Tài liệu Báo cáo khoa học: 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 docx

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.

However, activation via t hese signaling events was abso-
lutely dependent on actin cytoskeleton integrity. In partic-
ular, RhoA-dependent regulation of the CTGF/CCN2 gene
was concomitant to i ncreased polymerization o f a ctin
microfilaments resulting in d ecreased G- to F-actin ratio a nd
appeared to be achieved at the t ranscriptional level. The p38
signaling pathway was RhoA-independent and led to
CTGF/CCN2 mRNA stab ilization. Use of actin-binding
drugs showed that the actual physical state of monomeric
G-actin i s a critical determinant for CTGF/CCN2 gene
induction. These data indicate that distinct cytoskeletally
based signaling events within the intracellular signaling
machinery affect either transcriptionally or post-transcrip-
tionally the expression of the CTGF/CCN2 gene in smooth
muscle cells.
Keywords: actin cytoskeleton; CTGF/CCN2; p38 MAP
kinase; Rho GTPase; smooth muscle cells.
Connective tissue growth factor (CTGF) also known as
CCN2 was identified as an immediate early responsive gene
activated by growth factors in connective tissue c ell types
[1,2]. It encodes 349 amino acids of which the first 26 residues
are a presumptive signal p eptide for secretion of t he protein,
which b elongs t o a family of extracellular m atrix-associated,
cysteine-rich heparin-binding proteins. CTGF/CCN2 is a
potent inducer of extracellular matrix protein (ECM)
expression, particularly fibrillar and b asement membrane
collagens [3]. Studies of diseas ed tiss ues f rom human clinical
specimens and animal models established a direct correlation
between high levels of expression of CTGF/CCN2 and
excessive accumulation and deposition of type I collagen in

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
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

growth factor.
*Present a d dress: I nstitute for Environmental Medicine, U niversity o f
Pennsylvania, Philadelphia, PA, USA.
(Received 23 August 2004, revised 24 September 2004,
accepted 28 September 2004)
Eur. J. Biochem. 271, 4436–4450 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04382.x
TGF-b. Consistent with this, the expression of CTGF/
CCN2 is either not or minimally affected upon stimulation
of cultured smooth muscle cells (SMCs) by TGF-b,whereas
fibroblastic cells are affected [3,8,10]. Similarly, the applica-
tion of mechanical forces seems to upregulate the CTGF/
CCN2 gene in fibroblasts but either downregulates its
expression in endothelial cells or does not affect it in SMCs,
indicating that the regulatory mechanisms of the CTGF/
CCN2 gene are cell-type specific and likely depend on
specific intracellular signaling e vents w ithin t he cells [11–13].
Current models of eukaryotic gene regulation s uggest the
existence o f an i ntracellular communication network among
signaling molecules that converts a given stimulus into
activation or inhibition of the expression of specific genes
[14]. The two major signaling molecule groups, Rho
GTPases and mitogen-activated protein (MAP) kinases
form the pillars of this signal transduction network. The Rho
GTPase proteins, of which the best-characterized members
are RhoA, Cdc42 and Rac1, regulate a w ide variety of cell
functions by acting as biological timers that initiate and
terminate specific cell functions. They regulate actin cyto-
skeletal reorganization a nd gene expression either directly or
via the activation of m embers of the MAP kinase family. T he
latter relay, amplify and integrate signals from diverse

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

Avanti (Alabaster, AL, USA). Chemical inhibitors were
purchased from Calbiochem (San Diego, CA, USA). All
other chemicals used were of reagent gr ade. Y- 27632
inhibitor was kindly provided by T. Kondo (Welfide Corp.,
Osaka, Japan). Anti-CTGF/CCN2 s era have been described
elsewhere [12,17]. Anti-phospho-p38, anti-phospho-JNK,
and a nti-phospho-Akt/PKB were from New England Bio-
labs (Beverly, MA, USA). Radioactive materials such as
[
32
P]UTP[aP] and [
32
P]dCTP[aP] were purchased from
NEN Life Science Products (Boston, MA, USA)
1
.
Cell culture and drug treatments
Primary cultures of SMCs were prepared from the bladders
of mid- to late-gestational fetal calves as previously
described [12,18]. Freshly isolated cells were phenotypically
characterized using muscle-specific antibodies against
smooth musc le actin and myosin. Cells were maintained
in DMEM supplemented with 10% (v/v) fetal bovine s erum
(FBS) and antibiotics in a humidified atmosphere contain-
ing 5% (v/v) CO
2
in air at 37 °C. Cells from passages 2–8
were used for the experiments. For most experiments,
cells were grown to subconfluence e ither i n 25-cm
2

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

)1
) a nd the cells were
harvestedafter0,0.5,1,2and4h.TotalRNAwaspurified
and analyzed b y northern blot hybridization and phosphor-
imaging densitometry. The relative amounts of normalized
mRNA were plotted as a function of time and the slope of
this curve was used to calculate the interval period within
which half of the original amount of mRN A had decayed.
Nuclear run-on assay
Subconfluent cells were left untreated or stimulated with
S1P, anisomycin or FBS for 1 h. Experiments with
Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4437
pharmacological inhibitors were performed as described
above. Cells were subsequently washed twice with NaCl/P
i
,
trypsinized and centrifuged at 4 °C. The cellular pellet w as
resuspended in buffer containing 10 m
M
Tris/HCl (pH 7 .4),
10 m
M
NaCl, 3 m
M
MgCl
2
, and 0.5% (v/v) Nonidet P-40
allowing swelling and lysis of the cell membrane. The lysate
was recentrifuged at 300 g at 4 °C and the resulting nuclear
pellet was resuspended in 150 lL of buffer containing

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

Z-probe nylon membrane using a slot blot apparatus (Bio-
Rad). The membran e was U V-irradiated and p rehybridized
as described above for northern blotting. Equal amounts of
the purified radiolabeled transcripts (10
6
c.p.m.) were
resuspended in hybridization solution. Hybridization with
the slot-blotted DNA probes was carried out for 4 8 h at
42 °C. The membranes were then washed under s tringent
conditions before phosphorimager scanning of the hybridi-
zation signals.
Transient transfection and coexpression experiments
Cultured cells were plated at a d ensity of 1 · 10
5
cm
)2
in
60-mm tissue culture dishes and maintained in medium
containing 10% serum for 18 h. Cells were transfected with
the indicated expression vector using Fugene6 Transfection
Reagent (Roche Diagnostics, Mannheim, Germany)
according to the manufacturer’s specifications. The
Fugene6–DNA mixture plus serum-free medium was left
on cells for 3 h. Cells were allowed to recover in fresh
medium containing 10% (v/v) serum. The next day, the
experimental treatments were performed as described in the
text. Cells were th en washed three times with ice-cold NaCl/
P
i
and total RNA w as isolated and analyzed by northern

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-

luminescence (Amersham Bioscience Inc., Piscataway, NJ,
USA)
2
. For immunodetection of phosphorylated proteins,
SDS sample buffer was added directly to the cells, which
were subsequently scraped o ff the plate and subjected to
denaturing SDS/PAGE under reducing conditions. For
immunohistochemical analyses, cells were plated on glass
cover slips, treated with the indic ated drugs, fixed i n 2%
(v/v) formaldehyde/NaCl/P
i
for 30 min, permeabilized in
0.1% (v/v) Triton X-100 at room temperature for 5 min
and stained with rhodamine–phalloidin (Cytoskeleton,
Inc., Denver, CO). Images were acquired using a Bio-
Rad 1024 MDC laser scanning confocal imaging system.
RhoA-, Cdc42- and Rac1-GTP pull-down assays
Measurement of GTP-bound Rho GTPases was per-
formed using the activation assay kit (Upstate Biotech-
nology, Lake Placid, N Y, and C ytoskeleton Inc.),
following the manufacturer’s instructions. Briefly, cells
were lysed in buffer containing 50 m
M
Tris, pH 7.2, 1%
(v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate,
0.1% (w/v) SDS, 500 m
M
NaCl, 10 m
M
MgCl

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

EGTA, 5% (v/v)
lyceral, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) Tri-
ton X-100, 0 .1% (v/v) Tween 20, 0.1% (v/v) 2 -mercapto-
ethanol and 0.001% (v/v) antifoam) and a protease
inhibitor cocktail followed by centrifugation for 1 h at
100 000 g to separate the F-actin from G-actin pool. The
pellet was resuspended in ice-cold water and incubated in
the presence of cytochalasin-D to dissociate F-actin.
Aliquots fro m b oth supernatant and pellet fractions were
separated by western blot, and actin was quantitated after
immunodetection analysis using a specific antiactin anti-
body and densitometric scanning. A ll steps were p er-
formed at 4 °C.
4438 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
Statistical analysis
Data were expressed a s mean ± SD. A paired Student’s
t-test was used to analyze differences between two groups,
and P-values of < 0.05 were considered significant.
Results
Modulation of
CTGF/CCN2
gene expression
As a basis for defining the signaling pathways regulating
CTGF/CCN2 gene expression in our system, we first
determined the response of primary cultures of SMCs to
various stimuli including S1P, a bioactive lysolipid and
G-protein-coupled receptor agonist, anisomycin, a geno-
toxic agent that mimics the effects of stress stimuli and FBS
that is enriched in mitogenic growth factors. Cultured
SMCs were exposed to either S1P (10 l

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

(data not shown).
However, because a nisomycin i s a lso an i nhibitor o f p rotein
synthesis at concentrations above 40 ng ÆmL
)1
,weper-
formed our studies with a concentration of 1 0 ngÆmL
)1
that
efficiently turned on specific signaling pathways and caused
no apparent cell death over 24 h [22]. Also, incubation of
the cells with a combination of serum and either S1P or
anisomycin, did not have an additive effect on CTGF/
CCN2 mRNA l evels but incubation o f the cells with
anisomycin further augmented S1P-mediated increase in
A
0 1 6 (hrs)
CTGF
Incubation Time (hrs)
c c c s an ser s an ser
GAPDH
B
CTGF
CTGF
CTGF
+ S1P
+ Anisomycin
+ Serum
0 1 4 6 12 hrs
C
CTGF mRNA Levels (%)

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

,anisomycin
(an) at a concentration of 10 ngÆmL
)1
or 5% (v/v) FBS (ser) for the
indicated periods. T otal RN A w as isolated and subjected to n orthe rn
blot hyb ridization analysis. To control f or un eq ual R NA loading, the
blot was hybridized with a specific GAPDH DNA probe . CTGF/
CCN2 mRNA levels were normalized to those of GAPDH and the
graphical representation of the results of phosphorimage scans of the
mRNA hybridization signals is shown as well. To compare mRNA
expression from different experiments, mRNA levels of control cells
were set to 100%. Data represent means ± SD (n ¼ 3). (B) CTGF/
CCN2 protein expression in cells stimulated with S1P, anisomycin or
serum. CTGF/CCN2 protein was detected in cellular lysates by
western blot with an antibody directed against human CTGF/CCN2
protein. Im munodetectio n w as p erformed by enhanced chemilumi-
nescence. (C) Cells were treated for 1 h with S1P, anisomycin or
serum or a combination of S1P and serum (s/ser), serum and aniso-
mycin (ser/an) or S1P and anisomycin (s/an). Data are average of three
independent expe riments.
Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4439
CTGF/CCN2 mRNA, suggesting the involvement of
separate and, perhaps, independent signaling mechanisms
(Fig. 1C) .
CTGF/CCN2
gene regulation via Rho GTPase signaling
The role o f Rho family proteins in CTGF /CCN2 expres sion
was investigated using toxin B from Clostridium d ifficile,
which glucosylates Rho family proteins, thereby causing
their inactivation, and the Y-27632 compound, a p yridine

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
*

*
*
*
*
Control
500
450
400
350
300
250
200
150
100
50
0
S1P
Anisomycin
Serum
Fig. 2. CTGF/CCN2 gene expression is sensitive to Rho GTPase
inhibitors. (A) Cells were p retreated for 30 min with either toxin B
(10 ngÆmL
)1
) or Y -27632 (10 l
M
) prior t o the addition of e ither 10 l
M
S1P ( s), 10 ngÆmL
)1
anisomycin (an) or 5% (v/v) F BS (ser). One h our

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

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

-
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

1
D
N
-
hR
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
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
-

R
h
o
A
0
100
200
300
400
500
600
C
T
G
F
m
R
N
A
L
e
v
e
l
s
(
%
)
*
*

*
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

Fig. 3A, stimulation of SMCs with S1P induced a s ixfold
increase in the a mount o f GTP-RhoA but did not affect the
cellular levels of Cdc42-GTP or Rac1-GTP. Stimulation
with FBS induced Rho GTPase activation by increasing
GTP loading of RhoA, Cdc42 and R ac1 r aising the
possibility that the enhanced activity of these GTPases,
either individually or collectively, enhanced CTGF/CCN2
expression in serum-treated cells. Stimulation with FBS
caused a relatively sustained increase of GTP-RhoA com-
paredwiththetransientincreaseinGTP-Cdc42andGTP-
Rac1, t he levels of which returned to those in control cells
within 15 min of stimulation. This activation pattern is
mechanistically consistent with the kinetic parameters of
translocation t o the cell membrane o f these GTPases [25]. In
contrast, a nisomycin h ad no effect on the a ctivation of these
Rho GTPases (data not shown). To further investigate the
individual contribution of the Rho G TPases to CTGF /
CCN2 exp ression, we transiently transfected cultured SMCs
with the dominant n egative forms DN-RhoA, DN-Cdc42
or DN-Rac1. Figure 3B shows that DN-RhoA reduced the
ability of S1P and s erum to induce the CTGF/CCN2 gene
by 31 and 40%, respectively (P<0.05). The dominant
negative form DN-Cdc42 reduced the transcript levels of
CTGF/CCN2 in se rum-treated cells ()35%) only, but did
not significantly affect S1P-induced CTGF/CCN2 expres-
sion. In contrast, D N-Rac1, had no effect on the expression
of CTGF/CCN2 whic hever stimulus was used. Similarly,
neither of the DN-GTPase forms had an effect on
anisomycin-induced CTGF/CC N2 expression. Therefore,
both RhoA and Cdc42 play a significant role in serum-

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–

phalloidin, which labels actin stress fibers (Fig. 4).
Control untreated cells had fairly well-developed stress
fibers, whereas S1P- and serum-treated cells showed
enhanced actin stress fiber networks with highly organ-
ized microfilament bundles. Cells treated with serum
showed the most dramatic increase in the fluorescence
intensity of F-actin bundles compared with cells treated
with S1P, whereas exposure of the cells to anisomycin did
Control
+ Toxin B
+ Latrunculin B
+ S1P + Anisomycin
+ Serum
Fig. 4. Effects o f S1P, an is omycin and FBS on
actin stress fibers in SMCs and their modulation
by toxin B and latrunculin B. Cells were first
stimulated with e ither S1P, anisomycin or FBS
for 30 min and then fixed, permeabilized and
stained for F-actin with rhodamine-conju-
gated phalloidin. Th e effects of toxin B and
latrunculin B on the actin filaments was
examined by preincubating the cells with
either 10 ngÆmL
)1
toxin B or 0.5 l
M
latrun-
culin B for 30 min prior to the additio n of
either S1P, anisomycin or FBS for an addi-
tional 30 min.

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-

mycin, suggesting that an intact actin cytoskeleton is also
necessary for anisomycin signaling. Surprisingly, treatment
of the cells with latrunculin B alone induced an increase in
basal CTGF/CCN2 mRNA levels. To further examine
whether this effect was real or merely a non specific side
effect of the drug, we determined the kinetic parameters of
CTGF/CCN2 mRNA levels upon treatment of the cells
with latrunculin B alone. As shown in Fig. 5B, latruncu-
lin B i nduced a time-dependent increase in CTGF/CCN2
mRNA levels that peaked after 1 h and declined
progressively thereafter. Although unexpected, the modu-
lation of CTGF/CCN2 expression by latrunculin B sug-
gests t hat s equestration of G-actin m onomers by this
actin-binding drug is sufficient to modulate basal CTGF/
CCN2 expression, while disruption of actin filaments
interfered with stimulus-dependent induction of CTGF/
CCN2 expression.
The most physiologically conspicuous attribute of actin
is its ability t o exist in a dynamically regulated equilibrium
between the monomeric globular G-actin form and
polymeric filamentous F-actin [27]. Therefore, we tested
the ability of d rugs known t o affect actin polymerization
to modulate CTG F/CCN2 expression. We utilized jas-
plakinolide, a compound that induces actin polymeriza-
tion by increasing actin nucleation and stabilizing actin
filaments and swinholide A, a drug that sequesters
G-actin as dimers [28]. As shown in Fig. 6A, cells treated
with jasplakinolide assumed a diamond shape and
displayed thick F-actin bundles that aggregate at cell
margins consistent with the role of jasplakinolide as a

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

B
CTGF
300
250
200
150
100
50
0
0 0.5 1 2 4 8 hrs
CTGF mRNA Levels (%)
Incubation Time (hrs)
Fig. 5. Effects of latrun culin B on expre ssion of the CTGF/CCN2 gene
in SMCs. (A) Cells were pretreated with 0.5 l
M
latrunculin B (LtB) for
30 min prior to the addition of 1 0 l
M
S1P (s), 10 ngÆmL
)1
anisomycin
(an) or 5% serum (ser). Total RNA w as extracted and subjected t o
northern blot hybr idization analysis with CTGF/CCN2 a nd GAPDH
probes. The diagram is represe ntative of three independent experi-
ments with similar results. (B) The effects of latrunculin B a lone on
CTGF/CCN2 expression was determined by incubating the cells with
0.5 l
M
latruculin B for the indicated time periods. Total R NA w as
extracted and analyzed for the mRNA levels of CTGF/CCN2 .The

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

the control of a regulatory loop determined by the levels of
free G-actin, we investigated the possibility that changes in
CTGF/CCN2 expression upon exposure of the cells to
+ Jasplakinolide
Control
+ Swinholide A
CTGF
LtB + Jasplakinolide
GAPDH
0 0.5 1 2 4 8 16 24 hrs
0 0.5 1 2 4 8 16 24 hrs
Swinholide A +Jas
p
lakinolide
CTGF
Jasplakinolide
GAPDH
0 0.5 1 2 4 8 hrs
Swinholide A
0 0.5 1 2 4 8 hrs
A
B
C
Fig. 6. Effects of jasplakinolide and s winho-
lide A on actin stress fibers and CTGF/CCN2
expression. (A) Cells were stimulated with
either jasplakinolide (0.5 l
M
) o r swinholide A
(0.1 l

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

Y-27632 )) +– + – +
G-Actin/F-Actin 0.260 ± 0.023 0.181 ± 0.023* 0.22 ± 0.021 0.239 ± 0.018 0.223 ± 0.013 0.110 ± 0.034** 0.227 ± 0.019
*P < 0.05, **P < 0.01 versus control; P < 0.01 versus serum stimulation alone.
Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4443
various stimuli might reflect changes in the ratio of G- t o
F-actin. Cells were treated with v arious stimuli for 30 min
and fractionated cell extracts containing nonpolymerized
globular actin (G-actin) and actin engaged in polymerized
microfilament (F-actin) were prepared and analyzed for
G- and F-actin contents. As shown in Table 1, there was a
significant decrease of G- to F-actin ratio in cells treated
with either S1P or FBS compared with control untreated
cells in dicating that a larger pool of total actin exists as
filamentous actin in the stimulated cells. However, the
G- to F-actin ratio seemed to significantly increase as the
G-actin levels i ncrease when the cells were pretre ated with
RhoA kinase inhibitor (Y-27632) prior to serum stimula-
tion. Similarly, the pool of F-actin in S1P-treated cells was
consistently lower than that after pretreatment with
Y-27632 although n o significant d ifferences were seen,
probably due to the moderate sensitivity of t he methodo-
logy used. Also, treatment of the cells with either TNF-a or
UV-irradiation that neither induced RhoA activation nor
CTGF/CCN2 expression did not significantly alter the
G- to F-actin ratio (data not shown). T his indicates that
CTGF expression is sensitive to changes in the G- to
F-actin ratio and that RhoA GTPase pathway contributes,
at least in part, to the recruitment of actin into actin
polymerized filaments. Moreover, treatment of t he cells
with anisomycin did not significantly alter t he G- to F-actin

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

P-p38
+ S1P + Anisomycin + Serum
P-JNK1/2
P-Akt
Total-Erk1/2
0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 min
0
100
200
300
400
500
600
Control
S1P
Anisomycin
Serum
C
T
G
F
m
R
N
A
L
e
v
e
l

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

Total-Erk1/2, respectively), phosphorylated
p38 (P-p38), phosphorylated JNK1/2 (P-
JNK1/2) and phosphorylated Akt/PKB (P-
Akt) using mon oclonal a ntibodies t hat
recognize specifically the phosphorylated
forms of these pro teins. (B) Cells w ere either
left untreated or pretreated for 30 min with
either Pd-09059 (20 l
M
), SB-203580 (10 l
M
),
SP-600125 (10 l
M
) or wortmanin (10 l
M
)
prior to the addition of 10 l
M
S1P (s),
10 ngÆmL
)1
anisomycin (an) or 5% FBS. One
hour later, total RNA was extracted and
subjected to northern blot analysis with
CTGF/CCN2 and GAPDH probes. The
CTGF/CCN2 hybridization signals were
normalized to th ose of GAPDH. S hown is the
percentage of the relative increase in mRNA
levels. The values are the means ± SD (n ¼

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-

mycin-induced CTGF/CCN2 gene expression by 30, 35 and
60%, respectively, suggesting an important ro le of p38 i n
CTGF/CCN2 gene expression (P<0.05). In agreement
with this, UV-irradiation of the cells (2.4 JÆm
)2
), although
inducing a strong activation of JNK1/2 and only a very
weak phosphorylation of p38, had no effects on C TGF
expression, which ruled out the potential involvement of
JNK1/2 in CTGF/CCN2 gene induction (data not shown).
Furthermore, inhibition of PI 3-kinase significantly reduced
the CTGF/CCN2 mRNA levels upo n stimulation with
anisomycin but did not affect the CTGF/CCN2 mRNA
levels in S1P- or serum-treated cells. T hese data in dicate that
serum- and S1P-induced CTGF/CCN2 expression signaling
overlap, albeit to various extent, at the level of p38 signali ng,
but are a ll independent of both Erk1/2 a nd JNK 1/2
signaling pathways.
The signaling components upstream of the p38 identified
thus far suggest a complex cell- and stimulus-dependent
regulation consistent with the d iversity of extracellular
stimuli that activate these pathways [20]. Both p38 and
JNK can be activated in vitro and in vivo by dual specificity
MAP kinase kinases (MKK) d epending on the cell s ystem
studied, although SAP/ERK kinase (SEK/MKK4) acti-
vates mostly JNK whereas MKK3 and MKK6 directly
activate p 38 [31]. We further examined the contribution of
p38 signaling to CTGF/CCN2 gene expression by trans-
fecting cells with the active forms CA-MKK3, CA-MKK6
or CA-MKK4. Expression of these kinases in the cells was

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

C
o
n
t
r
o
l
N
o
I
n
h
i
b
i
t
o
r
N
o
I
n
h
i
b
i
t
or
+
S

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

1
2
5
+
S
P
-
6
00
1
25
+
S
P
-
6
0
0
1
25
*
*
C
C
T
G
F
m
R
N

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

o
nt
or
l
N
o
I
n
h
i
bti
o
r
+
S
B
-
203
5
8
0
**
120
100
80
60
40
20
0
120

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

M
). To compare the CTGF/CCN2 mRNA
levels from different experiments, the stimulation b y CA-MK K3,
CA-MKK4 and CA-MKK6 w as set to 100%. Values are the average ±
SD of three expe riments. * P <0.05,**P < 0.01 compared with t he
cells transfected with the m utant forms and in cubated in the absen ce of
inhibitors. (C) Cells were transfected with the active fo rms CA-RhoA or
CA-Cdc42. After 24 h, cells were incubated for 6 h in serum-free
medium in the absence or presence of SB-203580 ( 10 l
M
). To compare
the CTGF/CCN2 mRNA levels f rom d ifferent experiments, the sti-
mulation by CA-Rho A and CA-Cdc42 was set to 100%. Values are the
average ± SD o f t hree e xperiments. **P < 0.01 compared w ith cells
transfected with t he mutant forms and incubated in the absence of
inhibitors.
Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4445
previously detected by western b lot analysis using anti-tag
sera [18]. As shown in Fig. 8A, overexpression of the active
forms of these kinases resulted in the activation of the
endogenous CTGF/CCN2 gene. MKK3, MKK6 and
MKK4 induced a 265, 187 and 275% increase of CTGF/
CCN2 mRNA levels, respectively. Treatment of either
CA-MKK3-, CA-MKK4- o r CA-MKK6-transfected cells
with the p 38 inhibitor, SB-20589, sig nificantly reduced
CTGF/CCN2 mRNA levels, whereas Pd-098059 and
SP-125600, which inhibit Erk1/2 and JNK, respectively,
did not significantly alter CTGF/CCN2 mRNA levels. The
ability of CA-MKK4 to increase CTGF/CCN2 mRNA
levels probably reflects the dual specificity of MKK4 for

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-

sure to either stimulus. Thus, RhoA GTPase pathway seems
to play a critical r ole in CTGF/CCN2 gene transcription,
+S1P
+Anisomycin
+Serum
+Y-27632 - - + -
+SB-203580 - - - +
G
A
P
D
H
C
T
G
F
C
T
G
F
C
T
G
F
1.35 1.23 1.40
2.5 1.64 2.75
3.29 2.8 3.5
4.6 2.9 4.31
Control
A

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

N
A
L
e
v
e
l
s
(
%
)
C
T
G
F
m
R
N
A
L
e
v
e
l
s
(
%
)
Incubation Time (hrs)
Incubation Time (hrs)

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

40
20
0
124 124
124
Fig. 9. The CTGF/CCN2 gene is transcriptionally regulated through RhoA GTPase and post-transcriptionally regulated through p38 signaling.
(A) N uclear run-on assay showing th e effects o f S1P, anisomycin a nd serum in the absence and presence of RhoA GTPase in hibito r Y - 27632 and
p38 inhibitor SB-203580 on the transcription rate of the CTGF/CCN2 gene. Nuclei were prepared from either control cells or those treated with
either S1P (10 l
M
), anisomycin (10 ngÆmL
)1
) or 5% serum for 1 h. Pharmacological inhibition of RhoA and p38 signaling was performed by
preincubating t he cells with the indicated d rugs for 30 min prior t o t he trea tment with various stimuli. The pre-mRNA was radiolabe led , i solated
andhybridizedtoCTGF/CCN2andGAPDHcDNAprobes,whichhadbeenslot blotted on nylon membranes. The hybridization signals for
CTGF/CCN2 were n ormalized to those of GAPDH. These experiments were p erformed in duplicate. (B) E ffects o f S 1P, anisomycin and FBS o n
the decay of CTGF/CCN2 mRNA was determined by treating the cells for 30 min with a control vehicle, S1P, anisomycin or FBS and further
incubating the cells with actinomycin D (10 lgÆmL
)1
) for the indicated periods. Likewise, the role of RhoA and p38 signaling was examined by
preincubating the ce lls with the indicated drugs for 30 min prior to treat ment with the v arious stimuli. For each time point, total RNA was prepared
and analyzed by northern blot hybridization. The CTGF/CCN2 mRNA levels prior to the addition of actinomycin D were set to 100%. Each point
is the means of two separate experiments.
4446 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
whereas p38 signaling minimally affects the transcriptional
control of the CTGF/CCN2 gene.
Next, we examined the CTGF/CCN2 mRNA turnover
by inhibiting new mRNA transcription with actinomycin
D upon stimulation of the cells with S1P, anisomycin or
FBS in the a bsence and in the presence of RhoA GTPase

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

transcriptional activation, phenotypic modulation of the
cells and oncogenic transformation. Evidence has previ-
ously been presented for the potential involvement of
small G-proteins in CTGF/CCN2 expression [32,33]. In
particular, Hahn et al. reported t hat activation of RhoA
GTPase by heptahelical receptor agonists induced the
expression of the CTGF/CCN2 gene in mesangial cells
and that disruption of the cytoskeleton by cytochalasin D
prevented such a n induction [32]. Our results concur and
significantly extend those studies in several important
ways. First, at the level of smooth muscle cells, the data
presented are consistent with a dual role of RhoA in the
cytoskeletal changes and transcriptional modulation o f
the CTGF/CCN2 gene. The overexpression of a consti-
tutively activated mutant of RhoA, which was shown in
separate experiments to induce the formation of stress
fibers of contractile actin and myosin filaments, upreg-
ulated expression of the endogenous CTGF/CCN2 gene
[34]. Second, only the separate pool of cytoskeletal actin
that contributes to stress fiber formation is critical for
CTGF/CCN2 expression because the active mutant Rac1
known to promote the p olymerization of cortical actin
did not affect expression of the CTGF/CCN2 gene [35].
Third, RhoA-actin signaling exerted bimodal modulation
of the CTGF/CCN2 gene expression; monomeric G-actin
inhibited CTGF/CC N2 gene induction, whereas F-actin
enhanced CTGF/CCN2 gene express ion. F ourth, a ctin
monomer-sequestering agents that mimic the physiologic
G-actin-binding proteins induced the expression of
the CTGF/CCN2 gene independent of RhoA activa-

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

hampered by a lack o f adequate tools to evaluate not only
total cellular PtdIns, but also local c oncentrations within the
cells. M eanwhile, u sing actin-binding drugs, we showed that
the express ion of the CTGF/CCN2 gene can be modulated
by either actin polymerization or the availa bility of poly-
merization competent G-actin referred to a s free b arbed-end
actin. Da ta from in vitro assays previously suggested t hat
major G-actin binding proteins (e.g. b-thymosins) selectively
affect the a vailability o f barb ed-end actin and determine the
level and distr ibution o f F -actin [34]. Therefore, it i s
tempting to speculate that interactions between monomeric
G-actin and actin-binding proteins are a potential target of
regulation by RhoA GTPase.
Furthermore, our data indicated that RhoA-mediated
CTGF/CCN2 express ion was carried out at the transcrip-
tional l evel, suggesting that CTGF p romoter activation is
critical for RhoA-dependent effects and additional mecha-
nisms that sen se actin d ynamics in t he cells may be involved
as well. RhoA was shown to activate s everal transcription
factors t hat play important roles in g rowth f actor r egulation
of gene expression, namely AP-1, NF-jB, GATA-4 and
serum response factor (SRF) [15,38]. Interestingly, treat-
ment of our cells with either curcumin or N-tosyl-
L
-
phenylalanine chloromethyl ketone, which inhibit AP-1
and/or NF-jB, dramatically affected the CTGF/CCN2
mRNA levels in japslakinolide-, S 1P- or FBS-treated cells,
whereas treatment with mithramycin, a Sp1 inhibitor, had
no effect (data not shown). Because the CTGF/CCN2

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

p38 i nhibitors had n o i mpact on t he induction of the CTGF /
CCN2 gene in fibroblasts and that instead, the ras/Erk
pathway is necessary for CTGF/CCN2 gene activation [41].
Hahn et al. reported that CTGF/CCN2 gene induction in
mesangial fibroblasts was independent of both Erk1/2 and
p38 MAP kinase activation [32]. Supporting the conclusion
that p38 can upregulate expression of the CTGF/CCN2
gene, however, is the observation that administration of
FR-167653, a highly specific inhibitor of p38, suppressed
expression of the CTGF/CCN2 gene in a murine m odel of
bleomycin-induced pulmonar y fibrosis, suggesting that p 38-
dependent regulation of CTGF/CC N2 expression may be an
in vivo active mechanism as well [42]. We have no ready
explanation for the differences between our results and
those of o ther laboratories but they may be due to cell type-
specific or species variations.
The p38-dependent activation of the CTGF/CCN2 gene
although RhoA GTPase-independent required an intact
cytoskeleton, and, at least in part, the upstream activation
of PtdIns 3-kinase. Inhibition of PtdIns 3-kinase reduced
both p38 and PKB/Akt phosphorylation in anisomycin-
treated ce lls (Fig. 10). These data are in line with previous
observations indicating that physiological activators of the
p38 and PtdIns 3-kinase pathways including thr ombin,
dexamethasone, angiotensin II and prostaglandins stimulate
the expression of CTGF/CCN2 efficiently [8,43–45]. The
role of the cytoskeleton is important, particularly in the
compartmentalization of the cytoplasm and organization of
specialized zones for sustained s ignaling b etween cell s urface
and nucleus. In fact, many lines of evidence indicate that the

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

and IFN-c transcripts that were reported to mediate the
post-transcriptional effects of p38 [48]. However, specificity
cannot be explained in terms of the p resence or absence of
AREs because several proto-oncogene mRNAs contain
AREs but are not responsive to the p38 pathway [49].
Instead, it may be necessary to consider the contexts of
RNA sequence or secondary structures in which the
AU-rich motifs are found. In particular, p38 activation
was reported to release labile transcripts s uch a s those of
TNF-a and Cox-2 from a state of translational arrest
imposed by AREs within the 3¢-UTR by regulating
deadenylation rather t han d ecay of the mRNA body
[50–52]. It was also suggested that p38 stabilizes mRNA
by targeting putative ARE-binding proteins. However,
despite t he identification of several ARE-binding proteins, it
is unclear which ( if any) provides a link between p38 and the
Fig. 10. Model of signal tra nsduction pathways involved in the inducible
CTGF/CCN2 gene expression by either S1P, FBS or anisomycin.
4448 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271) Ó FEBS 2004
AREs. Much further work is required to precisely ascertain
which specific mRNA decay steps and ARE-binding
proteins are t argeted by the p38 signaling pathway. Our
study showed that the C TGF/CCN2 mRNA can be used as
a model of labile RNA to establish the potential role of the
AREs and ARE-binding proteins and their significance for
CTGF/CCN2 mRNA regulation b y the p38 pathway.
Accordingly, elucidation of whether the types of signal-
dependent gene expression described for other labile
mRNAs are un ique or relevant for the CTGF/CCN2
mRNA is warranted.

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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