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Isoflavone attenuates vascular contraction through
inhibition of RhoA/Rho-kinase signaling pathway
Young Mi Seok, Inji Baek, Yong-Hoon Kim, Yeon-Shin Jeong, In-Jung Lee,
Dong Hyun Shin, Young Hyun Hwang and In Kyeom Kim*
Institute (I.K.K.), Kyungpook National University School of Medicine, Daegu,
700-422, Republic of Korea; Department of Biochemistry, College of Natural
Sciences (Y-H.K.), Division of Plant Biosciences, College of Agriculture and Life
Sciences (Y-S.J., I-J.L., D.H.S., Y.H.H.), Kyungpook National University, Daegu,
702-701, Republic of Korea
1
Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
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Department of Pharmacology (Y.M.S., I.B., I.K.K.), Cardiovascular Research
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Running title
Isoflavone inhibits RhoA/Rho-kinase pathway
*Correspondence and Proofs
InKyeom Kim, M.D., Ph.D.
Department of Pharmacology
Kyungpook National University School of Medicine
Tel: +82-53-420-4833
Fax: +82-53-426-7345
E-mail: [email protected]
Number of pages: 26
Number of tables: 0
Number of Figures: 6
References: 39
Number of Words in abstract: 217
Number of Words in introduction: 367
Number of Words in discussion: 1405
Abbreviations
myosin light chain (MLC20); myosin light chain phosphatase (MLCP); myosin
phosphatase targeting subunit 1 (MYPT1); protein kinase C (PKC); PKC-potentiated
inhibitory protein for heterotrimeric MLCP of 17 kDa (CPI17); phorbol 12, 13-dibutyrate
(PDBu).
Section assignment: Cardiovascular
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Daegu, 700-422, Republic of Korea
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Abstract
Isoflavones decrease blood pressure, improve lipid profiles, and restore vascular
function. We hypothesized that isoflavone attenuates vascular contraction by inhibiting
RhoA/Rho-kinase signaling pathway. Rat aortic rings were denuded of endothelium,
mounted in organ baths, and contracted with U46619, a thromboxane A2 analogue or
KCl 30 min after the pretreatment with genistein, daidzein or vehicle. We determined
the phosphorylation level of the myosin light chain (MLC20), myosin phosphatase
for heterotrimeric myosin light chain phosphatase of 17 kDa (CPI17) by means of the
Western blot. We also measured the amount of GTP RhoA as a marker regarding
RhoA activation. The cumulative additions of U46619 or KCl increased vascular
tension in a concentration-dependent manner, which were inhibited by pretreatment
with genistein or daidzein. Both U46619 (30 nM) and KCl (50 mM) increased MLC20
phosphorylation levels which were inhibited by genistein as well as daidzein.
Furthermore, both genistein and daidzein decreased the amount of GTP RhoA
activated by either U46619 or KCl. U46619 (30 nM) increased phosphorylation of the
MYPT1Thr855 and CPI17Thr38, which were also inhibited by genistein or daidzein.
However, neither genistein nor daidzein inhibited PDBu-induced vascular contraction
and CPI17 phosphorylation. In conclusion, isoflavone attenuates vascular contraction,
at least in part, through inhibition of the RhoA/Rho-kinase signaling pathway.
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targeting subunit 1 (MYPT1) and protein kinase C (PKC)-potentiated inhibitory protein
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Introduction
Isoflavones have been a subject of intensive researches that have evaluated their
possible hypocholesterolemic effects, antioxidant effects, and estrogen-like effects on
blood vessels (Kapiotis et al., 1997; Nestel et al., 1997). Investigations in regards to
animals and in humans have suggested that dietary soy protein containing isoflavones
reduce blood pressure, improve lipid profiles, and restore vascular function (Teede et
al., 2001). The 3 major isoflavones found in soybeans are genistein (4',5,7-
methoxydaidzein). Soy protein containing isoflavones has been observed to have
several beneficial effects on cardiovascular health; a meta-analysis study showed that
total cholesterol decreased by 9.3%, triglyceride by 10.5% and low-density lipoprotein
cholesterol by 12.9%, when an average of 47 g of soy protein was consumed daily
(Anderson et al., 1995). Soybean isoflavones were suggested to inhibit proliferation of
vascular smooth muscle and thus contribute to the prevention of atherosclerotic
cardiovascular diseases (Pan et al., 2001).
The small guanosine triphosphatase RhoA plays an important role as a molecular
switch in the enhancement of Ca2+ sensitivity of smooth muscle contraction (Hirata et
al., 1992). Activated RhoA increases the Ca2+ sensitivity of both MLC phosphorylation
and contractions in smooth muscle (Gong et al., 1995). Rho-kinase is an effector of the
small GTP-binding protein RhoA and is a major cellular regulator for agonist-induced
Ca2+-sensitization in smooth muscle contraction. Rho-kinase directly phosphorylates
the myosin phosphatase targeting subunit 1 (MYPT1) at Thr855 of myosin light-chain
phosphatase (MLCP) and consequently inhibits phosphatase activity. Rho-kinase also
phosphorylates protein kinase C (PKC)-potentiated inhibitory protein for heterotrimeric
MLCP of 17 kDa (CPI17) (Eto et al., 1995), which directly inhibits the catalytic domain
of MLCP. CPI17 can be phosphorylated by both Rho-kinase and PKC.
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trihydroxyisoflavone), daidzein (4',7-dihydroxyisoflavone), and glycitein (6-
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Genistein and daidzein prevented agonist-induced vascular contraction in isolated
rat aortic rings (Vera et al., 2005). There are few reports exhibiting whether isoflavones
exert vasorelaxation by modulating the RhoA/Rho-kinase signaling pathway or
phosphorylation of MYPT1 or CPI17.
It was hypothesized that isoflavone attenuates vascular contraction by inhibiting the
RhoA/Rho-kinase signaling pathway. Therefore, the purpose of present study was to
investigate the inhibitory effects of isoflavones on RhoA activation and subsequent
induced by U46619 or KCl.
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phosphorylation of MYPT1 or CPI17, as well as on vascular smooth muscle contraction
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Materials and Methods
Materials
U46619, genistein, daidzein, PDBu, Ro31-8220, KCl and phenylephrine were
obtained from the Sigma Chemical Co (St. Louis, MO, USA). Stock solutions of
U46619, genistein, daidzein, PDBu and Ro31-8220 were prepared in DMSO. All other
reagents were analytical grade.
Tissue preparation and tension measurement
Animals published by the US National Institute of Health (Health, 1996). Male SpragueDawley rats, weighing 320-350 g, were used. With animals under anesthesia (sodium
pentobarbital 50 mg kg-1 i.p.), the thoracic aorta was immediately excised and
immersed in an ice-cold, modified Krebs solution composed of (in mM) NaCl, 115.0;
KCl, 4.7; CaCl2, 2.5; MgCl2, 1.2; NaHCO3, 25.0; KH2PO4, 1.2; and dextrose, 10.0. The
aorta was cleaned of all adherent connective tissue on wet filter paper, soaked in the
Krebs-bicarbonate solution and cut into four ring segments (4 mm in length) as
described by Seok et al (Seok et al., 2006). All rings were denuded of endothelium by
gently rubbing the internal surface with the edge of forceps. Two stainless-steel
triangles were inserted through each vessel ring. Care was taken to avoid rubbing the
endothelial surface of the vessels that had intact endothelium. Each aortic ring was
suspended in a water-jacketed organ bath (22 ml) maintained at 37°C and aerated with
a mixture of 95% O2 and 5% CO2. One triangle was anchored to a stationary support,
and the other was connected to an isometric force transducer (Grass FT03C, Quincy,
Mass., USA). The rings were stretched passively by imposing the optimal resting
tension, 2.0 g, which was maintained throughout the experiment. Each ring was
equilibrated in the organ bath solution for 90 min before the experiment involving the
contractile response to 50 mM of KCl. Isometric contractions were recorded using a
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The investigation is in accordance with Guide for the Care and Use of Laboratory
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computerized data acquisition system (PowerLab/8SP, ADInstruments, Castle Hill,
NSW, Australia).
For the contractile response, aortic rings were pretreated with 10, 30 and 100 µM
genistein or daidzein for 30 min, and subjected to cumulative addition of U46619 or
KCl.
Western blot
After functional study, muscle strips were quickly immersed in acetone containing
aortic rings were washed in acetone containing 5 mM DTT to remove TCA, air-dried
and stored at -80°C until use. Previously stored samples were homogenized in a buffer
containing 320 mM sucrose, 50 mM TRIS, 1 mM EDTA, 1% Triton X-100, 1 mM DTT,
and protease inhibitors leupeptin (10 µg/ml), trypsin inhibitor (10 µg/ml), aprotinin (2
µg/ml), phenylmethylsulphonyl fluoride (PMSF; 100 µg/ml) and phosphatase inhibitor
β-glycerophosphate (50 mM). Protein-matched samples (Bradford assay) were
electrophoresed (SDS-PAGE), transferred to nitrocellulose membranes, and subjected
to an immunoblot with a pMYPT1 antibody (1:4,000, Upstate Biotechnology, Lake
Placid, NY, USA) and a pCPI17 antibody (1:250, Santa Cruz Biotechnology, Inc) that
detects phosphorylated MYPT1 and CPI17. Anti-rabbit IgG and anti-goat IgG,
conjugated with horseradish peroxidase, were used as secondary antibody (1:4,000,
Sigma, St. Louis, MO, USA and 1:1,000, Santa Cruz Biotechnology, Inc). The bands
containing pMYPT1 and pCPI17 were detected with enhanced chemiluminescence
(ECL) visualized on films. The nitrocellulose membranes were striped of the pMYPT1
and pCPI17 antibody and reblotted with total form of MYPT1 antibody (1:4,000, BD
Biosciences Pharmingen, San Diego, CA, USA) and CPI17 antibody (1:500, Upstate
Biotechnology, Lake Placid, NY, USA). Anti-mouse IgG and anti-rabbit IgG conjugated
with horseradish peroxidase, were used as secondary antibody (1:4,000, Sigma, St.
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10% trichloroacetic acid (TCA) and 10 mM dithiothreitol (DTT) precooled to -80°C. The
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Louis, MO, USA and 1:1,000, Upstate Biotechnology, Lake Placid, NY, USA).
MLC20 phosphorylation
Muscle strips were quick frozen by immersion in acetone containing 10% TCA and 10
mM DTT precooled to -80°C. Muscles were washed four times with acetone containing
5 M DTT for 15 min each to remove TCA and were soaked 1 hr with frequent vortex in
100 µl of urea sample buffer containing 20 mM Tris base/23 mM glycine (pH 8.6), 8.0
M urea, 10 mM DTT, 10% glycerol, and 0.04% bromphenol blue. 12.5% polyacrylamide
running buffer consists of 20 mM Tris base/23 mM glycine (pH 8.6), 2.3 M thioglycolate,
and 2.3 mM DTT. The urea-extracted samples (12 µl) were electrophoresed at 300 V
for 6 hours, transferred to nitrocellulose membranes, and subjected to immunoblotting
with a specific myosin light chain 20 antibody (1:2,000, Sigma, St. Louis, MO, USA).
Anti-mouse IgM (goat), conjugated with horseradish peroxidase, was used as a
secondary antibody (1:4,000, Stressgen, Victoria, BC Columbia, Canada). The bands
containing myosin light chains were visualized with enhanced chemiluminescence
(ECL) on films, and then analyzed by NIH image as described (Kim et al., 2004).
Assay for RhoA activation: Muscle strips were quick frozen by liquid nitrogen, and
stored at -80 °C. For RhoA activation assays, the procedure followed the
manufacture’s instruction regarding RhoA G-LISATM Activation Assay (Cytoskeleton Inc,
Denver, CO, USA ). Briefly, previously stored samples were homogenized in a lysis
buffer, and were centrifuged at 13,200 rpm for 15 min at 4°C. The supernatant,
containing 37 µg of proteins, was transferred into a plate and equal volumes of ice-cold
binding buffer mixtures were added into each well. The plate was shaken on a cold
orbital microplate shaker (300 rpm) for 30 min at 4°C, flicked out of the solution from
wells, and incubated with diluted anti-RhoA primary antibodies followed by secondary
antibodies on a microplate shaker (300 rpm) at room temperature for 45 min each. The
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gels containing 40% glycerol were pre-electrophoresed for 30 min at 200 V. The
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plate was incubated with an HRP detection reagent for 15 min at 37°C and, after
addition of an HRP stop buffer, the absorbance was immediately recorded at 490 nm.
Statistical Analysis
Data are expressed as mean ± SEM and were analyzed by repeated measures
ANOVA or one-way ANOVA followed by the post-hoc Tukey’s test in order to determine
statistical significance. P values of less than 0.05 were regarded as significant.
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Results
Effect of pretreatment with genistein or daidzein on the U46619- or KCl-induced
contractions
Thirty minutes after the pretreatment with genistein, daidzein or vehicle, the
concentration-response relationships to U46619 or KCl in endothelium-denuded aortic
rings were obtained by means of a cumulative addition of the chemicals (Fig. 1). The
tension is expressed as a percentage to initial contractions in response to 50 mM KCl.
response curves rightward (Fig. 1A). The contractile response to KCl was inhibited by
pretreatment with either genistein or daidzein (Fig. 1B).
Inhibitory effect of genistein or daidzein on U46619- or KCl-induced MLC20
phosphorylation
To determine whether isoflavones affect U46619 (30 nM)- or KCl (50 mM)-induced
MLC20 phosphorylation, aortic rings were pretreated with genistein, daidzein or vehicle
for 30 min before addition of U46619 or KCl. Both genistein (30 and 100 µM) and
daidzein (30 and 100 µM) decreased U46619- or KCl-induced MLC20 phosphorylation
(Fig. 2).
Inhibitory effect of genistein or daidzein on U46619- or KCl-induced RhoA
activation
After pretreatment with genistein, daidzein or vehicle for 30 minutes, the addition of
U46619 (30 nM) or KCl (50 mM) induced contractions, reaching a plateau in 10 min.
We freeze-clamped aortic rings contracted with U46619 or KCl and determined the
amounts of GTP-RhoA by using RhoA G-LISATM Activation Assay. U46619 or KCl
increased GTP-RhoA as compared with the basal level (2.60 ± 0.55 fold or 1.83 ±
0.10 fold, respectively) in endothelium-denuded aortic rings (Fig. 3). Both genistein
(100 µM) and daidzein (100 µM) almost completely suppressed U46619- or KCl-
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Pretreatment with genistein or daidzein shifted U46619-induced concentration-
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induced GTP-Rho (Fig. 3).
Inhibitory effect of genistein or daidzein on U46619-induced MYPT1Thr855
phosphorylation
U46619 induced contractions in the rat caudal artery, accompanied by Rho kinasedependent phosphorylation of MYPT1 at Thr855, without affecting the level of
phosphorylation at Thr697 (Wilson et al., 2005; Tsai and Jiang, 2006; Jeon et al.,
2007). It was examined whether U46619 induced phosphorylation of MYPT1Thr855 in the
MYPT1Thr855. If U46619-induced Rho-kinase activation is inhibited by isoflavone, the
phosphorylation level of MYPT1Thr855 should decrease. U46619 (30 nM) was applied 30
min after the pretreatment with 30 or 100 µM isoflavones (genistein or daidzein). As
shown in Fig. 4, the phosphorylation level of MYPT1Thr855 significantly decreased by 30
or 100 µM genistein (1.32 ± 0.29 and 0.39 ± 0.13, respectively) or 30 or 100 µM
daidzein (1.55 ± 0.31 and 0.85 ± 0.19, respectively).
Inhibitory effect of genistein or daidzein on U46619-induced CPI17Thr38
phosphorylation
CPI17 can be phosphorylated at Thr38 in rat aorta (Fig. 5). The phosphorylation
level of CPI17Thr38 was significantly decreased by 30 and 100 µM genistein (0.55 ±
0.08 and 0.10 ± 0.04, respectively) and 30 and 100 µM daidzein (0.60 ± 0.13 and 0.08
± 0.04, respectively) when compared to the phosphorylation level induced by U46619
alone.
Effect of Ro31-8220, genistein or daidzein on PDBu-induced contraction and
CPI17Thr38 phosphorylation
Thirty minutes after the pretreatment with Ro31-8220 (1.0 µM), genistein, daidzein or
vehicle, aortic rings were challenged with an addition of 0.1 µM phorbol 12, 13dibutyrate (PDBu). The tension is expressed as a percentage to initial contractions in
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rat aorta. Fig. 4 shows that U46619 (30 nM) increased the phosphorylation level of
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response to 50 mM KCl. Pretreatment with Ro31-8220, a PKC inhibitor, inhibited the
vascular contractility and phosphorylation of CPI17Thr38 in response to PDBu. However,
both genistein and daidzein had no effect on the PDBu-induced vascular contraction
and CPI17 phosphorylation (Fig. 6).
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Discussion
The present study demonstrates that isoflavones attenuate vascular contraction
through inhibition of the RhoA/Rho-kinase signaling pathway. The isoflavones
attenuated the activation of RhoA and subsequent Rho-kinase-dependent
phosphorylation of MYPT1 and CPI17. However, isoflavones did not affect
phosphorylation of CPI17 induced by an activation of PKC.
U46619, a thromboxane A2 mimetic, as well as KCl activated RhoA, increased
genistein and daidzein (Fig. 1, 2, and 3). RhoA/Rho-kinase activation plays an
important role in the tonic component of KCl- or U46619-induced contraction and
MLC20 phosphorylation (Mita et al., 2002; Wilson et al., 2005). Both genistein and
daidzein also inhibited U46619-induced phosphorylation of MYPT1Thr855 and CPI17Thr38
(Fig. 4 and 5), which are substrates of Rho-kinase. These results indicate that
isoflavones suppress the U46619- or KCl-induced RhoA/Rho-kinase signaling pathway
and are in agreement with reports that genistein strongly inhibited U46619- or
vanadate-induced RhoA activation in the smooth muscles of rabbits or guinea pigs
(Sakurada et al., 2001; Mori and Tsushima, 2004).
The activity of myosin phosphatase decreases when PKC-potentiated inhibitory
protein for heterotrimeric MLCP of 17 kDa (CPI17) or MYPT1 is phosphorylated (Feng
et al., 1999; Ito et al., 2004). We examined the level of phosphorylated MYPT1Thr855,
which is an inhibitory site and is phosphorylated by the Rho-kinase. Activation of Rhokinase by U46619 or phenylephrine, an α1-adrenergic agonist, phsophorylates MYPT1
at Thr855, but not MYPT1 at Thr697 (Wilson et al., 2005; Tsai and Jiang, 2006; Jeon et
al., 2007). The phosphorylation of MYPT1Thr697 is independent of the stimulation of Gproteins, Rho-kinase or PKC (Kitazawa et al., 2003; Niiro et al., 2003). We observed
that both genistein and daidzein inhibited MYPT1Thr855 phosphorylation induced by
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phosphorylation of MLC20, and induced contraction which were inhibited by both
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U46619 (Fig. 4) or by phenylephrine (data not shown). Altogether these results suggest
that Rho-kinase mediates MYPT1Thr855 phosphorylation in response to the activation of
receptors coupled to G12/13 or Gq/11.
CPI17 is another potential mediator of Ca2+ sensitization that is independent of
MYPT1 phosphorylation. CPI17 is a 17-kDa peptide, first isolated from porcine aorta
(Eto et al., 1995), in which phosphorylation enhances its potency for inhibiting the
catalytic subunit of myosin phosphatase (Seko et al., 2003; Pang et al., 2005).Pang et
the PKC inhibitor GF109203X, but was abolished by a Rho-kinase inhibitor Y-27632
(Pang et al., 2005), suggesting that Rho-kinase is involved in U46619-induced CPI17
phosphorylation and contractions. These reports showed that U46619 caused CPI17
phosphorylation through activation of the RhoA/Rho-kinase pathway. Our data also
showed that U46619 significantly increased CPI17Thr38 phosphorylation, which was
inhibited by both genistein and daidzein (Fig. 5). Daidzein inhibited CPI17Thr38
phosphorylation induced by phenylephrine (data not shown).
Genistein and daidzein are structurally very similar, differing only in the substitution
at position 5 (a hydroxyl group absent in daidzein) (Nakashima et al., 1991). The
difference shows that genistein is a well known inhibitor of a broad range of tyrosine
kinase, whereas daidzein lacks inhibitory activity regarding the tyrosine kinase.
However, both genistein and daidzein inhibited Rho-kinase and vascular contractions,
suggesting that the inhibitory action of genistein or daidzein is independent of tyrosine
kinase inhibition.
Isoflavones inhibited vascular contractions and MLC20 phosphorylation induced by
U46619 or KCl. Genistein and daidzein rightward shifted concentration-response
curves to U46619 and to KCl in endothelium-denuded aortic rings. These data are
consistent with previous reports that have used several vascular beds from male
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al. have shown that the U46619-induced CPI17 phosphorylation was not affected by
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Wistar Kyoto rats and spontaneously hypertensive rats (Vera et al., 2005) as well as
normotensive animals (Martinez et al., 2000; Janssen et al., 2001).
Flavonoids including genistein not only antagonized thromboxane A2 receptors in
platelets and smooth muscle cells but also impaired U46619-induced calcium
mobilization in a concentration-dependent manner (Guerrero et al., 2007). In Fig. 1A,
daidzein shifted the concentration-response curves rightward with similar Emax,
whereas genistein shifted the concentration-response curves rightward with Emax
competitively antagonizes binding of U46619 to the receptor. A radioligand
displacement study may be needed to elucidate whether daidzein is a competitive
antagonist to U46619. However, the flavone, similarly to genistein, shifted the
concentration-response curves rightward with decrease in Emax (Jeon et al., 2007),
suggesting that flavonoids may have two or more mechanisms to inhibit Rho-kinase
pathway.
In order to figure out the target sites of isoflavone regarding the Rho-kinase
signaling pathway, we contracted aorta with KCl which activates Rho-kinase by means
of a different mechanism. KCl induces membrane depolarization and the subsequent
influx of extracellular Ca2+ (Mita et al., 2002). Isoflavones also inhibited vascular
contraction induced by KCl. The fact that there were dose-dependent genistein- and
daidzein-induced inhibition of KCl-mediated contractions supports the notion that the
inhibitory targets of isoflavones with respect to vascular contractions are located
somewhere in the Rho-kinase signaling pathway besides thromboxane A2 receptors.
Although isoflavones suppressed GTP RhoA activation induced by U46619
and KCl, the molecular mechanism by which isoflavones inhibit RhoA activation
remains to be elucidated. GDP dissociation inhibitors (RhoGDI), guanine nucleotide
exchange factors (GEFs), Rho GTPase activating proteins (RhoGAPs), and GTP RhoA
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being decreased. The inhibitory patterns imply that daidzein, rather than genistein,
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are all upstream of the Rho-kinase pathway. In a resting state, GDP RhoA resides in
cytosol with its prenylated, hydrophobic tail buried within its partner known as RhoGDI.
Activation of receptors coupled to certain trimeric G proteins (Gαq, Gα12,13) leads,
through the activity of GEFs, to the exchange of GTP for GDP on RhoA. GTP RhoA
associates with the membrane, where it interacts with Rho-kinase in order to initiate
signaling cascades. When RhoGAPs catalyze hydrolysis of GTP bound to RhoA, GDP
RhoA reassociates with RhoGDI (Somlyo and Somlyo, 2003). In addition, a report has
Rho activation in vascular smooth muscles (Wang et al., 2006). Several studies implied
that PI3K class II α-isoform (PI3K-C2α) plays a role in the Ca2+- dependent Rho
activation (Stein and Waterfield, 2000; Azam et al., 2007). Isoflavone may suppress
GTP RhoA activation, induced by U46619 or KCl, through interaction with GEFs,
RhoGAPs, RhoGDI or PI3K.
The phosphorylation of CPI17Thr38 was shown to be mediated by both Rho-kinase
and PKC in vascular as well as other smooth muscle tissues (Eto et al., 2001; Niiro et
al., 2003). PDBu-induced contraction and CPI17Thr38 phosphorylation were suppressed
by Ro31-8220, an inhibitor of PKC, but insensitive to genistein or daidzein (Fig. 6).
These results are consistent with previous reports showing that the PKC inhibitor
GF109203X, but not the Rho-kinase inhibitor Y27632, abolished only PDBu-induced
CPI17Thr38 phosphorylation. In addition, GF109203X did not inhibit thrombin- or
U46619-induced CPI17Thr38 phosphorylation (Pang et al., 2005; Azam et al., 2007).
These observations suggest that genistein and daidzein inhibited the phosphorylation
of CPI17Thr38 induced by Rho-kinase, but not PKC.
MLC20 is known to be phosphorylated by MLCK, Rho-kinase (Somlyo and Somlyo,
2003) and some of the Ca2+-independent kinases including zipper-interacting protein
kinase (ZIPK), integrin-linked kinase (ILK), and citron kinase. (Deng et al., 2001). ZIPK
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suggested that a phosphoinositide 3-kinase (PI3K) is essential for the Ca2+-dependent
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and ILK contract smooth muscles in a Ca2+-independent way (Deng et al., 2001) and
are also able to diphosphorylate MLC20, all of which were not inhibited by the Rhokinase inhibitor (Kiss et al., 2002).
Activation of Rho-kinase by U46619 or KCl inhibits myosin light chain phosphatase
through phosphorylation of MYPT1Thr855, leading to an increased MLC20
phosphorylation and contraction (Sakurada et al., 2003; Wilson et al., 2005). U46619
or KCl each monophosphorylated MLC20, which was inhibited by genistein or daidzein.
phosphorylation induced by either vanadate (Mori and Tsushima, 2004) or
angiotension II (Rattan et al., 2003).
The soybean isoflavones, genistein, daidzein and glycitein, may have potential
benefits in regards to human health maintenance due to their biological effects (Pan et
al., 2001). Isoflavones (phytoestrogens) have gained considerable attention for their
potential role in improving risk factors regarding cardiovascular diseases (Sacks et al.,
2006). Genistein, daidzein and glycitein inhibit the growth and DNA synthesis of aortic
smooth muscle cells in stroke-prone spontaneously hypertensive rats (Pan et al., 2001).
It has recently been proven that activation of the RhoA/Rho-kinase signal transduction
pathway is one of the principal mechanisms of vasoconstriction in arterial hypertension,
and that this pathway is a novel therapeutic target for regulating arterial hypertension
(Johns et al., 2000).
In conclusion, isoflavone attenuates vascular contraction by inhibiting RhoA/Rhokinase signaling. These results suggest that isoflavone inhibited vascular contractions
in response to U46619 or KCl, through dysinhibition of MLCP, by inhibiting activation of
RhoA and the subsequent phosphorylation of MYPT1Thr855 and CPI17Thr38.
17
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This result is in accordance with the finding that genistein decreased MLC20
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Footnotes
This study was supported by the Korea Research Foundation Grant funded by the
Korean Government (MOEHRD, Basic Research Promotion Fund) (KRF-2006-511E00011), the Technology Development Program for Agriculture and Forestry, Ministry
of Agriculture and Forestry, Republic of Korea, and the Brain Korea 21 Project in 2008.
Pharmacology. Kyungpook National University School of Medicine. Daegu.
Republic of Korea. E-mail: [email protected]
23
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Send reprint requests to: InKyeom Kim, M.D., Ph.D. Department of
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Legends for figures
Fig. 1. Effect of genistein and daidzein on U46619 (A)- or KCl (B)-induced contraction.
U46619 or KCl was each added cumulatively to elicit tension 30 min after pretreatment
with genistein, daidzein or vehicle to denuded aortic rings. Developed tension is
expressed as a percentage of the maximum contraction to 50 mM KCl. Data are
expressed as the means of 5 experiments with vertical bars showing SEM. * P < 0.05,
** P < 0.01 as compared to vehicle (repeated measures ANOVA).
MLC20 phosphorylation in rat aorta. U46619 (30 nM) or KCl (50 mM) were each added
to elicit tension 30 min after pretreatment with genistein, daidzein or vehicle to
denuded aortic rings. Phosphorylation of MLC20 was measured when the tension
reached plateaus and was expressed as a percentage of the total MLC20. Data are
expressed as the means of 5 experiments with vertical bars showing SEM. ## P < 0.01
as compared to the basal. * P < 0.05, ** P < 0.01 as compared to U46619 or KCl alone.
Fig. 3. Inhibitory effect of genistein and daidzein on U46619 (A)- or KCl (B)-induced
RhoA activation in rat aorta. U46619 (30 nM) or KCl (50 mM) were each added to elicit
tension 30 min after pretreatment with genistein, daidzein or vehicle to denuded aortic
rings. RhoA activation was assessed by RhoA G-LISATM Activation Assay. Absorbance
at the control (OD of around 0.4) was expressed as 1 arbitrary unit. Data were
expressed as the means of 4 experiments with vertical bars showing SEM. ## P < 0.01
as compared to the basal. ** P < 0.01 as compared to U46619 or KCl alone.
Fig. 4. Inhibitory effect of genistein (A) and daidzein (B) on U46619-induced MYPT1
phosphorylation in rat aorta. U46619 (30 nM) was added to elicit tension 30 min after
24
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Fig. 2. Inhibitory effect of genistein and daidzein on U46619 (A)- or KCl (B)-induced
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pretreatment with genistein, daidzein or vehicle to denuded aortic rings. MYPT1
phosphorylation at Thr855 was assessed by Western blots. Upper and lower bands in
representative Western blots were probed with anti-p-MYPT1 and anti-total MYPT1
antibodies, respectively. The ratio of density of phosphorylated MYPT1 (upper) to that
of the total MYPT1 (lower) regarding the control was expressed as 1 arbitrary unit.
Data are expressed as the means of 5 experiments with vertical bars showing SEM. ##
P < 0.01 as compared to the basal. * P < 0.05, ** P < 0.01 as compared to U46619
Fig. 5. Inhibitory effect of genistein (A) and daidzein (B) on U46619-induced CPI17
phosphorylation in rat aorta. U46619 (30 nM) was added to elicit tension 30 min after
pretreatment with genistein, daidzein or vehicle to denuded aortic rings. CPI17
phosphorylation at Thr38 was assessed by means of Western blots. Upper and lower
bands in representative Western blots were probed with anti-p-CPI17 and anti-total
CPI17 antibodies, respectively. The ratio of density of phosphorylated CPI17 (upper) to
that of the total CPI17 (lower) for treatment with 30 nM U46619 alone was expressed
as 1 arbitrary unit. Data are expressed as the means of 4 experiments with vertical
bars showing SEM. ## P < 0.01 as compared to the basal. * P < 0.05, ** P < 0.01 as
compared to U46619 alone.
Fig. 6. Effect of protein Kinase C inhibitor Ro31-8220, genistein or daidzein on PDBuinduced contraction and CPI17 phosphorylation in rat aorta. PDBu (0.1 µM) was added
to elicit tension 30 min after pretreatment with Ro31-8220, genistein, daidzein or
vehicle to denuded aortic rings. (A) Developed tension was expressed as a percentage
of the maximum contraction to 50 mM KCl. (B) CPI17 phosphorylation at Thr38 was
assessed by means of Western blots after PDBu treatment. Upper and lower bands in
25
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alone.
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representative Western blots were probed with anti-p-CPI17 and anti-total CPI17
antibodies, respectively. The ratio of density of p-CPI17 (upper) to that of total CPI17
(lower) for treatment with PDBu was expressed as 1 arbitrary unit. Data are expressed
as the means of 4 experiments with vertical bars showing SEM. ** P < 0.01 as
compared to PDBu alone.
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26
Fig. 1
140
** **
Vehicle
10 µM Daidzein
30 µM Daidzein
100 µM Daidzein
160
120
100
80
60
40
20
0
140
120
100
80
60
40
20
0
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0
U46619 (-log M)
KCl 50 mM contraction (%)
100
** **
80
60
40
20
0
0
10
20
KCl (mM)
30
Vehicle
10 µM Daidzein
30 µM Daidzein
100 µM Daidzein
120
KCl 50 mM contraction (%)
Vehicle
10 µM Genistein
30 µM Genistein
100 µM Genistein
120
U46619 (-log M)
40
50
100
** **
80
60
40
20
0
0
10
20
KCl (mM)
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(B)
* **
30
40
50
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KCl 50 mM contraction (%)
160
Vehicle
10 µM Genistein
30 µM Genistein
100 µM Genistein
KCl 50 mM contraction (%)
(A)
Fig. 2
MLC20
phosphorylation (%)
MLC20
p-MLC20
50
##
50
##
40
40
*
30
20
**
10
*
30
**
20
10
0
Genistein (µM) 0
U46619 (30 nM) -
0
0
+
30
+
100
+
Daidzein (µM)
0
-
0
+
30
+
100
+
(B)
40
40
##
*
20
*
20
**
**
10
10
0
Genistein (µM)
KCl (50 mM)
##
30
30
0
0
-
0
+
30
+
100
+
Daidzein (µM)
0
-
0
+
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MLC20
phosphorylation (%)
MLC20
p-MLC20
30
+
100
+
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(A)
Fig. 3
(B) 3
4
##
2
**
** **
**
1
##
2
**
** **
1
**
0
0
+
10
+
30
+
100
+
10
+
30
+
100
+
Genistein (µM)
Daidzein (µM)
KCl (50 mM)
-
+
10
+
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Genistein (µM) Daidzein (µM)
U46619 (30 nM) -
Arbitrary unit
Arbitrary unit
3
30
+
100
+
10
+
30
+
100
+
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(A)
Fig. 4
(B)
p-MYPT1Thr855
p-MYPT1Thr855
3.0
##
2.5
2.0
*
1.5
**
1.0
0.5
0.0
Genistein (µM)
U46619 (30 nM)
0
-
0
+
30
+
100
+
Fold increase over control
MYPT1
3.0
##
2.5
2.0
*
1.5
**
1.0
0.5
0.0
Daidzein (µM)
U46619 (30 nM)
0
-
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Fold increase over control
MYPT1
0
+
30
+
100
+
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(A)
CPI17
CPI17
0.6
0.4
0.4
**
0.2
**
0.2
0.0
0.0
100
+
30
+
0
+
0
Daidzein (µM)
U46619 (30 nM)
*
*
0.6
100
+
1.0
1.0
##
Fold decrease over U46619
p-CPI17Thr38
0.8
0.8
30
+
0
+
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0
Genistein (µM)
U46619 (30 nM)
p-CPI17Thr38
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(B)
(A)
1.2
##
1.2
Fold decrease over U46619
Fig. 5
Fig. 6
(B)
p-CPI17Thr38
Fold decrease over PDBu
150
100
50
**
0
Ro31-8220 (1 µM ) Genistein (µM)
Daidzein (µM )
PDBu (0.1 µM)
+
+
+
30
+
100
+
30
+
100
+
1.2
1.0
0.8
0.6
0.4
**
0.2
0.0
Ro31-8220 (1 µM ) Genistein (µM)
Daidzein (µM )
PDBu (0.1 µM)
-
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KCl 50 mM contraction (%)
CPI17
200
+
+
+
30
+
100 30
+
+
100
+
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(A)