Serotonin stimulates mitogen-activated protein kinase activity
through the formation of superoxide anion
Sheu-Ling
Lee,
Wei-Wei
Wang,
Geraldine A.
Finlay, and
Barry L.
Fanburg
Pulmonary and Critical Care Division, Department of Medicine, Tupper
Research Institute, and New England Medical Center, Tufts University
School of Medicine, Boston, Massachusetts 02111
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ABSTRACT |
Our previous studies have shown that, through an
active transport process, serotonin (5-HT) rapidly elevates
O
2· formation, stimulates protein
phosphorylation, and enhances proliferation of bovine pulmonary artery
smooth muscle cells (SMCs). We presently show that 1 µM 5-HT also
rapidly elevates phosphorylation and activation of the
mitogen-activated protein (MAP) kinases extracellular signal-regulated
kinase (ERK) 1 and ERK2 of SMCs, and the enhanced phosphorylation is
blocked by the antioxidants Tiron,
N-acetyl-L-cysteine (NAC),
and Ginkgo biloba extract. Inhibition
of MAP kinase with PD-98059 failed to block enhanced
O
2· formation by 5-HT. Chinese
hamster lung fibroblasts (CCL-39 cells), which demonstrate both 5-HT
transporter and receptor activity, showed a similar response to 5-HT
(i.e., enhanced mitogenesis, O
2· formation, and ERK1 and ERK2 phosphorylation and activation). Unlike
SMCs, they also responded to 5-HT receptor agonists. We conclude that
downstream signaling of MAP kinase is a generalized cellular response
to 5-HT that occurs secondary to
O
2· formation and may be initiated
by either the 5-HT transporter or receptor depending on the cell type.
signal transduction; mitogen-activated protein kinase; superoxide; smooth muscle cell; fibroblast
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INTRODUCTION |
REACTIVE OXYGEN SPECIES (ROS) have now been recognized
to be mediators of cellular proliferation for various types of
nonphagocytic cells (4, 20, 32, 34, 35, 37, 42). The intracellular release of ROS in response to ligand stimulation acts as a second messenger in signal transduction, leading to cellular growth (11, 19,
43). However, the downstream targets of endogenous ROS are largely
unknown. Mitogen-activated protein (MAP) kinases are known to provide
an important intermediate signal transduction pathway in response to
growth factors or other stimuli that lead to cellular proliferation or
hypertrophy (7, 24). The best-characterized component of this pathway
is the extracellular signal-regulated kinase (ERK) cascade (5, 13, 21).
The regulation of the activation of MAP kinase signal by exogenous
oxidants has been reported for neutrophils (10) and various other cell
types (1, 15, 41, 42), including smooth muscle cells (SMCs; see Ref. 3). In addition to the effects of exogenous ROS, inhibition of
intracellular ROS by either chemical or enzymatic antioxidants has been
shown to inhibit MAP kinase signal transduction (22, 42).
Serotonin (5-HT) acts as a mitogen in both vascular and nonvascular
cells through multiple intermediate mechanisms that have been proposed
for this action (9). We have previously shown that superoxide mediates
5-HT-induced mitogenesis and that the superoxide formation is
downstream to tyrosine phosphorylation of GTPase-activating protein
(GAP) activated by 5-HT (31, 32). We have also found that initial
signaling for SMCs from the bovine pulmonary artery occurs via active
transport of 5-HT. Other investigators have reported that mitogenesis
of other cell types produced by 5-HT is dependent upon action on 5-HT
receptors (9). To try to sort out this possible discrepancy and to
better identify downstream signaling pathways of 5-HT in this study, we
have compared 5-HT-related properties of bovine pulmonary artery SMCs
with those of Chinese hamster lung fibroblasts (CCL-39 cells), which
have been previously identified by other investigators to respond to
5-HT to produce mitogenesis through a 5-HT receptor(s) (39, 40).
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MATERIALS AND METHODS |
Reagents. Ginkgo
biloba extract (GK) 501 (batch no. 91196) was a gift
from Dr. Fabio Soldati (Pharmaton, Lugano, Switzerland). Diphenyliodonium (DPI) was from ICN Pharmaceuticals (Costa Mesa, CA).
Phospho-specific p44/42 MAP kinase
(Thr202/Tyr204)
antibody was from New England BioLabs (Beverly, MA). ERK1 polyclonal antibody was from Santa Crutz Biotechnology (Santa Cruz, CA). CGS-120668 maleate,
(±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI HCl),
-methylserotonin maleate (m-5-HT),
ketanserin tartrate, pirenperone, propranolol hydrochloride, and
clomipramine were from RBI (Natick, MA). PD-98059
(2'-amino-3'-methoxyflavone) was from
Calbiochem-Novabiochem International (San Diego, CA). Human recombinant
basic fibroblast growth factor
(FGF2) was from Promega
(Madison, WI). All other reagents were from Sigma Chemical (St. Louis, MO).
Cell culture. SMCs from bovine
pulmonary artery were isolated and cultured by a modification of the
method of Ross as previously described (30). In these experiments,
third- to fifth-passage SMCs were used. Chinese hamster lung
fibroblasts CCL-39 cells were obtained from American Type Culture
Collection (Manassas, VA) and were cultured in McCoy's 5A medium with
10% FBS.
Incorporation of
[3H]thymidine.
The protocol used has been described in detail (30). In brief, plated
SMCs were cultured for 72 h in RPMI medium containing 10% FBS followed
by 72 h of growth arrest in medium containing 0.1% FBS. SMCs were then
incubated at a density of 0.1 × 106 cells/35-mm petri dish with
and without 1 µM 5-HT in the same medium for 20 h before being
labeled with
[methyl-3H]thymidine
(0.1 mCi/ml, specific activity 20 Ci/mmol; New England Nuclear, Boston,
MA) for 4 h. Iproniazid (an inhibitor of degradation of 5-HT by
monoamine oxidase) or other inhibitors were added 30 min before the
5-HT. These agents alone at the concentrations reported did not alter
the incorporation of
[3H]thymidine by SMCs.
After labeling, experiments were terminated by aspiration of medium and
washing the cellular monolayer first with ice-cold PBS and then with
cold 6% TCA. Cells were then dissolved in 0.2 N NaOH, and
radioactivity was counted. The only modification to this protocol used
for CCL-39 cells was that cells were growth arrested in McCoy's medium
without FBS for 24 h followed by incubation with or without 5-HT in the
presence or absence of inhibitors for 24 h and labeled with
[3H]thymidine (0.5 mCi/ml) for the final 4 h.
Measurement of superoxide anion production in intact
cells by a lucigenin-enhanced chemiluminescence assay.
SMCs and CCL-39 cells were cultured in 100-mm petri dishes and growth
arrested in medium containing 0.1% FBS. The assay was done as
previously described (16, 32). 5-HT and other reagents were first added directly to the cellular monolayer. Cells were then trypsinized, pelleted by centrifugation, and resuspended in PBS containing 10 mM
glucose and 1 mg/ml BSA. 5-HT and/or other reagents were then again
added to the cellular suspensions in the cuvette. Cellular suspensions
were loaded into a luminometer, and lucigenin (final concentration 500 µM) was automatically injected to start the reaction. A 15-s
dark-adaptive period was carried out before each sample reading in the
luminometer. Photoemission was recorded with 60-s integration for
5-10 min with a Lumac Biocounter M2010 (Lumac System, Titusville,
FL). Buffer blank, lucigenin, or other reagents used alone in these
studies produce negligible chemiluminescence. Data are expressed as
degree of stimulation of chemiluminescence relative to control.
Preparation of whole cell extracts for
electrophoresis. Cells were grown in 100-mm petri
dishes to confluency and were growth arrested in medium containing
0.1% FBS. The cells were preincubated with inhibitors for 1-2 h
before the addition of 1 µM 5-HT or other stimuli for periods
indicated in RESULTS. Cellular monolayers were then washed
two times with ice-cold PBS. Cell lysates were obtained by incubating
the cellular monolayer in 1 ml of cell lysis buffer (47) containing 50 mM Tris · HCl, pH 7.5, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 40 µg/ml
phenylmethylsulfonyl fluoride (PMSF), 0.07 µg/ml pepstatin, 1%
Nonidet P-40, 150 mM NaCl, and 5 mM EDTA for 10 min at 4°C. The
insoluble material was removed by centrifugation (14,000 g, 2 min), and the supernatant
fraction was used for analysis. Twenty to fifty micrograms of protein
of the whole cell lysate were subjected to SDS-PAGE on a 10% slab gel.
Immunoblotting with anti-phospho-specific p44/p42 MAP
kinase antibody. After electrophoresis, gel proteins
were electrophoretically transferred to a polyvinylidene difluoride
(PVDF) membrane (Tropifluor, Tropix, Bedford, MA, or Immobilon-P,
Millipore, Bedford, MA). After transfer, nonspecific PVDF binding sites
were blocked with 5% HiPure liquid gelatin (Norland, New Brunswick,
NJ) in buffer, pH 7.4, containing 75 mM sodium phosphate, 70 mM NaCl,
0.02% sodium azide, and 0.1% Tween 20. Blocking was done for 1 h at
ambient temperature. The membrane was then treated overnight with a
1:1,000 dilution of alkaline phosphatase-conjugated
anti-phospho-specific p44/42 MAP kinase antibody in blocking buffer at
4°C. Nitroblock (Tropix) was used according to the manufacturer's
instructions. Subsequent washing, detection with the chemiluminescent
substrate CSPD (disodium
3-(4-methoxyspiro[1,2-dioxetane-3,2'(5'-chloro)-tricyclo[3.3.1.13,7]decan]-4-yl)phenyl
phosphate; Tropix), and film exposure were done as described previously
(31). Densitometry was performed with a Millipore densitometer with
Visage v4.6p software (Millipore). Data are expressed as
degree of stimulation compared with control sample.
Measurement of 5-HT uptake. 5-HT
uptake was measured as previously described (27, 28). The effect of
hypothermia was tested by carrying out uptake experiments in the cold
room at 4°C. Media used were equilibrated at 4°C before
measuring uptake. For assessment of uptake in
Na2+-free medium, NaCl in PBS was
replaced by LiCl, and
Na2HPO4 · 7H2O
was replaced by tris(hydroxymethyl)aminomethane. The pH of the final
solution was adjusted to 7.4 by adding 1 N KOH. The CCL-39 cell
monolayer was rinsed two times with PBS containing 15 mM dextrose (pH
7.4) and was incubated for 30 min in this solution containing 0.1 mM
iproniazid to block monoamine oxidase activity. 5-[3H]HT
(5-[1,2-3H(N)]hydroxytryptamine
creatinine sulfate, specific activity 30 Ci/mmol; New England Nuclear)
was added from a stock solution containing ascorbic acid (10 µg/ml)
and EDTA (10 µg/ml) as antioxidants. The 5-HT concentration used in
this study was 15 nM. The uptake of 5-HT by CCL-39 cells was saturated
in 20 min; therefore, incubations were carried out for 10 min. Under
these conditions, radioactivity taken up by cells was no more than 1%
of that in the medium. After the incubation, media were removed, and
monolayers were washed three times with ice-cold PBS plus 0.1 mM
imipramine (5-HT uptake inhibitor). Cells were dissolved in 0.2 N NaOH,
and the radioactivity was counted.
MAP kinase activity assay. MAP kinase
activity was measured in immune complexes using myelin basic protein as
the substrate (44). The whole cell lysates obtained as described in
Preparation of whole cell extracts for electrophoresis were
used for immunoprecipitation (IP) for kinase assay. IP was performed by
binding ERK1 polyclonal antibody to protein A-Sepharose beads
(Repligen, Cambridge, MA), and 50 µg protein of cell lysates were
added. This mixture was gently rocked at 4°C for 1 h. The
precipitated immune complex was then washed one time with Triton lysis
buffer (20 mM Tris buffer, pH 7.4, 137 mM NaCl, 2 mM EDTA, pH 7.4, 1%
Triton X-100, 25 mM
-glycerophosphate, 1 mM sodium orthovanadate, 2 mM sodium pyrophosphate, 10% glycerol, 0.2 mM PMSF with 1 µg/ml
leupeptin, and 1 µg/ml pepstatin) and one time with kinase assay
buffer (125 mM HEPES, pH 7.5, 100 mM
-glycerophosphate, 12.5 mM
MgCl2, 100 mM
p-nitrophenyl phosphate, and 0.5 mM
sodium orthovanadate) plus protease inhibitors. The pellet was then
resuspended in 20 µl of kinase assay buffer supplemented with 0.1 mg/ml of myelin basic protein (Sigma), 25 µM ATP, and 10 µCi
[
-32P]ATP (New
England Nuclear) and incubated at 30°C for 20 min. The reaction was
terminated by addition of 7 µl of 4× Laemmli sample buffer.
Samples were then boiled for 5-10 min, centrifuged, and
resolved by 12.5% SDS-PAGE. Gels were dried and quantitated by
ImageQuant software on a PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). Data are expressed as degree of kinase activity relative to
quiescent control.
Quantitation. Each treatment was
carried out in at least triplicate experiments, and a representative
experiment is shown in Figs. 1-10. Values are means ± SD
(n = 4). Unpaired Student's t-test was used for determination of
the significance of the results.
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RESULTS |
We have previously reported that 5-HT elevates superoxide formation,
stimulates protein phosphorylation, and enhances proliferation of
bovine pulmonary artery SMCs in culture (30-32). Exposure of these
SMCs to 1 µM 5-HT also rapidly elevated phosphorylation of both p44
and p42 (ERK1 and ERK2) MAP kinases by 7- to 10-fold within 5 min (Fig.
1A).
This elevation returned to baseline by 1 h. Omission of
Na+ from the cellular medium
prevented the stimulation of the MAP kinases by 5-HT (Fig.
1B). PD-98059, a specific inhibitor
of MAP kinase kinase (2, 8), dose dependently inhibited stimulation of
DNA synthesis by 5-HT (Fig. 2). Thus
5-HT-induced mitogenesis of SMCs appears to occur via signaling through
a MAP kinase-dependent pathway. Antioxidants such as Tiron
[4,5-dihydroxy-(1,3-benzene disulfonic acid)], NAC, and GK
dose dependently inhibited 5-HT-stimulated DNA synthesis of SMCs (23,
32, 33) and also blocked the 5-HT-elevated phosphorylation of ERK1 and
ERK2, as shown in Fig. 3. However,
the MAP kinase inhibitor PD-98059, which had no quenching effect on
O
2· generated from interaction of
xanthine (60 µM) with xanthine oxidase (2 mU/ml; unpublished data),
also failed to show any effect on
O
2· released from SMCs incubated
with 5-HT.

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Fig. 1.
A: time course of serotonin
(5-HT)-induced phosphorylation of p44/p42 mitogen-activated protein
(MAP) kinase of smooth muscle cells (SMCs) [extracellular
signal-regulated kinase (ERK) 1 (open bars)/ERK2 (filled bars),
respectively]. Immunoblotting and densitometry were done as
described in MATERIALS AND METHODS. Data are expressed as
degree of stimulation compared with control (Contl) sample.
B: lack of influence of 5-HT on
phosphorylation of p44/p42 when SMCs are incubated in
Na+-free medium.
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Fig. 2.
Inhibition by PD-98059 of 5-HT-induced DNA synthesis of SMCs.
Experiments were done as described in MATERIALS AND
METHODS. Values are means ± SD
(n = 4 dishes), cpm, Counts/min; TdR,
thymidine. Significantly different (P < 0.05) from:
* quiescent control; ** 5-HT alone.
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Fig. 3.
Inhibitory effect of PD-98059 (PD; 10 µM), Tiron (2 mM),
N-acetyl-L-cysteine (NAC;
10 mM), and Ginkgo biloba (GK; 200 µg/ml) on 5-HT-induced phosphorylation of p44/p42 of SMCs.
Experiments were done as described in MATERIALS AND
METHODS.
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With the use of various inhibitors of 5-HT uptake, we have previously
concluded that 5-HT produces its mitogenic effect on vascular SMCs
through action on a 5-HT transporter (30). On the other hand, various
other studies have reported that 5-HT produces a mitogenic response
through action on 5-HT receptors (9). One such report is that of Seuwen
and colleagues (39, 40), which showed that 5-HT stimulates mitogenesis
of CCL-39 fibroblasts by action on a
5-HT1B receptor. We have confirmed the mitogenic effect of 5-HT on CCL-39 cells (Fig.
4) and have also found that this effect is
blocked by a number of inhibitors of 5-HT transport, including
imipramine, clomipramine, and fluoxetine (Fig. 4). This observation
prompted us to study the 5-HT transport activity of CCL-39 cells. As
shown in Fig.
5A, CCL-39
cells, like SMCs, rapidly accumulate 5-HT, and the uptake is blocked by
a variety of agents and conditions that are known to inhibit 5-HT transport (28, 29), including the use of
Na2+-free medium, cold
temperature, and agents such as imipramine, propranolol, ketanserin,
and verapamil (Fig. 5B).

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Fig. 4.
Effect of 5-HT transporter inhibitors [imipramine (IMP),
clomipramine (Clom), and fluoxetine (Flu)] and
5-HT2 receptor antagonists
[ketanserin (Ket) and pirenperone (Pir)] on 5-HT-induced
DNA synthesis of CCL-39 cells. Experiments were done as noted in
MATERIALS AND METHODS. Concentrations of inhibitors as
follows: 0.1 mM for imipramine and 10 µM for the others. Values are
means ± SD (n = 4 dishes).
Significantly different (P < 0.05) from: * quiescent
control; ** 5-HT alone.
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Fig. 5.
5-HT uptake by CCL-39 cells. A: time
course of 5-HT uptake of CCL-39 cells at 15 nM 5-HT.
B: 5-HT transport by CCL-39 cells is
inhibited by Na+-free medium, cold
temperature, and uptake inhibitors such as imipramine (0.1 mM),
propanolol (Prop), ketanserin, and verapamil (Verp) at 10 µM
concentration. Values are means ± SD
(n = 4 dishes). * Significantly
different from control, P < 0.05.
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DOI and m-5-HT are 5-HT analogs that are thought to act through
stimulation of a 5-HT2 receptor.
Contrary to their failure to stimulate proliferation of SMCs (Fig.
6), these agents produced a two- to
threefold stimulation of proliferation of CCL-39 cells (Fig.
7). The CCL-39 cells also showed a
mitogenic response to CGS-12066B dimaleate, a specific
5-HT1B-receptor agonist (Fig. 7).

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Fig. 6.
Failure of stimulation of mitogenic response of SMCs by 5-HT receptor
agonists [CGS (5-HT1B receptor),
(±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), and
-methylserotonin maleate (m-5-HT;
5-HT2 receptor)] in contrast to a
6- to 7-fold stimulation of DNA synthesis induced by 5-HT. Values are
means ± SD (n = 4 dishes).
* Significantly different from quiescent control, P < 0.05.
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Fig. 7.
Inhibitory effect of PD-98059 on the mitogenic response of CCL-39 cells
to 5-HT, CGS-120668 (CGS), DOI, and m-5-HT. Experiments were done as
noted in MATERIALS AND METHODS. Values are means ± SD
(n = 4 dishes). Significantly
different (P < 0.05) from:
* quiescent control; ** stimulated control.
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Both 5-HT and m-5-HT produced a rapid increase in phosphorylation of
ERK1 and ERK2 by 10- to 20-fold in CCL-39 cells (Fig. 8A). All
of these stimulations of ERK1 and ERK2 were blocked by both 5-HT uptake
inhibitors (imipramine, clomipramine, and fluoxetine) and
5-HT2-receptor antagonists
(ketanserin and pirenperone) as shown in Fig. 8,
Aa and
Ab, respectively. MAP kinase activity of CCL-39 cells was elevated by 5-HT and m-5-HT 5.5- and 3.4-fold, respectively; and this stimulation was inhibited by 5-HT transporter or
receptor inhibitors (Fig.
8B).


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Fig. 8.
Inhibitory effect of 5-HT transporter inhibitors (imipramine,
clomipramine, and fluoxetine) and
5-HT2-receptor antagonists
(ketanserin and pireperone) on phosphorylation of p44/p42
(A) and induced MAP kinase activity
(B) by 5-HT (lanes
2 and
4-6)
and m-5-HT (lanes 3,
7, and
8) vs. control (lane
1) in CCL-39 cells. Treatments in the immunoblot of
B correspond with those in the
densitometric results. Experiments were done as noted in
MATERIALS AND METHODS. Concentrations of inhibitors as
follows: 0.1 mM imipramine and 10 µM for the others. Densitometric
results in A are phosphorylation of
5-HT ± inhibitors (a) and m-5-HT ± receptor antagonists (b). Open
bars, p44; hatched bars, p42.
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We assessed the importance of MAP kinase in the proliferative process
of CCL-39 cells stimulated by 5-HT and 5-HT agonists. As shown in Fig.
7, [3H]thymidine
incorporation of these cells induced by 5-HT and receptor agonists was
inhibited by PD-98059, a known inhibitor of MAP kinase.
We have previously reported that
O
2· is important in 5-HT signaling
of mitogenesis in SMCs (32). Therefore, we tested a variety of
antioxidants and an inhibitor of NAD(P)H oxidase (DPI) on the responses
of CCL-39 cells to 5-HT. DPI alone stimulated
[3H]thymidine
incorporation. Tiron, DPI, GK, and NAC dose dependently inhibited both
[3H]thymidine
incorporation and phosphorylation of ERK1 and ERK2 induced by 5-HT in
CCL-39 cells (Fig. 9,
A-D). Similar to our observations
with SMCs (30), 5-HT also acted synergistically with 10 ng/ml
FGF2 in stimulating DNA synthesis
and activating ERK1 and ERK2 of CCL-39 cells (Fig.
10, A
and B). Tiron, NAC, DPI, and GK
inhibited O
2· generation from this cell type when it was incubated with 5-HT plus
FGF2 (Fig.
10C). These antioxidants also
inhibited both DNA synthesis (Fig.
10A) and the activation of ERK1 and
ERK2 (Fig. 10B). PD-98059, however, showed no effect on release of O
2·
from CCL-39 cells produced by treatments with either 5-HT (data not shown) or with 5-HT plus FGF2
(Fig. 10C) but highly inhibited DNA synthesis induced by these agents.




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Fig. 9.
Inhibitory effect of antioxidants (Tiron, NAC, and GK) and
diphenyliodonium (DPI) on 5-HT-induced DNA synthesis
(A and
B) and phosphorylation of p44/p42
(C) of CCL-39 cells induced by
either 5-HT (a) or m-5-HT
(b). Open bars, p44; hatched bars,
p42. Concentrations of inhibitors in C
as follows: 10 µM PD-98059, 150 µg/ml GK, 2 mM Tiron, and 10 mM
NAC. DPI (0.1 mM) inhibits phosphorylation of p44/42 as shown in
D. Experiments were done as noted in
MATERIALS AND METHODS. Values are means ± SD
(n = 4 dishes). Significantly
different (P < 0.05) from: * quiescent control;
** 5-HT alone.
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Fig. 10.
Effect of 1 µM 5-HT plus 10 ng/ml fibroblast growth factor
(FGF2) on DNA synthesis
(A) and phosphorylation of p44/p42
of CCL-39 cells (B). Antioxidants (2 mM Tiron, 10 mM NAC, and 150 µg/ml GK) and 0.1 mM DPI inhibit
stimulated DNA synthesis, phosphorylation of p44/p42, and
O 2· release from CCL-39 cells
incubated with 5-HT plus FGF2.
However, PD-98059 (10 µM) fails to show any inhibitory effect on
O 2· release
(C). Experiments were done as noted
in MATERIALS AND METHODS. Values are means ± SD
(n = 4 dishes). Significantly
different (P < 0.05) from: * quiescent control;
** 5-HT plus FGF2.
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DISCUSSION |
From the studies presented and other data we have published, some
general statements can be made about at least part of the sequence of
events whereby 5-HT signals cells to proliferate. Depending upon the
cell type, the data indicate that the initial signal occurs either
through internalization of 5-HT via the 5-HT transporter or by way of a
5-HT receptor on the cell surface. GAP is phosphorylated early in the
series of subsequent events (31), then
O
2· is formed intracellularly (32), possibly through activation of
p21ras (38). In response to the
O
2· formation, MAP kinases,
specifically ERK1 and ERK2, are phosphorylated and activated.
Whether the initiating signal occurs through a 5-HT transporter or a
5-HT receptor has been controversial. The 5-HT transporter and the 5-HT
receptor(s) are different molecules that have now been cloned
separately (9). Their functional properties in cells have been largely
defined with the use of well-characterized transport inhibitors or
receptor antagonists along with use of other known features of an
active transport process, such as dependence on
Na+ and inhibition by cold. All of
our data obtained with bovine pulmonary artery SMCs have clearly
defined the participation of a 5-HT transporter, internalization of
5-HT, and a requirement for a pertussis toxin-insensitive G protein (9)
in the initiation of a proliferation process for this cell type (30).
Because other cells, including Chinese hamster lung fibroblasts, have
been described by other investigators to show a proliferative response
to 5-HT through a more conventional cell membrane receptor action (as
has been described for growth factors in general), we undertook in the
present experiments to study for the first time the ability of the
CCL-39 fibroblasts to actively transport 5-HT. These studies showed
that the CCL-39 cells, indeed, actively transport 5-HT and that the
uptake process for these cells is also, like that of the bovine
pulmonary artery SMCs, a mechanism by which proliferation is initiated.
A confusing factor for these cells is the observation that, unlike
SMCs, they also show a mitogenic response to purported 5-HT receptor
agonists, and the mitogenic response to 5-HT is inhibited by 5-HT
receptor antagonists. The results of these pharmacological studies are
somewhat difficult to interpret but suggest that more than one 5-HT
receptor type may exist for CCL-39 cells and may be responsive to 5-HT.
Why this cell type may react to 5-HT and show a proliferative response through either a 5-HT receptor or transporter is unclear. The transporter action appears to predominate, since all methods to inhibit
transport of 5-HT, including exposure to cold and removal of
Na+ from the medium, strongly
block cellular proliferation caused by 5-HT.
Regardless of the initiating mechanism, both SMCs and the CCL-39
fibroblasts show stimulation of formation of
O
2· by 5-HT, and uses of
antioxidants such as Tiron, NAC, and GK all block the proliferative
process. DPI, which is a nonspecific inhibitor of NAD(P)H oxidase, also
blocks the process, suggesting that a NAD(P)H oxidase may participate
in the signaling mechanism. Similarly, both cell types rapidly respond
to 5-HT by showing enhanced phosphorylation of the MAP kinases, ERK1,
ERK2, and MAP kinase activity, which are blocked by antioxidants.
Activation of MAP kinase is generally believed to be regulated by Ras
protein (6, 17). 5-HT activation of ERK1 and ERK2 has been reported to
occur in bovine tracheal SMCs and CHO-K1 fibroblasts through actions on
5-HT1A,
5-HT2, and
5-HT2B receptors (14, 18, 26) and
in 5-HT-induced vascular contraction through a
5-HT2A receptor (12, 45, 46). Our studies show that this action can also happen through a 5-HT transporter.
It was recently recognized that superoxide relays the oncogenic message
of Ras protein for cellular growth (36). Lander et al. (25) proposed
that p21ras is a common signaling
target of ROS and cellular redox stress. Our data show that
antioxidants have no effect on the 5-HT-induced elevation of tyrosine
phosphorylation of GAP (33) but do inhibit MAP kinase activation. This
finding is consistent with previous observations that NAC suppresses
the phosphorylation of MAP kinase induced by platelet-derived growth
factor (42) and nerve growth factor (22) in cell types different from
ours. Inhibition of 5-HT-induced cellular proliferation by the MAP
kinase inhibitor PD-98059 supports the role of MAP kinase in the
proliferative process produced by 5-HT. Similarly, failure to block
5-HT-induced O
2· formation with
PD-98059 indicates that O
2·
formation is upstream to phosphorylation of ERK1 and ERK2. Thus these
and our other previous data (31-33) strongly suggest that
O
2· formation is downstream to
tyrosine phosphorylation of GAP and upstream to MAP kinase activation.
Superoxide formation appears to be a critical step in signaling
5-HT-induced cellular growth.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart, Lung, and Blood
Institute Grant HL-32723.
 |
FOOTNOTES |
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: S.-L. Lee, New
England Medical Center, Pulmonary and Critical Care Division, 750 Washington St., NEMC #265, Boston, MA 02111.
Received 28 December 1998; accepted in final form 6 April 1999.
 |
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