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

doi:10.1111/j.1432-1033.2004.04382.x

Eur. J. Biochem. 271, 4436–4450 (2004) (cid:1) FEBS 2004

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 Chowdhury1,* and Brahim Chaqour2 1Department of Anatomy and Cell Biology, University of Pennsylvania, PA, USA; 2Department of Anatomy and Cell Biology, State University of New York (SUNY) Downstate Medical Center, Brooklyn, NY, USA

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/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 signaling mechanisms. phosphatidyl inositol 3-kinase

Connective tissue growth factor (CTGF) also known as CCN2 was 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,

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)

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/ CCN2 in 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

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1 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

to late-gestational

Primary cultures of SMCs were prepared from the bladders fetal calves as previously of mid- 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) CO2 in air at 37 (cid:2)C. Cells from passages 2–8 were used for the experiments. For most experiments, cells were grown to subconfluence either in 25-cm2 culture 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

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 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/ 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 obstructive diseases and atherosclerosis [8,15,16].

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

Materials and methods

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

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

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 Subconfluent cells were left untreated or stimulated with S1P, anisomycin or FBS for 1 h. Experiments with

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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) . 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

pharmacological inhibitors were performed as described above. Cells were subsequently washed twice with NaCl/Pi, trypsinized and centrifuged at 4 (cid:2)C. The cellular pellet was resuspended in buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 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 (cid:2)C 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 mM dithiothreitol and 50% (v/v) glycerol. In vitro tran- scription was then performed with the suspended nuclei at 30 (cid:2)C for 30 min in a buffer containing 10 mM Hepes (pH 8.3), 5 mM MgCl2, 300 mM KCl, 50 mM EDTA, 1 mM dithiothreitol, 0.1 mM rCTP, 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 (106 c.p.m.) were resuspended in hybridization solution. Hybridization with the slot-blotted DNA probes was carried out for 48 h at 42 (cid:2)C. The membranes were then washed under stringent conditions before phosphorimager scanning of the hybridi- zation signals.

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- these GTPases. The pitate the GTP-bound forms of 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-actin invitroassay

Transient transfection and coexpression experiments Cultured cells were plated at a density of 1 · 105 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 (Roche Diagnostics, Mannheim, Germany) Reagent 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/ Pi and 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 105 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 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 conditions. After dishes under normal culture cell 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 4 (cid:2)C.

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Statistical analysis

Results

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.

Modulation of CTGF/CCN2 gene expression

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)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 levels but incubation of the cells with anisomycin further augmented S1P-mediated increase in

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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

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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 lM, anisomycin (an) at a concentration of 10 ngÆmL)1 or 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 anisomycin (s/an). Data are average of three independent experiments.

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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 inhibition of Rho GTPase signaling induced maximal [23,24]. These data pinpoint to an important role for RhoA GTPase signaling in CTGF/CCN2 gene regulation.

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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 lM 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.

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 lM) prior to the addition of either 10 lM S1P (s), 10 ngÆmL)1 anisomycin (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.

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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].

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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)1 toxin B or 0.5 lM latrun- culin B for 30 min prior to the addition of either S1P, anisomycin or FBS for an addi- tional 30 min.

thoroughly 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 GTP-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/ CCN2 expression, 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, the DN-GTPase forms had an effect on neither of 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. 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

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with S1P, anisomycin or serum similarly disrupted the morphology of the actin cytoskeleton.

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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 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

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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/ CCN2 expression.

Fig. 5. Effects of latrunculin B on expression of the CTGF/CCN2 gene in SMCs. (A) Cells were pretreated with 0.5 lM latrunculin B (LtB) for 30 min prior to the addition of 10 lM S1P (s), 10 ngÆmL)1 anisomycin (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 lM 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.

respectively, and

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 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 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 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, 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- the CTGF/ merization, enhanced basal expression of CCN2 gene suggests that a key determinant factor of the effects of actin on CTGF/CCN2 expression is the actual

Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4443

(cid:1) FEBS 2004

A

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LtB + Jasplakinolide

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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 lM) or swinholide A (0.1 lM) 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 lM) or swinholide A (0.1 lM) prior to the addition of jasplakinolide (0.5 lM). 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.

the pool 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.

temporarily, unavailable for least Changes in G-actin/F-actin ratio correlate with RhoA GTPase activation

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 lM) 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(cid:1)

*P < 0.05, **P < 0.01 versus control; (cid:1)P < 0.01 versus serum stimulation alone.

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 physiologic 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 free G-actin but via different mechanisms. levels of Jasplakinolide depletes 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 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

4444 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271)

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ratio, suggesting that RhoA/actin-independent signaling mechanisms are involved in anisomycin-induced CTGF/ CCN2 expression.

CTGF/CCN2gene regulation through MAP kinase signaling

various 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 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

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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 (P-p38), phosphorylated JNK1/2 (P- 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 lM), SB-203580 (10 lM), SP-600125 (10 lM) or wortmanin (10 lM) prior to the addition of 10 lM 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 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(*).

Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4445

(cid:1) FEBS 2004

3-kinase (PtdIns 3-kinase) that acts either downstream or upstream of the MAP kinases.

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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

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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/ 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 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.

0

The signaling components upstream of the p38 identified thus far suggest a complex cell- and stimulus-dependent regulation consistent with the diversity of extracellular

NoInhibtior NoInhibitor +SP-600125 +SB-203580 NoInhibitor +SB-203580 +SP-600125 +SB-203580 Control +SP-600125

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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 lM) or SP-600125 (10 lM). 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 lM). 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.

4446 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271)

(cid:1) FEBS 2004

Role of RhoA GTPase and p38 in transcriptional and post-transcriptional regulation of the CTGF/CCN2gene

previously detected by western blot 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- or CA-MKK6-transfected cells with the p38 inhibitor, SB-20589, significantly 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 p38 and JNK1/2.

In order to determine whether CTGF/CCN2 expression occurs via increased transcription and/or by stabilization of the CTGF transcripts and the 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 the presence of RhoA GTPase and p38 inhibitors (Y-27632 and SB-203580, respectively). As shown 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 role in CTGF/CCN2 gene transcription, Meanwhile, because p38 is a potential downstream target of RhoA and Cdc42, we examined the effects of SB-203580, a p38 inhibitor on CTGF/CCN2 expression in CA-RhoA and CA-Cdc42-transfected cells. As shown 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.

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Fig. 9. The CTGF/CCN2 gene is transcriptionally regulated through RhoA GTPase and post-transcriptionally regulated through p38 signaling. (A) Nuclear run-on assay showing the effects of S1P, anisomycin and serum in the absence and presence of RhoA GTPase inhibitor 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 lM), anisomycin (10 ngÆmL)1) or 5% serum for 1 h. Pharmacological inhibition of RhoA and p38 signaling was performed by preincubating the cells with the indicated drugs for 30 min prior to the treatment with various stimuli. The pre-mRNA was radiolabeled, isolated and hybridized to CTGF/CCN2 and GAPDH cDNA probes, which had been slot blotted on nylon membranes. The hybridization signals for CTGF/CCN2 were normalized to those of GAPDH. These experiments were performed in duplicate. (B) Effects of S1P, anisomycin and FBS on 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 cells with the indicated drugs for 30 min prior to treatment with the various 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.

Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4447

(cid:1) FEBS 2004

whereas p38 signaling minimally affects the transcriptional control of the CTGF/CCN2 gene.

induced the

of the CTGF/CCN2 gene expression; monomeric G-actin inhibited CTGF/CCN2 gene induction, whereas F-actin enhanced CTGF/CCN2 gene expression. Fourth, actin monomer-sequestering agents that mimic the physiologic G-actin-binding proteins 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.

Discussion

3 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 absence and in the presence of RhoA GTPase and p38 inhibitors. 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, respectively. This indicates that an mRNA stabilizing effect is involved the CTGF/CCN2 gene as well. in the regulation of Pretreatment of the cells with Y-27632 inhibitor did not dramatically alter the 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 signaling mechanisms.

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 mediating 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 and increases acto-myosin-based contractility [27]. The resulting contractile forces are thought to contribute to the formation of stress fibers and focal contacts. In addition, RhoA-kinase also activates Lin11/Isl-1/Mec3 (LIM) 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 RhoA 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 of adequate tools to evaluate not only total cellular PtdIns, but also local concentrations within the cells. Meanwhile, using actin-binding drugs, we showed that the expression of the CTGF/CCN2 gene can be modulated by either actin polymerization or the availability of poly- merization competent G-actin referred to as free barbed-end actin. Data from in vitro assays previously suggested that major G-actin binding proteins (e.g. b-thymosins) selectively affect the availability of barbed-end actin and determine the level and distribution of F-actin [34]. Therefore, it is tempting to speculate that interactions between monomeric G-actin and actin-binding proteins are a potential target of regulation by RhoA GTPase.

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 the 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 GTPases play a pivotal 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 that 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 an induction [32]. Our results concur and important significantly extend those studies in several 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 of 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 polymerization of cortical actin did not affect expression of the CTGF/CCN2 gene [35]. Third, RhoA-actin signaling exerted bimodal modulation Furthermore, our data indicated that RhoA-mediated CTGF/CCN2 expression was carried out at the transcrip- tional level, suggesting that CTGF promoter activation is critical for RhoA-dependent effects and additional mecha- nisms that sense actin dynamics in the cells may be involved as well. RhoA was shown to activate several transcription factors that play important roles in growth factor regulation 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-, S1P- or FBS-treated cells, whereas treatment with mithramycin, a Sp1 inhibitor, had no effect (data not shown). Because the CTGF/CCN2

4448 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271)

(cid:1) FEBS 2004

those of other laboratories but they may be due to cell type- specific or species variations.

promoter contains several AP-1 and NF-jB binding sites, it is conceivable that the actin cytoskeleton architecture orchestrated by RhoA regulates the CTGF/CCN2 gene by acting as a catalytic surface and/or protein cofactor 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 and 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.

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 cells (Fig. 10). These data are in line with previous observations indicating that physiological activators of the p38 and PtdIns 3-kinase pathways including thrombin, 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 signaling between cell surface 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 signaling machinery components such as p38 and PtdIns 3-kinase involved in intracellular communications [46].

than by transcription of

Fig. 10. Model of signal transduction pathways involved in the inducible CTGF/CCN2 gene expression by either S1P, FBS or anisomycin.

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 and that signaling mechanisms via p38 MAP kinase were involved as well. 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 reported by other laboratories. Leask et al. found that p38 inhibitors had no impact on the 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 model of bleomycin-induced pulmonary fibrosis, suggesting that p38- dependent regulation of CTGF/CCN2 expression may be an in vivo active mechanism as well [42]. We have no ready explanation for the differences between our results and

Furthermore, the p38-dependent increase in CTGF/ CCN2 expression is mediated by stabilization of CTGF/ CCN2 mRNA rather the CTGF/CCN2 gene. This post-transcriptional control pro- vides an additional means of increasing the expression of the gene and ensuring that its levels remain within a critical range. It also enables rapid changes in CTGF/CCN2 mRNA levels in response to stimuli and provides a mechanism for prompt termination of the protein synthesis. These data add 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 growth 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 target an mRNA for rapid deadenylation and degradation and may even enhance mRNA decapping [47]. Interestingly, the 3¢-untranslated region (3¢-UTR) of the CTGF/CCN2 gene contains three AUUUA pentamers 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 presence 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 such as those of TNF-a and Cox-2 from a state of translational arrest imposed by AREs within the 3¢-UTR by regulating deadenylation rather than decay of the mRNA body [50–52]. It was also suggested that p38 stabilizes mRNA by targeting putative ARE-binding proteins. However, despite the identification of several ARE-binding proteins, it is unclear which (if any) provides a link between p38 and the

Regulatory mechanisms of the CTGF/CCN2 gene (Eur. J. Biochem. 271) 4449

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angiotensin-converting enzyme inhibition in diabetic apolipopro- tein E-deficient mice. Circulation 106, 246–253.

8. Chaqour, B., Whitbeck, C., Han, J.S., Macarak, E., Horan, P., Chichester, P. & Levin, R. (2002) Cyr61 and CTGF are molecular markers of bladder wall remodeling after outlet obstruction. Am. J. Physiol. Endocrinol. Metab. 283, E765–E774.

9. Oemar, B.S., Werner, A., Garnier, J.M., Do, D.D., Godoy, N., Nauck, M., Marz, W., Rupp, J., Pech, M. & Luscher, T.F. (1997) Human connective tissue growth factor is expressed in advanced atherosclerotic lesions. Circulation 95, 831–839.

10. Mori, T., Kawara, S., Shinozaki, M., Hayashi, N., Kakinuma, T., Igarashi, A., Takigawa, M., Nakanishi, T. & Takehara, K. (1999) Role and interaction of connective tissue growth factor with transforming growth factor-beta in persistent fibrosis: a mouse fibrosis model. J. Cell Physiol. 181, 153–159.

AREs. Much further work is required to precisely ascertain which specific mRNA decay steps and ARE-binding proteins are targeted by the p38 signaling pathway. Our study showed that the CTGF/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 by the p38 pathway. Accordingly, elucidation of whether the types of signal- dependent gene expression described for other labile mRNAs are unique or relevant for the CTGF/CCN2 mRNA is warranted.

11. Schild, C. & Trueb, B. (2002) Mechanical stress is required for high-level expression of connective tissue growth factor. Exp. Cell Res. 274, 83–91.

12. Tamura, I., Rosenbloom, J., Macarak, E. & Chaqour, B. (2001) Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways. Am. J. Physiol. Cell Physiol. 281, C1524–C1532.

activates CTGF/CCN2

13. McCormick, S.M., Eskin, S.G., McIntire, L.V., Teng, C.L., Lu, C.M., Russell, C.G. & Chittur, K.K. (2001) DNA microarray reveals changes in gene expression of shear stressed human um- bilical vein endothelial cells. Proc. Natl Acad. Sci. USA 98, 8955– 8960.

Acknowledgements

14. Kyriakis, J.M. & Avruch, J. (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81, 807–869.

15. Charron, F., Tsimiklis, G., Arcand, M., Robitaille, L., Liang, Q., Molkentin, J.D., Meloche, S. & Nemer, M. (2001) Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA. Genes Dev. 15, 2702–2719.

This study is supported by the 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. Hall (University College, London, UK) for the generous gifts of the vectors encoding constitutively active forms of RhoA, Cdc42 and Rac; to Dr J.H. Han (The Scripps Institute, CA) for providing CaMKK3 and CaMKK6 constructs and to Dr A. Morrison for providing us with the active form of MKK4.

References

16. Lau, L.F. & Lam, S.C. (1999) The CCN family of angiogenic regulators: the integrin connection. Exp. Cell Res. 248, 44–57. 17. Perbal, B., Martinerie, C., Sainson, R., Werner, M., He, B. & Roizman, B. (1999) The C-terminal domain of the regulatory protein NOVH is sufficient to promote interaction with fibulin 1C: a clue for a role of NOVH in cell-adhesion signaling. Proc. Natl Acad. Sci. USA 96, 869–874.

1. Igarashi, A., Okochi, H., Bradham, D.M. & Grotendorst, G.R. (1993) Regulation of connective tissue growth factor gene expression in human skin fibroblasts and during wound repair. Mol. Biol. Cell 4, 637–645.

18. Han, J.S., Macarak, E., Rosenbloom, J., Chung, K.C. & Chaqour, B. (2003) Regulation of Cyr61/CCN1 gene expression through RhoA GTPase and p38MAPK signaling pathways. Eur. J. Biochem. 270, 3408–3421.

2. Takigawa, M. (2003) CTGF/Hcs24 as a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells. Drug News Perspect. 16, 11–21.

19. Guan, Z., Buckman, S.Y., Pentland, A.P., Templeton, D.J. & Morrison, A.R. (1998) Induction of cyclooxygenase-2 by the activated MEKK1 (fi) SEK1/MKK4 (fi) p38 mitogen-activated protein kinase pathway. J. Biol. Chem. 273, 12901–12908.

20. Ono, K. & Han, J. (2000) The p38 signal transduction pathway:

3. Leask, A. & Abraham, D.J. (2003) The role of connective tissue growth factor, a multifunctional matricellular protein, in fibro- blast biology. Biochem. Cell Biol. 81, 355–363.

activation and function. Cell Signal. 12, 1–13.

21. Wahab, N.A., Brinkman, H. & Mason, R.M. (2001) Uptake and intracellular transport of the connective tissue growth factor: a potential mode of action. Biochem. J. 359, 89–97.

4. Chen, M.M., Lam, A., Abraham, J.A., Schreiner, G.F. & Joly, A.H. (2000) CTGF expression is induced by TGF-beta in cardiac fibroblasts and cardiac myocytes: a potential role in heart fibrosis. J. Mol. Cell Cardiol. 32, 1805–1819.

22. Laursen, N.B., Kessler, R., Frohli, E. & Klemenz, R. (1998) Effects of ras transformation on the induction of the IL-1 receptor related T1 gene in response to mitogens, anisomycin, IL-1 and TNFalpha. Oncogene 16, 575–586.

5. Yokoi, H., Sugawara, A., Mukoyama, M., Mori, K., Makino, H., Suganami, T., Nagae, T., Yahata, K., Fujinaga, Y., Tanaka, I. & Nakao, K. (2001) Role of connective tissue growth factor in profibrotic action of transforming growth factor-beta: a potential target for preventing renal fibrosis. Am. J. Kidney Dis. 38, S134– S138.

23. Somlyo, A.P. & Somlyo, A.V. (1998) From pharmacomechanical coupling to G-proteins and myosin phosphatase. Acta Physiol. Scand. 164, 437–448.

6. Black, J.L., Burgess, J.K. & Johnson, P.R. (2003) Airway smooth muscle – its relationship to the extracellular matrix. Respir. Physiol. Neurobiol. 137, 339–346.

24. Uehata, M., Ishizaki, T., Satoh, H., Ono, T., Kawahara, T., Morishita, T., Tamakawa, H., Yamagami, K., Inui, J., Maekawa, M. & Narumiya, S. (1997) Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389, 990–994.

7. Candido, R., Jandeleit-Dahm, K.A., Cao, Z., Nesteroff, S.P., Burns, W.C., Twigg, S.M., Dilley, R.J., Cooper, M.E. & Allen, (2002) Prevention of accelerated atherosclerosis by T.J.

In conclusion, this study demonstrates a critical role of 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 expression through actin-dependent mechanisms, whereas Cdc42-mediated p38 activation enhanced the stability 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.

4450 I. Chowdhury and B. Chaqour (Eur. J. Biochem. 271)

(cid:1) FEBS 2004

its induction by transforming growth factor-beta 2 in fibroblasts. J. Biol. Chem. 278, 13008–13015.

25. Li, S., Chen, B.P., Azuma, N., Hu, Y.L., Wu, S.Z., Sumpio, B.E., Shyy, J.Y. & Chien, S. (1999) Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress. J. Clin. Invest. 103, 1141–1150.

42. Matsuoka, H., Arai, T., Mori, M., Goya, S., Kida, H., Morishita, H., Fujiwara, H., Tachibana, I., Osaki, T. & Hayashi, S. (2002) A p38 MAPK inhibitor, FR-167653, ameliorates murine bleomycin- induced pulmonary fibrosis. Am. J. Physiol. Lung Cell Mol. Physiol. 283, L103–L112.

26. Teramoto, H., Malek, R.L., Behbahani, B., Castellone, M.D., Lee, N.H. & Gutkind, J.S. (2003) Identification of H-Ras, RhoA, Rac1 and Cdc42 responsive genes. Oncogene 22, 2689–2697. 27. Carpenter, C.L. (2000) Actin cytoskeleton and cell signaling. Crit.

Care Med. 28, N94–N99.

43. Ahmed, M.S., Oie, E., Vinge, L.E., Yndestad, A., Oystein, A.G., Andersson, Y., Attramadal, T. & Attramadal, H. (2004) Con- nective tissue growth factor – a novel mediator of angiotensin II- stimulated cardiac fibroblast activation in heart failure in rats. J. Mol. Cell Cardiol. 36, 393–404.

28. Spector, I., Braet, F., Shochet, N.R. & Bubb, M.R. (1999) New anti-actin drugs in the study of the organization and function of the actin cytoskeleton. Microsc. Res. Techn. 47, 18–37.

29. Sotiropoulos, A., Gineitis, D., Copeland, J. & Treisman, R. (1999) Signal-regulated activation of serum response factor is mediated by changes in actin dynamics. Cell 98, 159–169.

44. Pendurthi, U.R., Allen, K.E., Ezban, M. & Rao, L.V. (2000) Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa · tissue factor-induced signal transduction. J. Biol. Chem. 275, 14632–14641.

30. Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. (2000) Speci- ficity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105.

31. Hagemann, C. & Blank, J.L. (2001) The ups and downs of MEK

kinase interactions. Cell Signal. 13, 863–875.

(2000)

45. Suzuma, K., Naruse, K., Suzuma, I., Takahara, N., Ueki, K., Aiello, L.P. & King, G.L. (2000) Vascular endothelial growth factor induces expression of connective tissue growth factor via KDR, Flt1, and phosphatidylinositol 3-kinase-akt-dependent pathways in retinal vascular cells. J. Biol. Chem. 275, 40725– 40731.

32. Hahn, A., Heusinger-Ribeiro, J., Lanz, T., Zenkel, S. & Goppelt-Struebe, M. Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J. Biol. Chem. 275, 37429–37435.

46. Janmey, P.A. (1998) The cytoskeleton and cell signaling: compo- nent localization and mechanical coupling. Physiol. Rev. 78, 763– 781.

33. Heusinger-Ribeiro, J., Eberlein, M., Wahab, N.A. & Goppelt- Struebe, M. (2001) Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J. Am. Soc. Nephrol. 12, 1853–1861.

34. Ridley, A.J.

(2001) Rho family proteins: coordinating cell

47. Clark, A.R., Dean, J.L. & Saklatvala, J. (2003) Post-transcrip- tional regulation of gene expression by mitogen-activated protein kinase p38. FEBS Lett. 546, 37–44.

responses. Trends Cell Biol. 11, 471–477.

48. Kubota, S., Hattori, T., Nakanishi, T. & Takigawa, M. (1999) Involvement of cis-acting repressive element(s) in the 3¢-untrans- lated region of human connective tissue growth factor gene. FEBS Lett. 450, 84–88.

35. Weed, S.A., Y. & Parsons, J.T. (1998) Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1. J. Cell Sci. 111, 2433–2443.

36. Ren, X.D., Kiosses, W.B. & Schwartz, M.A. (1999) Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J. 18, 578–585.

49. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C.Y., Shyu, A.B., Muller, M., Gaestel, M., Resch, K. & Holtmann, H. (1999) The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18, 4969– 4980.

37. Hilpela, P., Vartiainen, M.K. & Lappalainen, P. (2004) Regula- tion of the actin cytoskeleton by PI(4,5),P2 and PI(3,4,5),P3. Curr. Top. Microbiol. Immunol. 282, 117–163.

38. Chang, J.H., Pratt, J.C., Sawasdikosol, S., Kapeller, R. & Burakoff, S.J. (1998) The small GTP-binding protein Rho potentiates AP-1 transcription in T cells. Mol. Cell Biol. 18, 4986– 4993.

50. Dean, J.L., Sarsfield, S.J., Tsounakou, E. & Saklatvala, J. (2003) p38 Mitogen-activated protein kinase stabilizes mRNAs that contain cyclooxygenase-2 and tumor necrosis factor AU-rich ele- ments by inhibiting deadenylation. J. Biol. Chem. 278, 39470– 39476.

39. Blom, I.E., Goldschmeding, R. & Leask, A. (2002) Gene regula- tion of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biol. 21, 473–482.

51. Kraatz, J., Clair, L., Rodriguez, J.L. & West, M.A. (1999) Macrophage TNF secretion in endotoxin tolerance: role of SAPK, p38, and MAPK. J. Surg. Res. 83, 158–164.

40. Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Court- neidge, S.A. & Alberts, A.S. (2000) Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling. Mol. Cell 5, 13–25.

52. Paste, M., Huez, G. & Kruys, V. (2003) Deadenylation of inter- feron-beta mRNA is mediated by both the AU-rich element in the 3¢-untranslated region and an instability sequence in the coding region. Eur. J. Biochem. 270, 1590–1597.

41. Leask, A., Holmes, A., Black, C.M. & Abraham, D.J. (2003) Connective tissue growth factor gene regulation. Requirements for