(Received for publication, July 6, 1995; and in revised form, December 4, 1995)
From the
The family of serotonin 5-HT2 receptors stimulates the
phospholipase C second messenger pathway via the subunit of the
G
GTP-binding protein. Here, we show that agonist
stimulation of the 5-HT2B receptor subtype stably expressed in the
mouse fibroblast LMTK
cell line causes a rapid and
transient activation of the proto-oncogene product p21
as measured by an increase in GTP-bound Ras in response to
serotonin. Furthermore, 5-HT2B receptor stimulation activates
p42
/p44
(ERK2/ERK1)
mitogen-activated protein kinases as assayed by phosphorylation of
myelin basic protein. Antibodies against p21
,
G
, -
, or -
subunits of the
GTP-binding protein inhibit MAP kinase-dependent phosphorylation. The
MAP kinase activation is correlated with a stimulation of cell division
by serotonin. In addition to this mitogenic action, transforming
activity of serotonin is mediated by the 5-HT2B receptor since its
expression in LMTK
cells is absolutely required for
foci formation and for these foci to form tumors in nude mice. Finally,
we detected expression of the 5-HT2B receptor in spontaneous human and Mastomys natalensis carcinoid tumors and, similar to the
5-HT2B receptor transfected cells, the Mastomys tumor cells
are also responsive to serotonin with similar coupling to
p21
activation.
Serotonin (5-hydroxytryptamine, 5-HT) ()is one of the
best known examples of a neurotransmitter that mediates a wide variety
of physiological effects, including peripheral and central actions,
through the binding to multiple receptor subtypes(1) . The
major sites of 5-HT synthesis and storage are located in the periphery,
in gut enterochromaffin cells and blood platelets, respectively. The
large diversity of 5-HT functions is paralleled by the pharmacological
complexity of 5-HT receptors. At least four classes of 5-HT receptors
have been distinguished pharmacologically, reflecting the second
messenger system to which the receptor is coupled. The family including
5-HT1 and 5-HT5 subtypes of receptors interacts negatively with
adenylyl cyclase, the 5-HT2 subfamily of receptors is coupled to the
activation of the phospholipase C-
, the 5-HT3 receptor is a
ligand-gated ion channel, and the family, including 5-HT4, 5-HT6, and
5-HT7 subtypes of receptors, activates adenylyl cyclase(2) .
5-HT2 receptors mediate many of the central and peripheral
physiological functions of 5-HT. Cardiovascular effects include
contraction of blood vessels and shape change in platelets; central
nervous system effects include neuronal sensitization to tactile
stimuli and mediation of hallucinogenic effects of lysergic acid
diethylamide and related phenylisopropylamine hallucinogens. The most
characterized 5-HT2 receptor subtypes are the 5-HT2A (formerly 5-HT2)
and the 5-HT2C (formerly 5-HT1C) both of which stimulate phospholipase
C-. Many investigators have observed that some peripheral
5-HT2-like effects of 5-HT are mediated by ``atypical''
receptors(3) . In the mouse, we cloned a new member (5-HT2B) of
the 5-HT2 family which is mainly expressed in the cardiovascular
system, gut, and developing brain(4) . This mouse 5-HT2B
receptor shares the highest degree of homology with the other 5-HT2B
receptors cloned from the rat fundus (5, 6) and, more
recently, from human libraries (7, 8, 9) (for
review, see (10) ) and from Drosophila(11) .
5-HT, detected early in embryonic development(12) ,
participates in craniofacial (13) and cardiovascular
morphogenesis (14, 15) by unknown molecular
mechanisms. We have investigated the growth factor properties mediated
by the 5-HT2B receptor since (i) both Drosophila 5-HT2 and
mouse 5-HT2B receptors are expressed during embryogenesis (11, 16) , (ii) the mitogenic activity of 5-HT has
been linked mainly to 5-HT2 receptor-dependent stimulation of
phospholipase C-/protein kinase C; when expressed at high density
in NIH3T3 fibroblasts, both 5-HT2C and 5-HT2A have mitogenic effects
induced by 5-HT(17, 18) . In mammalian cells, the
ability to activate the mitogen-activated protein (MAP) kinase cascade
is a feature common to many extracellular stimuli, including growth
factors, hormones, and neurotransmitters, leading to transcription
factor phosphorylation and to cell division(19) . The signaling
pathways that activate the MAP kinase cascade use receptor tyrosine
kinase or non-receptor tyrosine kinases. Other stimuli activate
GTP-binding protein (G-protein)-coupled receptors generating second
messengers or activating ion channels (20) .
The present report concerns studies on the mitogenic and transforming activity of the mouse 5-HT2B receptor. This receptor mediates 5-HT stimulation of MAP kinase through the Ras pathway and can be classified as ligand-dependent proto-oncogene since expression of the 5-HT2B receptor is necessary and sufficient to induce tumor formation in nude mice. In addition, expression of the 5-HT2B receptor is detected in vivo in spontaneous human and Mastomys natalensis carcinoid tumors (CT).
All
antibodies are from Santa Cruz Biotechnology (dilution 1/1000) and
include a rat monoclonal antibody against v-Ha-Ras (259) which also
recognizes Ki- and N-Ras p21, rabbit antiserum against the
amino-terminal sequence of mouse G/11 (E17), rabbit
polyclonal antibody against the carboxyl terminus of MAP
kinase-p44
(C16) which also reacts with p42, rabbit
antiserum against the amino-terminal sequence of bovine G
2 (A16),
and rabbit polyclonal antibody against the carboxyl terminus of mouse
G
1 (T20) which also reacts with G
1,-2,-3,-4.
Figure 1:
5-HT induced GTPase activation. A, kinetics of GTPase activation by 5-HT, the whole-cell
GTPase activity was measured as described in (21) and (22) by monitoring the release of
[P]P
from
[
-
P]GTP in response to 5-HT activation.
GTPase activity is expressed as femtomoles of GTP hydrolyzed per mg of
protein per min. Time starts with 5-HT addition. 5-HT (10 nM)
stimulations of LMTK
(
) or of LM5 (
)
cells have been obtained in four independent experiments. The
5-HT-induced LM5 curve presents peaks at 1.5 min and at 5 min. B, kinetics of Ras activation in response to 5-HT, the
activation of Ras-GTPase activity was determined by analyzing the
ratios of GTP to GDP bound to immunoprecipitated Ras from control and
stimulated cells; the bound [
P]GTP and
[
P]GDP were resolved using
polyethyleneimine-cellulose thin layer chromatography and quantitation
of radiolabeled GDP and GTP. 5-HT (10 nM) stimulations of
LMTK
(
) or of LM5 (
) cells or of LM5
cells in the presence of 1 nM ritanserin (x) have been
performed four times independently. The LM5 cells present a peak at 5
min after 5-HT addition. The left scale is the amount of
GTP-bound Ras as the percentage of GTP plus GDP-bound Ras, and the right scale is the same values converted in GTPase activity
expressed as percentage of the maximal total GTPase activity of Fig. 1A. C and D, the cellular level
of GTPase activity was investigated on LMTK
(white boxes) or LM5 (gray boxes) cells without
treatment (
) or after incubation with 5-HT alone (
), 5-HT
plus anti-Ras antibodies (
), 5-HT plus anti-G
antibodies (diamond divided lengthwise), 5-HT plus
anti-
antibodies (
), 5-HT plus anti-
antibodies
(
), 5-HT plus anti-
and -
antibodies (diamond
divided widthwise). The experiments have been performed four
times. Specific antibodies were used at a final concentration of 2
ng/ml which is within the range of the physiological concentrations of
total IgG (around 10 mg/ml serum). The incubation in the same
conditions with preimmune serum was ineffective (not shown). C, blocking of the 5-HT-induced GTPase activation at 5 min. D, blocking of the 5-HT-induced GTPase activation at 10
min.
In several
signaling paradigms thought to involve the function of the Ras protein,
stimulation with growth factors leads to the rapid accumulation of Ras
protein in its GTP-bound (i.e. active) state. The
immunoprecipitation by anti-Ras antibodies of extracts of LM5 cells
indicates that stimulation by 5-HT, transiently and specifically,
increases the amount of the GTP-bound Ras complex (Fig. 1B). This activity is not seen in the parental
cell line nor in presence of the antagonist ritanserin. Interestingly,
the LM5 cell basal level of Ras GTPase activity is more than three
times higher than that of LMTK cells. Compared to
basal levels of LM5 cells, the increase in GTP-bound Ras induced by
5-HT is nearly 4-fold at 5 min, 78.2% ± 4.3 (n =
4) of which is sensitive to
antibodies (not shown). By 30
min after agonist addition, GTP-bound Ras returns to basal levels.
Figure 2:
5-HT-induced MAP kinase activity. MAP
kinase activity was measured on quiescent cells extracts by
purification onto Mono Q columns in four independent experiments. A, kinetics of MAP kinase activation, MBP phosphorylation
levels were essayed in duplicate using protein extracts obtained at
various times after 5-HT addition to quiescent LM5 () cells
alone or in the presence of 1 nM ritanserin (x). The
5-HT-induced LM5 cell curve presents a peak at 10 min. B,
blocking of MAP kinase activity at 10 min, the cellular level of MAP
kinase activity was investigated on LMTK
(white
boxes) or LM5 (gray boxes) cells after 10 min without
treatment (
) or after a 10-min incubation with 5-HT alone
(
), 5-HT plus ritanserin (x), 5-HT plus anti-G
antibodies (diamond divided lengthwise), 5-HT plus
anti-
and -
antibodies (diamond divided widthwise),
or 5-HT plus anti-Ras antibodies (
). The blockers of the GTPase
activity also block the MAP kinase
activity.
Since stimulation of MAP kinases
can lead to cell division(31) , we investigated further the
effect of 5-HT on the rate of LM5 cell division. This analysis
indicates that 5-HT acts through the 5-HT2B receptor on the steady
state number of cells: both 5-HT2 agonists 5-HT (1 µM) and
DOI (0.1 µM) stimulate cell growth of the LM5 clone (but
not of the LMTK cells). These effects can be
inhibited by incubation of agonists together with 1 µM ritanserin (Fig. 3A). Interestingly, in the
absence of agonist and in 5-HT-free serum, the LM5 cells have a cell
division rate that is 2-fold greater than the LMTK
cells, and this rate is reduced by the antagonist ritanserin
alone (Fig. 3A).
Figure 3:
Mitogenic action of 5-HT. No treatment
() or treatment in the presence of 1 nM ritanserin
(+), 1 nM DOI (
), 10 nM 5-HT (
), 1
nM DOI plus 1 nM ritanserin (
), or 10 nM 5-HT plus 1 nM ritanserin (x) of LMTK
(white boxes) or of LM5 (gray boxes) cells were
used to assess the effect on cell growth or on foci formation. A, cellular division assay, cells plated at an initial density
of 10
cells/35-mm dish, in 10% 5-HT-free FCS medium, and
transferred after 24 h to medium containing 0.6% 5-HT-free FCS, with or
without serotonergic drugs as indicated, were counted after 5 days.
Each sample of four independent experiments was counted twice. Stars indicate statistically significant differences. The
results are expressed as the fold increase in cell per day. The
unstimulated LM5 cells have about twice the cell division rate of the
parental LMTK
cells, ritanserin alone inhibits the
LM5 cells division rate, whereas agonists stimulate this rate. B, focus formation assay, foci were scored on confluent cells
after 2 or 3 weeks in medium containing 3% 5-HT-free FCS with or
without serotonergic drugs as indicated in 4 independent experiments.
Spontaneous foci appear in LM5 cells, but agonists enhance the number
of foci.
We then investigated receptor
expression in tumors by examining the presence of 5-HT2B-specific
binding on tumor-derived cell lines. Interestingly, 2 weeks of
treatment of nude mice-bearing tumors with the antagonist ritanserin
induces a significant reduction in tumor size, suggesting the presence
of the 5-HT2-like receptors on these tumors (Table 1). DOI
binding under nonsaturating conditions (2 nM [I]DOI) indicates a relative value of
receptor densities. We, therefore, examined the persistence of the
5-HT2B receptor expression by plating the tumor-derived cells and
estimating the receptor density from the DOI binding values. All
tumor-derived cell lines expressed a significantly (p <
0.01, KS test) greater density of 5-HT2B receptors than the parental
LM5 cell line (Table 1). This suggests that the growth of these
tumors is associated with selective expansion of cells which express
the highest number of 5-HT2B receptors (Table 1). Furthermore,
all cell lines derived from tumors retain the ability to form foci,
with a rough correlation between the number of foci and the density of
5-HT2B receptors (Table 1). To test whether the formation of foci
requires activation of 5-HT2B receptors, tumor-derived cell lines were
plated on a lawn of LMTK
cells in the presence or
absence of antagonists, ritanserin, or mesulergine for 2 weeks. As for
the parental foci, the ability of the tumor-derived cell lines to form
foci appears almost (95%) completely blocked by antagonists (Table 1). Thus, foci formation by these tumor-derived cells
remain ligand-dependent.
Figure 4:
Immunohistochemistry of anti-5-HT2B
receptor antibody on tissue sections. Tissue sections were incubated in
the presence of anti 5-HT2B receptor antibodies and revealed by
secondary antibodies coupled to peroxidase. Similar experiments
performed after incubation of the serum with the immunizing peptide
reveals no signal. The original magnifications were 200. A, human CT section treated with anti-5-HT2B antibodies,
neuroendocrine-like structure expressing the 5-HT2B receptor protein
are surrounded by normal tissue. Similar 5-HT2B positive staining has
been observed in three out of three independent human tumor samples. B, Mastomys CT section treated with anti 5-HT2B
antibodies, similar structures are observed in all samples examined. C, normal nude mice tissue section treated with anti 5-HT2B
antibodies, no 5-HT2B receptor expression can be detected. D,
nude mice tumor section treated with anti-5-HT2B antibodies, 5-HT2B
receptors are seen in the four observed tumors and present a
fibrosarcoma-like tumor organization.
Figure 5:
Ras activation in Mastomys CT by
5-HT. The Ras-GTPase activity was determined by analyzing the amount of
GTP and GDP bound to immunoprecipitated Ras from control and stimulated
cells as in Fig. 1B. Stimulation of LMTK (white boxes), of LM5 (gray boxes), or of Mastomys dissociated CT cells (black boxes) alone
(
) or in the presence of 10 nM 5-HT (
) or of 10
nM 5-HT plus 1 nM ritanserin (x) have been performed
4 times. The results, after 5 min of stimulation (peak of Fig. 1B), are expressed as femtomoles of GTP bound per
mg of protein per min. The LMTK
cells are not
responsive to 5-HT, in LM5 cells as in Fig. 1B, the
amount of GTP-bound Ras is stimulated 4 times by 5-HT and blocked by
ritanserin, whereas in the Mastomys tumor cells the amount of
GTP-bound Ras is stimulated 2.2 times and is also blocked by
ritanserin. We notice that the level of active Ras in the Mastomys tumor cells is nearly as high as that of stimulated LM5 cells,
indicating that Ras is already activated in these tumor
cells.
The aim of this study is to characterize further biochemical
signals transduced by 5-HT in 5-HT2B receptor transfected cells (LM5).
The early GTPase activation involves G,
-
, and -
subunits (1.5 min) (Fig. 1, A-D). The late phase of GTPase
activation (5 min) is resistant to anti-G
antibodies
and, therefore, implicates secondary activation of other G-proteins:
35% of this late GTPase is sensitive to anti-Ras and to anti-
antibodies (Fig. 1, A, C, and D). In
addition, 5-HT2B agonists stimulate the accumulation of the GTP-bound
form of Ras with similar kinetics peaking at 5 min after 5-HT
stimulation (Fig. 1B). These results suggest that Ras
is involved in the secondary phase of GTPase activation and that the
subunit is mediating this effect. The amount of GTP-bound
Ras is stimulated 4-fold by 5-HT, and this is blocked in the presence
of ritanserin (Fig. 1B). However, the Ras-GTPase
activity determined by immunoprecipitation of Ras (Fig. 1B and Fig. 5) represents only 18% of the maximal total
cellular GTPase activity (Fig. 1A), which in turn
corresponds to approximately half of the GTPase activity blocked by
anti-Ras antibodies (Fig. 1D). This indicates that
other processes, that are inhibited by anti-Ras antibodies, may also
participate in the 5-HT2B-dependent GTPase stimulation. Agonist
stimulation of heterotrimeric G-proteins involves dissociation of the
G
subunit from the receptor(36) . The
subunit, subsequently released from
G
(37, 38) , stimulates the Ras-GTPase
activity, raising, at least in part, the late GTPase activity. The Ras
stimulation by the
subunit remains to be dissected out.
Candidates to mediate this stimulation are proteins containing
plekstrin homology domain since
subunits have affinity for
such domains at the carboxyl terminus of
-adrenergic
kinase(39) . This includes the GTPase activating proteins,
whose noncatalytic domain has been reported to interfere with
transformation induced by G-protein-coupled receptors (40, 41) or other protein kinases (see (42) for review).
In LM5 cells, 5-HT triggers the activation
of p42/p44
(ERK2/ERK1) MAP kinase cascade (Fig. 2A). This ``extracellular signal-regulated
kinase'' pathway is classically used by growth factor stimulation
of intracellular tyrosine kinase activity(43) . This cascade
leads to cell division(31) , differentiation, and/or
transformation and can be potentiated by stimulation of
G
(44) . Ligands for multiple G-protein coupled
receptors have been shown to stimulate tyrosine kinase
activity(42, 45, 46, 47) , and
angiotensin II AT1 receptor recently has been reported to stimulate
directly the Jak/STAT pathway in rat aortic smooth muscle(48) .
The contribution of the 5-HT2B receptor to tyrosine kinase activation
remains to be investigated. However, MAP kinase activity has been
reported to be stimulated by G
, a pertussis toxin-sensitive
G-protein(47, 49) , and by G
(50, 51, 52) or inhibited by G
(see (53) for review). Furthermore, 90% of the
5-HT-induced MAP kinase activation in LM5 cells is specifically blocked
by antibodies against either G
, -
, -
subunit or Ras (Fig. 2B), also suggesting that
the dissociation of the G
and -
as well as
Ras activation are involved in this stimulation. In contrast to other
receptor
systems(52, 54, 55, 56, 57) ,
the contribution of the phospholipase C-
stimulation to MAP kinase
activation seems only minor. Nevertheless, our results add 5-HT via the
5-HT2B receptors to the growing list of receptors, including
-adrenergic, bombesin, m1-5 acetylcholine
muscarinic, prostaglandin, and thrombin, which activate Ras by
G
(58, 59, 60) or MAP
kinases by G
and -
subunit(51, 52) .
The stimulation of the 5-HT2B
receptor not only triggers the MAP kinase cascade, but also activates
the LM5 cell division rate (Fig. 3A). This is confirmed
by the enhancement of [H]thymidine incorporation
in response to agonists (data not shown). Therefore, these data
demonstrate that 5-HT has a mitogenic effect on LM5 cells. This
mitogenic effect probably results from MAP kinase stimulation via Ras,
G
, and -
, as already shown in other systems
by use of the dominant negative form of Ras (58) or of
constitutively active G
(44, 61) . In
addition to short-term effects, MAP kinase activation may lead to
long-term effects, including cell transformation and/or
differentiation. LM5 cells grown to confluence are forming foci, an
effect which is stimulated by agonist and inhibited by antagonist (Fig. 3B). Surprisingly, the potency of antagonist is
greater with regard to the ability to inhibit foci formation than with
the rate of cell division (Fig. 3, A and B).
This difference may be related to different responses in different
physiological states (exponential growth versus quiescent) and
probably corresponds to short-term effects versus long-term
effects involving different effectors of the receptor. In addition, all
nude mice-derived tumor cells express more 5-HT2B binding sites than
LM5 cells. This indicates that a threshold level of receptor expression
(1 pmol/mg of protein) is probably necessary for transformation.
However, these events remain 5-HT and 5-HT2B receptor-dependent (Table 1). Although the contribution of Ras stimulation to the
long-term alterations is probable, since activated Ras is known to
transform NIH3T3 fibroblasts (62) and since expression of a
dominant negative form of Ras inhibits the muscarinic m5
receptor-dependent transformation(60) , additional transduction
mechanisms are probably involved. Nevertheless, expression of the
5-HT2B receptor triggers both the 5-HT-mediated mitogenicity and the
transformed phenotype of the LMTK
cells, indicating
that the 5-HT2B receptor behaves as a ligand-dependent proto-oncogene
for the LMTK
cell line.
Interestingly, the
unstimulated LM5 cells have a higher cell division rate than the
parental LMTK cells. This basal activity is partially
reduced in the presence of the antagonist ritanserin (Fig. 3A) (inverse agonist) and probably corresponds to
an intrinsic activity of the receptor which has also been shown for the
5-HT2C receptor(63) . Similarly, in unstimulated LM5 cells,
foci spontaneously appear and the basal level of Ras activity is
elevated suggesting intrinsic activity of the 5-HT2B receptor.
Therefore, intrinsic activity and a high level of expression may be two
important parameters for tumor development. Tumor regression in nude
mice induced by ritanserin treatment may open lines of investigation to
develop new types of therapeutic agents.
In several aspects, the
oncogenic properties of the 5-HT2B receptor seems to differ from those
described for the other 5-HT2 receptors: very high levels of 5-HT2A-2C
receptor expression are required in NIH3T3 cells to induce foci
formation (>10 pmol of receptor/mg of protein), cells derived from
nude mice tumors induced by these foci are 5-HT
independent(17, 18) , and expression of the 5-HT2C
receptor does not transform the CCL39 cell line(64) . Several
factors may be responsible for these differences. (i) Use of
5-HT-depleted serum (<1 nM 5-HT) in this report may be
important for the full activity of the receptor. (ii) The different
cell lines (LMTK, NIH3T3, or CCL39) may express
different levels of G-proteins (65) or different combinations
of G-protein subunits(66) , some of which may be crucial for
transformation; the
subunit has also been implicated
in G
-mediated Ras activation(51) . (iii) Despite
common coupling to phospholipase C-
, the ability to stimulate Ras
has not been reported for the 5-HT2A or 5-HT2C receptors. (iv) The
carboxyl-terminal region of the third intracellular loop, close to the
sixth transmembrane domain, contains important amino acids involved in
coupling to different G
subtypes of G-protein (67) and is not conserved among 5-HT2 receptors. This region
controls the G
-dependent mitogenicity of the m1-5
muscarinic receptors (65) and, when mutated, leads to
constitutive activation of the G
by the
-adrenergic receptor(68) .
Furthermore,
5-HT which is present early in embryonic development (12) participates in craniofacial (13) and
cardiovascular morphogenesis(14, 15) . Therefore, some
5-HT trophic functions during embryogenesis may be controlled by the
mitogenic and transforming properties of 5-HT2B receptor. Its embryonic
expression starts earlier than the 5-HT2A or 5-HT2C receptors (69) and is located in heart primordia and in neural fold
before neural tube closure(4, 16) . In addition, the
5-HT2B receptor, which is expressed by immortalized
teratocarcinoma-derived cells 1C11 before complete serotonergic
cAMP-induced differentiation()(21) , seems to have
an autocrine function.
Finally, the 5-HT2B receptor is
expressed in tumors from both human and M. natalensis species (Fig. 4, B and C). The Mastomys CT
expresses a 5-HT2B receptor very similar to the mouse receptor, ()and the human 5-HT2B receptor cDNA has been found in a
cDNA library made from mRNA of a human CT(16) . In addition,
the pharmacology of the Mastomys CT indicates a high level of
5-HT2B receptor expression.
Therefore, this report makes,
for the first time, a parallel between induced tumorigenicity by
expression of the 5-HT2B receptor in nontransformed fibroblasts and its
expression by spontaneous tumors, which are both coupled to Ras
activation (Fig. 5). However, the participation of the 5-HT2B
receptor in enterochromaffin cell malignant transformation remains to
be proven.