Raf-1, did
not. The discrepant effects of p301 expression and forskolin are
consistent with the notion that the dominant-negative Raf-1 sequesters
Ras, thereby nonspecifically blocking both Raf-1 and other
Ras-dependent activators of MEK. Nevertheless, the presence of
persistent, albeit reduced, ERK2 activity in PDGF-treated Raf p301
transfectants suggests that PDGF activation of MAP kinase can indeed
occur in a Raf-1-independent manner. As in human (27) and
canine (28) tracheal smooth muscle cells, histamine induces
cytosolic Ca
Volume 270,
Number 34,
Issue of August 25, pp. 19908-19913, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-methyl-5-HT and reduced by
pretreatment with either phorbol 12,13-dibutyrate or forskolin,
suggesting activation of the 5-HT
receptor, protein kinase
C, and Raf-1, respectively. Raf-1 activation was confirmed by
measurement of Raf-1 activity, and the requirement of Raf-1 for
5-HT-induced MAP kinase activation was demonstrated by transient
transfection of cells with a dominant-negative allele of Raf-1.
Histamine pretreatment significantly inhibited 5-HT and insulin-derived
growth factor-1-induced MAP kinase activation. Attenuation of MAP
kinase activation was reversed by cimetidine, mimicked by forskolin,
and accompanied by cAMP accumulation and inhibition of Raf-1,
suggesting activation of the H receptor and cAMP-dependent
protein kinase A. However, histamine treatment inhibited Raf-1 but not
MAP kinase activation following treatment with either platelet-derived
growth factor or epidermal growth factor, implying a Raf-1-independent
MAP kinase activation pathway. In summary, our data suggest a model
whereby 5-HT activates MAP kinase via a protein kinase C/Raf-1 pathway,
and histamine attenuates MAP kinase activation by serotonin via
activation of cAMP-dependent protein kinase A and inhibition of Raf-1.
)kinase, a family of 40-46-kDa cytosolic
serine/threonine kinases, which participate in the transduction of
mitogenic and differentiation-promoting signals to the cell nucleus, in
cultured bovine tracheal myocytes(3, 4) . A variety of
substances activate MAP kinase in these cells, including
platelet-derived growth factor (PDGF), epidermal growth factor (EGF),
insulin-like growth factor-1 (IGF-1), 5-hydroxytryptamine (5-HT), and
hydrogen peroxide, suggesting that MAP kinase occupies a central
position in a complex signaling system regulating airway smooth muscle
cell proliferation. receptor activates a G protein that is positively coupled to
phospholipase C, whereas stimulation of the 5-HT receptor
activates a G
protein negatively coupled to adenylate
cyclase (13, 14, 15, 16) .
Stimulation of MAP kinase could occur by either pathway, the first via
protein kinase C and Raf-1 activation (17, 18, 19) and the second by blocking
protein kinase A-mediated inhibition of
Raf-1(20, 21, 22, 23, 24, 25) . release in human (27) and canine
tracheal myocytes via its H
receptor subtype (28) and stimulate cAMP synthesis in cultured guinea pig
tracheal smooth muscle cells via its H receptor
subtype(29) . We have demonstrated that Ca flux activates MAP kinase in human foreskin
fibroblasts(30) , whereas cAMP accumulation suppresses MAP
kinase by inhibiting Raf-1
activation(20, 21, 22, 23, 24, 25) .
Thus, the net effect of histamine treatment on MAP kinase activation in
cultured airway smooth muscle cells may depend on the relative
stimulation of the two pathways.
Materials
Anti-human -smooth muscle actin,
5-HT, histamine, myelin basic protein, goat anti-rabbit IgG, isobutyl
methylxanthine, phorbol 12,13-dibutyrate (PDBu), pertussis toxin, and
forskolin were purchased from Sigma. -Methyl-5-HT and cimetidine
were obtained from Research Biochemicals Inc. PDGF and EGF were
obtained from Upstate Biotechnology. IGF-1 was from Becton-Dickinson.
Biotinylated horse anti-mouse IgG and ABC reagent (avidin plus
biotinylated horseradish peroxidase) were purchased from Vectastain
Laboratories. [-
P]ATP was obtained from
DuPont NEN. For Western analyses of MAP kinase activation, a rabbit
antiserum (antibody 283) raised against a peptide fragment of ERK1
(amino acid residues 283-306) was used as the primary
antibody(31) ; MAP kinase bands were visualized using an
enhanced chemiluminescence kit from Amersham Corp. For in vitro phosphorylation assays, antibodies against Raf-1 and the
hemagglutinin epitope tag were purchased from Santa Cruz and Babco,
respectively. A plasmid coding for an inactive MAP
kinase/ERK-activating kinase (MEK-1) was obtained from Dr. Gary
Johnson(24) , and the p301-1 dominant-negative Raf-1 was
supplied by Dr. David Foster(32) . A hemagglutinin-tagged p42
MAP kinase expression vector was constructed by ligating a DNA fragment
encoding the influenza hemagglutinin epitope to the 5` end of murine
p42 MAP kinase cDNA. The resulting cDNA was ligated into the expression
vector pcDNAI Neo (Invitrogen). Lipofectamine was purchased from Life
Technologies, Inc.
Cell Culture
Bovine trachea smooth muscle cells
were cultured as described previously(3, 4) .
Confluent flasks exhibited the typical ``hill and valley''
appearance under phase contrast microscopy and exhibit specific
immunostaining with an antibody against -smooth muscle actin.Preparation of Cell Extracts for Analysis of MAP Kinase
Activation
Bovine tracheal smooth muscle cell cultures in 100-mm
plates were serum-starved by incubation in DMEM for 24 h. After
incubation with the relevant stimulus, cells were washed with cold
phosphate-buffered saline and incubated with 0.3-0.5 ml of a
homogenization solution consisting of 50 mM
-glycerophosphate, pH 7.4, 1 mM EGTA, 1 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, and
0.1 mM sodium vanadate. Cells were scraped off culture plates
and lysed by passing through a 26-gauge needle 10 times. The homogenate
was centrifuged (14,000 rpm for 10 min at 4 °C), and supernatant
was transferred to a microcentrifuge tube.Kinase Renaturation Assay
All isoforms of the MAP
kinase family will phosphorylate myelin basic protein (MBP) in
vitro on a threonine residue(33) . Thus, after
electrophoretic resolution on a MBP-impregnated polyacrylamide gel, MAP
kinases may be renatured to active form and detected by phosphorylation
of MBP(3, 4, 30, 32) . Cell extracts
(5-10 µg of protein/lane) were resolved on a 10%
SDS-polyacrylamide gel with 0.1 mg/ml of MBP copolymerized in the
running gel. After electrophoresis the gel was washed twice with 50
mM Hepes, pH 7.4, and 5 mM mercaptoethanol plus 30%
isopropyl alcohol to remove SDS (1 h each at room temperature). After
another wash in 50 mM Hepes and mercaptoethanol alone (1 h),
gels were incubated in 6 M guanidine HCl plus 5 mM
mercaptoethanol to denature proteins (two washes, 1 h each). Next, gels
were equilibrated twice in renaturation buffer containing 50 mM Hepes, pH 7.4, 5 mM mercaptoethanol, and 0.04% Tween 20
(total incubation time approximately 16 h, each at 4 °C), The
renatured gels were then incubated in a buffer containing 25 mM HEPES, 10 mM MgCl, 10 mM
MnCl, and 90 µM sodium vanadate for 30 min.
The phosphorylation step was conducted by setting each gel in 10 ml of
the aforementioned buffer supplemented with 5 mM mercaptoethanol, 50 µM ATP, and 150-200 µCi
of [-
P]ATP for a period of 1 h at 30
°C. The reaction was stopped by washing in 5% trichloroacetic acid
and 10 mM sodium pyrophosphate. Gels were stained with
Coomassie Blue, destained, and dried. Autoradiograms were developed by
exposing Kodak X-Omat film to the dried gel. Quantitation of MBP
phosphorylation by MAP kinases was measured by optical scanning (Ambis
image analyzer, San Diego, CA).
Western Blotting of MAP Kinases
Cell extracts were
resolved on a 10% SDS-polyacrylamide gel and transferred to
nitrocellulose using a semidry transfer unit (Hoefer, San Francisco,
CA). After incubation with rabbit anti-MAP kinase antiserum (antibody
283), immunoblots were amplified and visualized using goat anti-rabbit
IgG and an enhanced chemiluminescence kit. A shift in the MAP kinase
bands toward a slower mobility reflects the phosphorylation of MAP
kinases at threonine and tyrosine residues, which is required for
enzyme activity (34) .Raf-1 Kinase Assay
Raf-1 kinase activity was
analyzed by immune complex kinase assay(24) . After incubation
with the appropriate stimulus, cells were washed twice and lysed in 500
µl of buffer consisting of 10 mM Tris HCl, pH 7.4, 1%
Triton X-100, 1 mM EDTA, 150 mM NaCl, 50 mM NaF, 0.1% bovine serum albumin, 20 µg/ml aprotinin, 200
µM Na
VO
, and 0.2 mM
phenylmethylsulfonyl fluoride. After centrifugation (14,000 rpm for 10
min at 4 °C), supernatants were incubated with anti-c-Raf-1
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 90 min at 4
°C. Protein A-Sepharose was added for another 30 min. Next, after
addition of an equal volume of lysis buffer plus 10% sucrose, the
Sepharose beads were allowed to settle, and the mixtures were
centrifuged for 2 min at 4000 rpm. Each pellet was then washed twice
with lysis buffer, twice with PAN (10 mM Pipes, pH 7.0, 100
mM NaCl, 20 µg/ml aprotinin) plus 0.5% Nonidet P-40, and
finally twice with PAN alone. Each pellet was then resuspended in 20
µl of kinase buffer (20 mM Pipes, pH 7.0, 10 mM MnCl, 20 µg/ml aprotinin, and 200 µM
NaVO) containing approximately 1 ng of
kinase-inactive MEK. The immune complex kinase reaction was initiated
by adding 10 µCi of [-
P]ATP to the
mixture, which was then incubated at 30 °C for 20 min. The reaction
was stopped by adding Laemmli buffer and boiling for 2-5 min.
Samples were resolved on a 10% SDS gel, and the MEK phosphorylation was
assessed by optical scanning.
Transient Transfection of Bovine Tracheal
Myocytes
Cells were seeded into 100-mm plates at a density of
510
cells/plate and incubated in 10% fetal bovine
serum/DMEM for 24 h. After rinsing, cells were incubated with a
liposome solution consisting of serum- and antibiotic-free medium,
plasmid DNA (total of 8 µg/plate), and Lipofectamine (40
µl/plate). After 5 h, the liposome solution was replaced with 10%
fetal bovine serum/DMEM. Cells were co-transfected with plasmids
encoding a hemagglutinin-tagged murine ERK2 and either the dominant
inhibitory Raf-1 p301-1 or calf thymus DNA. Forty-eight hours
after transfection, cells were serum-starved in DMEM. The next day,
cells were treated with the relevant stimulus and harvested for
analysis of ERK2 kinase activity.Epitope-tagged ERK2 Kinase Assay
ERK2 kinase
activity was assessed by immunoprecipitation of the epitope tag
followed by in vitro phosphorylation assay(35) .
Transfected cells were stimulated and lysed with 0.5 ml of cold lysis
buffer containing 50 mM Tris-HCl, pH 7.5, 1% (w/v) Triton
X-100, 40 mM -glycerophosphate, 100 mM NaCl, 50
mM NaF, 2 mM EDTA, 200 µM sodium
orthovanadate, and 0.2 mM phenylmethylsulfonyl fluoride.
Insoluble materials were removed by centrifugation at 14,000 rpm for 10
min at 4 °C. Cell lysates were then incubated with 25 µl of
protein A precoupled with the antibody 12CA5 specific to the
hemagglutinin epitope. After overnight incubation at 4 °C, the
immune complexes were washed four times with the lysis buffer and once
with the kinase buffer containing 20 mM Hepes, pH 7.4, 10
mM MgCl, 1 mM dithiothreitol, 200
µM NaVO, and 10 mMp-nitrophenyl phosphate. The immune complex kinase activity
was measured by phosphorylation of MBP. Immune complexes were
resuspended in a final volume of 40 µl of kinase buffer with 0.25
mg/ml MBP and 50 µM ATP (5 µCi of
[-
P]ATP). Mixtures were incubated at 30
°C for 20 min. The reactions were terminated by adding 20 µl of
Laemmli buffer. Samples were resolved on a 10% SDS gel, and an
autoradiogram was developed from the dried gel. The MBP bands were
excised from the gel, and radioactivity was counted by liquid
scintillation.
Enzyme-linked Immunoassay for Cyclic AMP
After
pretreatment for 15 min with isobutyl methylxanthine, cells were
exposed to histamine
(10-10
M) for 15
min. The medium was then aspirated, and the monolayer was exposed to
0.1 N hydrochloric acid overnight. The cAMP content of each
plate was then measured by enzyme-linked immunosorbent assay (Amersham
Corp.), and the results were normalized to cell number.
Effects of 5-HT and Histamine on MAP Kinase Activity in
Bovine Tracheal Myocytes
Bovine tracheal smooth muscle cell
extracts were electrophoresed on a MBP-impregnated polyacrylamide gel,
renatured to active form, and detected by phosphorylation of the
substrate MBP. As we have shown previously(4) , 5-HT induced an
approximately 4-fold increase in the activation of both the 42- and
44-kDa MAP kinases (Fig. 1A). Unlike 5-HT, histamine
failed to stimulate MAP kinase in bovine tracheal myocytes, and in some
instances, it appeared to reduce MAP kinase activation below that
observed at base line. Immunoblotting with anti-MAP kinase antiserum
antibody 283 demonstrated a slight upward shift in the position of the
MAP kinase bands following 5-HT treatment, confirming phosphorylation
of the enzymes (Fig. 1B).
Mechanism of MAP Kinase Activation following 5-HT
Stimulation
We assessed the effects of a specific 5-HT receptor agonist, -methyl-5-HT, on MAP kinase activation (Fig. 2A). Stimulation with -methyl-5-HT-activated
MAP kinase, consistent with the notion that 5-HT activates MAP kinase
via this receptor subtype. Stimulation of the 5-HT receptor
activates a G protein, which is positively coupled to phospholipase
C(13, 14, 15, 16) ; activation of
phospholipase C, in turn, could lead to MAP kinase activation via
successive activation of protein kinase C and
Raf-1(17, 18, 19) . Pretreatment with PDBu
(200 ng/ml for 24 h) reduced 5-HT-induced activation of both the 42-
and 44-kDa MAP kinase homologues (Fig. 2, A and B), demonstrating that 5-HT-induced stimulation of MAP kinase
is in part dependent on protein kinase C. On the other hand,
pretreatment with pertussis toxin (100 ng/ml for 4 h), an inhibitor of
the G protein, had little effect on the kinase activity
induced by 5-HT (Fig. 2A). The failure of pertussis
toxin to inhibit MAP kinase activation suggests that stimulation of the
G-linked 5-HT receptor subtype is not involved
in 5-HT-induced MAP kinase activation.
-methyl-5-HT (10M), pertussis toxin (100 ng/ml for 4 h), and forskolin
(50 µM for 15 min) on MAP kinase activity. The potency of
the pertussis toxin was confirmed by demonstrating that a corresponding
treatment inhibited thrombin-induced MAP kinase activation in these
cells (data not shown). Similar results were obtained in two separate
experiments. C, control sample. Panel B,
autoradiogram of a representative experiment demonstrating the effect
of phorbol ester pretreatment on the time course of MAP kinase
activation by 5-HT. Cells were incubated with PDBu (200 ng/ml) 24 h
prior to stimulation with 5-HT. PDBu pretreatment also prevented
phorbol ester-induced kinase activation, confirming the effectiveness
of chronic PDBu treatment in down-regulating protein kinase C activity
(data not shown). Similar results were obtained in two separate
experiments. Panel C, the activation of Raf-1 by 5-HT was
confirmed by measurement of Raf-1 kinase activity. Cells were
stimulated with 5-HT, lysed, and immunoprecipitated with an antibody
specific for Raf-1. The kinase activity of Raf-1 was measured by in
vitro phosphorylation assay using kinase-inactive MEK-1 as
substrate. Panel D, autoradiogram of ERK2 activation in bovine
tracheal smooth muscle cells transiently co-transfected with an
epitope-tagged murine ERK2 and the p301-1 dominant-negative
Raf-1. Expression of the mutant Raf-1 returned 5-HT-induced ERK2
activity to base line, whereas PDGF-induced ERK2 activation was only
partially reduced.
Effect of Histamine on MAP Kinase Activation by 5-HT and
Peptide Growth Factors
The absence of MAP kinase activation
following histamine exposure led us to test whether histamine might
inhibit MAP kinase activation via its stimulatory effect on
cAMP(29) . To test this, we pretreated cells with histamine
(10M for 15 min) or forskolin, which
directly activates adenylate cyclase activity, and assessed MAP kinase
activation following stimulation with 5-HT, IGF-1, EGF, and PDGF (30
ng/ml). Pretreatment with either forskolin or histamine nearly
abolished 5-HT and IGF-1-induced MAP kinase activity (Fig. 3, A and B). These data demonstrate that MAP kinase
activity in airway smooth muscle cells can be negatively regulated both
by histamine and by forskolin, presumably via cAMP. Pretreatment with
these agents had no significant effect on EGF and PDGF-induced MAP
kinase activation, however (Fig. 3, B and C).
Mechanism of MAP Kinase Inhibition following Histamine
Treatment
Histamine has been shown to stimulate cyclic cAMP
synthesis in cultured guinea pig tracheal smooth muscle cells via its
H receptor subtype(29) . The inhibition of MAP
kinase activation by histamine and forskolin, together with previous
data demonstrating the inhibition of Raf-1 activation by
cAMP(20, 21, 22, 23, 24, 25) ,
led us to assess the effects of H receptor blockade on MAP
kinase activity. Co-incubation of bovine tracheal smooth muscle cells
with both histamine and cimetidine prior to 5-HT treatment prevented
the reduction in MAP kinase activation observed after pretreatment with
histamine alone (Fig. 4A), suggesting that the
inhibitory effect of histamine on MAP kinase activation is mediated
though stimulation of the H receptor subtype. To confirm
increased adenylate cyclase activity after H receptor
stimulation, we measured alterations in intracellular cAMP
concentration following histamine treatment. Histamine increased
intracellular cAMP levels in a concentration-dependent manner (Fig. 4B). Finally, we assessed the effect of histamine
pretreatment on Raf-1 kinase activity by 5-HT as well as by the peptide
growth factors IGF-1, PDGF, and EGF. Histamine pretreatment inhibited
the activation of Raf-1 by each substance tested (Fig. 4, C and D). The inhibitory effect of histamine on PDGF- and
EGF-induced Raf-1 activity contrasts with the inhibitory effect of
histamine on MAP kinase activation, which did not extend to these
growth factors (see above). Forskolin pretreatment had similar,
negative effects on Raf-1 kinase activity (data not shown).
receptor
blockade on MAP kinase activity. Cells were incubated with either
histamine (HIST) or both histamine and cimetidine (CIM) prior to 5-HT treatment. Similar results were obtained
in three separate experiments. Panel B, alterations in
intracellular cAMP concentration following histamine treatment. For
each group, the results are expressed as mean ± S.E. of two
different experiments. Panel C, effects of histamine
pretreatment on Raf-1 kinase activity induced by 5-HT as well as by the
peptide growth factors EGF, IGF-1, and PDGF. Raf-1 activity was
assessed as described in the Fig. 2legend. C, control
sample. Panel D, quantification of Raf-1 activity was
performed by scintillation counting of the optical scanning. For each
group, the results are expressed as mean ± S.E. of at least
three different experiments; *, p < 0.05, paired t test.
receptor activates a G
protein that is positively coupled to phospholipase
C(13, 14, 15, 16) . Activation of
phospholipase C, in turn, induces the formation of inositol
triphosphate, intracellular Ca release, and protein
kinase C activation. Protein kinase C has been demonstrated to activate
the serine/threonine kinase Raf-1 by direct
phosphorylation(18) , and activation of Raf-1 may stimulate MAP
kinase(19) . In our study, pretreatment of tracheal myocyte
cultures with PDBu, which down-regulates protein kinase C activity,
substantially reduced 5-HT-induced MAP kinase activation, implying the
importance of the phospholipase C/protein kinase C/Raf-1 pathway for
MAP kinase activation in this instance. The observed reduction in MAP
kinase activation with forskolin pretreatment, which has been shown to
inhibit Raf-1 activation by
Ras(20, 21, 22, 23, 24, 25) ,
further supports the role of Raf-1 in 5-HT-induced MAP kinase
activation. Activation of Raf-1 by 5-HT was confirmed by measurement of
Raf-1 kinase activity using a kinase-inactive MEK-1 as substrate.
Finally, transient transfection of bovine tracheal smooth muscle cells
with a dominant-negative mutant of Raf-1 (p301-1) abolished
5-HT-induced ERK2 activity, establishing the requirement of Raf-1 for
MAP kinase activation following 5-HT treatment.
receptor also activates a G
protein
negatively coupled to adenylate cyclase (13, 14, 15, 16) . Compounds such as
forskolin that increase cAMP and activate protein kinase A decrease MAP
kinase activation by inhibiting Raf-1
activity(20, 21, 22, 23, 24, 25) ;
therefore, stimulation of the G-linked 5-HT receptor should inhibit cAMP accumulation and enhance MAP kinase
activity. In our study, pretreatment with pertussis toxin failed to
abolish MAP kinase activation following 5-HT stimulation, suggesting
that G
subunit stimulation is not essential for activation. )Nevertheless, we cannot rule
out a limited role for MEK kinase in 5-HT-induced activation of MAP
kinase, since the sensitivity of MEK kinase to cAMP has not been
established. receptor
subtype(29) . Such stimulation would tend to inhibit MAP kinase
activation via cAMP-dependent protein kinase
A(20, 21, 22, 23, 24, 25) .
In this study, we confirmed that histamine stimulates cAMP accumulation
in bovine tracheal myocytes. Further, we demonstrated that inhibition
of 5-HT-induced MAP kinase activity by histamine was blocked by the
H receptor antagonist cimetidine. Finally, pretreatment
with either histamine or forskolin inhibited 5-HT and growth
factor-induced Raf-1 kinase activity. Taken together, these data
strongly suggest that histamine attenuates MAP kinase activity via
activation of cAMP-dependent protein kinase A, with subsequent
inhibition of Raf-1.)In a BALB/c 3T3 derivative stably transfected
with p301-1, treatment with EGF but not IGF-1 was effective in
activating MAP kinase, despite the absence of functional
Raf-1(32) . The exact pathway(s) by which activation of MAP
kinase may occur independently of Raf-1 are unclear. As noted above, it
has been suggested that MEK and MAP kinase may be activated by MEK
kinase(36) . Alternatively, it has recently been shown that
B-Raf, rather than Raf-1, may be the major activator of MEK in NIH3T3
fibroblasts(37) . However, B-Raf activity, like Raf-1
activation, appears to be cAMP-sensitive(37, 38) ,
suggesting that B-Raf could not have been responsible for the
Raf-1-independent, cAMP-insensitive activation of MAP kinase observed
here. release in bovine tracheal myocytes. (
)The observation that histamine fails to activate MAP
kinase in bovine tracheal myocytes appears to contrast with our
previous findings that both thapsigargin, a
non-12-O-tetradecanoylphorbol 13-acetate type tumor promoter
that acts through the mobilization of cytosolic Ca,
and the calcium ionophore ionomycin induce
Ca
-dependent MAP kinase activation in human foreskin
fibroblasts(30) . However, later studies from our laboratory
demonstrated that activation of MAP kinase by Ca
occurs via a Raf-1-dependent pathway(32) . Thus, while it
is conceivable that under some conditions histamine might favor MAP
kinase activation by inducing Ca
release and Raf-1
activation, the inhibitory effects of histamine-induced cAMP release on
Raf-1 activity predominate in this system.
))))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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