Regulation of connective tissue growth factor (CTGF/CCN2) gene
transcription and mRNA stability in smooth muscle cells
Involvement of RhoA GTPase and p38 MAP kinase and sensitivity to actin dynamics
Ibrul Chowdhury
1,
* and Brahim Chaqour
2
1
Department of Anatomy and Cell Biology, University of Pennsylvania, PA, USA;
2
Department of Anatomy and Cell Biology,
State University of New York (SUNY) Downstate Medical Center, Brooklyn, NY, USA
Connective tissue growth factor (CTGF/CCN2) is an
immediate early gene-encoded polypeptide modulating cell
growth and collagen synthesis. The importance of CTGF/
CCN2 function is highlighted by its d isregulation in fibrotic
disorders. In this study, we investigated the r egulation and
signaling pathways that are required for various stimuli of
intracellular signaling events to induce the expression of the
endogenous CTGF/CCN2 gene in smooth muscle cells.
Incubation with the bioactive lysolipid sphingosine 1-phos-
phate (S1P) produced a threefold increase , whereas stimu-
lation with either fetal bovine serum or anisomycin induced
an even stronger act ivation (eightfold) of CTG F/CCN2
expression. Using a combination of pathway-specific inhib-
itors and mutant forms of signaling molecules, we found that
S1P- and fetal bovine serum-induced CTGF/CCN2 expres-
sion were dependent on both RhoA GTPase and p38
mitogen-activated protein kinase transduction pathways ,
whereas the e ffects of anisomycin largely involved p38 and
phosphatidyl inositol 3-kinase signaling mechanisms.

fibrotic tissue areas suggesting a potential role of CTGF /
CCN2 in the p athogenesis of fibrosis. T hus, CTGF/CCN2
emerged not only as a useful prognostic and diagnostic
marker of tissue fibrosis, but also as a viable t herapeutic
target. Early studies revealed that CTGF/CCN2 may act, in
part, as a downstream mediator of the profibrotic effects of
transforming growth factor (TGF)-b which, itself, is a potent
inducer of CTGF/CCN2 expression in fibroblasts [4,5].
We, and others, have previously shown that aberrant
expression of CTGF/CCN2 occurs during the pathological
remodeling of smooth muscle-rich tissues associated with
bladder obstructive diseases, atherosclerosis, restenosis and
airway smooth muscle in a sthma [6–9]. However, in many
cases, upregulation of the CTGF/CCN2 gene is neither
preceded nor accompanied by a concomitant i ncrease in
TGF-b expression and/or activity suggesting that CTGF/
CCN2 i s not systematically a downstream effector of
Correspondence to B. Chaqour, Department of Anatomy and Cell
Biology, SUNY Downstate M edical Center, 450 Clarkson Avenue,
Box 5, Br ooklyn, NY 11203–2098, USA. Fax: +1 718 270 3732,
Tel.: +1 718 270 8285, E-mail: brah i [email protected]
Abbreviations: RE, AU-rich element; CA, constitutively active kinase;
CTGF/CCN2, connective t issue g rowth fa ctor; DMEM, Dulbecco’s
modified Eagle’s medium; DN, dominant negative ki nase; EC M,
extracellular matrix; FB S, f etal bo vine s erum; GA PDH, g lyceralde-
hyde-3-phosphate dehydrogenase; IFN, interferon; IL, interleukin;
JNK, c-Jun N-terminal ki nase; MAP, mito gen-activated protein;
MKK, MAP k inase k inases; S1P, s phingosine 1 -phosphate; SMC,
smooth muscle cell; S RF, s erum response fa ctor; TGF, tr ansforming
growth factor; UTR, untranslated region; VEGF, vascular endothelial

stimuli, thereby controlling the genomic and physiological
response o f t he cells . The MAP k inase p athway was
subdivided into t he extracellular-regulated k inase (Erk1/2),
the c-Jun N-terminal kinase (JNK) and the 38-kDa MAP
kinase (p38). The Erk1/2 pathway is largely regulated by the
GTPase Ras and was implicated in TGF-b-induced CTGF/
CCN2 expression, while members of the Rho GTPase family
regulate the JNK and p38 MAP kinases. The role of these
signaling molecules is prominent in the regulation of cell
cycle and cell differentiation particularly in stress-related
pathologies including hypertension, bladder o bstructive
diseases and atherosclerosis [8,15,16].
We undertook this study to investigate t he role of Rho
GTPase and MAP kinase signaling pathways in the modu-
lation of the CTGF/CCN2 gene in respo nse to dive rse
extracellular stimuli known for their ability to activate the
Rho GTPase and/or MAP kinase signaling molecules in
SMCs. We found that RhoA–actin signaling transcription-
ally affects t he CTGF/CCN2 expression, w hile the p 38 MAP
kinase modulates the CTGF/CCN2 gene at the level of
mRNA stability. However, all s ignals depend on the a ctin
cytoskeleton integrity. In partic ular, t he G-actin levels
modulate CTGF/CCN2 gene expression and suffice for its
activation indicating that the actin cytoskeleton is a conver-
gence point for signals emanating from various stimuli.
Materials and methods
Materials
Dulbecco’s modified Eagle’s medium (DMEM) was
obtained from Life Technologies, Inc. (Grand Island, NY,
USA). Sphingosine 1-phosphate (S1P) were obtained from

culture
flasks or 60-mm dishes. Twenty-four hours later, cells were
washed with DMEM to remove traces of se rum, placed in
serum-free medium and stimulated with exogenous factors
as indicated in the text. To test the effects of specific
inhibitors of signaling molecules, the cells were left in the
presence of a g iven inhibitor a t least 30 min followed by the
addition of chemical stimuli for an additional 1 h.
RNA isolation and northern blot analysis
Total RNA was extracted from cells using TRIzol Reagent
from Invitrogen. A sample containing 12 lgoftotalRNA
was fractionated by electrophoresis in 1% (w/v) agarose/
formaldehyde gel, transferredtoZeta-Probenylonfilters
(Bio-Rad, R ichmond, CA, USA) and hybridized with
radiolabeled cDNA probes a s described previously [12].
Total RNA loading a nd transfer were evalua ted by p robing
with a glyceraldehyde-3-phosphate dehydrogenase (GAP-
DH) cDNA probe. The filters were analyzed by phosphori-
maging and hybridization signals were quantified to
determine t he relative amounts of CTGF/CCN2 mRNA
(Molecular Dynamics, Sunnyvale, CA, USA). The mRNA
levels were analyzed in duplicate and normalized to
equivalent values f or GAPDH t o c ompensate for variations
in loading and transfer.
mRNA stability assay
Cells were cultured in tissue culture flasks a s described above
and either preincubated or not with pharmacological inhib-
itors and further treated with various stimuli for 30 min. The
culture medium was then replaced with serum-free medium
containing actinomycin D (10 lgÆmL

20 m
M
Tris/HCl (pH 8.0), 75 m
M
NaCl, 0.5 m
M
EDTA,
1m
M
dithiothreitol and 50% (v/v) glycerol. In vitro tran-
scription was then performed with the suspended nuclei at
30 °C for 30 min in a buffer containing 10 m
M
Hepes
(pH 8.3) , 5 m
M
MgCl2, 300 m
M
KCl, 50 m
M
EDTA, 1 m
M
dithiothreitol, 0.1 m
M
rCTP, rATP, rGTP and 250 lCi of
[
32
P]UTP[aP]. The radiolabeled RNA was extracted from
the nuclei. Equal amounts (2.5 lg) of CTGF/CCN2 and
GAPDH cDNA probes w ere vacuum transferred onto a

blot as described above. Transfection efficiency was evalu-
ated using fluorescence microscopy in cells cotransfected
with plasmid containing the green fluorescent protein gene
(pEGFP-N1) from Clontech. The transfection efficiency
varied bet ween 35 and 45% using 1 lg of pEGFP-N1 per
10
5
cells.
Expression vectors
Plasmids encoding constitutively active (CA) and dominant
negative (DN) kinases and GTPases were use in this study.
These include CA-RhoA, CA-Cdc42, CA-Rac1 and their
respective D N forms and the corresponding empty v ector as
described previously [18]. Other expression vectors used
include CA-MKK3, CA-MKK4 and CA-MKK6 [19,20].
Immunoblotting, immunodetection and
immunohistochemical analyses
For western blot analyses, ce lls were cultured in 35-mm
dishes under normal cell culture conditions. After
incubation with various stimuli, the cells were washed
twice with NaCl/P
i
and cell l ysates were prepared by
harvesting the cells in 0.1% (v/v) Triton X-100 lysis
buffer. Protein concentration was determined by using the
Bradford protein a ssay (Bio-Rad). Protein samples (20 lg)
were separated by 10% (w/v) SDS/PAGE, transferred to
nitrocellulose membranes and further incubated overnight
with the primary antibody as indicated in the text.
Immunodetection was performed by enhanced chemi-

2
and a
cocktail of protease inhibitors (Roche). Specific Rho and
Cdc42/Rac-binding domains were used to affinity preci-
pitate the GTP-bound forms of these GTPases. The
precipitated complexes were then fractioned by electro-
phoresis and detected by immunoblot analysis, using a
polyclonal anti-Rho (-A, -B, -C), Cdc42 and Rac1 Igs.
Total RhoA, Cdc42 and Rac1 in each lysate were
determined by western b lotting.
G-Actin/F-actin
in vitro
assay
Determination o f the amount of filamentous (F-actin)
content compared with free globular actin (G-actin)
content was performed using the F-actin/G-actin in vivo
assay kit from Cytoskeleton according t o the manufac-
turer’s instructions. Briefly, upon exposure to various
stimuli and/or inhibitors, the cells were homogenized in
cell lysis and F-actin stabilization buffer [50 m
M
Pipes,
50 m
M
NaCl, 5 m
M
MgCl
2
,5m
M

M
), anisomycin
(10 n gÆmL
)1
) or FBS (5%). As s hown i n F ig. 1 A, treatment
of the cells with S1P induced only a moderate and
monophasic increase in C TGF/CCN2 transcripts, whereas
either anisomycin or FBS induced a strong and biphasic
increase in the steady-state levels of CTGF/CCN2 mRNA.
Maximum stimulation was i nduced by seru m with five- a nd
ninefold increases in CTGF/CCN2 mRNA levels after 1
and 6 h, respectively. Nearly similar in creases were observed
in anisomycin-treated cells, a nd a 3.1-fold transient stimu-
lation was observed in S1P-treated cells. Similarly, the
CTGF/CCN2 protein levels, analyzed by western blotting,
increased upon stimulation with S1P, anisomycin o r FBS,
although the increase seemed to occur in a time-dependent
manner and not biphasically like the mRNA, probably
because of differences between the half-lives of CTGF/
CCN2 mRNA and protein (Fig. 1B); protein turnover
being slower that that of the mRNA [21]. Meanwhile, the
micromolar concentration of S1P used in our experiments
was within the range reported t o occur e ither physiologically
or in serum. Low S1P concentrations (in the nanomolar or
picomolar range) were without effects (data not shown).
Higher concentrations were not used to avoid potential
nonspecific and/or toxic effects. In contrast, anisomycin
induced CTGF/CCN2 expression over a wide range of
concentrations e.g. 1–100 ngÆmL
)1

CTGF mRNA Levels (%)
0 s an ser s/ser ser/an s/an
1000
900
800
S1P
Anisomycin
Serum
700
600
600
500
400
300
200
100
0
500
400
300
200
100
0
0
0.5
124616
Fig. 1. Stim ulation of CTGF /CCN2 gene expression by S1P, aniso-
mycin and fetal bovine s erum. (A) Cells were left untreated as a c on trol
(C) or treated with S 1P (s) at a concentration of 10 l
M

derivative that specifically targets RhoA GTPase signaling.
As shown in Fig. 2, treatment of the cells with toxin B
significantly altere d S1P-, anisomycin- a nd serum-induced
CTGF/CCN2 expression. When the cells were pretreated
with the inhibitor Y -27632, serum- and S1P-induced CTGF/
CCN2 expression was significantly reduced, while aniso-
mycin-induced CTGF/CCN2 expression was not as much
affected (P<0.05). Both toxin B and the Y-27632 inhibitor
were used at a concentration that selectively and effectively
induced maximal i nhibition of Rho GTPase signaling
[23,24]. These data pinpoint to an important role for RhoA
GTPase signaling in CTGF/CCN2 gene regulation.
Incubation of various cell types with stimulatory agents
triggers several signal-transduction pat hways that culminate
in the a ctivation of RhoA, Cdc42 and Rac1, the most
A
B
CTGF
ToxB
- - - - + + + - - -
ToxB
- - - - + + + - - -
Y-27632
- - - - - - - + + +
Y-27632
- - - - - - + + +
GAPDH
CTGF mRNA Levels (%)
c s an ser s an ser s an sr
*

later, total R NA was e xtracted and subjected to no rthern blot an alysis
with CTGF/CCN2 and GAPDH probes. Shown is the percentage of
therelativeincreaseinmRNAlevels.Thevaluesarethemeans±SD
(n ¼ 3). *P < 0.05 compared with stimulated cells in the absence of
inhibitors.
A
Serum - + + +
S1P - + + +
GTP-RhoA
0 5 10 15 min
0 5 10 15 min
GTP-Cdc42
GTP-Cdc42
GTP-Rac1
CTGF
GAPDH
GTP-Rac1
Total-RhoA
Total-Cdc42 Total-Cdc42
Total-Rac1
Total-Rac1
Total-RhoA
GTP-RhoA
B
C
D
N
-
R
h

-
R
h
o
A
E
m
p
t
y
V
e
c
t
o
r
D
N
-
C
d
c
4
2
D
N
-
R
a
c

a
c
1
E
m
p
t
y
V
e
c
t
o
r
C
A
-
C
d
c
4
2
AC
-
R
a
c
1
AC
-

*
control
S1P
Anisomycin
Serum
Fig. 3. Effects of RhoA, Cdc42 and Rac1 on the expression of the CTGF/
CCN2 gene. (A) Immunoblot analyses of RhoA, Cdc42 and Rac1
activation by S1P and FBS. Cells were s timulated with either 1 0 l
M
S1P
or 5% serum for the indicated periods and t he amount of GTP-loaded
RhoA, Cdc42 and Rac1 was determined by pull-down assay as des-
cribed in Mate rials a nd m ethod s. T otal a mount o f R hoA, C dc42 an d
Rac1 in the same s am ples was determined b y western blot and immu-
nodetection analyses. (B) Cultured cells were transfected with th e
dominant negative forms DN-Rho A, DN-Cdc42 or DN -Rac1. Control
cells were transfected with the pCDNA3 empty vector. Twenty-four
hours later, the cells were stimulated for 1 h with either S1P, anisom ycin
or FBS and the mRNA levels of the endogenous CTGF/CCN2 gene
were determined by northern blot h ybridization analysis. Shown is t he
percentage of the relative i ncrease i n mRNA levels. The values are the
means ± SD (n ¼ 3). * P < 0.05 compared w ith s timulated cells that
were transfected with t he empty vector. (C) C ells were transfected with
the constitutively active forms CA-RhoA, CA-Cdc42 or CA-Rac1.
Twenty-four hours later, the cells were incubated in serum-free medium
for 8 h and the mRNA levels of the endogen ous CTGF/CCN2 gene
were determined by northern blot hybridization. The diagram is rep-
resentative o f three separate experiments w ith nearly similar results.
4440 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
thoroughly studied Rho GTPase proteins [14]. As shown in

induced CTGF/CCN2 expression, whereas only RhoA
seems to be involved in S1P-induced CTGF/CCN2 mRNA
levels.
To further establish the specificity of action of Rho
GTPases on CTGF/CCN2 expression, we examined the
ability of the constitutively active form s of Rho GTPases t o
enhance the expression of the endogenous CTGF/CCN2
gene. As shown in Fig. 3C, transfection of the cells with
CA-RhoA and C A-Cdc42 induced a 215 and 1 75% increase
in CTGF/CCN2 mRNA levels, respectively (P<0.05).
Conversely, the active form CA-Rac1 f ailed to affect the
expression of CTGF/CCN2, thus corroborating the previ-
ous data obtained with the dominant negative form of
Rac1. The relatively potent activation of the endogenous
CTGF/CCN2 gene by the active mutants of RhoA and
Cdc42 may simply reflect the ability of Rho GTPases when
activated individually to recruit, perhaps nonspecifically,
signaling mechanisms more effectively t han when they
are simultaneously activated in response to an external
stimulus [26].
Actin polymerization inhibitors affect
CTGF
/
CCN2
expression
Increasing amounts of e vidence support a n obligatory
role for the actin cytoskeleton in the regulation of specific
genes by small GTPase proteins. The m orphology of the
actin cytoskeleton upon treatment of t he cells with S1P,
anisomycin or serum was visualized with rhodamine–

Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4441
not result in dramatic changes in stress fiber intensity.
However, preincubation of the cells with toxin B
dramatically altered the existing stress fiber network
independent of the applied stimulus. Treatment of the
cells with the Y-27632 inhibitor altered the cytoskeleton
integrity as w ell (data not shown). A lso, almost total
disruption of the actin cytoskeletal organization was
observed when the cells were pretreated with latruncu-
lin B, a toxin that disrupts the actin cytoskeleton by
sequestering G-actin monomers, therefore inhibiting actin
polymerization ( Fig. 4). T reatment of the cells with
latrunculin B alone completely depolymerized stress
fibers. These cells showed no spatial organization of
F-actin other than a few marginal patches and contained
unusual F-actin patches rather than organized microfila-
ment b undles. Stimulation of latrunculin B-treated cells
with S1P, anisomycin or serum similarly disrupted the
morphology of the actin cyto skeleton.
To determine whether a ctin cytoskeleton organization is
critical for CTGF/CCN2 gene expression, we examined
the effects of latrunculin B on CTGF/CCN2 mRNA
levels in response to various stimuli. As shown in Fig. 5A,
stimulation of latrunculin B-tre ated cells with either S1P
or serum dramatically decreased the expression levels of
CTGF/CCN2 by a factor of 2.9 and 3.2, respectively,
indicating a causal relationship between CTGF/CCN2
gene induction and actin tre admilling. In addition, treat-
ment of the cells with latrunculin B significantly reduced
the CTGF/CCN2 m RNA levels i n response to aniso-

stabilizer of F-actin. In contrast, treatment of the cells
with swinholid e A did not affect the intensity of F-actin
stress fibers in the b asal state. However, F-actin bundles
appear shorter and contained s ignificantly l ess b ranching,
consistent with the r ole of swinholide A as a p romoter of
G-actin dimerization. Interestingly, both jasplakino-
lide and swinholide A activated CTGF/CCN2 expres-
sion in a time-dependent manner, albeit to different
extents (Fig. 6B). The CTGF/CCN2 mRNA levels were
increased six- and threefold after 1–2 h in the presence of
jasplakinolide and swinholide A, respectively, an d
decreased rapidly thereafter. Jasplakinolide and swinho-
lide A were used at concentrations (1 l
M
and 1 0 n
M
,
respectively) that exhibit optimal effects on actin dynamics
[29]. However, t he observation that swinholide A, which
promotes actin monomer dimerization rather than poly-
merization, enhanced basal expression of the CTGF/
CCN2 gene suggests that a key d eterminant factor of the
effects of actin on CTGF/CCN2 expression is the actual
CTGF
LtB - + - - - + + +
LtB - + + + + +
c c s an ser s an ser
GAPDH
GAPDH
A

CTGF/CCN2 hybridization signals were normalized to those of
GAPDH. Values are means ± SD from three experiments.
4442 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
physiologic states of G-actin monomers within the cells.
Correspondingly, both latrunculin B and jasplakionolide
increased t he expression of CTGF/CCN2 even though
they exert opposite effects on F-actin. Considering the
specific effects of these drugs, they actually all decrease the
levels of free G-actin but via different mechanisms.
Jasplakinolide depletes the pool of free G-actin by
promoting actin polymerization and stabilizing the resul-
tant actin filaments, whereas latrunculin B and swinho-
lide A directly sequester free G-actin and render G-actin
monomers, at least temporarily, unavailable f or the
polymerization process. In agreement with these observa-
tions, pretreatment of the cells with either latrunculin B or
swinholide A delayed jasplakinolide-induced CTGF/CCN2
expression but did not block it. This is consistent with the
fact that these drugs bind reversibly to different types of
actin targets (Fig. 6C). I n addition, jasplakinolide and
swinholide A had no effects on the expression of TGF-b1,
a potent inducer of CTGF/CC N2 expression, although
their effects on the a ctivation o f p re-existing T GF-b1
protein is unknown (data not shown). The pharmacolo-
gical effects of these drugs are only partially understood,
and some of their unknown effects may affect gene
expression as well.
Changes in G-actin/F-actin ratio correlate with RhoA
GTPase activation
Because the expression of CTGF/CCN2 seemed to be under

M
) for 30 min and then fixed, p ermea-
bilized and stained for F-actin with rhodam-
ine-conjugated phalloidin. (B) The kinetics of
CTGF/CCN2 mRNA accumulation in jas-
plakinolide- and swinholide A-treated cells
were determined for the indicated time peri-
ods. Total RNA was extrac ted an d analyzed
by northern blot hybridization. The diagram is
representative of three independent experi-
ments with similar results. (C) Cells were pre-
treated for 15 min with either latrunculin B
(0.5 l
M
) or swinholide A (0.1 l
M
) prior to the
addition of jasplak inolide (0.5 l
M
). Total
RNA w as extracted at the indicated t imes an d
analyzed by northern blot hybridizatio n. The
diagrams are representative of three se parate
experimen ts w ith s im ilar r esul ts.
Table 1. Effects o f S1P, anisomycin and fetal serum on the G- t o F-actin ratio. G- to F-actin r atio was determined upon stimulation of the cells with
either S1P, anisomycin or fetal serum for 30 min. The role of RhoA GTPase was assessed by pre-treating the cells with Rho kinase inhibitor,
Y-27632 (10 l
M
) for 30 min prior to the addition of various stimuli. Values are the means ± SD of four experiments.
Control +S1P +Anisomycin +Serum

ratio, suggesting that RhoA /actin-independent signaling
mechanisms are involved in anisomycin-induced CTGF/
CCN2 exp ression.
CTGF
/
CCN2
gene regulation through MAP kinase
signaling
Because Rho GTPases r egulate cytoskeletal reorganization
and g ene e xp ression e ither directly or throu gh the activation
of members of the MAP kinase family, we investigated
whether CTGF/CCN2 expression is mediated via signaling
molecules of the MAP kinase signal transduction network.
S1P stimulation induced the phosphorylation of E rk1/2 a nd
p38 o n ly, whereas FBS or anisomycin stimulation s eemed to
induce that of JNK1/2 as well (Fig. 7A). Differences in the
kinetic parameters of activation of these kinases i n S1P-,
anisomycin- a nd serum-treate d cells were observed. Bec ause
the p rotein levels of MAP kinases remain unchanged
throughout the course of stimulation , dephosphorylation b y
phsophatases would be the key factor in the type of pattern
of activation of the MAP kinase in response to various
stimuli. Activation o f p38 and JNK1/2 appeared substan-
tially stronger in anisomycin-treated cells relative to that
in serum-stimulated cells. In addition , serum, S1P or
anisomycin induced the phosphorylation of PKB/Akt, a
well-known downstream effector of phosphatidylinositol
A
B
P-Erk1/2

s
(
%
)
c s an ser s an ser s an ser s an ser s an ser
*
*
*
*
*
*
No Inhibitor
+ Pd-098059 + SB-203580
+ SP-600125
+ Wortmanin
No Inhibitor
+ Pd-098059
+ SB-203580
+ SP-600125
+ Wortmanin
Fig. 7. Effects of MAP kinase and
PtdIns 3-kinase inhibitors on S1P-, aniso-
mycin- and FBS-induced CTGF/CCN2
expression. (A) Cells were treated for the
indicated periods with S1P (s), anisomycin
(an) or serum (ser), lysed and 20 lgofeach
protein lysate were subjected to SDS–PAGE.
Proteins were transferred to nitrocellulose
membrane and immunoblotted for phos-
phorylated and total Erk1/2 (P-Erk1/2 and

3). For each stimulus, the mRNA levels of
CTGF/CCN2 were compared in the presence
and in the absence of the drugs. Inhibition was
significant with P < 0.05(*).
4444 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
3-kinase (PtdIns 3-kinase) that acts either downstream or
upstream of the MAP k inases.
To determine the role of these signaling molecules in
CTGF/CCN2 expression, cells we re pretreated for 30 min
with Pd- 098059 (20 l
M
), SB-20856 (10 l
M
), SP-60 0125
(10 l
M
), or worthmanin (10 l
M
), which inhibit Erk1/2, p38,
JNK1/2, and PtdIns 3-k inase, respectively. These inhibitors
were used at a c oncentration that s pecifically and e ffectively
induced maximal inhibition of Erk1/2, p38, JNK1/2 and
PtdIns 3-kinase [18,30]. The incubation was further contin-
ued in the presence of S1P, anisomycin or serum for an
additional 1 h. As shown in Fig. 7B, Pd-098059 minimally
affected S1P-, anisomycin- and serum-induced CTGF/
CCN2 gene expression indicating that inducible CTGF/
CCN2 gene expression is independent of the Ras signaling
pathway. In contrast, exposure of the cells to the p38
inhibitor significantly reduced serum-, S1P- and aniso-

A
B
CTGF
GAPDH
C
T
G
F
m
R
N
A
L
e
v
e
l
s
(
%
)
N
o
I
n
h
i
bt
i
or

B
-
2
0
3
5
80
+
S
B
-
2
0
3
5
8
0
+S
B
-
20
3
58
0
+
S
P
-
6
00

A
L
e
v
e
l
s
(
%
)
+
S
B
-
2
0
3
5
80
N
o
I
n
h
i
bi
t
o
r
C

100
80
60
40
20
0
Empty Vector
Empty Vector
Empty Vector
CA-MKK3
CA-MKK4
CA-MKK6
CA-MKK3
CA-RhoA
CA-Cdc42
CA-MKK4
CA-MKK6
Fig. 8. Effects of p38 on CTGF/CCN2 expression. (A) Cells were
transfected with expression vect ors encoding the active forms
CA-MKK3, CA-MKK4 or C A-MKK6. C ontrol ce lls w ere t ransfected
with the pCDNA3 empty vector. Twenty-four hours later, cells were
incubated in s erum-free m edium for 6 h. T otal RNA was extracted and
the CTGF/CCN2 mRNA levels were analyzed by northern blot
hybridization. The diagram shown is representative of three separate
experiments. (B) Cells were transfected with the indicated expression
vectors as described in (A). A fter 2 4 h, cells were incubated for 6 h in
serum-free medium in the absence or in the presence of SB-203580
(10 l
M
) or S P-600125 (10 l

both p 38 and JNK1/2.
Meanwhile, because p 38 is a potential downstream target
of RhoA and Cdc42, we examined the effects of SB-20 3580,
a p 38 inhibitor on CTGF /CCN2 expression in CA-RhoA
and CA-Cdc42-transfected c ells. As s hown in Fig. 8B,
expression of CTGF/CCN2 was not significantly affected in
CA-RhoA-transfected cells but was nearly abrogated in
CA-Cdc42-transfected cells indicating a preponderant role
of p38 in Cdc42 signaling as well.
Role of RhoA GTPase and p38 in transcriptional and
post-transcriptional regulation of the
CTGF/CCN2
gene
In order t o determine whether CTGF/CCN2 expression
occurs via increased transcription and/or by stabilization of
the CTGF transcripts and t he role of RhoA GTPase and
p38 signaling in such a regulation, nuclear run-on assays
and message stability analyses were carried out. The
transcription rate of the CTGF/CCN2 gene was determined
upon stimulation of the cells with S1P, anisomycin or FBS
in the absence and in t he presence of RhoA GTPase and p38
inhibitors (Y-27632 and S B-203580, r espectively). A s s hown
in Fig. 9A, the CTGF/CCN2 gene transcription rate was
increased by 85, 140 and 240% upon stimulation with S1P,
anisomycin and serum, respectively. Interestingly, preincu-
bation of the cells with Y-27632 reduced the CTGF/CCN2
transcription rate by 75, 21 and 55% upon stimulation of
the cells with S1P, anisomycin and FBS, respectively. In
contrast, pretreatment of the cells with SB-203580 did not
dramatically affect CTGF/CCN2 transcription upon expo-

B
CTGF
GAPDH
CTGF
GAPDH
CTGF
GAPDH
C
T
G
F
m
R
N
A
L
e
v
e
l
s
(
%
)
C
T
G
F
m
R

Incubation Time (hrs)
Control Control
+ Serum +SB-203580
+ Serum +Y27632
+ Serum
Control
+ An
+ An +Y27632
+ An +SB-203580
SIP
+SIP + Y27632
+SIP + SB-203580
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60

and p38 inhib itors. As shown in Fig. 9B, stimulation of
the cells with S1P, anisomycin or FBS prolonged the half-
life of CTGF/CCN2 mRNA as the mRNA decay curve
was steeper in the stimulated cells than in control cells. In
the absence of exogenous stimuli, the observed half-life
was 1.5 h, whereas in the presence of S1P, anisomycin and
FBS, the half-life averaged 2.3, 3.6 and 3.1 h, respective ly.
This indicates that an mRNA stabilizing effect is involved
in the regulation of the CTGF/CCN2 gene as well.
Pretreatment of the cells with Y-27632 i nhibitor did not
dramatically alter t he mRNA decay in the stimulated cell.
In contrast, preincubation with SB-203580 reversed the
slow decline of CTGF/CCN2 mRNA, particularly in
anisomycin- and FBS-treated cells with half-lives decreas-
ing to 1.96 and 2.1 h, respectively. Taken together, these
results suggest that increased expression of CTGF/CCN2
elicited via the Rho GTPase pathway is achieved mainly at
the transcriptional level, whereas post-transcriptional regu-
lation at the level of mRNA stability seems to occur via
p38 s ignaling mechanisms.
Discussion
This study has focused on the identification of intracel-
lular signaling events that are involved in the activation
of the endogenous CTGF/CCN2 gene in cultured SMCs.
One of t he key findings in our study is that RhoA
GTPase activation mediated both the organization of the
actin cytoskeleton and the superinduction of the CTGF/
CCN2 gene. Rho-like G TPases play a p ivotal role
in orchestrating changes in the actin cytoskeleton in
response to various stimuli and have been implicated in

tion because, unlike cytochalasin D , neither latruculin B
nor swinholide A or jasplakinolide reportedly activate
RhoA GTPases [28,36]. Fifth, the control level of RhoA/
actin-mediated CTGF/CCN2 gene activation is transcrip-
tional.
The downstream elements of pathways via which RhoA
regulates cytoskeletal organization and gene expression are
poorly understood. Thus far, more than 20 RhoA targets
have been identified, begging the question of which was
responsible for mediatin g actin reorganization and ulti-
mately gene expression [34]. Among RhoA targets, RhoA-
associated kinase, which is inhibited by the Y-27632
inhibitor, seemed to concomitantly alter actin stress forma-
tion and CTGF /CCN2 expression. Functionally, RhoA-
associated kinase directly phosphorylates myosin light
chains and negatively regulates myosin phosphatases a nd
increases acto-myosin-based contractility [27]. The resulting
contractile forces are thought to contribute t o the formation
of stress fibers and f ocal contacts . I n a ddition, RhoA-kinase
also activates Lin11/Isl-1/Mec3 (LIM)
3
kinase, which subse-
quently phosphorylates cofilin and inhibits actin-depolym-
erizing activity, thus contributing to actin fiber stabilization
[27,29]. However, whether these signaling pathways directly
affect actin polymerization and F-actin rearrangement is
unknown. Recent studies indicate that regulation of PtdIns
metabolism by R hoA GTPase is likely involved because the
increase in PtdIns turnover often correlates with increase in
F-actin levels within the cells [37]. However, studies are

Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4447
promoter contains several A P-1 and N F-jB binding sites, it
is conceivable that the actin cytoskeleton architecture
orchestrated by RhoA regu lates the CTGF/CCN2 gene by
acting as a catalytic surface and/or protein c ofactor for these
transcription factors [39]. The exact mechanism by which
RhoA activates these transcription factors is just beginning
to be elucidated. In particular, RhoA-mediated SRF
activation was recently shown to require the actin-polymer-
ization-inducing activity of Diaphanous family proteins
[40]. In addition, there is some evidence to suggest that
G-actin monomers shuttle between the nucleus and the
cytoplasm a nd modulate the activity of transcription factors
either via direct physical interactions or by sequestering
cofactors required for their activation [29]. The final
understanding of the underlying mechanisms is still forth-
coming.
Another important advance provided in our study is that
RhoA-actin signaling was sufficient but not necessary for
the regulation of the CTGF/CCN2 gene an d that signaling
mechanisms via p38 MAP kinase were i nvolve d a s w ell. The
p38 MAP kinase seemed to act as a downstream effector of
Cdc42, but not RhoA or Rac1, even though all three
GTPases were reported to be potential activators of p38. In
fact, the upstream molecular components that feed into the
p38 pathway are diverse and cell-type specific and it is not
excluded that, in smooth muscle cells, Rac1 recruits
additional signaling pathways that prevent CTGF/CCN2
expression. Our findings are, however, in variance with
those r eported by other laboratories. Leask et al. f ound that

cytoskeletal architecture systematically undergoes rapid and
dramatic conformational changes in response to cell
stimulation and serves as a major scaffolding element for
the s ignaling machinery components such a s p38 and
PtdIns 3-kinase involved in intracellular communications
[46].
Furthermore, the p38-dependent increase in CTGF/
CCN2 expression is mediated by stabilization of CTGF/
CCN2 m RNA rather t han by t ranscription of the
CTGF/CCN2 gene. This post-transcriptional control pro-
vides a n additional means of i ncreasing the exp ression of the
gene and ensuring t hat its levels remain within a critical
range. It also enables rapid ch anges in CTGF/CCN2
mRNA levels in response to stimuli and provides a
mechanism for prompt termination of the pr otein s ynthesis.
These data a dd to the growing body of information
supporting a preponderant role of p38 in the regulation of
gene expression at the level of mRNA stability. The p38
MAP kinase is now known to stabilize a wide range of
mRNAs including those encoding TNF-a,interferon
(IFN)-c, interleukin (IL)-1b,IL-8,MIP-1a, Cox-2 vascular
endothelial g rowth factor (VEGF) and matrix metallopro-
teinase-1 and -3 [47]. The best characterized p38-regulated
mRNAs contains AU-rich elements (AREs) consisting of
multiple, frequently overlapping copies of the AUUUA
motif that t arget an mRNA for rapid deadenylation and
degradation and may even enhance mRNA decapping [47].
Interestingly, the 3¢-untranslated region (3¢-UTR)ofthe
CTGF/CCN2 gene contains three A UUUA p entamers as
well as other mRNA destabilizing motifs found in TNF-a

In conclusion, this study demonstrates a critical role o f
Rho GTPases and p38 MAP kinase in regulating the
endogenous CTGF /CCN2 gene in SMCs and the level of
control at which such regulation occurs. RhoA transcrip-
tionally activates CTGF/CCN2 expression th rough
actin-dependent mechanisms, w hereas Cdc42-mediated
p38 activation e nhanced the s tability of CTGF/CCN2
mRNA. Our findings confirmed the validity of the pre-
diction that CTGF/CCN2 regulation is fundamentally
distinct from that previously reported in fibroblasts.
Further work is needed to delineate the specific mecha-
nisms of this regulation.
Acknowledgements
This study is supported by t he grant from the National Institutes of
Health and National Institute of Diabetes, Digestive and Kidney
Diseases R01-DK060572 (to B. Chaqour). The critical technical
assistance of Q. Sha was greatly appreciated. We are grateful to Dr
A. H all (University C ollege, London, UK) for t he generous gifts of the
vectors e ncoding c onstitutively active forms o f RhoA, Cdc42 and Rac;
to Dr J.H. Han (The Scripps Institute, CA) for providing CaMKK3
and CaMKK6 const ructs and to D r A. Morrison for providing u s with
theactiveformofMKK4.
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