Tài liệu Báo cáo khoa học: Regulation of connective tissue growth factor (CTGF/CCN2) gene transcription and mRNA stability in smooth muscle cells Involvement of RhoA GTPase and p38 MAP kinase and sensitivity to actin dynamics docx

Eur J Biochem 271, 4436–4450 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04382.x 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 Department 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 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-phosphate (S1P) produced a threefold increase, whereas stimulation with either fetal bovine serum or anisomycin induced an even stronger activation (eightfold) of CTGF/CCN2 expression Using a combination of pathway-specific inhibitors and mutant forms of signaling molecules, we found that S1P- and fetal bovine serum-induced CTGF/CCN2 expression 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 absolutely dependent on actin cytoskeleton integrity In particular, 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-transcriptionally the expression of the CTGF/CCN2 gene in smooth muscle cells Connective tissue growth factor (CTGF) also known as CCN2 was identified as an immediate early responsive gene activated by growth factors in connective tissue 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/ 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 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, glyceraldehyde-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) Keywords: actin cytoskeleton; CTGF/CCN2; p38 MAP kinase; Rho GTPase; smooth muscle cells Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4437 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 application 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 cytoskeletal 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] We undertook this study to investigate the role of Rho GTPase and MAP kinase signaling pathways in the modulation 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 transcriptionally 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 convergence 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 Biolabs (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 containing 5% (v/v) CO2 in air at 37 °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 followed by the addition of chemical stimuli for an additional 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 (GAPDH) cDNA probe The filters were analyzed by phosphorimaging 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 inhibitors and further treated with various stimuli for 30 The culture medium was then replaced with serum-free medium containing actinomycin D (10 lgỈmL)1) and the cells were harvested after 0, 0.5, 1, and h Total RNA was purified and analyzed by northern blot hybridization and phosphorimaging 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 h Experiments with Ó FEBS 2004 4438 I Chowdhury and B Chaqour (Eur J Biochem 271) pharmacological inhibitors were performed as described above Cells were subsequently washed twice with NaCl/Pi, trypsinized and centrifuged at °C The cellular pellet was resuspended in buffer containing 10 mM Tris/HCl (pH 7.4), 10 mM NaCl, 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 °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, mM dithiothreitol and 50% (v/v) glycerol In vitro transcription was then performed with the suspended nuclei at 30 °C for 30 in a buffer containing 10 mM Hepes (pH 8.3), mM MgCl2, 300 mM KCl, 50 mM EDTA, 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 (BioRad) 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 °C The membranes were then washed under stringent conditions before phosphorimager scanning of the hybridization signals Transient transfection and coexpression experiments Cultured cells were plated at a density of · 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 Reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s specifications The Fugene6–DNA mixture plus serum-free medium was left on cells for 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 evaluated 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 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 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 chemiluminescence (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 and stained with rhodamine–phalloidin (Cytoskeleton, Inc., Denver, CO) Images were acquired using a BioRad 1024 MDC laser scanning confocal imaging system RhoA-, Cdc42- and Rac1-GTP pull-down assays Measurement of GTP-bound Rho GTPases was performed using the activation assay kit (Upstate Biotechnology, 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 precipitate the GTP-bound forms of these GTPases The precipitated complexes were then fractioned by electrophoresis 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 in 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 manufacturer’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, mM MgCl2, mM EGTA, 5% (v/v) lyceral, 0.1% (v/v) Nonidet P-40, 0.1% (v/v) Triton X-100, 0.1% (v/v) Tween 20, 0.1% (v/v) 2-mercaptoethanol and 0.001% (v/v) antifoam) and a protease inhibitor cocktail followed by centrifugation for 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 antibody and densitometric scanning All steps were performed at °C Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4439 Statistical 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 of CTGF/CCN2 gene expression As a basis for defining the signaling pathways regulating CTGF/CCN2 gene expression in our system, we first determined the response of primary cultures of SMCs to various stimuli including S1P, a bioactive lysolipid and G-protein-coupled receptor agonist, anisomycin, a genotoxic 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 ngỈmL)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 and h, respectively Nearly similar increases were observed in anisomycin-treated cells, and a 3.1-fold transient stimulation 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 ngỈmL)1 (data not shown) However, because anisomycin is also an inhibitor of protein synthesis at concentrations above 40 ngỈmL)1, we performed our studies with a concentration of 10 ngỈmL)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 A c c c s an ser s an ser CTGF GAPDH 1000 CTGF mRNA Levels (%) Ó FEBS 2004 (hrs) 900 800 S1P Anisomycin Serum 700 600 500 400 300 200 100 0 0.5 16 Incubation Time (hrs) B + S1P CTGF + Anisomycin CTGF + Serum CTGF C 12 hrs 600 CTGF mRNA Levels (%) Fig Stimulation of CTGF/CCN2 gene expression by S1P, anisomycin 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 ngỈmL)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 chemiluminescence (C) Cells were treated for h with S1P, anisomycin or serum or a combination of S1P and serum (s/ser), serum and anisomycin (ser/an) or S1P and anisomycin (s/an) Data are average of three independent experiments 500 400 300 200 100 0 s an ser s/ser ser/an s/an Ó FEBS 2004 4440 I Chowdhury and B Chaqour (Eur J Biochem 271) CTGF/CCN2 mRNA, suggesting the involvement of separate and, perhaps, independent signaling mechanisms (Fig 1C) A 0 10 15 GTP-RhoA Total-RhoA GTP-Cdc42 Total-Cdc42 GTP-Cdc42 Total-Cdc42 GTP-Rac1 Total-Rac1 10 15 GTP-RhoA Total-RhoA GTP-Rac1 CTGF/CCN2 gene regulation via Rho GTPase signaling Total-Rac1 Serum - + B 600 + + S1P - + + + control S1P Anisomycin Serum 500 CTGF mRNA Levels (%) 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 anisomycin-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 400 * * 300 * 200 100 c A Em pty DNVec DN -R tor -C h o DN dc4A -R Em ac1 pt y Ve DN c DN -Rhtor -C o DN dc4A -R ac1 Em pty DNVec DN -R tor -C h o DN dc4A -R ac s an ser s an ser s an sr CTGF C GAPDH - - - + - 500 + - + + Control S1P Anisomycin Serum 450 CTGF mRNA Levels (%) + + - - 400 GAPDH Ve CA ctor -R CA hoA -C d CA c42 -R ac1 B - Em pty ToxB Y-27632 CTGF 350 * 300 250 * 200 * * * 150 100 50 ToxB Y-27632 - - - - - - + + + - - - - - + + + Fig CTGF/CCN2 gene expression is sensitive to Rho GTPase inhibitors (A) Cells were pretreated for 30 with either toxin B (10 ngỈmL)1) or Y-27632 (10 lM) prior to the addition of either 10 lM S1P (s), 10 ngỈmL)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 Fig 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 described in Materials and methods Total amount of RhoA, Cdc42 and Rac1 in the same samples was determined by western blot and immunodetection 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 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 h and the mRNA levels of the endogenous CTGF/CCN2 gene were determined by northern blot hybridization The diagram is representative of three separate experiments with nearly similar results Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4441 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 compared with the transient increase in GTP-Cdc42 and GTPRac1, the levels of which returned to those in control cells within 15 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 expression 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 seruminduced CTGF/CCN2 expression, whereas only RhoA seems to be involved in S1P-induced CTGF/CCN2 mRNA levels + Latrunculin B Fig 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 and then fixed, permeabilized and stained for F-actin with rhodamine-conjugated phalloidin The effects of toxin B and latrunculin B on the actin filaments was examined by preincubating the cells with either 10 ngỈmL)1 toxin B or 0.5 lM latrunculin B for 30 prior to the addition of either S1P, anisomycin or FBS for an additional 30 + Toxin B Control 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 previous 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 affect CTGF /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 organized 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 + S1P + Anisomycin + Serum 4442 I Chowdhury and B Chaqour (Eur J Biochem 271) 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 latrunculin 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 microfilament bundles Stimulation of latrunculin B-treated cells c c s LtB - + - A an ser s an ser + + CTGF GAPDH B 0.5 - - + hrs + + + CTGF GAPDH LtB - + + CTGF mRNA Levels (%) 300 250 200 150 100 50 Incubation Time (hrs) Fig 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 prior to the addition of 10 lM S1P (s), 10 ngỈmL)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 experiments 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 Ó FEBS 2004 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, treatment of the cells with latrunculin B significantly reduced the CTGF/CCN2 mRNA levels in response to anisomycin, 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, latrunculin B induced a time-dependent increase in CTGF/CCN2 mRNA levels that peaked after h and declined progressively thereafter Although unexpected, the modulation of CTGF/CCN2 expression by latrunculin B suggests 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 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 jasplakinolide, a compound that induces actin polymerization 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 jasplakinolide and swinholide A activated CTGF/CCN2 expression 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 swinholide 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 polymerization, enhanced basal expression of the CTGF/ CCN2 gene suggests that a key determinant factor of the effects of actin on CTGF/CCN2 expression is the actual Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4443 A Fig Effects of jasplakinolide and swinholide 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 and then fixed, permeabilized and stained for F-actin with rhodamine-conjugated phalloidin (B) The kinetics of CTGF/CCN2 mRNA accumulation in jasplakinolide- and swinholide A-treated cells were determined for the indicated time periods Total RNA was extracted and analyzed by northern blot hybridization The diagram is representative of three independent experiments with similar results (C) Cells were pretreated for 15 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 Control + Jasplakinolide + Swinholide A B CTGF GAPDH 0.5 Jasplakinolide hrs 0.5 Swinholide A hrs C CTGF GAPDH 0.5 16 24 hrs 0.5 16 24 hrs LtB + Jasplakinolide Swinholide A +Jasplakinolide 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 pharmacological effects of these drugs are only partially understood, and some of their unknown effects may affect gene expression as well 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 levels of free G-actin but via different mechanisms Jasplakinolide depletes the pool of free G-actin by promoting actin polymerization and stabilizing the resultant actin filaments, whereas latrunculin B and swinholide A directly sequester free G-actin and render G-actin monomers, at least temporarily, unavailable for the polymerization process In agreement with these observations, 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 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 Table 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 The role of RhoA GTPase was assessed by pre-treating the cells with Rho kinase inhibitor, Y-27632 (10 lM) for 30 prior to the addition of various stimuli Values are the means ± SD of four experiments Control +S1P +Anisomycin Y-27632 ) ) + – + – + G-Actin/F-Actin 0.260 ± 0.023 0.181 ± 0.023* 0.22 ± 0.021 0.239 ± 0.018 0.223 ± 0.013 0.110 ± 0.034** 0.227 ± 0.019  *P < 0.05, **P < 0.01 versus control;  P < 0.01 versus serum stimulation alone +Serum Ó FEBS 2004 4444 I Chowdhury and B Chaqour (Eur J Biochem 271) various stimuli might reflect changes in the ratio of G- to F-actin Cells were treated with various stimuli for 30 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 stimulation 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 methodology 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/ CCN2 expression 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 substantially 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 P-Erk1/2 P-p38 P-JNK1/2 P-Akt Total-Erk1/2 c B 600 10 15 20 + S1P 10 15 20 10 15 20 + Anisomycin + Serum s an ser s an ser s an ser s an ser s an ser No Inhibitor + Pd-098059 + SB-203580 + SP-600125 + Wortmanin Control S1P Anisomycin Serum CTGF mRNA Levels (%) 500 * 400 * 300 * 200 * * * 100 No Inhibitor + Pd-098059 + SB-203580 + SP-600125 + Wortmanin Fig Effects of MAP kinase and PtdIns 3-kinase inhibitors on S1P-, anisomycin- 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 phosphorylated and total Erk1/2 (P-Erk1/2 and Total-Erk1/2, respectively), phosphorylated p38 (P-p38), phosphorylated JNK1/2 (PJNK1/2) and phosphorylated Akt/PKB (PAkt) using monoclonal antibodies that recognize specifically the phosphorylated forms of these proteins (B) Cells were either left untreated or pretreated for 30 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 ngỈmL)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 3-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 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 continued in the presence of S1P, anisomycin or serum for an additional 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 anisomycin-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 JỈm)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) activates mostly JNK whereas MKK3 and MKK6 directly activate p38 [31] We further examined the contribution of p38 signaling to CTGF/CCN2 gene expression by transfecting cells with the active forms CA-MKK3, CA-MKK6 or CA-MKK4 Expression of these kinases in the cells was Em pt y A CA Ve c to r -M CA KK -M CA KK -M KK CTGF GAPDH B Empty Vector CA-MKK3 CA-MKK4 CA-MKK6 120 CTGF mRNA Levels (%) Ó FEBS 2004 100 80 60 * * 40 20 120 Empty Vector CA-RhoA CA-Cdc42 100 80 60 ** 40 35 bi to In hi No r 58 +S B20 bi to In hi No Co nt ro l r 20 +S B20 C CTGF mRNA Levels (%) Fig 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 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 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 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 stimulation 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 C No ont In rol h +S ibit or B2 +S 035 P- 80 No 001 I n 25 h +S ibit or B2 +S 035 80 P6 No 001 I n 25 h +S ibit or B2 +S 035 P60 01 25 Ó FEBS 2004 4446 I Chowdhury and B Chaqour (Eur J Biochem 271) 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 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 Role of RhoA GTPase and p38 in transcriptional and post-transcriptional regulation of the CTGF/CCN2 gene 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, preincubation 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 exposure to either stimulus Thus, RhoA GTPase pathway seems to play a critical role in CTGF/CCN2 gene transcription, A 2.8 3.5 2.9 4.31 20 120 + Control + An + An +Y27632 + An +SB-203580 100 80 60 40 20 Incubation Time (hrs) 120 Control + Serum + Serum +Y27632 + Serum +SB-203580 100 CTGF mRNA Levels (%) 40 2.75 + - CTGF mRNA Levels (%) CTGF mRNA Levels (%) 60 GF Control SIP +SIP + Y27632 +SIP + SB-203580 Incubation Time (hrs) 1.64 - 80 1.40 4.6 - 1.23 3.29 +Serum 100 CTGF GAPDH 2.5 +Anisomycin 120 CTGF GAPDH 1.35 +S1P B CT Control +Y-27632 +SB-203580 GF CT H GF PD CT GA CTGF GAPDH 80 60 40 20 Incubation Time (hrs) Fig 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 ngỈmL)1) or 5% serum for h Pharmacological inhibition of RhoA and p38 signaling was performed by preincubating the cells with the indicated drugs for 30 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 with a control vehicle, S1P, anisomycin or FBS and further incubating the cells with actinomycin D (10 lgỈmL)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 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 Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4447 whereas p38 signaling minimally affects the transcriptional control of the CTGF/CCN2 gene Next, we examined the CTGF/CCN2 mRNA turnover by inhibiting new mRNA transcription with actinomycin D upon stimulation of the cells with S1P, anisomycin or FBS in the 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 halflife 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 in the regulation of the CTGF/CCN2 gene as well 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 decreasing 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 regulation at the level of mRNA stability seems to occur via p38 signaling mechanisms Discussion This study has focused on the identification of intracellular 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 previously 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 significantly extend those studies in several important ways First, at the level of smooth muscle cells, the data presented are consistent with a dual role of RhoA in the cytoskeletal changes and transcriptional modulation of the CTGF/CCN2 gene The overexpression of a constitutively activated mutant of RhoA, which was shown in separate experiments to induce the formation of stress fibers of contractile actin and myosin filaments, upregulated 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 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 induced the expression of the CTGF/CCN2 gene independent of RhoA activation 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 transcriptional 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 ultimately gene expression [34] Among RhoA targets, RhoAassociated kinase, which is inhibited by the Y-27632 inhibitor, seemed to concomitantly alter actin stress formation and CTGF/CCN2 expression Functionally, RhoAassociated 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 subsequently phosphorylates cofilin and inhibits actin-depolymerizing 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 polymerization 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 Furthermore, our data indicated that RhoA-mediated CTGF/CCN2 expression was carried out at the transcriptional level, suggesting that CTGF promoter activation is critical for RhoA-dependent effects and additional mechanisms 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, treatment of our cells with either curcumin or N-tosyl-Lphenylalanine 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) 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-polymerization-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 forthcoming 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 p38dependent 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 Fig 10 Model of signal transduction pathways involved in the inducible CTGF/CCN2 gene expression by either S1P, FBS or anisomycin Ó FEBS 2004 those of other laboratories but they may be due to cell typespecific or species variations The p38-dependent activation of the CTGF/CCN2 gene although RhoA GTPase-independent required an intact cytoskeleton, and, at least in part, the upstream activation of PtdIns 3-kinase Inhibition of PtdIns 3-kinase reduced both p38 and PKB/Akt phosphorylation in anisomycintreated 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] Furthermore, the p38-dependent increase in CTGF/ CCN2 expression is mediated by stabilization of CTGF/ CCN2 mRNA rather than by transcription of the CTGF/CCN2 gene This post-transcriptional control provides 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 metalloproteinase-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 Ó FEBS 2004 Regulatory mechanisms of the CTGF/CCN2 gene (Eur J Biochem 271) 4449 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 signaldependent gene expression described for other labile mRNAs are unique or relevant for the CTGF/CCN2 mRNA is warranted 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 transcriptionally activates CTGF/CCN2 expression through actin-dependent mechanisms, whereas Cdc42-mediated p38 activation enhanced the stability of CTGF/CCN2 mRNA Our findings confirmed the validity of the prediction that CTGF/CCN2 regulation is fundamentally distinct from that previously reported in fibroblasts Further work is needed to delineate the specific mechanisms of this regulation Acknowledgements 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 10 11 12 13 14 15 16 17 References 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 Takigawa, M (2003) CTGF/Hcs24 as a multifunctional growth factor for fibroblasts, chondrocytes and vascular endothelial cells Drug News Perspect 16, 11–21 Leask, A & Abraham, D.J (2003) The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology Biochem Cell Biol 81, 355–363 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 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 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 Candido, R., Jandeleit-Dahm, K.A., Cao, Z., Nesteroff, S.P., Burns, W.C., Twigg, S.M., Dilley, R.J., Cooper, M.E & Allen, T.J (2002) Prevention of accelerated atherosclerosis by 18 19 20 21 22 23 24 angiotensin-converting enzyme inhibition in diabetic apolipoprotein E-deficient mice Circulation 106, 246–253 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 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 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 Schild, C & Trueb, B (2002) Mechanical stress is required for high-level expression of connective tissue growth factor Exp Cell Res 274, 83–91 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 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 umbilical vein endothelial cells Proc Natl Acad Sci USA 98, 8955– 8960 Kyriakis, J.M & Avruch, J (2001) Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation Physiol Rev 81, 807–869 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 Lau, L.F & Lam, S.C (1999) The CCN family of angiogenic regulators: the integrin connection Exp Cell Res 248, 44–57 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 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 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 Ono, K & Han, J (2000) The p38 signal transduction pathway: activation and function Cell Signal 12, 1–13 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 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 Somlyo, A.P & Somlyo, A.V (1998) From pharmacomechanical coupling to G-proteins and myosin phosphatase Acta Physiol Scand 164, 437–448 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 Ó FEBS 2004 4450 I Chowdhury and B Chaqour (Eur J Biochem 271) 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 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 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 30 Davies, S.P., Reddy, H., Caivano, M & Cohen, P (2000) Specificity 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 32 Hahn, A., Heusinger-Ribeiro, J., Lanz, T., Zenkel, S & Goppelt-Struebe, M (2000) 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 33 Heusinger-Ribeiro, J., Eberlein, M., Wahab, N.A & GoppeltStruebe, 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 responses Trends Cell Biol 11, 471–477 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 37 Hilpela, P., Vartiainen, M.K & Lappalainen, P (2004) Regulation 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 39 Blom, I.E., Goldschmeding, R & Leask, A (2002) Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biol 21, 473–482 40 Tominaga, T., Sahai, E., Chardin, P., McCormick, F., Courtneidge, S.A & Alberts, A.S (2000) Diaphanous-related formins bridge Rho GTPase and Src tyrosine kinase signaling Mol Cell 5, 13–25 41 Leask, A., Holmes, A., Black, C.M & Abraham, D.J (2003) Connective tissue growth factor gene regulation Requirements for 42 43 44 45 46 47 48 49 50 51 52 its induction by transforming growth factor-beta in fibroblasts J Biol Chem 278, 13008–13015 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 bleomycininduced pulmonary fibrosis Am J Physiol Lung Cell Mol Physiol 283, L103–L112 Ahmed, M.S., Oie, E., Vinge, L.E., Yndestad, A., Oystein, A.G., Andersson, Y., Attramadal, T & Attramadal, H (2004) Connective tissue growth factor – a novel mediator of angiotensin IIstimulated cardiac fibroblast activation in heart failure in rats J Mol Cell Cardiol 36, 393–404 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 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 Janmey, P.A (1998) The cytoskeleton and cell signaling: component localization and mechanical coupling Physiol Rev 78, 763– 781 Clark, A.R., Dean, J.L & Saklatvala, J (2003) Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38 FEBS Lett 546, 37–44 Kubota, S., Hattori, T., Nakanishi, T & Takigawa, M (1999) Involvement of cis-acting repressive element(s) in the 3¢-untranslated region of human connective tissue growth factor gene FEBS Lett 450, 84–88 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 and an AU-rich region-targeted mechanism EMBO J 18, 4969– 4980 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 elements by inhibiting deadenylation J Biol Chem 278, 39470– 39476 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 Paste, M., Huez, G & Kruys, V (2003) Deadenylation of interferon-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  ... 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... 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... 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