© 1999 American Heart Association, Inc.
Original Contribution |
Rho and Rho Kinase Mediate Thrombin-Stimulated Vascular Smooth Muscle Cell DNA Synthesis and Migration
From the University of California, San Diego, Department of Pharmacology, La Jolla.
Correspondence to Joan Heller Brown, University of California, San Diego, Department of Pharmacology, 9500 Gilman Dr, La Jolla, CA 92093-0636. E-mail jhbrown{at}ucsd.edu
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Abstract—Aberrant regulation of smooth muscle cell proliferation and migration is associated with the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. To elucidate molecular mechanisms that regulate proliferation and migration of vascular smooth muscle cells, we determined whether signaling through the small G protein Rho is involved in thrombin- and phenylephrine-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). Thrombin and the thrombin peptide SFLLRNP stimulated DNA synthesis of RASMCs as measured by [3H]thymidine incorporation. Both ligands also increased cell migration as measured by the Boyden chamber method. L-Phenylephrine failed to induce either of these responses but increased inositol phosphate accumulation and mitogen-activated protein kinase activation in these cells, which indicated that the cells were responsive to
1-adrenergic stimulation. The C3 exoenzyme, which
ADP-ribosylates and inactivates Rho, fully inhibited both
thrombin-stimulated proliferation and migration but had no effect on
inositol phosphate accumulation. In addition, Y-27632, an
inhibitor of the Rho effector p160ROCK/Rho kinase,
decreased thrombin-stimulated DNA synthesis and migration. To directly
examine Rho activation, Rho-[35S]GTP
S binding was
measured. The addition of the thrombin peptide SFLLRNP, but not
phenylephrine, to RASMC lysates resulted in a significant
increase in Rho-[35S]GTPS binding. Thrombin and
SFLLRNP, but not phenylephrine, also increased
membrane-associated Rho in intact RASMCs, consistent with
selective activation of Rho by thrombin. These results indicate that
thrombin activates Rho in RASMCs and establish Rho as a
critical mediator of thrombin receptor effects on DNA synthesis and
cell migration in these cells.
Key Words: Rho • thrombin • vascular smooth muscle • DNA synthesis • cell migration
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Introduction |
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The low-molecular-weight G protein Rho is a member of the Rho family of small GTPases that also includes Rac and Cdc42. These GTPases act as molecular switches to regulate cellular functions, the best characterized of which are changes in the actin cytoskeleton. An increasing body of evidence has revealed that some heterotrimeric G protein-coupled receptors (GPCRs) signal through small G proteins such as Rho. GPCR agonists such as lysophosphatidic acid (LPA),1 carbachol,2 and phenylephrine3 have been shown to increase levels of membrane-associated Rho or decrease cytosolic Rho, which is indicative of Rho activation. Studies from our laboratory have demonstrated a role for Rho in
1-adrenergic
receptor (1AdrR)–induced
hypertrophy and gene expression in cardiac
myocytes4 5 and in thrombin-induced mitogenesis and actin
cytoskeletal rearrangement in astrocytoma cells.6 In
addition, Rho has been implicated in GPCR-induced cytoskeletal
responses,7 8 in cell transformation,9 10 and
in activation of phospholipase D in other systems.11
It appears that Rho is also the mediator of
Ca2+-sensitization in vascular smooth muscle
(VSM). This is a phenomenon by which VSM contractility
can be increased by agonists independent of increases in intracellular
Ca2+.12 13 The observation that
increases in contractility are elicited by GTP and GTP
analogs in permeabilized preparations suggested the
involvement of G proteins in this response.12 The C3
exoenzyme from Clostridium botulinum, which specifically ADP
ribosylates and inactivates Rho, inhibits
1AdrR and guanine
nucleotide–dependent contraction of
permeabilized blood vessels.14 15 In
addition, C3 blocks the associated increase in myosin light chain
phosphorylation in permeabilized smooth
muscle cells.16 The signaling cascade elicited by Rho that
results in Ca2+-sensitization involves the
Rho-dependent kinases p160ROCK and Rho kinase. Rho kinase has been
shown to phosphorylate the myosin-binding subunit of myosin
light chain phosphatase, thus inhibiting its function and allowing for
increases in levels of phosphorylated
myosin.17 Studies performed in
permeabilized vessels reveal that the addition of the
catalytic subunit of Rho kinase elicits contraction.18 In
addition, Y-27632, an inhibitor of p160ROCK and Rho kinase,
blocks contractions in response to phenylephrine (PE) in
intact rabbit aorta and GTPS in permeabilized rabbit
aorta.19
Although results from the above-mentioned studies provide evidence for Rho and Rho kinase involvement in vascular contractility, the role of Rho and Rho kinase in other vascular responses such as mitogenesis and cell migration has not been investigated. Understanding the mechanisms of vascular smooth muscle cell (VSMC) proliferation and migration is critical to evaluation of the involvement of these processes in the pathophysiology of vascular disorders such as hypertension, atherosclerosis, restenosis, and graft rejection. The possibility that Rho is involved in vascular proliferation and migration is suggested by the involvement of Rho in the growth of nonvascular cells in response to heterotrimeric G protein receptor stimulation4 6 and in the migration of endothelial cells in response to mechanical strain or tyrosine kinase growth factors.20 21
Altered responsiveness to thrombin and catecholamines has been implicated in the pathophysiology of vascular diseases. These agonists act on heterotrimeric G protein–linked receptors to regulate vascular contraction and have been reported to affect vascular growth responses.22 23 24 25 26 27 28 29 30 Thrombin also appears to regulate vascular cell migration.31 The studies presented here used rat aortic smooth muscle cells (RASMCs) to determine whether thrombin or PE signals through the small G protein Rho and through Rho kinase to regulate vascular mitogenesis and cell migration.
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Materials and Methods |
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Materials
The Rho kinase inhibitor Y-27632 was a gift from Yoshitomi Pharmaceutical Industries, Ltd (Iruma-shi, Saitama, Japan). The monoclonal RhoA antibody was obtained from Santa Cruz Biotechnology.
C3 Exoenzyme and Rho ADP Ribosylation
The method used for preparation of the glutathione S-transferase
(GST) fusion protein has been previously described.6
Briefly, cDNA for the GST-C3 fusion protein (a gift from Dr J.
Meinkoth, University of Pennsylvania) was used to transform
Escherichia coli strain JM 109 to produce the protein for
purification. After 3 hours, the cells were lysed, and extracts were
clarified and incubated with GSH-Sepharose. The C3 toxin protein was
cleaved from GST by overnight incubation with thrombin. Thrombin was
removed by incubation with p-aminobenzamidine-Sepharose. The
supernatant was concentrated to 5 mg/mL protein with a Centricon-10
(Amicon Inc, Beverly, Mass). Pretreatment of cells with 40
µg/mL C3 toxin was performed for 12 to 16 hours.
To measure the extent of in vivo ADP ribosylation of Rho after 12 to 16 hours of extracellular treatment with 40 µg/mL C3, cells were rinsed in PBS and lysed in 0.1 mol/L phosphate buffer, pH 7.9, that contained 1% Triton X-100. Cells were scraped, and the lysate was centrifuged at 4°C for 5 minutes at 12 000g. The supernatant was collected, and in vitro ribosylation was performed at 30°C for 30 minutes in a reaction buffer that contained 100 mmol/L Tris-HCl, 5 mmol/L MgCl2, 30 mmol/L thymidine, 0.2 mmol/L GTP, 5 mmol/L DTT, 5 µmol/L NAD, 15 µCi/mL [32P]NAD, and 10 µg/mL C3. The reaction was stopped with 4x Laemmli buffer and the samples boiled. Proteins were separated by SDS-PAGE, and the radioactivity was quantified on an AMBIS radioanalytic scanner.
Cell Culture Conditions
RASMCs were obtained from Dr Wolfgang Dillmann's laboratory,
University of California, San Diego. These cells were isolated from
thoracic aortas of 8- to 12-week-old male Sprague-Dawley rats as
described.32 Cells were grown in high glucose DMEM
supplemented with 10% FCS, penicillin (100 U/mL), and streptomycin
(100 µg/mL) and kept in a 37°C humidified incubator at 5%
CO2. RASMCs between passages 5 and 12 were used
for all of the following experiments.
Rho-[35S]GTPS Binding
The Rho-[35S]GTPS binding assay was a
modification of that developed in the Manning laboratory for measuring
[35S]GTPS binding to -subunits of
heterotrimeric G proteins.33 RASMCs (10-cm plates) were
grown to confluency in 20% FCS. Cells were washed with ice-cold PBS
followed by incomplete lysis buffer that contained 50
mmol/L Tris-HCl, 10 mmol/L MgCl2, 2
mmol/L EDTA, and 100 mmol/L NaCl. Cells were lysed and scraped in
complete lysis buffer that in addition contained 0.3 µmol/L GDP,
20 µg/mL aprotinin, 10 µg/mL leupeptin, and 0.1 mmol/L PMSF.
The lysate was passed through a 27-gauge needle 15 times and spun at
500g for 5 minutes at 4°C to remove unbroken cells. The
supernatant was collected and diluted to 20 µg of protein per 50
µL. The reaction was initiated by the addition of the cell lysate (20
µg of protein) to [35S]GTPS (final
concentration, 300 nmol/L) in the presence or absence of agonist. The
reaction was terminated by the addition of 600 µL of ice-cold
immunoprecipitation (IP) buffer that contained 50 mmol/L Tris,
20 mmol/L MgCl2, 150 mmol/L NaCl, 0.5%
NP-40, 20 µg/ml aprotinin, 100 µmol/L GDP, and 100
µmol/L GTP. Samples were shaken at 4°C for 30 minutes before IP
overnight with monoclonal RhoA antibody and protein A Sepharose. The
IPs were washed 3 times with IP buffer and 1 final time with IP buffer
that lacked NP-40. Samples were then boiled in 500 µL of 0.5% SDS
for 1 minute before addition of scintillant and quantitation with a
liquid scintillation counter.
Measurement of Rho Distribution
Plates (10 cm) of 75% confluent RASMCs were washed twice with
HEPES-buffered DMEM that contained 0.1% BSA and changed to buffered
media with or without agonist for 3 minutes at 37°C. The reaction was
stopped by washing the plates with ice-cold PBS followed by the
addition of incomplete lysis buffer that contained 10 mmol/L
HEPES, 2 mmol/L EDTA, and 1 mmol/L
MgCl2. Plates were then placed on ice, and the
cells were treated with 800 µL of complete lysis buffer (complete
lysis buffer also contained 10 mmol/L
Na4P2O7,
500 µmol/L Na3VO4,
10 µg/mL leupeptin, 10 µg/mL antipain, 1 mmol/L DTT, and
1 mmol/L PMSF) for 15 minutes. Cells were then scraped,
triturated, transferred to microfuge tubes, and centrifuged at
4°C for 5 minutes at 500g. The low-speed pellet was
discarded, and the supernatant was centrifuged at 4°C for 30
minutes at 37 000g to generate membrane and cytosolic
fractions. The membrane pellet was resuspended in lysis buffer and
fractions were boiled in Laemmli buffer. Proteins were separated with
the use of SDS-PAGE, transferred to Immobilon membranes, and blocked
with 3% BSA at room temperature for 1 hour. Blots were then incubated
in mouse anti-Rho A (1:1000) followed by horseradish
peroxidase–labeled goat anti-mouse (1:2000) and subsequent enhanced
chemiluminescence. Blots were exposed to film that was analyzed
by densitometry.
[3H]Thymidine Incorporation
Cells were set on 12-well plates (22 mm) and allowed to
grow in 20% FCS for 8 days until confluent. Cells were serum-starved
for 24 hours before the addition of agonists for 48 hours.
[3H]Thymidine (1 µCi/mL) was added for the
last 24 hours of agonist stimulation. Cells were washed twice with
ice-cold PBS and fixed in methanol for 10 minutes. Cells were washed
twice with 10% trichloroacetic acid, once with 0.5%
trichloroacetic acid, and dissolved in 0.5 mL 1N NaOH for 30 minutes at
room temperature followed by the addition of 0.5 mL 1N HCl.
Radioactivity was quantitated using a liquid scintillation counter.
Cell Migration
Cell migration was performed using the Boyden chamber method as
described.31 Briefly, the lower well of the chemotaxis
chamber was filled with 100 µL of DMEM that contained the appropriate
concentration of agonist or vehicle. An 8-µm polycarbonate filter was
placed between the upper and lower chambers. Cells were trypsinized,
counted with a hemacytometer, and resuspended in serum-free DMEM to a
concentration of 5x104 cells per 200 µL. This
volume was then added to the upper chamber. After incubation for 24
hours at 37°C in a 5% CO2 incubator, filters
were removed and cells on the top surface of the membrane (which had
not migrated through the filter) were removed with a cotton swab.
Membranes were next washed with PBS, fixed with methanol, stained with
hematoxylin, and mounted on glass slides. Cells were counted with the
use of a Zeiss-Axiophot microscope with a Zeiss Plan-Apochromat 63x
oil immersion lens.
Adenoviral Studies
The adenoviral vectors that encode LacZ, activated L63
RhoA, and activated V12 Ras were prepared as described
previously.5 RASMCs were plated at
1x104 cells per well on 12-well plates and grown
for 4 days before being serum-starved. The following morning, cells
were infected with the adenovirus at titers ranging from 250 to 750
viral particles per cell. Twenty-four hours after infection, cells were
washed with serum-free media. [3H]Thymidine (1
µCi/mL) was added 
30 hours after infection, and
[3H]thymidine incorporation was measured (as
described above) 52 hours after infection.
Statistics
Data were analyzed by ANOVA. Postanalysis was
performed with the use of the Tukey test, with P<0.05.
Probability values are given in the figure legends.
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Results |
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To verify that the RASMCs used in our studies contained functional receptors for thrombin and PE, the ability of these agonists to stimulate phospholipase C (PLC) and activate MAP kinase was examined. Maximal concentrations of either agonist (0.5 U/mL thrombin and 100 µmol/L PE) produced similar increases in total [3H]inositol phosphate formation, and both agonists elicited significant increases in MAP kinase activity (data not shown). The response to PE was blocked by 0.3 µmol/L prazosin hydrochloride but not by 2 µmol/L propranolol hydrochloride. These data confirm the existence of thrombin and
1AdrRs coupled to the
activation of PLC and MAP kinase in these cells as previously shown by
other groups.34 35 36 37 38
Experiments were next performed to determine whether these agonists
stimulated DNA synthesis in RASMCs. When cells were grown to confluence
and serum-starved for 1 day before the addition of agonist, thrombin
(0.5 U/mL) consistently increased
[3H]thymidine incorporation (Figure 1A
). Smaller responses were observed in
nonconfluent cells or cells starved for 2 to 3 days. Unlike thrombin,
PE (100 µmol/L) did not stimulate
[3H]thymidine incorporation (Figure 1A).
A range of concentrations of PE (100 nmol/L to 100 µmol/L) was
tested on [3H]thymidine incorporation with
similar negative results (data not shown). DNA synthesis was also
assessed by immunocytochemical analysis with bromodeoxyuridine.
Sixty-five percent of cells stimulated with thrombin (0.5 U/mL) were
found to incorporate bromodeoxyuridine compared with 19% of control
cells (data not shown). Migration of RASMCs in response to the two
PLC-coupled agonists was also examined with the use of the Boyden
chamber method (Figure 1B). Analogous to the findings for DNA
synthesis, thrombin (0.5 U/mL) but not PE (100 µmol/L) was able
to increase cell migration.
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Similar experiments were also performed with the thrombin
receptor–activating peptide SFLLRNP, which lacks the proteolytic
activity of thrombin but mimics the effects of the tethered ligand
exposed after cleavage of the thrombin receptor with thrombin. Although
this peptide sequence is based on that of the human receptor and
differs from the rat receptor by 1 amino acid, it was clearly equal to
or more efficacious than thrombin at eliciting the responses of
interest. Figure 1A
and 1B show that the thrombin receptor
peptide (100 µmol/L) stimulates both DNA synthesis and migration
of RASMCs. These results confirm that the observed stimulation of DNA
synthesis and cell migration is mediated through the cell surface
thrombin receptor.
To determine whether the small G protein Rho is involved in
thrombin-stimulated DNA synthesis and the migration of RASMCs, the C3
exoenzyme from C botulinum was used to ADP-ribosylate and
inactivate Rho. C3 exoenzyme treatment (40 µg/mL C3 toxin
for 12 to 16 hours) fully inhibited both thrombin-stimulated DNA
synthesis and migration of RASMCs (Figure 1A and 1B). This was
associated with ribosylation of at least 50% of cellular Rho (data not
shown). Thrombin-stimulated accumulation of inositol phosphates was not
inhibited (data not shown), which indicated that C3 exoenzyme treatment
does not nonspecifically block receptor function. This observation
further indicates that C3 inhibits DNA synthesis and migration without
blocking the generation of PLC-derived second messengers.
Rho kinase and the closely related p160ROCK are putative effectors of
Rho that we considered possible candidates for mediating
thrombin-stimulated VSMC mitogenesis and migration. Recently, the agent
Y-27632 has been shown to specifically inhibit these Rho-dependent
kinases.19 In RASMCs, treatment with 10 µmol/L
Y-27632 prevented thrombin from producing significant increases in
[3H]thymidine incorporation and cell migration
(Figure 2).
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To directly examine the ability of the GPCR agonists to
activate Rho, Rho-[35S]GTPS binding
was measured in RASMC lysates. The thrombin peptide SFLLRNP produced
significant increases in Rho-[35S]GTPS
binding, although PE did not (Figure 3).
Increases in membrane-bound Rho are also associated with Rho
activation. To further investigate the ability of these GPCR agonists
to activate Rho in intact RASMCs, we measured changes in
membrane-associated Rho. In unstimulated RASMCs, 20% of Rho was
membrane-bound and 80% was cytosolic (data not shown). When stimulated
with 0.5 U/mL thrombin or 100 µmol/L thrombin peptide, an
approximate doubling in membrane-associated Rho was seen (Figure 4A). Concomitant decreases in cytosolic
Rho were not consistently observed (data not shown), probably
because a large fraction of the total Rho protein still remained
cytosolic. No effect of 100 µmol/L PE on levels of
membrane-associated Rho was observed (Figure 4B). The addition
of 2 µmol/L propranolol hydrochloride had no effect
on the inability of PE to induce RASMC DNA synthesis, migration, or
activation of Rho (data not shown), indicating possible ßAdrR effects
of PE are not responsible for the observed lack of PE-mediated
responses. Levels of membrane-associated Rac were assessed and found to
be unaffected in two separate experiments in which thrombin increased
levels of membrane-associated Rho (Figure 4C). Thus, selective
activation of Rho appears to correlate with the ability of agonists to
induce DNA synthesis and migration.
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The ability of activated Rho to induce DNA synthesis in RASMCs
was examined by infection of cells with an activated adenoviral
RhoA expression plasmid. Cells infected with activated RhoA
alone demonstrated no increase in [3H]thymidine
incorporation when compared with infection with LacZ (Figure 5). Conversely, infection with
activated Ras produced an 3-fold increase in
[3H]thymidine incorporation. Furthermore,
activated Rho and activated Ras expressed together had
a synergistic effect on [3H]thymidine
incorporation in RASMCs.
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Discussion |
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Evidence is rapidly accumulating that suggests a role for Rho in heterotrimeric GPCR-mediated signaling. For example, Rho has been shown to be involved in GPCR activation of phospholipase D,11 stress fiber formation,7 8 and Ca2+-sensitization associated with the tonic component of VSM contraction.14 15 16 Only a few reports demonstrate increases in Rho-GTP binding on Rho activation,39 40 although a fair number of studies have shown increases in membrane-associated Rho or decreases in cytosolic Rho.1 2 3 41 42 In this report we examined both Rho-[35S]GTP
S binding and Rho distribution
to determine whether GPCR agonists activate Rho in intact
RASMCs. Our data provides the first demonstration that Rho is
activated by GPCR stimulation in intact VSMCs, a phenomenon
previously demonstrated by guanine nucleotide and agonist
treatment in permeabilized vessels.3 This
is also the first report of Rho activation by thrombin in smooth
muscle. Both thrombin and PE couple to the activation of PLC, which presumably results in increases in intracellular Ca2+ and activation of protein kinase C. Thus, the observation that thrombin activates Rho, although PE does not, indicates that PLC-generated second messengers are not sufficient to induce this response. Rho-dependent changes in cytoskeletal reorganization also appear to be independent of Ca2+ and protein kinase C in other systems.6 8 43 Early work from our laboratory has shown that although thrombin and carbachol both activate PLC in 1321N1 astrocytoma cells, only thrombin mediates Rho-dependent cytoskeletal rearrangement6 and DNA synthesis.44 Similarly in PC12 cells, two PLC-coupled receptor agonists, LPA and bradykinin, differ in their ability to induce cytoskeletal change.45 These data are consistent with our current observation that the PLC pathway appears insufficient to activate Rho.
We hypothesize that the ability of receptor agonists to
activate Rho reflects selective receptor-coupling to a
heterotrimeric G protein other than or in addition to
Gq. Recent reports suggest that the thrombin
receptor can also signal through the pertussis toxin-insensitive
G12 and G13 proteins
to mediate cellular responses. Thrombin receptor stimulation has been
shown to increase GTP labeling of
G12/13,33 46 and
G12/13 is required for thrombin-stimulated DNA
synthesis44 47 and cell migration.48 The
relationship between G12 and Rho has not yet
been elucidated, although overexpression of activated
G12 or activated
G13 was shown to increase Rho-GTP binding in
COS-7 cells.40 Most importantly,
G13 has been shown to activate a
guanine nucleotide exchange factor for Rho, which suggests
a pathway for activation of Rho by this G protein.49 50
Studies in our laboratory are currently underway to determine if
selective coupling to G12/13 in addition to
Gq/PLC is required for thrombin to
activate Rho and the subsequent induction of DNA synthesis and
migration of RASMCs.
The balance in favor of activation of Rho by a given GPCR is probably
mediated by a complex network of signals that result from activation of
a variety of G proteins and/or protein kinases. Cell-specific signaling
to Rho may occur not only as a result of differential coupling of
receptors to G proteins but also as a result of differential
availability and/or coupling of G proteins to guanine
nucleotide exchange factors, GDP dissociation
inhibitors, and GTPase activating proteins. Thus, although
1AdrRs have been shown to mediate
Ca2+-sensitization, a Rho-dependent process, in
VSM,12 14 15 18 19 51 vessel-specific differences in G
protein coupling such as those described above52 53 could
account for the lack of observed PE activation of Rho in RASMCs. In
addition, previous studies on 1AdrR regulation
of Rho and Ca2+-sensitization were performed
using permeabilized preparations. Exogenous guanine
nucleotides added back to the permeabilized
vessels may promiscuously activate G proteins and allow for
activation of receptor-mediated pathways (ie,
1AdrR coupling to
Ga12/13) not effectively engaged by receptor
activation in the intact system.
A role for Rho and Rho-mediated myosin phosphorylation in the Ca2+-sensitization component of VSM contraction has been extensively investigated and supported by studies from several laboratories including the Somlyo and Kaibuchi laboratories.3 13 17 18 54 In contrast, the role of Rho in DNA synthesis and the migration of VSMCs has not been investigated. Our finding that C3 fully blocks thrombin stimulation of these responses in RASMC provides the first evidence that Rho is involved in VSMC proliferation and migration. Our data also suggest that although Rho is necessary for thrombin-stimulated DNA synthesis, activation of this small G protein by itself is not sufficient to induce DNA synthesis in RASMCs. Instead, Rho appears to potentiate the effects of Ras, which is in agreement with results from other laboratories that demonstrate concerted effects of Rho activation on Ras-stimulated cell cycle progression.55
Although other investigators have demonstrated Rho involvement in cellular growth4 6 9 10 and migration responses,20 21 the downstream effectors of these responses have not been identified. GTP-bound Rho has been shown to bind to and activate Rho kinase54 56 57 as well as other putative Rho effectors.54 58 59 60 61 Recent studies from our laboratory have demonstrated that cardiomyocyte hypertrophy and myofibrillar assembly are blocked by inhibitory mutants of Rho kinase, which suggests a role for this Rho effector in cellular growth responses.5 A recent report suggests that the binding of Rho to Rho kinase correlates with its ability to produce cell transformation.62 Our current results further support this idea and suggest that Rho kinase is involved in thrombin stimulation of both VSMC proliferation and migration.
In summary, results from these studies indicate that agonist stimulation of the G protein–coupled thrombin receptor activates the small G protein Rho in lysed and intact RASMCs. We further demonstrate that Rho is required for both thrombin-stimulated vascular proliferation and migration. With the use of the inhibitor Y-27632, we suggest that Rho kinase is required for thrombin-stimulated VSMC DNA synthesis and migration. Interestingly, VSMC proliferation,63 64 migration,65 and Ca2+-sensitization66 are all responses that have been reported to be enhanced in experimental hypertension. A recent report demonstrated that acute administration of the Rho kinase inhibitor Y-27632 reduced blood pressure in three different experimental forms of hypertension.19 This acute reduction in blood pressure is presumably the result of decreases in Ca2+-sensitization. Current studies provide evidence for a role for Rho and Rho kinase in two additional processes, vascular cell proliferation and migration, both of which have been implicated in the pathophysiology of atherosclerosis, restenosis, and graft rejection.67 68 69 70 Therefore, a central role for disturbances in Rho and Rho kinase function in these vascular disorders warrants additional investigation.
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Acknowledgments |
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This work was done during the tenure of a research fellowship from the American Heart Association, Western States Affiliate, to T.M.S. and supported by NIH grants HL-28143 and GM-36927 to J.H.B. D.D.K. was a recipient of a summer fellowship from the American Society for Pharmacology and Experimental Therapeutics. We thank David Goldstein and G. Todd Giles for their assistance in preparing the manuscript. We also thank Yoshitomi Pharmaceutical Industries, Ltd, for their generous gift of Y-27632.
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Footnotes |
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This manuscript was originally submitted on August 13, 1998, resubmitted on January 26, 1999, and accepted on March 12, 1999.
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R. Blindt, A.-K. Bosserhoff, J. Dammers, N. Krott, L. Demircan, R. Hoffmann, P. Hanrath, C. Weber, and F. Vogt Downregulation of N-cadherin in the neointima stimulates migration of smooth muscle cells by RhoA deactivation Cardiovasc Res, April 1, 2004; 62(1): 212 - 222. [Abstract] [Full Text] [PDF]
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H. Kawamura, K. Yokote, S. Asaumi, K. Kobayashi, M. Fujimoto, Y. Maezawa, Y. Saito, and S. Mori High Glucose-Induced Upregulation of Osteopontin Is Mediated via Rho/Rho Kinase Pathway in Cultured Rat Aortic Smooth Muscle Cells Arterioscler Thromb Vasc Biol, February 1, 2004; 24(2): 276 - 281. [Abstract] [Full Text]
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 A. I. Willis, D. Pierre-Paul, B. E. Sumpio, and V. Gahtan Vascular Smooth Muscle Cell Migration: Current Research and Clinical Implications Vascular and Endovascular Surgery, January 1, 2004; 38(1): 11 - 23. [Abstract] [PDF]
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Y. Matsumoto, T. Uwatoku, K. Oi, K. Abe, T. Hattori, K. Morishige, Y. Eto, Y. Fukumoto, K.-i. Nakamura, Y. Shibata, et al. Long-Term Inhibition of Rho-Kinase Suppresses Neointimal Formation After Stent Implantation in Porcine Coronary Arteries: Involvement of Multiple Mechanisms Arterioscler Thromb Vasc Biol, January 1, 2004; 24(1): 181 - 186. [Abstract] [Full Text] [PDF]
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G.A. Stouffer and S.S. Smyth Effects of Thrombin on Interactions Between {beta}3-Integrins and Extracellular Matrix in Platelets and Vascular Cells Arterioscler Thromb Vasc Biol, November 1, 2003; 23(11): 1971 - 1978. [Abstract] [Full Text] [PDF]
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Z. Mallat, A. Gojova, V. Sauzeau, V. Brun, J.-S. Silvestre, B. Esposito, R. Merval, H. Groux, G. Loirand, and A. Tedgui Rho-Associated Protein Kinase Contributes to Early Atherosclerotic Lesion Formation in Mice Circ. Res., October 31, 2003; 93(9): 884 - 888. [Abstract] [Full Text] [PDF]
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M. Higashi, H. Shimokawa, T. Hattori, J. Hiroki, Y. Mukai, K. Morikawa, T. Ichiki, S. Takahashi, and A. Takeshita Long-Term Inhibition of Rho-Kinase Suppresses Angiotensin II-Induced Cardiovascular Hypertrophy in Rats In Vivo: Effect on Endothelial NAD(P)H Oxidase System Circ. Res., October 17, 2003; 93(8): 767 - 775. [Abstract] [Full Text] [PDF]
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E. R. Lazarowski, R. C. Boucher, and T. K. Harden Mechanisms of Release of Nucleotides and Integration of Their Action as P2X- and P2Y-Receptor Activating Molecules Mol. Pharmacol., October 1, 2003; 64(4): 785 - 795. [Full Text] [PDF]
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 A. P. SOMLYO and A. V. SOMLYO Ca2+ Sensitivity of Smooth Muscle and Nonmuscle Myosin II: Modulated by G Proteins, Kinases, and Myosin Phosphatase Physiol Rev, October 1, 2003; 83(4): 1325 - 1358. [Abstract] [Full Text] [PDF]
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G. S. Bogatkevich, E. Tourkina, C. S. Abrams, R. A. Harley, R. M. Silver, and A. Ludwicka-Bradley Contractile activity and smooth muscle {alpha}-actin organization in thrombin-induced human lung myofibroblasts Am J Physiol Lung Cell Mol Physiol, August 1, 2003; 285(2): L334 - L343. [Abstract] [Full Text] [PDF]
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 T.L. Ediger, N.A. Schulte, T.J. Murphy, and M.L. Toews Transcription factor activation and mitogenic synergism in airway smooth muscle cells Eur. Respir. J., May 1, 2003; 21(5): 759 - 769. [Abstract] [Full Text] [PDF]
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S. Pelletier, F. Duhamel, P. Coulombe, M. R. Popoff, and S. Meloche Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors Mol. Cell. Biol., February 15, 2003; 23(4): 1316 - 1333. [Abstract] [Full Text] [PDF]
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A. V. SOMLYO, C. PHELPS, C. DIPIERRO, M. ETO, P. READ, M. BARRETT, J. J. GIBSON, M. C. BURNITZ, C. MYERS, and A. P. SOMLYO Rho kinase and matrix metalloproteinase inhibitors cooperate to inhibit angiogenesis and growth of human prostate cancer xenotransplants FASEB J, February 1, 2003; 17(2): 223 - 234. [Abstract] [Full Text] [PDF]
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X.-F. Ming, H. Viswambharan, C. Barandier, J. Ruffieux, K. Kaibuchi, S. Rusconi, and Z. Yang Rho GTPase/Rho Kinase Negatively Regulates Endothelial Nitric Oxide Synthase Phosphorylation through the Inhibition of Protein Kinase B/Akt in Human Endothelial Cells Mol. Cell. Biol., December 15, 2002; 22(24): 8467 - 8477. [Abstract] [Full Text] [PDF]
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R. S. Ostrom, X. Liu, B. P. Head, C. Gregorian, T. M. Seasholtz, and P. A. Insel Localization of Adenylyl Cyclase Isoforms and G Protein-Coupled Receptors in Vascular Smooth Muscle Cells: Expression in Caveolin-Rich and Noncaveolin Domains Mol. Pharmacol., November 1, 2002; 62(5): 983 - 992. [Abstract] [Full Text] [PDF]
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Y. Chang, B. Ceacareanu, M. Dixit, N. Sreejayan, and A. Hassid Nitric Oxide-Induced Motility in Aortic Smooth Muscle Cells: Role of Protein Tyrosine Phosphatase SHP-2 and GTP-Binding Protein Rho Circ. Res., September 6, 2002; 91(5): 390 - 397. [Abstract] [Full Text] [PDF]
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L. Miao, Y. Dai, and J. Zhang Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H983 - H989. [Abstract] [Full Text] [PDF]
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N. Kobayashi, S. Horinaka, S.-i. Mita, S. Nakano, T. Honda, K. Yoshida, T. Kobayashi, and H. Matsuoka Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts Cardiovasc Res, September 1, 2002; 55(4): 757 - 767. [Abstract] [Full Text] [PDF]
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A. M. J. Buchan, C.-Y. Lin, J. Choi, and D. L. Barber Somatostatin, Acting at Receptor Subtype 1, Inhibits Rho Activity, the Assembly of Actin Stress Fibers, and Cell Migration J. Biol. Chem., August 2, 2002; 277(32): 28431 - 28438. [Abstract] [Full Text] [PDF]
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 N. Begum, O. A. Sandu, and N. Duddy Negative Regulation of Rho Signaling by Insulin and Its Impact on Actin Cytoskeleton Organization in Vascular Smooth Muscle Cells: Role of Nitric Oxide and Cyclic Guanosine Monophosphate Signaling Pathways Diabetes, July 1, 2002; 51(7): 2256 - 2263. [Abstract] [Full Text] [PDF]
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X. Li, L. Liu, J. C. Tupper, D. D. Bannerman, R. K. Winn, S. M. Sebti, A. D. Hamilton, and J. M. Harlan Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells J. Biol. Chem., May 3, 2002; 277(18): 15309 - 15316. [Abstract] [Full Text] [PDF]
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N. Kobayashi, S. Nakano, S.-i. Mita, T. Kobayashi, T. Honda, Y. Tsubokou, and H. Matsuoka Involvement of Rho-Kinase Pathway for Angiotensin II-Induced Plasminogen Activator Inhibitor-1 Gene Expression and Cardiovascular Remodeling in Hypertensive Rats J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 459 - 466. [Abstract] [Full Text] [PDF]
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F. Degraeve, M. Bolla, S. Blaie, C. Creminon, I. Quere, P. Boquet, S. Levy-Toledano, J. Bertoglio, and A. Habib Modulation of COX-2 Expression by Statins in Human Aortic Smooth Muscle Cells. INVOLVEMENT OF GERANYLGERANYLATED PROTEINS J. Biol. Chem., December 7, 2001; 276(50): 46849 - 46855. [Abstract] [Full Text] [PDF]
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 W. R. P. Agema, J. W. Jukema, S. N. Pimstone, and J. J. P. Kastelein Genetic aspects of restenosis after percutaneous coronary interventions;towards more tailored therapy Eur. Heart J., November 2, 2001; 22(22): 2058 - 2074. [PDF]
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L. M. Work, A. R. McPhaden, N. J. Pyne, S. Pyne, R. M. Wadsworth, and C. L. Wainwright Short-Term Local Delivery of an Inhibitor of Ras Farnesyltransferase Prevents Neointima Formation In Vivo After Porcine Coronary Balloon Angioplasty Circulation, September 25, 2001; 104(13): 1538 - 1543. [Abstract] [Full Text] [PDF]
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O. A. Sandu, M. Ito, and N. Begum Signal Transduction in Smooth Muscle: Selected Contribution: Insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle J Appl Physiol, September 1, 2001; 91(3): 1475 - 1482. [Abstract] [Full Text] [PDF]
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 T. Ishizuka, F. Okajima, M. Ishiwara, K. Iizuka, I. Ichimonji, T. Kawata, H. Tsukagoshi, K. Dobashi, T. Nakazawa, and M. Mori Sensitized Mast Cells Migrate Toward the Agen: A Response Regulated by p38 Mitogen-Activated Protein Kinase and Rho-Associated Coiled-Coil-Forming Protein Kinase J. Immunol., August 15, 2001; 167(4): 2298 - 2304. [Abstract] [Full Text] [PDF]
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H. Shimokawa, K. Morishige, K. Miyata, T. Kandabashi, Y. Eto, I. Ikegaki, T. Asano, K. Kaibuchi, and A. Takeshita Long-term inhibition of Rho-kinase induces a regression of arteriosclerotic coronary lesions in a porcine model in vivo Cardiovasc Res, July 1, 2001; 51(1): 169 - 177. [Abstract] [Full Text] [PDF]
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J. Sun, S. O. Marx, H.-J. Chen, M. Poon, A. R. Marks, and L. E. Rabbani Role for p27Kip1 in Vascular Smooth Muscle Cell Migration Circulation, June 19, 2001; 103(24): 2967 - 2972. [Abstract] [Full Text] [PDF]
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 S. R. Macfarlane, M. J. Seatter, T. Kanke, G. D. Hunter, and R. Plevin Proteinase-Activated Receptors Pharmacol. Rev., June 1, 2001; 53(2): 245 - 282. [Abstract] [Full Text] [PDF]
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C. Patterson, G. A. Stouffer, N. Madamanchi, and M. S. Runge New Tricks for Old Dogs : Nonthrombotic Effects of Thrombin in Vessel Wall Biology Circ. Res., May 25, 2001; 88(10): 987 - 997. [Abstract] [Full Text] [PDF]
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K. Morishige, H. Shimokawa, Y. Eto, T. Kandabashi, K. Miyata, Y. Matsumoto, M. Hoshijima, K. Kaibuchi, and A. Takeshita Adenovirus-Mediated Transfer of Dominant-Negative Rho-Kinase Induces a Regression of Coronary Arteriosclerosis in Pigs In Vivo Arterioscler Thromb Vasc Biol, April 1, 2001; 21(4): 548 - 554. [Abstract] [Full Text] [PDF]
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G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]
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U. Laufs, M. Endres, F. Custodis, K. Gertz, G. Nickenig, J. K. Liao, and M. Bohm Suppression of Endothelial Nitric Oxide Production After Withdrawal of Statin Treatment Is Mediated by Negative Feedback Regulation of Rho GTPase Gene Transcription Circulation, December 19, 2000; 102(25): 3104 - 3110. [Abstract] [Full Text] [PDF]
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H. Chen, U. Ikeda, M. Shimpo, M. Ikeda, S. Minota, and K. Shimada Fluvastatin Upregulates Inducible Nitric Oxide Synthase Expression in Cytokine-Stimulated Vascular Smooth Muscle Cells Hypertension, December 1, 2000; 36(6): 923 - 928. [Abstract] [Full Text] [PDF]
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D. E. Vatner and D. L. Kunze Prologue: low-molecular-weight GTPases in the heart and circulation Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1733 - H1735. [Full Text] [PDF]
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Y. Eto, H. Shimokawa, J. Hiroki, K. Morishige, T. Kandabashi, Y. Matsumoto, M. Amano, M. Hoshijima, K. Kaibuchi, and A. Takeshita Gene transfer of dominant negative Rho kinase suppresses neointimal formation after balloon injury in pigs Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1744 - H1750. [Abstract] [Full Text] [PDF]
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V. Sauzeau, H. le Jeune, C. Cario-Toumaniantz, N. Vaillant, A.-P. Gadeau, C. Desgranges, E. Scalbert, P. Chardin, P. Pacaud, and G. Loirand P2Y1, P2Y2, P2Y4, and P2Y6 receptors are coupled to Rho and Rho kinase activation in vascular myocytes Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1751 - H1761. [Abstract] [Full Text] [PDF]
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M. R. Morissette, V. P. Sah, C. C. Glembotski, and J. H. Brown The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1769 - H1774. [Abstract] [Full Text] [PDF]
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N. Sawada, H. Itoh, K. Ueyama, J. Yamashita, K. Doi, T.-H. Chun, M. Inoue, K. Masatsugu, T. Saito, Y. Fukunaga, et al. Inhibition of Rho-Associated Kinase Results in Suppression of Neointimal Formation of Balloon-Injured Arteries Circulation, May 2, 2000; 101(17): 2030 - 2033. [Abstract] [Full Text] [PDF]
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T. Ishizaki, M. Uehata, I. Tamechika, J. Keel, K. Nonomura, M. Maekawa, and S. Narumiya Pharmacological Properties of Y-27632, a Specific Inhibitor of Rho-Associated Kinases Mol. Pharmacol., May 1, 2000; 57(5): 976 - 983. [Abstract] [Full Text]
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 A. P Somlyo and A. V Somlyo Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II J. Physiol., January 15, 2000; 522(2): 177 - 185. [Abstract] [Full Text] [PDF]
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 D Nath, P. Slocombe, A Webster, P. Stephens, A. Docherty, and G Murphy Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1 )integrin, leading to a marked induction of fibroblast cell motility J. Cell Sci., January 6, 2000; 113(12): 2319 - 2328. [Abstract] [PDF]
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T. Yamakawa, S.-i. Tanaka, K. Numaguchi, Y. Yamakawa, E. D. Motley, S. Ichihara, and T. Inagami Involvement of Rho-Kinase in Angiotensin II-Induced Hypertrophy of Rat Vascular Smooth Muscle Cells Hypertension, January 1, 2000; 35(1): 313 - 318. [Abstract] [Full Text] [PDF]
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V. Sauzeau, H. Le Jeune, C. Cario-Toumaniantz, A. Smolenski, S. M. Lohmann, J. Bertoglio, P. Chardin, P. Pacaud, and G. Loirand Cyclic GMP-dependent Protein Kinase Signaling Pathway Inhibits RhoA-induced Ca2+ Sensitization of Contraction in Vascular Smooth Muscle J. Biol. Chem., July 7, 2000; 275(28): 21722 - 21729. [Abstract] [Full Text] [PDF]
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C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF]
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S. A. Sagi, T. M. Seasholtz, M. Kobiashvili, B. A. Wilson, D. Toksoz, and J. H. Brown Physical and Functional Interactions of Galpha q with Rho and Its Exchange Factors J. Biol. Chem., April 27, 2001; 276(18): 15445 - 15452. [Abstract] [Full Text] [PDF]
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T. M. Seasholtz, T. Zhang, M. R. Morissette, A. L. Howes, A. H. Yang, and J. H. Brown Increased Expression and Activity of RhoA Are Associated With Increased DNA Synthesis and Reduced p27Kip1 Expression in the Vasculature of Hypertensive Rats Circ. Res., September 14, 2001; 89(6): 488 - 495. [Abstract] [Full Text] [PDF]
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M. Eto, C. Barandier, L. Rathgeb, T. Kozai, H. Joch, Z. Yang, and T. F. Luscher Thrombin Suppresses Endothelial Nitric Oxide Synthase and Upregulates Endothelin-Converting Enzyme-1 Expression by Distinct Pathways: Role of Rho/ROCK and Mitogen-Activated Protein Kinase Circ. Res., September 28, 2001; 89(7): 583 - 590. [Abstract] [Full Text] [PDF]
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H. Chaulet, C. Desgranges, M.-A. Renault, F. Dupuch, G. Ezan, F. Peiretti, G. Loirand, P. Pacaud, and A.-P. Gadeau Extracellular Nucleotides Induce Arterial Smooth Muscle Cell Migration Via Osteopontin Circ. Res., October 26, 2001; 89(9): 772 - 778. [Abstract] [Full Text] [PDF]
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