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 acti
Trang 1Regulation 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 Chowdhury1,* and Brahim Chaqour2
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 disregulation in fibrotic
disorders In this study, we investigated the regulation 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 activation (eightfold) of CTGF/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 effects of anisomycin largely involved p38 and
phosphatidyl inositol 3-kinase signaling mechanisms
However, activation via these signaling events was abso-lutely dependent on actin cytoskeleton integrity In partic-ular, RhoA-dependent regulation of the CTGF/CCN2 gene was concomitant to increased polymerization of actin microfilaments resulting in decreased G- to F-actin ratio and appeared to be achieved at the transcriptional level The p38 signaling pathway was RhoA-independent and led to CTGF/CCN2 mRNA stabilization Use of actin-binding drugs showed that the actual physical state of monomeric G-actin is 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
CCN2was identified as an immediate early responsive gene
activated by growth factors in connective tissue cell types
[1,2] It encodes 349 amino acids of which the first 26 residues
are a presumptive signal peptide for secretion of the protein,
which belongs to a family of extracellular matrix-associated, cysteine-rich heparin-binding proteins CTGF/CCN2 is a potent inducer of extracellular matrix protein (ECM) expression, particularly fibrillar and basement membrane collagens [3] Studies of diseased tissues from 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/ CCN2in the pathogenesis of fibrosis Thus, CTGF/CCN2 emerged not only as a useful prognostic and diagnostic marker of tissue fibrosis, but also as a viable therapeutic 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 asthma [6–9] However, in many cases, upregulation of the CTGF/CCN2 gene is neither preceded nor accompanied by a concomitant increase in TGF-b expression and/or activity suggesting that CTGF/ CCN2 is not systematically a downstream effector of
Correspondence to B Chaqour, Department of Anatomy and Cell
Biology, SUNY Downstate Medical Center, 450 Clarkson Avenue,
Box 5, Brooklyn, NY 11203–2098, USA Fax: +1 718 270 3732,
Tel.: +1 718 270 8285, E-mail: brahim.chaqour@downstate.edu
Abbreviations: RE, AU-rich element; CA, constitutively active kinase;
CTGF/CCN2, connective tissue growth factor; DMEM, Dulbecco’s
modified Eagle’s medium; DN, dominant negative kinase; ECM,
extracellular matrix; FBS, fetal bovine serum; GAPDH,
glyceralde-hyde-3-phosphate dehydrogenase; IFN, interferon; IL, interleukin;
JNK, c-Jun N-terminal kinase; MAP, mitogen-activated protein;
MKK, MAP kinase kinases; S1P, sphingosine 1-phosphate; SMC,
smooth muscle cell; SRF, serum response factor; TGF, transforming
growth factor; UTR, untranslated region; VEGF, vascular endothelial
growth factor.
*Present address: Institute for Environmental Medicine, University of
Pennsylvania, Philadelphia, PA, USA.
(Received 23 August 2004, revised 24 September 2004,
accepted 28 September 2004)
Trang 2TGF-b Consistent with this, the expression of CTGF/
CCN2is 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 events within the cells [11–13]
Current models of eukaryotic gene regulation suggest the
existence of an intracellular 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 wide variety of cell
functions by acting as biological timers that initiate and
terminate specific cell functions They regulate actin
cyto-skeletal reorganization and gene expression either directly or
via the activation of members of the MAP kinase family The
latter relay, amplify and integrate signals from diverse
stimuli, thereby controlling the genomic and physiological
response of the cells The MAP kinase pathway was
subdivided into the extracellular-regulated kinase (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/
CCN2expression, 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 obstructive
diseases and atherosclerosis [8,15,16]
We undertook this study to investigate the role of Rho
GTPase and MAP kinase signaling pathways in the
modu-lation of the CTGF/CCN2 gene in response to diverse
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 the CTGF/CCN2 expression, while the p38 MAP
kinase modulates the CTGF/CCN2 gene at the level of
mRNA stability However, all signals depend on the actin
cytoskeleton integrity In particular, the 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 grade Y-27632
inhibitor was kindly provided by T Kondo (Welfide Corp.,
Osaka, Japan) Anti-CTGF/CCN2 sera have been described
elsewhere [12,17] Anti-phospho-p38, anti-phospho-JNK, and anti-phospho-Akt/PKB were from New England Bio-labs (Beverly, MA, USA) Radioactive materials such as [32P]UTP[aP] and [32P]dCTP[aP] were purchased from NEN Life Science Products (Boston, MA, USA)
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 muscle actin and myosin Cells were maintained
in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS) and antibiotics in a humidified atmosphere contain-ing 5% (v/v) CO2in air at 37C Cells from passages 2–8 were used for the experiments For most experiments, cells were grown to subconfluence either in 25-cm2culture flasks or 60-mm dishes Twenty-four hours later, cells were washed with DMEM to remove traces of serum, 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 given inhibitor at 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 lg of total RNA was fractionated by electrophoresis in 1% (w/v) agarose/ formaldehyde gel, transferred to Zeta-Probe nylon filters (Bio-Rad, Richmond, CA, USA) and hybridized with radiolabeled cDNA probes as described previously [12] Total RNA loading and transfer were evaluated by probing with a glyceraldehyde-3-phosphate dehydrogenase (GAP-DH) cDNA probe The filters were analyzed by phosphori-maging and hybridization signals were quantified to determine the relative amounts of CTGF/CCN2 mRNA (Molecular Dynamics, Sunnyvale, CA, USA) The mRNA levels were analyzed in duplicate and normalized to equivalent values for GAPDH to compensate for variations
in loading and transfer
mRNA stability assay Cells were cultured in tissue culture flasks as 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) and the cells were harvested after 0, 0.5, 1, 2 and 4 h Total RNA was purified and analyzed by 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 mRNA had decayed Nuclear run-on assay
Subconfluent cells were left untreated or stimulated with S1P, anisomycin or FBS for 1 h Experiments with
Trang 3pharmacological inhibitors were performed as described
above Cells were subsequently washed twice with NaCl/Pi,
trypsinized and centrifuged at 4C The cellular pellet was
resuspended in buffer containing 10 mMTris/HCl (pH 7.4),
10 mMNaCl, 3 mMMgCl2, and 0.5% (v/v) Nonidet P-40
allowing swelling and lysis of the cell membrane The lysate
was recentrifuged at 300 g at 4C and the resulting nuclear
pellet was resuspended in 150 lL of buffer containing
20 mM Tris/HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA,
1 mMdithiothreitol and 50% (v/v) glycerol In vitro
tran-scription was then performed with the suspended nuclei at
30C for 30 min in a buffer containing 10 mM Hepes
(pH 8.3), 5 mMMgCl2, 300 mMKCl, 50 mMEDTA, 1 mM
dithiothreitol, 0.1 mMrCTP, rATP, rGTP and 250 lCi of
[32P]UTP[aP] The radiolabeled RNA was extracted from
the nuclei Equal amounts (2.5 lg) of CTGF/CCN2 and
GAPDH cDNA probes were vacuum transferred onto a
Z-probe nylon membrane using a slot blot apparatus
(Bio-Rad) The membrane was UV-irradiated and prehybridized
as described above for northern blotting Equal amounts of
the purified radiolabeled transcripts (106c.p.m.) were
resuspended in hybridization solution Hybridization with
the slot-blotted DNA probes was carried out for 48 h at
42C The membranes were then washed under stringent
conditions before phosphorimager scanning of the
hybridi-zation signals
Transient transfection and coexpression experiments
Cultured cells were plated at a density of 1· 105cm)2in
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 then washed three times with ice-cold NaCl/
Piand total RNA was 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 between 35 and 45% using 1 lg of pEGFP-N1 per
105cells
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 DN forms and the corresponding empty vector 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, cells were cultured in 35-mm
dishes under normal cell culture conditions After
incubation with various stimuli, the cells were washed twice with NaCl/Pi and cell lysates 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 assay (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 off the plate and subjected to denaturing SDS/PAGE under reducing conditions For immunohistochemical analyses, cells were plated on glass cover slips, treated with the indicated drugs, fixed in 2% (v/v) formaldehyde/NaCl/Pi 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, NY, and Cytoskeleton Inc.), following the manufacturer’s instructions Briefly, cells were lysed in buffer containing 50 mM Tris, pH 7.2, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 500 mM NaCl, 10 mM MgCl2 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 blotting
G-Actin/F-actinin vitro assay Determination of 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 to 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 mM Pipes,
50 mM NaCl, 5 mM MgCl2, 5 mM 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 from both 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 All steps were per-formed at 4C
Trang 4Statistical analysis
Data were expressed as 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 ofCTGF/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 lM), anisomycin
(10 ngÆmL)1) or FBS (5%) As shown in Fig 1A, treatment
of the cells with S1P induced only a moderate and
monophasic increase in CTGF/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 induced by serum with five- and
ninefold increases in CTGF/CCN2 mRNA levels after 1
and 6 h, respectively Nearly similar increases were observed
in anisomycin-treated cells, and 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 or 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 to occur either 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 anisomycin is also an inhibitor of protein synthesis at concentrations above 40 ngÆmL)1, we per-formed our studies with a concentration of 10 ngÆmL)1that 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 levels but incubation of 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 mRN
0 s an ser s/ser ser/an s/an
1000 900
Anisomycin Serum 700
600
600
500
400
300
200
100
0
500 400 300 200 100 0
Fig 1 Stimulation of CTGF/CCN2 gene expression by S1P,
aniso-mycin and fetal bovine serum (A) Cells were left untreated as a control
(C) or treated with S1P (s) at a concentration of 10 l M , anisomycin
(an) at a concentration of 10 ngÆmL)1or 5% (v/v) FBS (ser) for the
indicated periods Total RNA was isolated and subjected to northern
blot hybridization analysis To control for unequal RNA 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 Immunodetection was performed 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 anisoaniso-mycin (s/an) Data are average of three
independent experiments.
Trang 5CTGF/CCN2 mRNA, suggesting the involvement of
separate and, perhaps, independent signaling mechanisms
(Fig 1C)
CTGF/CCN2 gene regulation via Rho GTPase signaling
The role of Rho family proteins in CTGF/CCN2 expression
was investigated using toxin B from Clostridium difficile,
which glucosylates Rho family proteins, thereby causing
their inactivation, and the Y-27632 compound, a pyridine
derivative that specifically targets RhoA GTPase signaling
As shown in Fig 2, treatment of the cells with toxin B
significantly altered S1P-, anisomycin- and 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 inhibition 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 pathways that culminate
in the activation of RhoA, Cdc42 and Rac1, the most
A
B
CTGF
ToxB + + +
-ToxB + + +
-Y-27632 - - - - - - - + + +
Y-27632 - - - - - - + + +
GAPDH
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 pretreated for 30 min with either toxin B
(10 ngÆmL)1) or Y-27632 (10 l M ) prior to the addition of either 10 l M
S1P (s), 10 ngÆmL)1anisomycin (an) or 5% (v/v) FBS (ser) One hour
later, total RNA was extracted and subjected to northern blot analysis
with CTGF/CCN2 and GAPDH probes Shown is the percentage of
the relative increase in mRNA levels The values are the means ± 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 -Rho A
Em
yV
ecto r
D -Cdc 42 D
-Ra c1 D -Rho A
Em
yV
ecto r
D -Cdc 42 D -Rac 1 D
-Rh oA
Em
yV ec r
D -Cdc 42 D -Rac 1
Em
yV
ecto r
C -Cdc
42
A-R
ac1
A-R
hoA
0 100 200 300 400 500 600
*
* *
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 stimulated with either 10 l M S1P
or 5% serum for the indicated periods and the amount of GTP-loaded RhoA, Cdc42 and Rac1 was determined by pull-down assay as des-cribed in Materials and methods Total amount of RhoA, Cdc42 and Rac1 in the same samples was determined by western blot and immu-nodetection analyses (B) Cultured cells were transfected with the dominant negative forms DN-RhoA, 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, anisomycin
or FBS and the mRNA levels of the endogenous CTGF/CCN2 gene were determined by northern blot hybridization analysis Shown is the percentage of the relative increase in mRNA levels The values are the means ± SD (n ¼ 3) *P < 0.05 compared with stimulated cells that were transfected with the empty vector (C) Cells 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 endogenous CTGF/CCN2 gene were determined by northern blot hybridization The diagram is rep-resentative of three separate experiments with nearly similar results.
Trang 6thoroughly studied Rho GTPase proteins [14] As shown in
Fig 3A, stimulation of SMCs with S1P induced a sixfold
increase in the amount of 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 Rac1 raising 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-pared with the transient increase in Cdc42 and
GTP-Rac1, the 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 to the cell membrane of these GTPases [25] In
contrast, anisomycin had no effect on the activation of these
Rho GTPases (data not shown) To further investigate the
individual contribution of the Rho GTPases to CTGF/
CCN2expression, we transiently transfected cultured SMCs
with the dominant negative forms DN-RhoA, DN-Cdc42
or DN-Rac1 Figure 3B shows that DN-RhoA reduced the
ability of S1P and serum 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 serum-treated cells ()35%) only, but did
not significantly affect S1P-induced CTGF/CCN2
expres-sion In contrast, DN-Rac1, had no effect on the expression
of CTGF/CCN2 whichever stimulus was used Similarly,
neither of the DN-GTPase forms had an effect on
anisomycin-induced CTGF/CCN2 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 forms of Rho GTPases to enhance the expression of the endogenous CTGF/CCN2 gene As shown in Fig 3C, transfection of the cells with CA-RhoA and CA-Cdc42 induced a 215 and 175% increase
in CTGF/CCN2 mRNA levels, respectively (P < 0.05) Conversely, the active form CA-Rac1 failed 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 than when they are simultaneously activated in response to an external stimulus [26]
Actin polymerization inhibitors affectCTGF /CCN2 expression
Increasing amounts of evidence support an obligatory role for the actin cytoskeleton in the regulation of specific genes by small GTPase proteins The morphology of the actin cytoskeleton upon treatment of the 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
xin B
+ Latrunculin B
+ S1P + Anisomycin + Serum
Fig 4 Effects of S1P, anisomycin and FBS on
actin stress fibers in SMCs and their modulation
by toxin B and latrunculin B Cells were first
stimulated with either S1P, anisomycin or FBS
for 30 min and then fixed, permeabilized and
stained for F-actin with
rhodamine-conju-gated phalloidin The effects of toxin B and
latrunculin B on the actin filaments was
examined by preincubating the cells with
either 10 ngÆmL)1toxin B or 0.5 l M
latrun-culin B for 30 min prior to the addition of
either S1P, anisomycin or FBS for an
addi-tional 30 min.
Trang 7not 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 well (data not shown) Also, 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) Treatment 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 bundles Stimulation of latrunculin B-treated cells
with S1P, anisomycin or serum similarly disrupted the morphology of the actin cytoskeleton
To determine whether actin 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-treated 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 treadmilling In addition, treat-ment of the cells with latrunculin B significantly reduced the CTGF/CCN2 mRNA levels in 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 nonspecific 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 induced 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 that sequestration of G-actin monomers 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/ CCN2expression
The most physiologically conspicuous attribute of actin
is its ability to 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 drugs known to affect actin polymerization
to modulate CTGF/CCN2 expression We utilized jas-plakinolide, a compound that induces actin polymeriza-tion by increasing actin nucleapolymeriza-tion 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 swinholide A did not affect the intensity of F-actin stress fibers in the basal state However, F-actin bundles appear shorter and contained significantly less branching, consistent with the role of swinholide A as a promoter of G-actin dimerization Interestingly, both jasplakino-lide and swinhojasplakino-lide 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, and decreased rapidly thereafter Jasplakinolide and swinho-lide A were used at concentrations (1 lM and 10 nM, respectively) that exhibit optimal effects on actin dynamics [29] However, the observation that swinholide A, which promotes actin monomer dimerization rather than poly-merization, enhanced basal expression of the CTGF/ CCN2gene suggests that a key determinant 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 mRN
Incubation Time (hrs) Fig 5 Effects of latrunculin B on expression 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 10 l M S1P (s), 10 ngÆmL)1anisomycin
(an) or 5% serum (ser) Total RNA was extracted and subjected to
northern blot hybridization analysis with CTGF/CCN2 and GAPDH
probes The diagram is representative of three independent
experi-ments with similar results (B) The effects of latrunculin B alone on
CTGF/CCN2 expression was determined by incubating the cells with
0.5 l M latruculin B for the indicated time periods Total RNA was
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.
Trang 8physiologic states of G-actin monomers within the cells.
Correspondingly, both latrunculin B and jasplakionolide
increased the 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 for 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) In addition, jasplakinolide and swinholide A had no effects on the expression of TGF-b1,
a potent inducer of CTGF/CCN2 expression, although their effects on the activation of pre-existing TGF-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
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 +Jasplakinolide
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
swinho-lide A on actin stress fibers and CTGF/CCN2
expression (A) Cells were stimulated with
either jasplakinolide (0.5 l M ) or swinholide A
(0.1 l M ) for 30 min and then fixed,
permea-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 extracted and 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 jasplakinolide (0.5 l M ) Total
RNA was extracted at the indicated times and
analyzed by northern blot hybridization The
diagrams are representative of three separate
experiments with similar results.
Table 1 Effects of S1P, anisomycin and fetal serum on the G- to F-actin ratio G- to F-actin ratio 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.
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.
Trang 9various stimuli might reflect changes in the ratio of G- to
F-actin Cells were treated with various 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 indicating 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 increase when the cells were pretreated 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 no significant differences were seen,
probably due to the moderate sensitivity of the
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) This 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 the cells
with anisomycin did not significantly alter the G- to F-actin
ratio, suggesting that RhoA/actin-independent signaling mechanisms are involved in anisomycin-induced CTGF/ CCN2expression
CTGF /CCN2 gene regulation through MAP kinase signaling
Because Rho GTPases regulate cytoskeletal reorganization and gene expression either directly or through 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 Erk1/2 and p38 only, whereas FBS or anisomycin stimulation seemed to induce that of JNK1/2 as well (Fig 7A) Differences in the kinetic parameters of activation of these kinases in S1P-, anisomycin- and serum-treated cells were observed Because the protein levels of MAP kinases remain unchanged throughout the course of stimulation, dephosphorylation by phsophatases would be the key factor in the type of pattern
of activation of the MAP kinase in response to various stimuli Activation of 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
AL
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 lg of each 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 p38), phosphorylated JNK1/2 JNK1/2) and phosphorylated Akt/PKB (P-Akt) using monoclonal antibodies that recognize specifically the phosphorylated forms of these proteins (B) Cells were 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)1anisomycin (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 those of GAPDH Shown 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(*).
Trang 103-kinase (PtdIns 3-kinase) that acts either downstream or
upstream of the MAP kinases
To determine the role of these signaling molecules in
CTGF/CCN2 expression, cells were pretreated for 30 min
with Pd-098059 (20 lM), SB-20856 (10 lM), SP-600125
(10 lM), or worthmanin (10 lM), which inhibit Erk1/2, p38,
JNK1/2, and PtdIns 3-kinase, respectively These inhibitors
were used at a concentration that specifically and effectively
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/
CCN2gene 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 role of p38 in
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 CTGF
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 upon stimulation with
anisomycin but did not affect the CTGF/CCN2 mRNA
levels in S1P- or serum-treated cells These data indicate that
serum- and S1P-induced CTGF/CCN2 expression signaling
overlap, albeit to various extent, at the level of p38 signaling,
but are all independent of both Erk1/2 and JNK1/2
signaling pathways
The signaling components upstream of the p38 identified
thus far suggest a complex cell- and stimulus-dependent
regulation consistent with the diversity 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) depending on the cell system studied, although SAP/ERK kinase (SEK/MKK4) acti-vates mostly JNK whereas MKK3 and MKK6 directly activate p38 [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
No
Inhi
btior
Con
trol
No
Inhi
tor
No
Inhi tor +SB
-203 580
+SB -203
580
+SB -20358 0
+S P-600
125
+S P-600 125
+S
P-6001 25
*
*
C
+SB -203 580
No
Inhi
tor
Cont
o r
No
Inhi
btior +SB -203
580
**
120
100
80
60
40
20
0
120
100
80
60
40
20
0
Empty Vector
Empty Vector
Empty V
ector CA-MKK3CA-MKK4CA-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 vectors encoding the active forms
CA-MKK3, CA-MKK4 or CA-MKK6 Control cells were transfected
with the pCDNA3 empty vector Twenty-four hours later, cells were
incubated in serum-free medium for 6 h Total 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) After 24 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 SP-600125 (10 l M ) To compare the CTGF/CCN2 mRNA
levels from different experiments, the stimulation by CA-MKK3,
CA-MKK4 and CA-MKK6 was set to 100% Values are the average ±
SD of three experiments *P < 0.05, **P < 0.01 compared with the
cells transfected with the mutant forms and incubated in the absence of
inhibitors (C) Cells were transfected with the active forms 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 from different experiments, the
sti-mulation by CA-RhoA and CA-Cdc42 was set to 100% Values are the
average ± SD of three experiments **P < 0.01 compared with cells
transfected with the mutant forms and incubated in the absence of
inhibitors.