381
Opposite Regulation of Gax Homeobox
Expression by Angiotensin II and
C-Type Natriuretic Peptide
Jun Yamashtta, Htrosht Itoh, Yoshthtro Ogawa, Naohtsa Tamura, Kazuhtko Takaya, Toshio Igalu,
Kentaro Dot, Tae-Hwa Chun, Mayumt Inoue, Ken Masatsugu, Kazuwa Nakao
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract Growth arrest-specific homeobox (Gax) gene was
isolated from rat aorta cDNA library and its expression was
largely confined to the cardrovascular trssues Gax gene was rapidly downregulated by platelet-derived growth factor m vascular
smooth muscle cells (VSMCs) and overexpressed Gax was reported to reduce the neomtlmal thtckenmg after balloon mJury m
vtvo We have demonstrated that angrotensm II (Ang II) sttmulates vascular growth In contrast, we also reported that C-type
natrmretrc peptrde (CNP) 1s secreted from vascular endothehal
cells to act as a novel endothelmm-derived relaxmg peptrde and
mhrbtts vascular growth via cGMP cascade In the present study,
we examined the effects of Ang II and CNP on Gax gene expression m VSMCs In quiescent rat aortrc VSMCs, Gax mRNA
(2 3 kb) level became neghgrble 6 hours after the addmon of Ang
II (10e6 mol/L) The mhlbttorv action of Ana II on Gax mRNA
expression (EDSo lo-” moli) was almost lompletely blocked
by an AT,R antagonist, CV11974 In contrast, CNP 10m6 mol/L
augmented Gax mRNA expression to exhibit 1 S-fold increase of
the control 12 hours after the sttmulatton This effect of CNP was
mtmrcked by the addition of 8-bromoadenosme 3’ 5’-cychc
monophosphate The addition of C-ANF[4-231, an atria1 natrturetlc pepttde-C receptor-specific agonist and devoid of sttmulatmgcGMP production,exhibitedno effecton Gax mRNA expression Srmultaneous admmlstratron of Ang II and CNP
revealed that CNP ( 1Om6mol/L) srgmficantly attenuated the mhtbrtory action of Ang II (lo-” mol/L) on Gax mRNA expression These results suggest that Gax 1s a common transcrrptton
factor involved m the slgnalmg pathway of vascular growth for
Ang II and CNP and regulates the cell cycle and/or phenotype of
VSMCs for vascular remodelmg m hypettenston and atherosclerosis (Hypertension. 1997;29[part 2]:381-387.)
Key Words l natrmretrc peptrdes l angtotensm II l
growtharrest-specifichomeobox
gene regulation
l
l
homeobox
l
growth
l
vascular smooth muscle cells
-
M
tgratton and proliferation
of VSMCs tn neomtlma are key events for the development
of
hypertensive
vascular compltcation,
atherosclerosis and coronary restenostsl-6 In contrast to
VSMCs with the contractile phenotype present m the in-
tact media of the vascular wall, m the proliferative neomtimal lesion, VSMCs exhibit the synthetic or embryonic
phenotype which is characterized by enhancedprohferative and synthetic actrvittes, reorganizatton of the cytoskeleton, and suppressedcontracttle activtty.*-6 Such alternation of phenotyprc and prohferatrve features of
VSMCs 1sthought to be modulated by various growth
factors and cytokmesrs7-‘0through the convergence of
their signaling pathways mto the network of transcrtptton
factors which govern the gene expressionsand phenotypic diversity of the cells. However, until now the mteraction between growth signalsand transcrrptton factors
concernmg the prohferatton and differentratton of
VSMCs hasnot been well understood
Gax gene was isolated from the adult rat aorta cDNA
library and its expression 1slargely confined to the cardiovascular tissuessuch as the aorta, heart, kidney and
lung.rr Gax gene was rapidly downregulated during
GO/G1 transition causedby PDGF or serum m cultured
From the Department of Medicine and Chmcal Science, Kyoto
Umversny Graduate School of Medicine, Japan
Correspondence to Huoshr Itoh, MD, PhD, Department of Medrcme and Cluucal Science, Kyoto Umversrty Graduate School of
Medrcme, 54 Shogorn Kawahara-cho, Sakyo-ku, Kyoto 606, Japan
E-mail huto@kuhp
kyoto-u ac up
0 1997 American
Heart Assoclatlon,
Inc
VSMCsi * and alsodownregulatedm the rat carotid artery
after balloon mJury.1z Recently, microinjected recombtnant Gax pepttde wasreported to inhibit mitogen-induced
entry into the S phasein VSMCsr3 and alsodemonstrated
that overexpressedGax reducedneomttmalthickening after balloon mJury.14These findings all together suggest
that Gax possesses
the crtttcal role m the growth mhtbrtton of VSMCs
We have been mvesttgatmg the growth control of
VSMCs by several vasoacttve substances.We and others
have demonstratedthat Ang II stimulates the growth of
VSMCs via the activation of PDGF, transforming growth
factor-p, and basic fibroblast growth factor 15On the
other hand, we and others reported that natrmretrc peptides inhibit the VSMC prolrferatron vta guanosme3’ 5’cyclic monophosphate(cGMP) cascadetriggered by the
acttvatton of the particulate guanylate cyclase which 1s
the natrmrettc pepttde receptor ttself 16-18
We also demonstrated that CNP, the thud member of the natnurettc
pepttde family, is secreted from vascular endothehal
cells19and possessesvascular growth mhrbttory action
via CNP-specific receptor, ANP-B receptor, highly expressedon VSMCs with the synthetic phenotype 2021
We,
thus, indicated that CNP 1sa novel endothelmm-derived
relaxing peptide involved m vascular remodeling m hypertension and other cardtovascular diseases,and proposed the existence of the vascular NPS similar to the
vascular RAS.22 Previously, we have demonstratedthat
the antagonistic relationshtp between the NPS and RAS
m blood pressurecontrol and blood fluid homeostasts,
both as an endocrine system and as a neuropeptidesystern.23We alsoreported that the vascular NPS is activated
382
Hypertension
Vol 29, No I, Part 2
JrmuurJ 1997
cDNA probe was obtained by cDNA synthesis and the polymerase chain reaction method using the total RNA of mesangial cells
derived from Wistar-Kyoto Rats with the sense, 5’-AGATGTCCTCCCCGCCAAGC-3’,
and antisense, 5’.TCCTGGGA
ATCTGAACTGTC-3’,
primers, corresponding to nucleotides
480-499 and 70 I-720 of rat Gax cDNA, I I respectively. The probe
was labeled by random-primed synthesis to the specific activity
of approximately 1 X 10” cpm/l,g. The filters were hybridized with
the “P-labeled probe at 42°C in 50% formamide, 5X SSC, 5~
Denhardt’s reagent, SO mmol/L sodium phosphate buffer (pH
6.8), 0.1% SDS and 100 mg/L heat-denatured salmon testis DNA,
and washed at 55°C in 0.1 X SSC and 0. I % SDS.” Imaging and
estimation of Gax mRNA expression were performed using image analyzer BAS 2000 (Fuji Photo Film Co. Ltd., Tokyo, Japan).
As a control, glyceraldehyde-3-phosphate dehydrogenase mRNA
level was determined as previously reported.26
Selected Abbreviations and Acronyms
8-Br-cGMP = &bromoadenosine-cGMP
Ang II = angiotensin II
ANP = atria1 natriuretic peptide
CNP = C-type natriuretic peptide
DSF = defined serum-free
Gax = growth arrest-specific homeobox gene
MAPK = mitogen-activated protein kinase
NPS = natriuretic peptide system
PCR = polymerase chain reaction
PDGF = platelet-derived growth factor
RAS = renin-angiotensin system
VSMC = vascular smooth muscle cell
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
in proliferative vascular lesions,suppressingfurther proliferation of VSMCs by antagonizing the action of the
vascular RAS.22
In the present study, to elucidate the role of Gax on
vascular growth control regulated by the antagonismbetween the RAS and NPS, we have examined the regulation of the Gax gene expressionby Ang II and CNP in
VSMCs.
Methods
Cell Culture
Cultured aortic VSMCs were derived from the explants of the
thoracic aortae of male Wistar rats as previously reported’4 and
were grown in Dulbecco’s modified Eagle’s medium (DMEM,
Flow Laboratories, Inc., Irvine, UK) supplemented with 10% fetal calf serum (FCS) (Hazleton Biologics,Inc., Lenexa,Kan.),
100 units/ml penicillin, and 100 pg/mL streptomycin. VSMCs
from 6th to 12th passages were used in the present study.
VSMCs were plated into IO-cm-diameter dishes for collection
of RNA. At about 70% confluent, cells were made quiescent by
the incubation for 48 hours in DSF medium containing insulin
(SX IO -’ mol/L), transferrin (5 pg/mL), and ascorbate (0.2
mmol/L).~s Then, the medium was replaced with fresh DSF medium and Ang 11was added. Cells were incubated for the various
times and harvested for RNA isolation. DMEM with 0.5% FCS
was used instead of DSF medium in the experiments in which
either CNP (Peptide Institute Inc., Osaka, Japan) or 8-Br-cGMP
(Sigma Chemical Co) was added.
Northern Blot Analysis
Total RNA from cultured VSMCs was prepared by the acid
guanidinium thiocyanate-phenol-chloroform
method using TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD.). Forty
p,g of total RNA per lane was electrophoresed on a formamidel
1.2% agarose gel and transferred to a nylon membrane filter. Gax
Gax
b. .z
c
mRNA
Vehtcle
All 106M
0
1
3 6 12 24(hr)
All results were expressed as mean-tSD with n=3 to 6. Statistical analysis of the data was performed using ANOVA. P<.O5
was considered significant. The experiments presented are the
representatives of at least two separate experiments.
Reagents
Human Ang 11 and human CNP were purchased from Peptide
institute Inc. 8-Bromo-cGMP was purchased from Sigma Chemical
Co. (St. Louis, MO.). CVll974, an active metabolite of TCV-116,
((A)-I-(cyclohexyloxycarbonyloxy)
ethyl 2-ethoxy-1-[[2’-( 1Htetrazol-S-yl) biphenyl-4-yl] methyl]- IH-benzimidazol-7-carboxylate}, which is an AT,R-selective non-peptide antagonist,*7 was
obtained from Takeda Chemical Industries, Ltd. (Osaka, Japan). des~Gln’8,Ser’9,Gly20,Leu2’,Gly2~]ANP-[4-23]-NH,(C-ANF[4-23])
was
purchased from Peninsula Laboratories Inc. Other reagents were purchased from standard commercial suppliers.
Results
Stimulation of VSMCs
a.
Statistical Analysis
g
ii
z 1.0
2
E
g 0.5
(II
.-iif
z
’
‘0
Regulation of Gax mRNA Expression by Ang II
The expression of Gax mRNA was analyzed in rat
VSMCs by Northern blot analysis.Gax mRNA of approximately 2.3 kilobaseswas detectedasa singlebandin quiescent VSMCs using the PCR-synthesized probe (see
“Methods”), being consistent with the reported size of
Gax mRNA, 2244 bases” (data not shown).
Fig 1 showsthe time courseof the effect of Ang II 1OA6
mol/L on Gax mRNA expression.Rapid and transient
downregulationof Gax mRNA wasobservedafter Ang II
stimulation. The downregulation occurred within 1 hour
after the stimulation and maximal at 6 hours when Gax
mRNA becamealmostnegligible. Gax mRNA level was
revealedto recover towards the baselinefrom 12 hoursto
24 hours. Fig 2 depicts the dose-dependent
effect of Ang
11on Gax mRNA expression.The left panel showsthe
1
~
1. Time course of the downregulation of
Gax mRNA expression after Ang II administratlon
” In VSMCs. a, Northern blot analysis for Gax
mRNA. Quiescent cells were stimulated by Ang
II 1Om6mol/L or vehicle and harvested after various incubation times for RNA isolation. Total
RNA (40 pg per lane) was separated on 1.2%
agarose gel and blotted to nylon membrane.
The filters were hybridized with the PCR-synthesized probe for Gax (see “Methods”). b,
Quantitative measurements of Gax mRNA levels
stimulated by Ang II 1OM6 mol/L (9 or vehicle
(0) as measured by image analyzer BAS2000
(see “Methods”). M indicates mol/L.
FIG
/
6
-O-
Vehicle
--c
All 1OW
12
18
Time (hr)
24
Yamaslzita et nl GeneRegulationby Ang II and CNP
Gax
FIG 2. Dose-dependent
effect of Ang II on Gax mRNA
expression.
a, Northern
blot analysis for Gax mRNA.
Quiescent
cells were stimulated
by vehicle or various
doses of Anq II (10 ‘* to IO-* mol/L) and harvested
3
hours after the stimulation.
Forty micrograms
of total
RNA per lane was aoolied for the analvsis. Lower panel
depicts the images biglyceraldehydel3-phosphaiedehydrogenase
mRNA. b, Quantitative
measurements
of
Gax mRNA levels stimulated
by Ang II, Bars represent
the mean?SD
(n=3). *P<.OO5
and **P<.OOi
vs vehicle-treated
cells. M indicates
mol/L.
mRNA
t
383
!
GJPDH
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
typical profile of Gax gene transcript obtained by Northern
blot analysis, and quantitative
measurements
of Gax
mRNA levels are demonstrated on the right panel. Ang II
lo-” to lo-’ mol/L suppressed Gax mRNA in the dosedependent fashion. The 50% effective dose for Gax downregulation 3 hours after the stimulation was approximately
lo-”
mol/L of Ang II. Suppression of Gax mRNA expression was observed from Ang 11 lo-‘* mol/L, and maximally 98% decrease of Gax gene transcript compared with
the control occurred by Ang II 10e8 mol/L.
To confirm the receptor subtype specificity of the inhibitory action of Ang II on Gax mRNA expression, we examined the effect of angiotensin
II type 1 receptor
(AT,-R) antagonist, CV11974. As shown in Fig 3, the
effect of Ang II-induced Gax mRNA downregulation
was
almost completely blocked by CVI 1974 10 ~6mol/L.
Regulation of Gax mRNA Expression by CNP
and S-Br-cGMP
Fig 4 shows the time course of Gax mRNA expression
after the administration
of CNP IO-’ mol/L and S-BrcGMP lo-’ mol/L. CNP 10m6 mol/L upregulated
Gax
mRNA expression 12 hours after the stimulation, and the
level of Gax mRNA tended to decrease 24 hours after the
stimulation, however, the level of Gax mRNA was still
higher than the control. The administration
of S-Br-cGMP
IO-? mol/L mimicked the effect of CNP on Gax mRNA
a.
AlllO’OM
-
CV 106M
--++
Gax
+
+
-
Interaction of Ang II and CNP on
Gax mRNA Expression
To confirm the opposite action of Ang II and CNP on
Gax mRNA expression, we examined the effect of the simultaneous administration
of Ang II and CNP. As shown
in Fig 7, 10. ’ mol/L of CNP attenuated the inhibitory action of Ang II lo-‘* to lo-’ mol/L on Gax mRNA expression 6 hours after the stimulation. The suppression of
Gax mRNA expression caused by loo-‘” mol/L of Ang II
was reversed approximately
60% by the administration
of
CNP 10m6 mol/L.
Discussion
mRNA
All
G3PDH
expression. Gax mRNA increased from 3 hours after the
stimulation, reaching a maximal level at 12 hours and also
showed the tendency to decrease 24 hours after the stimulation. The effects of CNP and 8-Br-cGMP
on Gax
mRNA
augmentation
were dose dependent
(Fig 5).
Twelve hours after the administration
of CNP or 8-BrcGMP, significant increase of Gax mRNA expression was
observed by lo-” mol/L of CNP and lo-” to 10-j mol/L
of 8-Br-cGMP,
respectively. The maximal augmentation
of Gax mRNA expression compared with the vehicle administration
was l&fold
and 2.5fold increases by IO-”
mol/L of CNP and 10m3 mol/L of S-Br-cGMP
(n=6),
respectively.
Next, we examined the effect of ANP-C receptor-specific agonist, C-ANF [4-231. ANP-C receptor is not coupled to guanylate cyclase and is proposed to have a major
role in the clearance of natriuretic peptide.“’ As shown in
Fig 6, 10m7 mol/L of C-ANF 14-231 exerted no significant
effect on Gax mRNA expression
12 hours after the
stimulation.
1O”‘M
CVIO’M
-
+
+
-
-
-
+
+
FIG 3. Inhibition
of Ang II-induced
suppression
of Gax mRNA
expression
by AT, -R antagonist
CVI 1974. a, Northern
blot analysis for Gax mRNA. Quiescent
VSMCs were treated with Ang II
(IO-”
mol/L) and/or CV11974
(CV) (10 6 mol/L) for 3 hours, and
their effects
on Gax mRNA
expression
were evaluated
by
Northern
blot analyses.
b, Quantitative
measurements
of Gax
mRNA expression
stimulated
by Ang II and/or CVl1974
as measured by image analyzer
BAS2000.
M indicates
mol/L.
The present study demonstrates
that Ang II, which
possesses the promoting
action on proliferation
and hypertrophy of VSMCs, potently induced rapid and transient downregulation
of Gax mRNA expression.
The
temporal profile of the downregulation
of Gax mRNA
expression caused by Ang II was similar to that induced
by PDGF and serum.” The present study also showed
that AT,-R
antagonist
CV11974
almost completely
blocked the inhibitory
action of Ang 11 on Gax mRNA
expression, suggesting that the effect of Ang II on Gax
gene expression is mediated via AT,-R. Ang II exhibits
proliferative
and hypertrophic
effects on VSMCs via Gprotein-coupled
AT,-R with multiple
signal transduction pathways, including the activation of phospholipase
C,28 calcium mobilization,29
the activation of protein ki-
384
Hypertension
of Gax mRNA exFIG 4. Time course of inductlon
pression
by CNP and 8-Br-cGMP.
After 48-hour
incubation
with DMEM
containing
0.5%
FCS,
VSMCs
were stimulated
by CNP (10e6 mol/L),
8-Br-cGMP
(1 Om3 mol/L), or vehicle. The cells were
harvested
at various time pornts for RNA isolation
and Northern
blot analyses
on Gax mRNA were
performed.
Forty micrograms
of total RNA per lane
was applied for the analysis. a, Representative
results of Northern
blot analyses.
b, Quantitative
measurements
of Gax mRNA levels stimulated
by
vehicle (o), CNP (0), and 8-Br-cGMP
(m) as measured by Image analyzer
BAS2000.
M indicates
mol/L.
a.
0 3 1224
Gax mRNA
(hr)
Vehicle
1
CNP
‘
II,
lO=M
1:
8E?r-cGMP
1
IOJM
$
0
18
6
12
Time (hr)
24
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
nasecl,‘0 the induction of proto-oncogenes,31,‘2
protein
tyrosine kinase phosphorylation,3’ the activation of
MAPK,‘4,“5 and the stimulation of Januskinase /STAT
(signal transducersand activators of transcription) pathway337 On the other hand, PDGF exerts the proliferative action mainly via the activation of the receptor
intrinsic protein tyrosine kinase,“* and other pathways
similar to AT,-R.38,39Recent study revealed that AT,-R
signal transduction pathway converges into the PDGFp receptor signaling cascadesand results in the activation of proteins upstream of ~21’“’ which initiates the
phosphorylation cascadestowards the MAPK activation.39 Similar action on Gax mRNA expression of Ang
II and PDGF suggeststhat the gene regulation of Gax
expression is under control of the molecules existed in
the downstream after the convergence of Ang II and
PDGF receptor signaling pathways, such as MAPK.
Ang II exerted quite potent effect on Gax mRNA suppression. Ang II lo-” mol/L and Ang II IO-’ mol/L
caused50% and 98% reduction of Gax mRNA expression
a,
2 WCWM)
$ f-6
G8xmRNA
-7
-6’
*a
_jl
I
:
d
s!
2 log@Br-cGW](M)
c.
c
*43’-6
Gax
3 hours after the stimulation, respectively. Previously, we
have demonstratedthat Ang II induced the growth of
VSMCs, but significant growth stimulation was observed
by more than lOmymol/L of Ang II.16 The reasonfor the
difference of sensitivity to Ang II betweenGax expression
and DNA synthesisis not known at present.The present
and previous studiesdemonstratedthat the suppressionof
Gax mRNA expressionis immediately early event in the
cell cycle progression,that is, that occurred at leastfrom
3 hours to 12 hours after the mitogen stimulation, corresponding to the period from GO/G1 transition to Gl/S
boundary.40To achieve further progressionof the cell cycle and initiation of DNA synthesis,the participation of
other factors should be necessaryin addition to the suppressionof Gax mRNA expressionmechanism.It can be,
however, at least saidthat the suppressionof Gax mRNA
expressionis a highly sensitivemarker of growth stimulation of VSMCs.
In contrastto the effect of Ang 11,CNP and S-Br-cGMP
upregulatedGax mRNA expression.It hasbeen reported
-5
d
l
-
i 3.0
P
L!
r
P
108
10-7
106
CNP (M)
t
-4 -3 ’
mRNA
G3PDH
c
r
g
106
104
8Br-cGMP
IO-4
103
(M)
effect of CNP and
FIG 5. Dose-dependent
8-Br-cGMP
on Gax mRNA expression.
After
48-hour
incubation
with DMEM
containing
0.5% FCS, VSMCs
were stimulated
by vehicle and various doses of CNP (1 O-* to 10m6
mol/L) [a and b] or 8-Br-cGMP
(1 O-” to 1 Om3
mol/L) [c and d] for 12 hours. Total RNA extraction
and Northern
blot analyses
were
performed
as described
in “Methods.”
a and
c, Representative
results of Northern
blot
analyses.
b and d, Quantitative
measurements of Gax mRNA levels. Bars represent
the meantSD
(n=3 to 6). *P<.O5, **P<.OOi
vs vehicle-treated-cells.
M Indicates mol/L.
Yamudzita
FIG 6. Effect of ANP-C receptor specific agonist, CANF[4-231
on Gax mRNA
expression.
After treatment
with
DMEM
containing
0.5%
FCS for 48 hours,
VSMCs
were treated
with
vehicle.
C-ANF14-231
(CANF) (iOm7 mok)
or ‘8-kcGMP
(10m3 mol/L) for 12
hours. Their effects on Gax
mRNA
expression
were
evaluated
by Northern
blot
analyses.
Gax mRNA
G3PDH
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
that Gax mRNA expressionwasdependenton the cell yuiescence,that is, Cax was expressedat low levels in subconfluent, but its mRNA level increasedwithin 8 hours
after the cellc are placed in serum-poormedium.40We
demonstratedhereinthat CNP upregulatedGax mRNA expressionin a similar time course with the serum starvation” in a dose-dependentmanner.The administrationof
S-Br-cGMP mimicked the effect of CNP, and C-ANF
14-231,the specific agonist for ANP-C receptor which is
not coupledwith guanylatecyclaseand exhibits no cGMP
genesisby natriuretic peptides in VSMCs,z’ exerted no
significant effect on Gax mRNA expression.All theseresults suggestthat CNP up-regulatesGax mRNA expression via the activation of cGMP cascade.This is, to our
knowledge, the first report that growth inhibitory signals
up-regulatethe Gax mRNA expression.As we have previously demonstrated,CNP possesses
the growth inhibitory effect on VSMCs via cGMP cascade.cGMP hasbeen
reportedto suppressthe proliferation of VSMCs by inhibiting the progressionfrom the Gl into S phaseof the cell
cycle.-II Indeed, recently we demonstratedthat overexpressionof CNP in VSMCs by adenovirussystemcaused
the Gl growth arrestof VSMCs, by flow cytometry analysiv and Northern blot analysisfor mRNA expressionsof
cyclins and cyclin-dependentkinasesrequiredfor the cell
cycle progressionin GI/S boundary.42The antiproliferalive action via cGMP pathway may be mediatedby Gax,
which might control the expressionsof the genesregulating Cl/S boundary.
We have proposedthe counterregulatoryeffects of NPS
on RAS not only in blood pressureand body fluid homeostasiv through vasorelaxation, diureyis and natriuresis,
a.
CNP
WW~)
GJPDH
nn
- -12-10
(-)
CNP
-8
lo%4
- -12-10
-8
et al
Gene Regulation
by Ang II and CNP
38.5
andinhibition of aldosteronesecretion,“‘,“4but alsoin vascular growth control.22 The present study revealed that
CNP also exerted the antagonistic effect on Ang II-induced suppressionof Gax mRNA expression,suggesting
that the antagonisticeffect between Ang II and CNP on
vascular growth is controlled via the regulation of Gax
geneexpression.Now we examinethe involvement of Gax
in the growth control of VSMCs by Ang II andCNP using
the antisenseoligonucleotidemethodfor Gax gene,aswe
previously reported.15Taking theseresultstogether, it is
indicated that Gax can be involved in the signalingpathway of vascular growth control by Ang II and CNP as a
common transcription factor and may act as an intracellular switch for controlling on and off of cell proliferation.
Homeobox gene family is well known to be important
in the regulation of organogenesis,
body plan formation in
embryo and cell differentiation as well as cell proliferation.40Somehomeoboxgenescontribute to the regulation
of both cell proliferation and differentiation. For example,
an intestine-specifichomeoboxgene,Cdx-2, leadsto arrest
of proliferation and inducesenterocyte-specificgene expressionsin an undifferentiated intestine cell line, IEC-6
cells.45Gax is a cardiovascular-specifichomeobox gene
that participates in the growth inhibition of VSMCs.
VSMCs are known to dedifferentiate into the synthetic or
embryonic phenotypewhen they re-enterthe cell cycle by
mitogen stimulation.8.46,47
Inferring from multiple functions of the homeobox gene family on cell proliferation
and differentiation, Gax may take someroles on differentiation of VSMCs aswell asthe growth inhibitory effect.
Indeed, the recent studiesdemonstratedthat overexpression of catalytic domain of cGMP-dependent protein kinase,which is one of the major signalingmoleculesfor
the cGMP cascade,leadsthe alternationof morphology of
cultured VSMCs into differentiated phenotype.“x In addition, CNP is alsoreported to stimulatethe differentiation
of osteoblastic
cells via cGMP cascade.“”It is, therefore,
suggestedthat CNP may promote not only the growth inhibition but also VSMC differentiation by upregulating
Gax geneexpressionvia cGMP cascade.Further study to
find target genesof Gax for VSMC differentiation should
be required.
Recently, Walsh et al reported that lransfer of Gax gene
into VSMCs after balloon injury hasbeenshownto inhibit
cell proliferation and neointima formation in vivo.14 For
gene therapy againstvascular proliferative diseasessuch
FIG 7. Interaction
of Ang II and CNP on Gax
mRNA expression.
Quiescent
VSMCs were simultaneously
treated with various doses of Ang
II (10 ” to lo-*
mol/L)
and/or
CNP (10 m6
mol/L) for 6 hours and their effects
on Gax
mRNA expression
were evaluated
by Northern
blotting.
a, Representative
results of Northern
blot analyses.
The four lanes on the left show
the effect of vehicle or Ang II (lo-‘*
to lOma
mol/L) without CNP administration,
whereasthe
four lanes on the right show the effect of simultaneous
administration
of CNP
(1 O--”
mol/L) and either vehicle or Ang II. b, Quantitative measurements
of Gax mRNA levels after
administration
of CNP (IO-” mol/L) and either
vehicle or Ang II (IO-”
mol/L). Bars represents
the mean&SD
(n=5 or 6). *P<.O5
vs corresponding
control values. M indicates
mol/L.
386
Hypertension
Vol29,
No 1, Part 2
January 1997
ashypertensivevascular comphcation,atherosclerosisand
coronary restenosls, common intracellular molecules
which affect the cell cycle progressionsuchasretmoblastoma gene,~53 and~21‘I” are recommendedasgood targets 14.50
The present study revealed that Gax can be a
commonregulator of VSMC growth control by many vascular growth promoting and mlubitmg substancesmcludmg Ang II and CNP, and may be involved m VSMC dlfferentiatlon. Gax is, therefore, a suitable candldate for
gene therapeutictarget for vascular diseases
In concluaon, we demonstratedthat Ang II and CNP
oppositely regulate Gax gene expressionm VSMCs The
findings suggestthat Gax 1sa commontranscriptionfactor
involved m the slgnalmgpathway of vascular growth for
Ang II and CNP, and regulatesthe cell cycle and/or phenotype of VSMCs for vascularremodelingm hypertension
and atherosclerosis
Acknowledgments
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
This work was supportedm part by researchgrantsfrom the
Japanese
Ministry of Education,Science,andCulture,theJapaneseMinistry of HealthandWelfareDisordersof AdrenalHormoneresearch
commlttee,theMolecularApproachfor thePathogeneslsof ImmunologicalDisorderresearchcommittee,the
SmokmgResearch
Foundation,the YamanouchlFoundationfor
Researchon Metabolic Disorders,the Salt ScienceResearch
Foundation,the UeharaMemorialFoundation,andtheJapanese
Societyfor Cardiovascular
Diseases
We thankHlsayoKlto and
Yuko Marl for thenexcellentsecretarial
work
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Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Opposite Regulation of Gax Homeobox Expression by Angiotensin II and C-Type
Natriuretic Peptide
Jun Yamashita, Hiroshi Itoh, Yoshihiro Ogawa, Naohisa Tamura, Kazuhiko Takaya, Toshio
Igaki, Kentaro Doi, Tae-Hwa Chun, Mayumi Inoue, Ken Masatsugu and Kazuwa Nakao
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension. 1997;29:381-385
doi: 10.1161/01.HYP.29.1.381
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