©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Histamine Antagonizes Serotonin and Growth Factor-induced Mitogen-activated Protein Kinase Activation in Bovine Tracheal Smooth Muscle Cells (*)

(Received for publication, June 5, 1995; and in revised form, June 26, 1995)

Marc B. Hershenson (2)(§) Tsung-Shu Oliver Chao (1) Mark K. Abe (2) Ignatius Gomes (1) Michael D. Kelleher (2) Julian Solway (3) Marsha Rich Rosner (1)

From the  (1)From theBen May Institute and the Department of Pharmacological and Physiological Sciences, the (2)Department of Pediatrics, and the (3)Department of Medicine, University of Chicago, Chicago, Illinois 60637-1470

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 alpha-methyl-5-HT and reduced by pretreatment with either phorbol 12,13-dibutyrate or forskolin, suggesting activation of the 5-HT(2) 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(2) 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.


INTRODUCTION

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) (^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.

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(2) receptor activates a G protein that is positively coupled to phospholipase C, whereas stimulation of the 5-HT receptor activates a G(i) 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(1) receptor subtype (28) and stimulate cAMP synthesis in cultured guinea pig tracheal smooth muscle cells via its H(2) 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.


EXPERIMENTAL PROCEDURES

Materials

Anti-human alpha-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. alpha-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 alpha-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 beta-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(2), 10 mM MnCl(2), 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(3)VO(4), 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(2), 20 µg/ml aprotinin, and 200 µM Na(3)VO(4)) 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 510^5 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 beta-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(2), 1 mM dithiothreitol, 200 µM Na(3)VO(4), 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 (10-10M) 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.


RESULTS

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).


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.



Mechanism of MAP Kinase Activation following 5-HT Stimulation

We assessed the effects of a specific 5-HT(2) receptor agonist, alpha-methyl-5-HT, on MAP kinase activation (Fig. 2A). Stimulation with alpha-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(2) 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(i) 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(i)-linked 5-HT receptor subtype is not involved in 5-HT-induced MAP kinase activation.


Figure 2: Panel A, kinase renaturation assay demonstrating the effects of alpha-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.



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).

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).


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.



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(2) 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(2) 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(2) receptor subtype. To confirm increased adenylate cyclase activity after H(2) 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).


Figure 4: Panel A, kinase renaturation examining the effects of H(2) 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.




DISCUSSION

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(2) 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(i) 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(i)-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(i) 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. (^2)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(2) 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(2) 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. (^3)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. (^4)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.


FOOTNOTES

*
This study was supported by a gift from the Cornelius Crane Trust (to M. R. R.), NCI (National Institutes of Health) Grant CA35541 (to M. R. R.), and National Institutes of Health Grants HL02731 (to M. B. H.), HL54685 (to M. B. H.), and HL48257 (to M. B. H and J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Wyler Children's Hospital, 5841 S. Maryland Ave., MC 4064, Chicago, IL 60637-1470. Tel.: 312-702-9659; FAX: 312-702-2488.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; PDGF, platelet-derived growth factor; EGF, epidermal growth factor; 5-HT, 5-hydroxytryptamine; PDBu, phorbol 12,13-dibutyrate; IGF-1, insulin-like growth factor-1; DMEM, Dulbecco's modified Eagle's medium; MBP, myelin basic protein; MEK, MAP kinase/ERK-activating kinase; Pipes, 1,4-piperazinediethanesulfonic acid.

(^2)
M. Hershenson and M. Rosner, data not shown.

(^3)
T-S. Chao, M. Abe, M. Hershenson, I. Gomes, and M. Rosner, unpublished data.

(^4)
M. Wylam, unpublished data.


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