(Received for publication, October 21, 1996, and in revised form, March 17, 1997)
From the Centre de Recherches sur l'Endocrinologie Moléculaire et le Développement, CNRS, UPR 1511, 9, rue Jules Hetzel, 92 190 Meudon, France
The neurotransmitter serotonin mediates a wide
variety of peripheral and central physiological effects through the
binding to multiple receptor subtypes (Wilkinson, L. O., and Dourish, C. T. (1991) in Serotonin Receptor Subtypes: Basic and Clinical Aspects (Peroutka, S. J., ed) Vol. 15, pp.147-210, Wiley-Liss, New York). Among them, serotonin 5-HT2A receptors are known
to activate the phospholipase C- second messenger pathway (Peroutka, S. J. (1995) Trends Neurosci. 18, 68-69). We identified
and localized in rat skeletal muscle myoblasts a functional serotonin
5-HT2A receptor. This receptor was detected on the plasma
membrane, in myoblasts, and at the level of T-tubules in contracting
myotubes. Binding of serotonin to its receptor increases the expression of genes involved in myogenic differentiation. Unexpectedly, the 5-HT2A receptor is able to activate another signaling
pathway; it triggers a rapid and transient tyrosine phosphorylation of Jak2 kinase in response to serotonin. Jak2 auto-phosphorylation is
followed by the tyrosine phosphorylation of STAT3 (signal
transducers and activators of
transcription) and its translocation into the nucleus. We
also find that the 5-HT2A receptor and STAT3 co-precipitate with Jak2, indicating that they are physically associated. We conclude
that the serotonin 5-HT2A receptor identified in skeletal muscle myoblasts is able to activate the intracellular phosphorylation pathway used by cytokines. The presence of serotonin receptors in
T-tubules suggests a role for serotonin in excitation-contraction coupling and (or) an effect in skeletal muscle fiber repairing.
Serotonin (5-hydroxytryptamine, 5-HT)1
is a neurotransmitter that mediates diverse central and peripheral
physiological responses by interacting with multiple serotonin receptor
subtypes (1). The physiological effects of serotonin are mediated by at
least four families of 5-HT receptors that have been distinguished
pharmacologically, depending on the second messenger they are coupled
to. The first family, including 5-HT1 and 5-HT5
receptor subtypes, interacts negatively with adenylate cyclase, whereas
the second family (5-HT2) is coupled to the activation of
phospholipase C-/protein kinase C. The 5-HT3 receptor is
a ligand-gated ion channel, and the family including 5-HT4,
5-HT6, and 5-HT7 receptor subtypes activates adenylate cyclase (2). The receptors of the 5-HT2 subfamily are implicated in many central physiological functions of serotonin, such as neuronal sensitization to tactile stimuli and mediation of
hallucinogenic effects of lysergic acid diethylamide, as well as in
peripheral cardiovascular effects, e.g. contraction of blood vessels and shape change in platelets. The 5-HT2A (formerly
5-HT2) receptor shares an overall 49% sequence identity
with the 5-HT2C (formerly 5-HT1C) receptor, but
they present different patterns of expression in the brain (3).
Transfection of both 5-HT2C and 5-HT2A
receptors in NIH3T3 fibroblasts results in cellular transformation and
focus formation (3). The mouse 5-HT2B receptor, mainly
expressed in the cardiovascular system, gut, and developing brain (4),
shares the highest degree of homology with the other 5-HT2B
receptors cloned from the rat fundus or from human libraries (5). The
5-HT2B receptor has been classified as a
ligand-dependent proto-oncogene, since its expression is
necessary and sufficient to induce tumor formation in nude mice
(6).
Serotonin 5-HT2A receptors have been mainly localized in frontal cortex (3), blood platelets (7), and aortic smooth muscle cells (8). To date, serotonin receptors have never been isolated in skeletal muscle cells. The present study reports the identification and localization of a functional skeletal muscle serotonin 5-HT2A receptor in rat fetal myoblasts. This receptor mediates the serotonin-induced up-regulation of the transcription factor myogenin, and the neuronal glucose transporter isoform GLUT3, both expressed during myogenic differentiation. Furthermore, we show that the skeletal muscle serotonin 5-HT2A receptor is capable of stimulating the Jak/STAT pathway.
Primary cultures of myoblasts from 19-day-old rat fetuses were grown as described previously (9) with the following modifications. Myoblasts were separated from muscle fibers by a treatment (20 min, 37 °C) with 0.15% protease (Sigma) in Ham's F-12 medium containing 10% fetal calf serum. Myoblasts were plated at a density of 1.5 × 104 cells/ml on gelatin-coated flasks, in minimal essential medium/199 medium (2/1) containing 10% horse serum and 0.2% Matrigel (Becton-Dickinson). Serum and Matrigel were removed 24 h before the beginning of the experiments. Media were purchased from Life Technologies, Inc.; fetal calf serum and horse serum were obtained from Boehringer Mannheim.
Cloning of a cDNA Coding for a Skeletal Muscle 5-HT2A ReceptorUsing total RNA isolated from
3-day-cultured fetal myoblasts as a template, first strand cDNA was
synthesized by reverse transcription, using a cDNA synthesis kit
according to the manufacturer's instructions (Perkin Elmer). For
amplification of the cDNA fragments by the PCR, 21-mer
oligonucleotide primers were designed, based on a portion of the third
intracytoplasmic loop of the rat 5-HT2A receptor cDNA
(3) (GenBankTM accession no. M30705[GenBank]). Forward (sense) primer was 5-ACC
TAC TTC CTG ACT ATC AAG-3
(nucleotides 816-837). Reverse (antisense)
primer was 3
-GCC CAG CAC CTT GCA CGC CTT-5
(nucleotides 1016-1037).
PCR fragments were subcloned into the BamHI-EcoRI
sites of a Bluescript pBS-SK(+) plasmid (Promega), amplified in XL1
Blue, and sequenced.
Myoblasts were treated
with 5·106 M serotonin hydrochloride
(Sigma) in the presence of the 5-HT2A receptor antagonist
ketanserin (10
6 M) in some experiments
(Research Biochemicals Inc.) Total RNA was extracted from the cells by
the guanidine thiocyanate method (10), then electrophoresed and
transferred as described previously (9). Northern blots were hybridized
with 32P-labeled cDNA probes coding for GLUT3, GLUT1 (a
gift of Dr. G. Bell, Howard Hughes Medical Institute, University of
Chicago, Chicago, IL), myogenin (provided by Dr. W. Wright,
Southwestern Medical Center, University of Texas, Dallas, TX), and
serotonin 5-HT2A receptor (cDNA cloned by reverse
transcription-PCR).
Fetal myoblasts were grown on gelatin-coated Permanox four-chamber slides (Lab-Tek, Nunc) for 3 days (fusioning myoblasts) or 8 days (contracting myotubes) as described previously (9). The serotonin 5-HT2A receptor was detected using a monoclonal anti-5-HT2A receptor antibody (PharMingen). Cells were incubated overnight at 4 °C in diluted antibody (dilution 1/500 in PBS, 0.2% gelatin), then washed three times with PBS, and treated for 1 h with fluorescein isothiocyanate-conjugated sheep anti-mouse IgG (dilution 1/128, Sigma). The slides were mounted in a glycerol/PBS mounting medium (Cityfluor), and confocal laser scanning microscopy was performed, using a Leica confocal imaging system (TCS-4D) and an immersion lens (63×, numerical aperture 1.4 plan Apochromat). A focal series of up to 18 sections apart (0.5 µm between two adjacent sections) was collected for each specimen and then processed to produce single composite images (extended focus). Micrographs were printed directly from the computer on a dye sublimation printer (Colorease, Eastman Kodak Co.).
AntibodiesPolyclonal anti-mouse Jak2 and anti-human/mouse Jak2/protein A-agarose were purchased from Upstate Biotechnology Inc. Monoclonal anti-STATs antibodies and biotinylated recombinant anti-phosphotyrosine (RC 20-B) were purchased from Transduction Laboratories. Anti-rabbit and anti-mouse Ig-peroxidase-linked whole antibodies were obtained from Amersham.
Protein Phosphorylation and Co-precipitation AssaysMyoblasts were exposed to 5·106
M serotonin for 0, 1, 5, 30, or 60 min in serum-deprived
minimal essential medium/199 medium at 37 °C. Cells were scraped on
ice in phosphate-buffered saline, then lysed for 1 h at 4 °C
with agitation, in 500 µl of ice-cold lysis buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 50 mM NaF, 50 mM
Na3P2O7, 1% Triton X-100, 0.4 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg·ml
1
pepstatin A, 2 µg·ml
1 leupeptin, and 5 µg·ml
1 aprotinin). The cell lysates were centrifuged
at 15,000 rpm at 4 °C for 30 min, to remove insoluble material.
Immunoprecipitation was performed overnight at 4 °C using 10 µg of
anti-Jak2/protein A-agarose, or 5 µg of biotinylated
anti-phosphotyrosine antibody (RC 20-B). Then the immune complexes were
precipitated for 2 h at 4 °C by addition of protein A-Sepharose
(Pharmacia), for anti-Jak2 antibody, or streptavidin immobilized on
agarose (Pierce) for biotinylated anti-phosphotyrosine antibody. The
immunoprecipitates were washed in cold lysis buffer, boiled in 2 × Laemmli's buffer, and separated on a 7.5% polyacrylamide-SDS gel,
then transferred electrophoretically to nitrocellulose membranes. The
membranes were blocked overnight at 4 °C with 5% nonfat dry milk in
TBST buffer (10 mM Tris-HCl, pH 7.4, 75 mM
NaCl, 1 mM EDTA, 0.1% Tween 20), or 3% bovine serum
albumin in TBST buffer, for anti-phosphotyrosine antibody. Membranes
were incubated with the relevant primary antibody, washed, incubated
with peroxidase-conjugated secondary antibody, and washed again. Blots
were developed using the ECL chemiluminescence reagents (Amersham).
Myoblasts were exposed
to 5·106 M serotonin for 0, 1, 5, 15, 30, or 60 min. Nuclei were isolated according to the protocol described by
Shapiro et al. (11), with the following modifications. Cells
were washed with ice-cold phosphate-buffered saline, recovered by
scraping, then pelleted and resuspended in 300 µl of hypotonic buffer
(10 mM Hepes, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.15 mM spermine, 10 mM KCl, 1 mM
dithiothreitol, 0.5 mM PMSF, 0.5 µg·ml
1
leupeptin, 0.5 µg·ml
1 pepstatin A, 1% aprotinin).
Cells were broken by 3 strokes of the tight pestle of a Dounce
homogenizer, after addition of hypotonic buffer containing 0.6%
Nonidet P-40. Sucrose restore buffer (50 mM Hepes, pH 7.9, 0.2 mM EDTA, 10 mM KCl, 1 mM
dithiothreitol, 0.5 mM PMSF, 1 µg·ml
1
pepstatin A, 2 µg·ml
1 leupeptin, and 5 µg·ml
1 aprotinin, 67.5% (w/v) sucrose) was rapidly
added and the homogenate mixed with 2 strokes of the loose pestle of
the homogenizer. After centrifugation (1 min at 12,000 × g, 4 °C), the nuclear pellet was resuspended in 10%
glycerol nuclear resuspension buffer. The nuclear resuspension was
treated according to Shapiro. Nuclear proteins (40 µg) were
electrophoresed on a 7.5% polyacrylamide-SDS gel, transferred to
nitrocellulose, and immunoblotted as described for immunoprecipitation
assays.
Using total RNA isolated from 3-day-cultured myoblasts
as a template, the first strand cDNA was synthesized by reverse
transcription, as described under "Materials and Methods." Since
the homology within the transmembrane domains of 5-HT2
receptors is over 80%, we chose, for amplification of the cDNA
fragment by PCR, two oligonucleotide primers in a portion of the
nonconserved domain of the 5-HT2A receptor, the third
intracytoplasmic loop (3). Sequencing of the amplified 221-base pair
cDNA fragment presented 100% homology with nucleotide 816-1037 of
the rat 5-HT2A receptor cDNA (3). Although
poly(A)+ RNA from the cortex revealed two discrete
transcripts between 5 and 6 kb, corresponding to 5-HT2A
receptor mRNAs (3), this cDNA recognized in total RNA (40 µg)
from rat fetal myoblasts a major 6.0-kb and a minor 4.0-kb transcript
(Fig. 1B).
Time Course of Serotonin Effect on GLUT3, GLUT1, and Myogenin mRNA Expression
The neuronal isoform GLUT3 is the main
glucose transporter isoform expressed during fusion of myoblasts into
myotubes, e.g. between day 3 and day 4 of culture (9).
Preliminary dose-response experiments showed that 5·106
M serotonin induced a maximal increase in mRNA
expression, in 3-day-cultured myoblasts (data not shown). A time course
of serotonin effect was performed after treatment of 3-day-cultured
myoblasts with 5·10
6 M serotonin, for times
ranging from 1.5 to 24 h. In control cells, GLUT3 mRNA
expression increased transiently between day 3 and day 4, as expected
(9). Nevertheless, a 2-3-fold increase in GLUT3 mRNA level was
observed as soon as 1.5 h after addition of serotonin to the
culture medium. The effect, maximal after 3 h, decreased to reach
the control level after 9 h. A similar time course was observed on
myogenin mRNA expression, whereas the ubiquitous glucose
transporter isoform GLUT1 was quite unaffected (Fig.
1A).
To determine if serotonin-induced up-regulation of mRNAs was mediated by the 5-HT2A receptor, myoblasts were exposed to serotonin in the absence or presence of serotonin receptor antagonists, and Northern blots were hybridized with 5-HT2A receptor, GLUT3 and myogenin cDNA probes. Within 6 h, serotonin induced a 5-fold increase in 5-HT2A receptor mRNAs, and a 2-3-fold increase in GLUT3 and myogenin mRNAs (Fig. 1B). This effect was completely abolished in the presence of 1 µM ketanserin, a specific 5-HT2A receptor antagonist (Fig. 1B), whereas zacopride, a 5-HT3 inhibitor, was inefficient (data not shown). Taken together, these results show that serotonin is able to increase the expression of genes expressed during myogenic differentiation, through the skeletal muscle 5-HT2A receptor.
Cellular Localization of the 5-HT2A ReceptorFetal myoblasts and contracting myotubes were stained
for the serotonin 5-HT2A receptor by immunofluorescence,
and images were analyzed by confocal laser scanning microscopy. The
5-HT2A receptor was localized on the plasma membrane, in
fusioning myoblasts (Fig. 2a). Later on
during myogenic differentiation, 5-HT2A receptors appeared
at the level of deep invaginations of the plasma membranes, the
transverse tubules (T-tubules), in contracting myotubes (Fig. 2,
b-h). T-tubules are known to transmit the electrical
impulses that trigger muscular contraction from the cell surface to the sarcoplasmic reticulum, and to facilitate the distribution of substrates to the myofibers (12). The presence of serotonin receptors
in T-tubules of contracting myotubes suggests a role for serotonin in
excitation-contraction coupling, and (or) a trophic role for the
skeletal muscle fiber.
Co-precipitation of the 5-HT2A Receptor with Jak2 Kinase
Serotonin increased gene expression through a very rapid
transduction pathway, since a 1-min stimulation of myoblasts by
serotonin was sufficient to induce a 50% increase in GLUT3 mRNA
levels 3 h later (data not shown). The intracellular Jak/STAT
pathway transduces very quickly the signal of many cytokines and
peptide growth factors (13). Ligand binding rapidly triggers tyrosine
phosphorylation of STATs (14, 15) by receptor-associated tyrosine
kinases of the Jak family (16-18). To investigate whether serotonin
was able to stimulate the Jak/STAT pathway, myoblasts were incubated with serotonin for 0, 5, 15, 30, or 60 min, and the association of Jak2
with the 5-HT2A receptor was assessed. Cell lysates from serotonin-treated myoblasts were immunoprecipitated with anti-Jak2 antibody, and, after SDS-PAGE electrophoresis, immunoblotted with anti-5-HT2A receptor antibody. This antibody revealed in
rat brain, used as a positive control, a strong band at 53 kDa, and a
minor band at 58 kDa, probably due to post-translational modifications (glycosylation or phosphorylation) of the receptor. In myoblasts, a
major band corresponding to 55 kDa was detected. The association of
Jak2 with the 5-HT2A receptor was observed within 5 min
after addition of serotonin, and was maximal after 15 min (Fig.
3A).
Serotonin-induced Phosphorylation of Jak2 Kinase
To determine whether Jak2 auto-phosphorylation was induced by serotonin stimulation, Western blots of cell lysates were immunoblotted with anti-phosphotyrosine antibody, then stripped and reprobed with anti-Jak2 antibody (Fig. 3B, a). In a second protocol, cell lysates were immunoprecipitated with anti-Jak2 antibody, then immunoblotted with anti-phosphotyrosine antibody (Fig. 3B, b). Tyrosine phosphorylation of Jak2 was maximal after 30 min and then decreased until 60 min. These results show that the 5-HT2A receptor cloned in skeletal muscle myoblasts is functional and activates a pathway never described for serotonin.
Phosphorylation of STAT Protein(s) Involved in Serotonin-mediated Signal TransductionThe activation of Jak kinases leads to the
recruitment and phosphorylation of STATs proteins (19, 20). To
determine which STATs proteins were phosphorylated after serotonin
treatment, cell lysates were immunoprecipitated with
anti-phosphotyrosine antibody and then immunoblotted with monoclonal
antibodies raised against STAT proteins. STAT1 /
(p91/84), STAT3
(p92), and STAT6 (interleukin-4-STAT) were expressed in fetal myoblasts
(Fig. 4, A-C). A431 cells, stimulated for 5 min with epidermal growth factor (100 ng/ml), were used as a positive
control for STAT1 and STAT3, and mouse 3T3 cells as a positive control
for STAT6. Tyrosine phosphorylation of STAT3 occurred within 5 min of
exposure of myoblasts to serotonin, and was maximal after 30 min (Fig.
4B). No tyrosine phosphorylation of STAT1 (Fig.
4A) or STAT6 (Fig. 4C) was observed. In our
study, only STAT3 was tyrosine-phosphorylated in response to binding of
serotonin to the 5-HT2A receptor.
Association of Jak2, 5-HT2A Receptor, and STAT3
To determine whether STAT3 was associated with the complex
Jak2 kinase-5-HT2A receptor in response to serotonin
stimulation, the same blot as in Fig. 3A was stripped and
then immunoblotted with anti-STAT3 antibody. Serotonin induced the
co-precipitation of STAT3 with Jak2 within 5 min (Fig.
5A). The simultaneous co-precipitation of the
5-HT2A receptor and STAT3 with Jak2 suggests that STAT3 is
associated to the receptor complex, in response to serotonin stimulation.
Nuclear Translocation of STAT3
The activated STATs dimerize and translocate to the nucleus, where they directly activate target genes (13). To test whether serotonin induced the nuclear translocation of STAT3 in myoblasts, cells were exposed to serotonin for 0, 1, 5, 15, 30, or 60 min, and nuclear protein extracts were electrophoresed. Western blots were immunoblotted with anti-STAT antibodies. Serotonin induced the nuclear translocation of STAT3 within 5 min, the presence of STAT3 in the nucleus remaining at least 60 min (Fig. 5B). In contrast, no translocation of STAT1 or STAT6 was observed (data not shown). In fetal myoblasts, serotonin triggers the recruitment of STAT3 to the Jak2-5-HT2A receptor complex, followed by the tyrosine phosphorylation of STAT3 and its translocation into the nucleus.
In this study, we tried to elucidate the mechanisms by which serotonin increased the expression of genes in relation with myogenic differentiation. We have shown that the effects of serotonin on skeletal muscle cells are mediated by a receptor of the 5-HT2 receptor subfamily, namely the 5-HT2A receptor. This receptor is located at the level of T-tubules in contracting myotubes.
The junctional T-tubules exert a dual role in muscle fibers, since they are involved both in facilitating substrate uptake, such as glucose (12), and excitation-contraction coupling. The presence of serotonin receptors in T-tubules underlies a trophic effect for serotonin, in skeletal muscle fibers, since serotonin increases glucose transport rate2 and GLUT3 glucose transporter expression in fetal myoblasts. Trophic effects of serotonin have already been described. Serotonin, detected early in embryonic development (21), plays a role in cranofacial (22) and cardiovascular morphogenesis (23, 24) by unknown molecular mechanisms. Some trophic functions of serotonin during embryogenesis have been related to the mitogenic and transforming properties of the 5-HT2B receptor (6). Nevertheless, the hypothesis that serotonin could play a role in excitation-contraction coupling must be considered.
The serotonin 5-HT2A receptor is a member of the G
protein-linked receptor superfamily known to activate the phospholipase C-/protein kinase C pathway (2). In fetal myoblasts, serotonin binding to the 5-HT2A receptor resulted in the stimulation
of another signaling pathway, the Jak/STAT pathway. The
serotonin-induced association of Jak2 to the 5-HT2A
receptor allowed the recruitment of STAT3 to the receptor complex and
its subsequent phosphorylation by the phosphorylated Jak2 kinase, and
led to STAT3 nuclear translocation. A similar result has been reported
in human interleukin-6-treated NJBC-T cells, where the rapid tyrosine
phosphorylation of STAT3 correlated with the rapid nuclear
translocation of STAT3, and the formation of STAT3-STAT3-DNA complexes
(25). STAT3 was reported to increase the expression of genes that are
expressed in response to tissue injury and inflammation, and are
referred as acute phase response genes (26). Some fetal myoblasts
persist in adult skeletal muscle as quiescent stem cells (muscle
satellite cells) that are capable of differentiation to repair muscle
fibers, in case of injury. In myoblasts, the nuclear translocation of
STAT3, in response to serotonin, might be related to the up-regulation
of myogenin, a transcription factor involved in myogenic
differentiation. This point requires further studies.
The serotonin 5-HT2A receptor we cloned in skeletal muscle is the second member of the seven-transmembrane domain G protein-coupled receptor superfamily able to activate the Jak/STAT pathway, after the angiotensin AT1 receptor, in rat aortic smooth muscle cells (27, 28). It is noteworthy that contractions of rat aortic smooth muscle cells are stimulated by serotonin through 5-HT2A receptors. It could be of great interest to determine if serotonin stimulates also the Jak/STAT pathway in these cells. Recently, a mouse 5-HT2B receptor transfected in fibroblasts was reported to induce serotonin stimulation of mitogen-activated protein kinase cascade (ERK2/ERK1) through the Ras pathway (6). Furthermore, serine/threonine phosphorylation mediated by the mitogen-activated protein kinases was shown to play a role in the activation of STATs, linking STAT and Ras pathways (25, 29).
In conclusion, our results show that the Jak/STAT pathway is implicated in the serotonin-induced increase in mRNAs coding for proteins involved in myogenic differentiation, suggesting that this pathway may play a role in skeletal muscle ontogenesis, and (or) repairing. This novel localization and signal transduction for serotonin 5-HT2A receptors deserve further studies, in particular a search for implication of serotonin in excitation-contraction coupling.
We thank Dr. M. Hamon for the gift of oligonucleotides and the Imagery Service of Jacques Monod Institute (CNRS) for confocal laser scanning microscopy.