(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 -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.
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) ()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.
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 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) .
Histamine is released into the airways by mast cells following
allergen exposure(26) . Histamine has been shown to induce
cytosolic Ca 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.
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 -methyl-5-HT (10
M), 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.
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 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.
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 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.
Stimulation of the
5-HT 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.
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. ()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.
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 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.
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. ()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.
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 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.