Molecular Medicine
Rho Kinase–Induced Nuclear Translocation of ERK1/ERK2
in Smooth Muscle Cell Mitogenesis Caused by Serotonin
Yinglin Liu, Yuichiro J. Suzuki, Regina M. Day, Barry L. Fanburg
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Abstract—There is now considerable evidence supporting a mitogenic action of serotonin (5-HT) on vascular smooth
muscle cells (SMC) that might participate in pulmonary hypertension (PH). Our previous studies have demonstrated that
5-HT–induced proliferation depends on the generation of reactive oxygen species and activation of extracellular
signal-regulated kinase (ERK) 1/ERK2. Activation of Rho kinase (ROCK) in SMC also may be important in PH. We
undertook the present study to assess the role of Rho A/ROCK and its possible relation to ERK1/ERK2 in 5-HT–induced
pulmonary artery SMC proliferation. We found that this stimulation of SMC proliferation requires Rho A/ROCK as
inhibition with Y27632, a ROCK inhibitor, or dominant negative (DN) mutant Rho A blocks 5-HT–induced
proliferation, cyclin D1 expression, phosphorylation of Elk, and the DNA binding of transcription factors, Egr-1 and
GATA-4. 5-HT activated ROCK, and the activation was blocked by GR 55562 and GR127935, 5-HT 1B/1D receptor
antagonists, but not by serotonin transport (SERT) inhibitors. Activation of Rho kinase by 5-HT was independent of
activation of ERK1/ERK2, and 5-HT activated ERK1/ERK2 independently of ROCK. Treatment of SMC with Y27632
and expression of DNRho A in cells blocked translocation of ERK1/ERK2 to the cellular nucleus. Depolymerization of
actin with cytochalasin D (CD) and latrunculin B (latB) failed to block the translocation of ERK, suggesting that the
actin cytoskeleton does not participate in the translocation. The studies show for the first time to our knowledge
combinational action of SERT and a 5-HT receptor in SMC growth and Rho A/ROCK participation in 5-HT receptor
1B/1D-mediated mitogenesis of vascular SMCs through an effect on cytoplasmic to nuclear translocation
of ERK1/ERK2. (Circ Res. 2004;95:579-586.)
Key Words: smooth muscle cells 䡲 serotonin 䡲 Rho kinase 䡲 ERK1/ERK2 䡲 pulmonary hypertension
I
n addition to its actions as a vasoconstrictor and neurotransmitter, serotonin (5-HT) is now recognized to be a
cellular mitogen.1–3 There is evidence that this mitogenic
action is initiated by active transport via a cell surface
transporter (SERT) of bovine, rat, and human pulmonary
vascular smooth muscle cells (SMC).1,4 – 6 For other cells, the
mitogenic action might be started through 1 or more of the
cell surface receptors for 5-HT. A hierarchy of cell signaling
responses occurs subsequent to ligation of the cell surface
transporter or receptor. It has been well-established that these
signaling responses include sequential activations of the
small GTPase coupled protein, Rac-1, NADPH oxidase
producing superoxide that is dismutated to H2O2, and extracellular signal-regulated kinase (ERK) 1/ERK2 MAP
kinase.2,7–9
The small GTPase Rho A and its effector, Rho kinase
(ROCK), also participate in cellular stress fiber formation and
cell cycle progression.10 –19 There has been limited study of
the relationship of Rho A and ROCK to 5-HT. One study
showed that Rho A bound to GTP is elevated in the rabbit
aortic vascular ring preparation treated with 5-HT.20 Another
study suggested the activation of Rho A and ROCK in
5-HT–induced contraction of the bovine middle cerebral
artery.21 Serotonin participates in pulmonary hypertension,5,22,23 and a polymorphism of the 5-HT transporter has
been proposed to be involved in pulmonary hypertension in
humans.24 Because agents that block ROCK are currently
available25,26 and may be useful in pulmonary hypertension,27–29 we have undertaken an investigation of the potential
participation of ROCK in pulmonary arterial SMC signaling
and proliferation produced by 5-HT. The results of our study
show that ROCK is activated by 5-HT and that its activation
is essential for SMC proliferation produced by 5-HT. Furthermore, with the use of a chemical inhibitor of ROCK and
a dominant negative mutant of Rho A, we found that neither
the activation of ROCK nor that of ERK1/ERK2 by 5-HT is
dependent on the other, but rather both pathways are required
to produce cellular proliferation. Both upregulate cyclin D1,
activate Elk, and cause DNA binding of Egr-1 and GATA-4,
transcription factors that participate in the mitogenic action of
5-HT on SMC. ROCK activation appears to depend on
ligation of 5-HT 1B/1D rather than SERT. Importantly,
ROCK participates in proliferation by causing translocation
of activated ERK1/ERK2 to the cellular nucleus. To our
Original received February 4, 2004; revision received July 7, 2004; accepted July 26, 2004.
From the Tufts-New England Medical Center, Pulmonary, Critical Care and Sleep Division, Tupper Research Institute, Boston, Mass.
Correspondence to Barry L. Fanburg, MD, Pulmonary, Critical Care and Sleep Division, Tufts-New England Medical Center, 750 Washington St, #257,
Boston, MA 02111. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000141428.53262.a4
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September 17, 2004
knowledge, this is the first demonstration that the translocation of ERK to the nucleus is dependent on ROCK for any
mitogenic event.
Materials and Methods
SMCs from bovine pulmonary artery were cultured in RPMI-1640
medium containing 10% fetal bovine serum and antibiotics. Thymidine uptake was determined with growth-arrested SMC treated with
1 ␮mol/L 5-HT, and supernatants of cell lysates were obtained for
analysis. DN Rho A cDNA gene insertion was performed with
adenoviral infections. Expressions of cyclin D1, Rho A, Erg-1, ERK,
p-ERK, p-Elk, p MYPT1, and MYPT1 were analyzed with Western
blots. Electrophoretic mobility shift assays were used to assess Egr-1
and GATA4. Immunochemistry was performed to localize intracellular ERK. F-actin stress fibers were visualized with a Zeiss
fluorescent microscopy. Specific details regarding methodology and
sources of materials used can be found in the online data supplement
available at http://circres.ahajournals.org.
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Results
5-HT Activates ROCK
We examined the effects of 5-HT on MYPT1, a myosin
phosphatase binding subunit, which is inactivated by phosphorylation by ROCK.25,30 –32 As shown in Figure 1A, 5-HT
(1 ␮mol/L) caused a transient phosphorylation of MYPT1,
with a peak in 10 to 15 minutes. The effect of lysophosphosphatidic acid, a known activator of ROCK, is shown for
comparison.33–35 Inactivation of MYPT1 by 5-HT was inhibited by the ROCK inhibitor, Y27632, in a dose-dependent
manner (Figure 1B). Bovine pulmonary artery SMC contain
both SERT and a variety of 5-HT receptors, including the
5-HT1B/1D receptor as determined by polymerase chain
reaction (unpublished data). We examined the influence of
SERT and 5-HT receptor inhibitors on ROCK activity.
Although inhibition of SERT had no influence on ROCK
activation, GR55562 and GR127935, known 5-HT1B/1D
receptor antagonists, blocked activation of ROCK by 5-HT
(online Figure IA, IB), and indicated that ROCK activation
by 5-HT occurred via action on this receptor as opposed to
SERT.
Figure 1. Stimulation of Rho kinase (ROCK) activity by 5-HT. A,
Activation by 5-HT of ROCK in growth-arrested bovine pulmonary arterial smooth muscle cells. B, Inhibition of ROCK activity
by ROCK inhibitor Y27632. Phosphorylation of MYPT1 (Thr696)
was determined by Western blot analysis using a phosphospecific antibody. **Significant difference from the untreated
control value at P⬍0.05. *Significant difference from 5-HT alone
control value P⬍0.05.
Role of ROCK in 5-HT–Induced Cell Growth
Treatment of cells with 5-HT caused an increase in thymidine
incorporation that was inhibited in a dose-dependent manner
by ROCK inhibitor Y27632 (Figure 2A). Similarly, 5-HT–
mediated cyclin D1 expression was inhibited by pretreatment
of cells with Y27632 (Figure 2B) and dominant negative Rho
A (Figure 2C).
To further determine the role of ROCK in cell growth
signaling mediated by 5-HT, we examined the effects of
Y27632 on the activation of transcription factors that control
cell mitogenesis. The E twenty-six domain transcription
factor Elk-1 is a direct target of the MAP kinase pathway.36
Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation.37–39 Elk participates in responses to ERK1/ERK2 in cellular proliferation.40
We found that treatment with 5-HT caused phosphorylation
of Elk-1 of SMC (online Figure IIA) and that the activation is
inhibited by Y27632 and U0126, inhibitors of ROCK and
MEK, respectively (online Figure IIB).
Egr-1 is a well-known immediate early response gene,
which has been shown to regulate cellular proliferation in
response to various growth factors.41– 43 We found that
treatment of SMC with 5-HT–induced transient Egr-1 activation as monitored by electrophoretic mobility shift assays
(online Figure IIC). The Egr-1 activation by 5-HT requires
the activation of both ERK and ROCK, because the induction
of Egr-1 activity was blocked by pretreatment of cells with
Y27632 or U0126 (online Figure IID).
We recently reported that transcription factor GATA-4,
another of the downstream effectors of ERK, plays a critical
role in 5-HT signaling for SMC mitogenesis.44 Thus, we
examined the effect of Y27632 on GATA-4 activity. We
found that although Y27632 had no effect on basal levels of
GATA-4 activity, this ROCK inhibitor blocked 5-HT–induced upregulation of GATA-4 (online Figure IIE). Taken
together, these results indicate that the Rho A/ROCK path-
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Figure 3. Activation of ERK1/ERK2 by 5-HT is independent of
ROCK activation. A, SMC were preincubated with 20 ␮mol/L
Y27632 or 10 ␮mol/L U0126 for 30 minutes, or infected with 10
pfu/live cell DNRho A for 48 hours, then treated with 1 ␮mol/L
5-HT for 5 minutes. Phosphorylation of ERK was determined by
Western blot analysis of the whole cell lysate using the
phospho-specific antibody. B, Densitometry measurements are
for the 44-kDa band. Bar graphs represent mean and vertical
bars represent the SD for n⫽4.
way plays a role in mitogenic signaling induced by 5-HT in
pulmonary artery SMC.
Interactions Between ERK and ROCK Pathways
Figure 2. 5-HT–induced smooth muscle cell proliferation and
cyclin D1 expression are Rho A/ROCK-dependent. A, smooth
muscle cells (SMC) were pretreated with Y27632 for 30 minutes and then incubated with 1 ␮mol/L 5-HT for 24 hours.
DNA synthesis was determined by monitoring [3H]thymidine
incorporation (n⫽8). B, Growth-arrested SMC were preincubated with 20 ␮mol/L Y27632 for 30 minutes. C, Infected
with DNRhoA (10 pfu/live cell) for 48 hours, then treated with
1 ␮mol/L 5-HT for 6 hours. The expression of cyclin D1 was
determined by Western blot analysis of the whole cell lysate.
**Significant difference from the untreated control value at
P⬍0.05. *Significant difference from 5-HT alone control value
P⬍0.05.
ERK has been shown to play an important role in 5-HT–
induced mitogenesis of SMC.1,7 As noted, inhibition of
ROCK diminished both cell proliferation and ERKdependent transcription factor activation induced by 5-HT in
SMC. We therefore examined the effect of the ROCK/Rho A
inhibitor Y27632 on 5-HT–induced ERK activation. Treatment of cells with 5-HT caused transient phosphorylation of
ERK1/2 (online Figure IIIA). This increase in ERK phosphorylation by 5-HT (Figure 3A, lane 3) was not influenced
by Y27632 (lane 4), whereas U0126, a MEK inhibitor,
completely inhibited the activation (lane 12). Similarly,
5-HT–induced ERK activation was not influenced by an
adenovirus expressing DNRho A (lane 8). These results
indicate that the Rho A/ROCK pathway is not upstream of the
MEK/ERK pathway in the 5-HT–induced signal transduction.
Densitometry of these results with and without 5-HT is
shown in Figure 3B.
To determine whether the MEK/ERK pathway might be
upstream to ROCK activation, we examined the effect of
U0126 on ROCK activation. U0126 effectively inhibited
5-HT–induced thymidine incorporation and upregulated cyclin D1 expression (online Figure IIIB, IIIC). However, as
shown in Figures 4 and 5, phosphorylation of MYPT1, a
ROCK target (lane 2), was not influenced by U0126 (lane 5).
Thus, although both the MEK/ERK and Rho A/ROCK
pathways are essential for 5-HT–induced proliferation of
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minutes of 5-HT treatment, persisted for 10 to 20 minutes,
and completely ceased by 40 minutes. Almost all of the cells,
which were treated with 5-HT for 5 minutes, showed that
ERK translocated to the nucleus (Figure 5C, third panel;
Figure 5D, second panel). This dramatic translocation of
ERK by 5-HT was not observed when cells were pretreated
with U0126, DNRho A, or Y27632 (Figure 5D). These results
demonstrate that ROCK regulates nuclear translocation of
ERK in the 5-HT signaling mechanism for SMC mitogenesis.
To assess a possible role of the cellular cytoskeleton in the
translocation of ERK1/ERK2 from the cytoplasm to the
nucleus under the regulation of ROCK, we examined the
5-HT–induced translocation in the presence of cytochalasin D
and latrunculin B, agents that depolymerize actin. Both
cytochalasin D and latrunculin B blocked cellular proliferation but failed to influence ERK phosphorylation (online
Figure IVA, IVB) or translocation of ERK1/ERK2 to the
cellular nucleus (online Figure IVC).
Discussion
Figure 4. 5-HT activation of ROCK does not require activation
of ERK1/ERK2. SMC were pretreated with 10 ␮mol/L U0126 for
30 minutes, vehicle control groups were pretreated with 0.1%
DMSO, and then SMC were stimulated with 1 ␮mol/L 5-HT for
15 minutes. The phosphorylation of MYPT1 (Thr696) was determined by Western blot with whole cell lysates. **Significant difference from the untreated control value at P⬍0.05.
SMC, Rho A/ROCK does not participate in the phosphorylation and activation of MEK/ERK and MEK/ERK does not
activate Rho A/ROCK.
ROCK Regulates ERK Translocation to
the Nucleus
Activation of ERK occurs in the cytoplasm, but to exert many
of its actions ERK must translocate into the nucleus. ERK has
been shown to activate various transcription factors by
translocating to the nucleus after being phosphorylated and
activated in the cytosol.45,46 To further determine the signaling role of Rho A/ROCK in 5-HT–induced mitogenesis of
SMC, we examined the effect of Y27632 on nuclear translocation of ERK induced by 5-HT. Treatment of SMC with
5-HT increased nuclear translocation of ERK as determined
by Western blot analysis of nuclear-rich cellular fractions. As
shown in Figure 5A, ERK nuclear translocation occurred
transiently with a peak at 5 minutes, consistent with its
phosphorylation. This induction of ERK nuclear translocation
by 5-HT was completely abrogated by pretreatment of cells
with Y27632 (Figure 5B). U0126, which blocks ERK activation, also caused a similar inhibition of the translocation to
the nucleus.
To confirm the experiments using nuclear-rich fractions,
we performed immunohistochemistry using ERK antibody to
directly visualize the ERK nuclear translocation. As shown in
Figure 5C, in untreated cells, much of the ERK signal was
dispersed in the cytosolic space around the nucleus. Treatment of cells with 5-HT caused the ERK protein to move into
the nucleus. This could be visualized as early as after 2
Serotonin produces pulmonary vascular SMC proliferation in
culture.2,3,6 Although some studies implicate actions on 5-HT
receptors in the proliferative process,47–50 others support a
hypothesis that the mitogenic action of 5-HT on vascular
SMC is associated with the 5-HT transporter (SERT).1 We
have performed extensive work demonstrating the participation of SERT in 5-HT–induced proliferation of bovine pulmonary artery SMC.2,3,8,9 5-HT activates a NADPH oxidase
to produce superoxide that rapidly dismutates to H2O2. This
H2O2 is responsible for activating the MAP kinase, ERK1/
ERK2.7,8 Although the precise mechanism by which this
occurs is not known, it may involve oxidation and inactivation of a relevant MAPK phosphatase.51–53 Activation of ERK
is responsible for downstream actions that increase DNA
binding of transcription factors, such as GATA-4,44 Egr-1,
and Elk-1, and enhance expression of factors important in cell
cycling, such as cyclin D1. SERT is important in the
production of hypoxia-induced pulmonary hypertension in
experimental animals4,54,55 and may participate in various
forms of pulmonary hypertension in humans.24
Rho A is a member of the Ras superfamily of GTP-binding
proteins. It cycles between a GDP-bound inactive state and a
GTP-bound active state, and has been found to participate in
cell growth, cell transformation, and change in shape and
actin organization.11,13–15,56 –59 The best-known downstream
effector of Rho A in SMC is ROCK. ROCK phosphorylates
a 130-kDa myosin phosphatase targeting (MYPT) subunit,
also known as myosin binding subunit (MBS), and concurrently inactivates a phosphatase.60,27 Rho A/ROCK has been
implicated in hypoxia-induced and monocrotaline-induced
pulmonary hypertension in experimental animal models.28,29,61 Rho A and ROCK have been studied in plateletderived growth factor-BB–induced proliferation of systemic
vascular SMC.62 Despite the recognition that Rho A/Rho
kinase participates in pulmonary hypertension, its role in
SMC signaling processes that may contribute to the hypertension (such as SMC proliferation produced by 5-HT) has
never been examined.
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We have found that SMC proliferation, cyclin D1 expression, Elk activation, and DNA binding of the transcription
factors, GATA-4 and Egr-1, are all dependent on activation
of ROCK by 5-HT. Activation of ERK1/ERK2 MAPK by
5-HT is independent of activation of ROCK and, conversely,
activation of ROCK by 5-HT is independent of activation of
ERK1/ERK2. Of interest, our studies with SERT and 5-HT
receptor inhibitors indicate that 5-HT 1B/1D receptors, but
not SERT, may be responsible for the activation of ROCK by
5-HT. These observations raise the interesting question of
whether SERT and a 5-HT receptor produce a combinatorial
action in response to 5-HT to result in a mitogenic effect. This
hypothesis is consistent with a requirement of the 5-HT1B
receptor for development of hypoxia-induced pulmonary
hypertension63 and recent studies suggesting interactions
between SERT and 5-HT receptors.64
We initiated an examination of mechanisms by which Rho
A/Rho kinase participates in the proliferative process produced by 5-HT. Although it has been recognized that growth
factors promote rapid nuclear translocation of ERK1/
ERK2,46,65 and that cellular actions of MAPK on gene
regulation require its entry into the nucleus, little is known
about the specific process by which this occurs. We have
found that translocation of ERK1/ERK2 to the nucleus with
5-HT stimulation requires the action of Rho A/Rho kinase,
although its activation does not. ROCK is known to participate in cell adhesion and assembly of cellular stress fibers,66
whereby altered nucleocytoplasmic trafficking of signaling
molecules may occur.67 Furthermore, a recent report showed
that cellular stretch and actin cytoskeleton configuration
participate in ERK translocation via Rho A activation in
cellular caveolae.68 Therefore, we undertook experimentation
to determine whether the actin cytoskeleton might participate
in the translocation of ERK we have observed. Although
disruption of the cytoskeleton with cytochalasin D and
latrunculin B blocked 5-HT–induced cell proliferation, ERK
phosphorylation and translocation of ERK was unaffected by
this treatment, indicating that translocation of ERK in our
cells does not depend on an intact actin cytoskeleton.
Activation by 5-HT of the transcription factor Elk-1 is
ROCK-dependent and ERK-dependent, providing supportive
evidence that movement of ERK into the nucleus requires
Figure 5. Effect of Rho A/ROCK on 5-HT-induced translocation
of ERK1/2 into nucleus. Western blot of nuclear ERK in 5-HT–
induced ERK translocation. A, Growth-arrested SMC were stimulated with 1 ␮mol/L 5-HT for the indicated time course. B,
SMC were pretreated with 20 mol/L Y27632 or 10 ␮mol/L
U0126 for 30 minutes, respectively, then incubated with
1 ␮mol/L 5-HT for 5 minutes. The level of ERK1/2 in the nucleus
was determined by Western blot analysis using the ERK antibody in the cellular nuclear extracts. ERK translocation induced
by 5-HT was detected by immunohistochemistry staining in
SMC. C, SMC were treated with 5-HT for the indicated time
course. D, Cells were pretreated with 10 ␮mol//L U 0126 or
20 ␮mol/ Y27632 for 30 minutes, or infected with DNRho A for
48 hours, then stimulated with 5-HT for 5 minutes. Localization
of ERK protein was visualized using ERK antibody and subsequent staining with FITC-conjugated goat antirabbit IgG antibody. **Significant difference from the untreated control value at
P⬍0.05. *Significant difference from 5-HT alone control value
P⬍0.05.
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September 17, 2004
to the cellular nucleus. A diagram showing this hypothesis is
presented in Figure 6. A polymerized actin cytoskeleton does
not appear to participate in this translocation. The activation
of ROCK by 5-HT is initiated through a 5-HT1B/1D receptor,
suggesting a concerted action of SERT and 5-HT receptor(s)
in cellular proliferation that is in need of further investigation.
Acknowledgments
This work was supported by NIH/NHLBI R01 grants HL32723
(B.L.F.), HL67340 (Y.J.S.), and HL72844 (Y.J.S.). R.M.D. is the
recipient of an American Heart Association National Center Scientist
Development Award.
References
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Figure 6. 5-HT–induced pulmonary vascular SMC proliferation
requires cooperation between ERK and ROCK pathways. ERK
activation occurs through 5-HT uptake by 5-HT transporter and
subsequent NADPH oxidase activation by which increased
superoxide anions stimulate ERK phosphorylation. ROCK activation occurs through 5-HT1B/1D receptor activation. Activated
ROCK facilitates phospho-ERK translocation into nucleus and
subsequent activation of nuclear transcription factors and cyclin
D1 expression. There also may be direct actions of the ROCK
pathway on transcription factors.
ROCK activation. As a direct target of the MAP kinase
pathways, transcription factor Elk-1 is known to be coupled
to ERK entry into the nucleus.69 Our studies show that in
nuclei isolated from Y27632-pretreated SMC, the activating
phosphorylation of Elk-1, which is catalyzed by ERK, was
strikingly diminished (online Figure IIA, IIB) and correlated
with a decrease in the abundance of activated ERK in the
nuclei (Figure 5). In contrast, Y27632 did not alter phosphorylation of ERK in the total cell lysate. Our data suggest that
impaired translocation of activated ERK to the nucleus by
inhibition of ROCK is responsible for the decreased Elk-1
phosphorylation in SMC.
This is the first time to our knowledge that nuclear
translocation of ERK has been reported to depend on Rho
A/Rho kinase for 5-HT. There has been a report suggesting
that ROCK-mediated signaling may be involved in ERKmediated p21Cip/waf1 induction in PMA-induced proapoptotic
TF-1 and D2 cells.70 However, unlike the results of our
studies, they report that upregulated ROCK signal interfered
with nuclear translocation of ERK. The process we observed
may be similar to that reported for cellular cytoplasm to
nucleus translocation of serum response factor produced by
exposure of tracheal SMC to serum,71 which also requires
Rho A/ROCK for the translocation. Also, it has been proposed previously that apoprotein D inhibits platelet-derived
growth factor-BB–induced vascular SMC proliferation by
prevention of phosphorylated ERK1/ERK2 movement to the
nucleus.72
From these studies, we hypothesize that Rho A and ROCK
play an important role in SMC proliferation produced by
5-HT through an influence on translocation of ERK1/ERK2
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Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Rho Kinase−Induced Nuclear Translocation of ERK1/ERK2 in Smooth Muscle Cell
Mitogenesis Caused by Serotonin
Yinglin Liu, Yuichiro J. Suzuki, Regina M. Day and Barry L. Fanburg
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 2004;95:579-586; originally published online August 5, 2004;
doi: 10.1161/01.RES.0000141428.53262.a4
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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CIRCRESAHA/2004/078923/R2 1
Activation of Rho kinase by serotonin Online supplement
ONLINE SUPPLEMENT
Material and Methods
Reagents
RPMI 1640 medium was purchased from GIBCO Laboratories (Grand Island, NY). Fetal
bovine serum (FBS), 5-HT, U0126, Citalopram, Imipramine, GR55562 and GR127935
were purchased from Sigma Chemical Co. (St. Louis, MO). Y27632, Cytochalasin D and
Latrunculin B were from Calbiochem Inc. (La Jolla, CA). Phospho-specific p42/44 MAP
kinase (Thr202/Tyr204) antibody and p42/44 MAP kinase antibody were from New
England Biolab (Beverly, MA). Anti-phospho-MYPT1(Thr696) rabbit polyclonal
antibody was purchased from Upstate Group, Inc. (Lake Placid, NY). Anti-MYPT-1,
anti-Egr-1, anti-Rho A, anti-Elk and anti-cyclin D1 rabbit polyclonal antibodies were
from Santa Cruz Biotech (San Diego, CA). Phospho-Elk-1 (ser383) rabbit polyclonal
antibody was from Cell Signaling Technology (Beverly, MA). [Methyl-3H]thymidine
(1mCi/ml, specific activity 6.7 Ci/mmol) was from New England Nuclear Corp. (Boston,
MA). Unifilter-96-well microplates were purchased from Perkin Elmer Life Sciences
(Boston, MA). Egr-1 and GATA-4 gel shift consensus oligonucleotides were from Santa
Cruz Biotech (Santa Cruz, CA). Adeno-X -null adenovirus was from Clontech (Palo
Alto, CA). Dominant negative mutant Rho A T19N adenovirus was a gift from Dr.
Tetsuaki Hirase (Kobe University Graduate School of Medicine, Kobe, Japan).
Cell culture
SMCs from bovine pulmonary artery were isolated by a modification of the method of
Ross as previously described 1 and were cultured in RPMI-1640 medium containing 10%
CIRCRESAHA/2004/078923/R2 2
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FBS, 1% penicillin/streptomycin and 0.5% amphotericin B. Cells from passage 3 to 10
were used in our study.
Incorporation of [3H]thymidine
SMCs seeded in 96-well plates were growth-arrested for 72 h in medium containing 0.1%
FBS. Cells were incubated with and without 1µM 5-HT in the same medium for 20 h
before being labeled with [methyl-3H]thymidine (20µCi/ml) for 4 h. In some
experiments, inhibitors were added 30 min before the 5-HT, 0.1%DMSO was added to
the vehicle control group. After labeling, experiments were terminated by aspiration of
medium and the cells were harvested into Unifilter-96-well microplates with a Parkard
harvester. Radioactivity was countered in a liquid microplate scintillation counter (Top
CountTM from PACKARD Instrument Company).
Preparation of whole cell lysates
The treated SMCs were rinsed with ice cold PBS, and then incubated for 15 min at 4°C
RIPA lysis buffer (50mmol/L Tris/HCl, pH 7.5, 150mmol/L NaCl, 2mmol/L EDTA, 1%
NP-40, 0.1% SDS, 0.5% sodium desoxycholate, 0.5mmol NaF, 0.01M Na3PO4, 2mmol/L
PMSF, 10µg/ml leupeptin, 20µg/ml aprotinin, 0.1mmol/L Na3VO4). Lysates were
centrifuged at 14,000×g for 10 min to collect supernatants.
Adenovirus-mediated gene transfer
Adenovirus-mediated gene transfer was implemented by adding to cells 10 plaqueforming units (pfu) of recombinant adenovirus. SMCs in 35mm dishes were infected
CIRCRESAHA/2004/078923/R2 3
Activation of Rho kinase by serotonin Online supplement
with 1ml of the serum-free medium containing the dominant negative (DN) Rho A
adenovirus for 2 h, control cells were infected with adeno-X -null adenovirus without
DNRho A cDNA insert (ad virus cont) and maintained for 48 h before performing
experiments2.
Preparation of cell nuclear extract
The growth-arrested SMCs were treated with 5-HT for indicated times and the nuclear
extracts from cells were prepared as previously described 3.
Western-blot analysis
Expression of cyclin D1, RhoA, Egr-1, ERK, p-ERK, p-Elk, Elk, p-MYPT1, MYPT1
were analyzed using rabbit polyclonal anti-cyclin D1, anti-RhoA, anti-Egr-1, anti-ERK,
anti-Elk-1, anti-MYPT1, anti-phospho-ERK1/2, anti- phospho-Elk-1(Ser383) and antiphospho-MYPT1(Thr696) antibodies, respectively. Immunoreactive bands were
visualized using horseradish peroxidase-conjugated secondary antibodies and subsequent
ECL detection.
Electrophoretic mobility shift assays (EMSA)
For EMSA for Egr-1, the binding reactions were performed at 25°C for 20 min in
5mmol/L Tris-HCl (pH 7.5), 37.5 mmol/L KCl, 2mmol/L EDTA, 20% glycerol, 1µg
poly(dI-dC)⋅poly(dI-dC), 32P-labeled double stranded oligonucleotide and 10µg protein
of nuclear extract. The double stranded oligonucleotide containing two Egr-1 consensus
elements (5’-GATCCAGCGGGGAGCGGGGGCGA-3’) was used. To perform EMSA
CIRCRESAHA/2004/078923/R2 4
Activation of Rho kinase by serotonin Online supplement
for GATA-4, binding reaction mixtures containing 10µg of protein of nuclear extract,
1µg poly(dI-dC)⋅ poly(dI-dC), 32P-labeled double stranded oligonucleotide probe
containing consensus GATA sequence (5’-ACATGATAACAGAAAGAGATAACTCT3’) in 100mmol/L NaCl, 1mmol/L EDTA, 1mmol/L dithiothreitol, 10% (v/v) glycerol,
and 20mmol/L Tris-HCl (pH 7.5) were incubated for 20 min at 25°C. Electrophoresis of
samples through a native 6% polyacrylamide gel was followed by autoradiography.
Immunocytochemistry
ERK1/2 tranlocation into the nucleus was identified by immunocytochemistry. SMCs
plated on glass coverslips, pre-coated with gelatin, were treated with 1µmol/L 5-HT for 5
and 10 min, and then fixed in 4% formaldehyde in PBS for 20 min, rinsed in PBS, and
permeabilized with 0.4% Triton/PBS for 5 min prior to staining. After washing with PBS
twice, the nonspecific staining on the slides was blocked with 1.5% goat serum/
1%BSA/PBS for 40min. Slides were incubated with 1:100 diluted rabbit ERK1/2
polyclonal antibody overnight at 4°C. Coverslips were rinsed with PBS twice and then
stained with FITC-conjugated goat anti-rabbit IgG for 60 min at ambient temperature.
Following the incubations, the coverslips were washed with PBS and mounted and
sealed. Slides were viewed on a Zeiss fluorescent microscope and photographs were
obtained digitally with Metamorph software.
F-actin visualization
Cells were plated in RPMI 1640 containing 10% FBS at a concentration of 2x105 cells
per dish in a 35mm cultural dish containing a glass coverslip. After 24 hours, the cells
were growth-arrested in RPMI 1640 containing 0.1% FBS for 48h. The cells were
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pretreated with either latrunculin B (1 µmol/L), cytochalasin D (0.5 µmol/L) or vehicle
for 30min, then stimulated with 5-HT 1µmol/L for 5min. The treated cells were fixed
with 4% paraformaldehyde, and permeabilized with 0.4% Triton X-100. For visualization
of actin stress fibers, cells were incubated with rhodamine-phalloidin 100µg/ml for 1 h.
Slides were viewed on a Zeiss fluorescent microscope and photographs were obtained
digitally with Metamorph software.
Statistical analysis
Means ± S.D. were calculated and statistically significant differences among groups were
determined by one-way ANOVA analysis with significance at p<0.05.
References
1. Lee SL, Wang WW, Moore BJ, Fanburg BL. Dual effect of serotonin on growth
of bovine pulmonary artery smooth muscle cells in culture. Circ Res.
1991;68:1362-1368.
2. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified
system for generating recombinant adenoviruses. Proc Natl Acad Sci U S A.
1998;95:2509-14.
3. Suzuki YJ, Day RM, Tan CC, Sandven TH, Liang Q, Molkentin JD, Fanburg BL.
Activation of GATA-4 by serotonin in pulmonary artery smooth muscle cells. J
Biol Chem. 2003;278:17525-17531.
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Figure legends
Online Figure 1. 5-HT activates ROCK via 5-HT1B/1D receptor but not 5-HT
transporter
A) SMCs were pre-treated with 5µmol/L GR55562, GR127935 or 10µmol/L Citalopram,
Imipramine for 30 min, then SMCs were stimulated with 1µmol/L 5-HT for 15 min. B)
SMCs were pre-treated with 5µmol/L GR55562 or GR127935 for 30 min, then treated
with 1µmol/L 5-HT for 6 h. The phosphorylation of MYPT1 (Thr696) and the expression
of cyclin D1 were determined by Western blot with whole cell lysates. (**) denotes a
significant difference from the untreated control value at p<0.05. (*) denotes a
significant difference from 5-HT alone control value p<0.05.
Online Figure 2. Inhibitory effect of Y-26732 on 5-HT-induced transcription factor
Elk, Egr-1, and GATA-4 activation.
Effect of Y27632 on 5-HT-induced Elk-1 phosphorylation. A) Growth-arrested SMCs
were stimulated with 1µmol/L 5-HT for the indicated time course. B) SMCs were pretreated with 20µmol/L Y27632 or 10µmol/L U0126 for 30 min, then treated with
1µmol/L 5-HT for 1 h. The level of phosphorylated Elk-1 in the nucleus was determined
by Western blot analysis using the p-Elk-1(Ser383) antibody in the cellular nuclear
extracts. C) Growth-arrested SMCs were treated with 1µmol/L 5-HT for indicated time
CIRCRESAHA/2004/078923/R2 7
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courses. D) SMCs were pre-treated with 10µmol/L Y27632 or 10µmol/L U0126 for 30
min, then treated with 1µmol/L 5-HT for 1 h. Egr-1 DNA binding activity in nuclear
protein was assayed by EMSA. E) SMCs were pre-treated with 10µmol/L Y27632 for 30
min, then treated with 1µmol/L 5-HT for 20h. Nuclear extracts were isolated and the
GATA-4 DNA-binding activities were monitored by EMSA. (**) denotes a significant
difference from the untreated control value at p<0.05. (*) denotes a significant difference
from 5-HT alone control value p<0.05.
Online Figure 3. The mitogenic action of 5-HT depends on the activation of
ERK1/ERK2 in SMCs
A) Growth-arrested SMCs were stimulated with 1µmol/L 5-HT for the indicated time
course. Phosphorylation of ERK was determined by Western blot analysis of the whole
cell lysate using the phospho-specific antibody. B) SMCs were pre-treated with
10µmol/L U0126 for 30 min, then incubated with 1µmol/L 5-HT for 24 h. DNA
synthesis was determined by monitoring [3H]thymidine incorporation. C) SMCs were
pre-treated with 10µ mol/L U0126 for 30 min, then SMCs were stimulated with 1µmol/L
5-HT for 6 h. The expression of cyclin D1 was determined by Western blot with whole
cell lysates. (**) denotes a significant difference from the untreated control value at
p<0.05. (*) denotes a significant difference from 5-HT alone control value p<0.05.
Online Figure 4. 5-HT-induced ERK1/2 translocation into nucleus does not require
intact actin cytoskeleton.
CIRCRESAHA/2004/078923/R2 8
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A) SMCs were pre-treated with CD 0.5µmol/L or Lat B 1µmol/L for 30 min, and then
incubated with 1µmol/L 5-HT for 24 h. DNA synthesis was determined by monitoring
[3H]thymidine incorporation. B) SMCs were preincubated with CD 0.5µmol/L or Lat B
1µmol/L for 30 min, then treated with 1µmol/L 5-HT for 5 min. ERK1/2 phosphorylation
was assayed by Western blot in cellular lysates. C) ERK1/2 translocation into nucleus
was assayed by immuno-histochemistry with rabbit polyclonal ERK1 antibody. The
distribution of f-actin was visualized with rhodamine-phalloidin staining and observed
under fluorescence microscopy. (**) denotes a significant difference from the untreated
control value at p<0.05. (*) denotes a significant difference from 5-HT alone control
value p<0.05.
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Online Figure 1
1A
p-MYPT1(Thr696)
MYPT1
-
p-MYPT1 (densitometry
5-HT 1µmol/L 15min
Citalopram 10µmol/L
Imipramine 10µmol/L
GR55562 5µmol/L
GR127935 5µmol/L
-
+
-
+
-
+
+
-
+
+
-
+
+
-
350
+
+
-
+
+
-
+
+
-
+
+
5-HT+inhibitor
300
250
**
200
150
*
100
*
50
G
R5
55
62
G
R1
27
93
5
m
in
e
Im
ip
ra
lo
pr
am
/L
Ci
ta
1u
m
ol
5H
T
un
t
re
a
te
d
0
1B
Cyclin D1
-
5-HT 1µmol/L 6h
GR55562 5µmol/L
GR127935 5µmol/L
+
-
+
+
-
+
+
Cyclin D1 (densitometry)
700
**
600
5-HT+inhibitor
500
400
300
*
200
*
100
0
untreated
5-HT
GR55562
GR5127935
+
+
CIRCRESAHA/2004/078923/R2 10
Activation of Rho kinase by serotonin Online supplement
Online Figure 2
2A
0
5
5-HT 1µmol/L
10 20 40 60
90
120
min
p-Elk-1(ser383)
p-Elk1 (densitometry)
Elk-1
1200
1000
**
**
800
**
**
600
400
200
0
0
5
10
20
40
60
90 120
(min)
2B
p-Elk-1(ser383)
Elk-1
5-HT 1µmol/L 1h
Y27632 20µmol/L
U0126 10µmol/L
-
+
-
+
+
-
+
+
p-ElK1 (densitometry)
1000
**
800
600
*
400
*
200
0
untreated
5HT
5HT+Y27632 5HT+U0126
CIRCRESAHA/2004/078923/R2 11
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2C
5-HT 1µmol/L 0 10 30 60 120 240 min
Egr-1
free probe
2D
5-HT 1µmol/L 1h
Y27632 10µmol/L
U0126 10µmol/L
-
+
-
+
-
+
+
-
+
+
Egr-1
free probe
CIRCRESAHA/2004/078923/R2 12
Activation of Rho kinase by serotonin Online supplement
2E
5-HT 1µmol/L 20h
Y27632 10µmol/L
-
+
+
-
+
+
GATA-4
free probe
Online Figure 3
3A
p-ERK
ERK
5-HT 1µmol/L
0
2
5
10
30
60
min
p-ERK1/2 (densitometry)
2500
**
2000
1500
**
**
1000
**
500
0
0
2
5
15
30
60
(min)
CIRCRESAHA/2004/078923/R2 13
Activation of Rho kinase by serotonin Online supplement
3B
**
**
20000
15000
*
10000
5000
10
uM
5H
5H
T+
D
T+
U
01
26
1u
M
5H
T
at
un
tre
M
SO
0
ed
thymidine incorporation (CPM)
25000
3C
cyclin D1
5-HT 1µmol/L 6h
U0126 10µmol/L
-
+
Cyclin D1 (densitometry)
700
+
-
+
+
**
600
500
400
300
200
100
**
*
0
untreated
U0126
5-HT
5-HT+U0126
CIRCRESAHA/2004/078923/R2 14
Activation of Rho kinase by serotonin Online supplement
Thymidine
incorporation (CPM)
Online Figure 4.
4A
5000
**
4000
**
3000
2000
1000
**
*
**
*
5H
5H
T
T+
D
M
SO
5H
T+
C
D
5H
T+
La
tB
La
tB
C
M
SO
D
at
ed
un
tre
D
0
4B.
5-HT 1µmol/L
CD 0.5µmol/L
Lat B 1µmol/L
-
+
-
+
+
-
+
+
p-ERK
ERK
ns
ns
p-ERK (densitometry)
300
**
250
200
150
100
50
0
untreated
5-HT
5-HT+CD 5-HT+LatB
CIRCRESAHA/2004/078923/R2 15
Activation of Rho kinase by serotonin Online supplement
4C
ERK
Untreated
5-HT 1µmol/L 5min
5-HT+ CD
0.5µmol/L
5-HT+ Lat B
1µmol/L
f-Actin