Matrix Metalloproteinase 2 deficient Fibroblasts Exhibit an Alteration in the Fibrotic Response to Connective Tissue Growth Factor/CCN2 because of an Increase in the Levels of Endogenous Fibronectin

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(1)THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 20, pp. 13551–13561, May 15, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.. Matrix Metalloproteinase-2-deficient Fibroblasts Exhibit an Alteration in the Fibrotic Response to Connective Tissue Growth Factor/CCN2 because of an Increase in the Levels of Endogenous Fibronectin* Received for publication, September 23, 2008, and in revised form, February 2, 2009 Published, JBC Papers in Press, March 9, 2009, DOI 10.1074/jbc.M807352200. Cristian A. Droppelmann, Jaime Gutiérrez, Cecilia Vial, and Enrique Brandan1 From the Centro de Regulación Celular y Patología, Centro de Regeneración y Envejecimiento, Departamento de Biología Celular y Molecular, Millenium Institute for Fundamental and Applied Biology, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile Matrix metalloproteinase-2 (MMP-2) is an important extracellular matrix remodeling enzyme, and it has been involved in different fibrotic disorders. The connective tissue growth factor (CTGF/CCN2), which is increased in these pathologies, induces the production of extracellular matrix proteins. To understand the fibrotic process observed in diverse pathologies, we analyzed the fibroblast response to CTGF when MMP-2 activity is inhibited. CTGF increased fibronectin (FN) amount, MMP-2 mRNA expression, and gelatinase activity in 3T3 cells. When MMP-2 activity was inhibited either by the metalloproteinase inhibitor GM-6001 or in MMP-2-deficient fibroblasts, an increase in the basal amount of FN together with a decrease of its levels in response to CTGF was observed. This paradoxical effect could be explained by the fact that the excess of FN could block the access to other ligands, such as CTGF, to integrins. This effect was emulated in fibroblasts by adding exogenous FN or RGDS peptides or using anti-integrin ␣V subunit-blocking antibodies. Additionally, in MMP-2-deficient cells CTGF did not induce the formation of stress fibers, focal adhesion sites, and ERK phosphorylation. Anti-integrin ␣V subunit-blocking antibodies inhibited ERK phosphorylation in control cells. Finally, in MMP-2-deficient cells, FN mRNA expression was not affected by CTGF, but degradation of 125I-FN was increased. These results suggest that expression, regulation, and activity of MMP-2 can play an important role in the initial steps of fibrosis and shows that FN levels can regulate the cellular response to CTGF.. Extracellular proteolysis is an essential physiological process that controls the immediate cellular environment and thus plays a key role in cellular behavior and survival (1). The members of the matrix metalloproteinase (MMP)2 family of zinc-de-. * This work was supported in part by Fondo de Financiamiento de Centros de Excelencia en Investigación (FONDAP-Biomedicine) Grant 13980001, Center for Aging and Regeneration (CARE) Grant PFB 12/2007, Muscular Dystrophy Association Grant 89419, FONDECYT Grant 3060100 (to C. D.), and funds from the Ministerio de Planificación y Cooperación (Chile). 1 To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, P.O. Box 114-D, Santiago, Chile. Fax: 56-2-635-5395; E-mail: ebrandan@bio.puc.cl. 2 The abbreviations used are: MMP, matrix metalloproteinase; CTGF, connective tissue growth factor; FN, fibronectin; ERK, extracellular signal-regulated kinase; TIMP, tissue inhibitors of metalloproteinase; ECM, extracellu-. MAY 15, 2009 • VOLUME 284 • NUMBER 20. pendent endopeptidases are major mediators of extracellular proteolysis by promoting the degradation of extracellular matrix (ECM) components and cell surface-associated proteins (2, 3). Each one of these enzymes is negatively regulated by tissue inhibitors of metalloproteinases (TIMPs) (4) and is secreted as a zymogen (pro-MMPs) that is activated in the extracellular space (5–7). This mechanism is an important form of regulation of gelatinase activity and in consequence, highly significant for ECM homeostasis. Among the members of the MMP family, the metalloproteinase type 2 (MMP-2 or gelatinase A) is known to be a key player in many physiological and pathological processes, such as cell migration, inflammation, angiogenesis, and fibrosis (8 –11). Fibrotic disorders are typified by excessive connective tissue and ECM deposition that precludes normal healing of different tissues. ECM accumulation can be explained in two ways: increasing expression and deposition of connective tissue proteins and/or decreasing degradation of ECM proteins (12). Transforming growth factor type ␤, a multifunctional cytokine, is strongly overexpressed, and it is associated to the pathogenesis of these diseases (13, 14). It stimulates the expression of connective tissue growth factor (CTGF/CCN2) (15), a cytokine that is responsible for transforming growth factor type ␤ fibrotic activity (16, 17). The role of CTGF in fibrosis has gained attention in recent years (16, 18 –22). CTGF overexpression is known to occur in a variety of fibrotic skin disorders (23, 24), renal (25), hepatic (26), and pulmonary fibrosis (27) and in muscles from patients with Duchenne muscular dystrophy (28). On the other hand, several pathologies involving fibrosis show an increase in MMP expression, including gelatinase A. Augmented expression of MMP-2 was found in submucous (29), skin (30), liver (31), and lung fibrosis (32, 33) and dystrophic myotubes from fibrotic muscles of Duchenne muscular dystrophy (34). It has been shown that transforming growth factor type ␤ induces an increase in the amount of MMP-2 in fibroblasts (35) and that CTGF induces MMP-2 expression in cultured renal interstitial fibroblasts (36). The putative role assigned to MMP-2 in fibrotic disorders is related to tissue regeneration because of the capacity of this enzyme to degrade basal lamina (37–39). Because MMP-2 expression is up-regular matrix; rqPCR, relative quantitative reverse transcription-PCR; shRNA, short hairpin RNA.. JOURNAL OF BIOLOGICAL CHEMISTRY. 13551.

(2) FN Excess Affects the CTGF Fibrotic Response lated in these pathologies but still a high ECM deposition is observed, we propose that this accumulation could be explained by a diminution of the MMP-2 enzymatic activity. In this article, we demonstrate that CTGF increases fibronectin (FN) amount, MMP-2 expression, and gelatinase activity in 3T3 fibroblasts. More significantly, we show that MMP-2deficient cells have an increased basal amount of FN and show a response to CTGF that is opposite to that of control cells. This paradoxical effect could be explained by the increase in the FN amount that blocks the integrins (at least integrins with ␣V subunit), which can act like CTGF receptors.. EXPERIMENTAL PROCEDURES Cell Culture—NIH-3T3 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum and penicillin/streptomycin in a 5% CO2 atmosphere. The cells were treated with recombinant purified CTGF (40) for 24 h in serum-free medium. The metalloproteinase inhibitor GM-6001 (BioMol) was used at 25 ␮M in the same conditions. The cells were treated with 200 nM FN (Sigma) for 24 h and then washed and incubated with CTGF in serum-free medium for 24 h. RGDS peptides (Sigma) were added at 0.2 mg/ml together with CTGF in serum-free medium for 24 h. In the experiments using blocking antibodies, the cells were pretreated with 5 ␮g/ml of anti-integrin-␣V (Santa Cruz Biotechnology) for 48 h and then incubated with CTGF for 15 min to evaluate phospho-ERK induction or 24 h to determine FN accumulation. RNA Isolation and Reverse Transcription—Total RNA was isolated from cell cultures using TRIzolTM reagent according to the manufacturer’s instructions (Invitrogen). Relative Quantitative PCR—Reverse transcriptase reaction was performed using Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Invitrogen). We designed PCR primers based on the previously published mouse mRNA sequence for MMP-2 (GI 47271505). The primers were: sense, 5⬘-GGCAACCCAGATGTGGCCAAC-3⬘, and antisense, 5⬘-AGATCTCACCACGGATCTGAGC-3⬘. For the FN expression experiments primers previously described were used (41). The relative quantitative reverse transcription-PCR (rqPCR) experiments were performed using a QuantumRNATM Classic II 18 S internal standard kit according to the manufacturer’s instructions (Ambion). Zymography—Gelatin zymography experiments were performed as described previously (42). Gelatinase Activity Assay—Gelatinase activity assays were performed as described previously using biotinylated gelatin as substrate (43). The enzymatic activity was represented as (A30 ⫺ A0/mg) ⫻ fV, where A30 is the absorbance of the sample after 30 min of incubation with the substrate; A0 is the absorbance of the sample at time 0; mg is the amount of protein/well, and fV is the ratio between the total volume of conditioned medium in the well and the volume used in the assay. MMP-2 Knockdown 3T3 Cells—Short hairpin (shRNA) expression vectors were constructed and packaged into recombinant lentivirus using the BLOCK-iT lentiviral RNA interfer-. 13552 JOURNAL OF BIOLOGICAL CHEMISTRY. ence expression system (Invitrogen) according to the manufacturer’s instructions. The target sequence for the shRNA against MMP-2 was 5⬘-AGTTGGCAGTGCAATACCTGA-3⬘ (44). The pLenti6-U6-shMMP2 vector and the packaging plasmid mixture were introduced into 293FT packaging cells provided within the kit. The recombinant lentiviruses in the conditioned medium were harvested and titrated using 3T3 cells. Transductions were performed at a multiplicity of infection of 50, and the cells were propagated and maintained with 10 ␮g/ml blasticidin. The target gene knockdown was confirmed by relative quantitative PCR and zymography. A stable cell line expressing a scramble sequence was made as control. Western Blot—Western blot experiments were performed as described previously (45). The antibodies used were anti-FN (Sigma) (1/10.000), anti-collagen III (Santa Cruz Biotechnology) (1/400), anti-phospho-ERK1/2 (Cell Signaling) (1/1000), anti-ERK1/2 (Cell Signaling) (1/1000), and anti-tubulin (Sigma) (1/10.000). Immunofluorescence—Cells to be immunostained were grown on coverslips. The medium was removed, and the coverslips were rinsed with phosphate-buffered saline. The cells were fixed with 4% paraformaldehyde for 20 min at 25 °C, incubated for 1 h in blocking solution (phosphate-buffered saline, pH 7.4, 8% bovine serum albumin), and then incubated with polyclonal anti-FN (Sigma) (1/1000) or anti-vinculin (Sigma) (1/250). The cells were rinsed with blocking solution and further incubated for 1 h with anti-rabbit Alexa 488-conjugated antibody (Invitrogen) (1/1000) or anti-mouse Alexa 568 (Invitrogen) (1/1000) diluted in blocking solution. For nuclear staining, the sections were treated with 1 ␮g/ml Hoechst 33258 in phosphate-buffered saline for 10 min. For actin staining, the cells were incubated with fluorescein-labeled phalloidin (Sigma) for 10 min. After rinsing, the coverslips were mounted using fluorescent mounting medium (DAKO) and viewed under a Nikon Diaphot microscope equipped for epifluorescence (45). Solid Phase Binding Assay—Binding experiments between FN and CTGF were performed as described previously (46). In brief, 96-well plates were coated over night at 4 °C with FN (Sigma). Then the plates were treated for 2 h with blocking solution (1% bovine serum albumin in phosphate-buffered saline) at room temperature and then incubated with rFLAGCTGF (46). Later, the wells were incubated for 2.5 h at 37 °C with anti-FLAG antibody (Sigma) and the appropriate horseradish peroxidase-conjugated secondary antibody. The binding was determined using ABTS peroxidase substrate (Calbiochem) reading at 405 nm. The KD for FN was calculated utilizing SigmaPlot 10 software. FN Degradation Analysis—The analysis of the putative degradation of FN by CTGF was performed using 125I-FN as substrate (47). 6 ng of radioactive FN (500,000 cpm) were incubated with 0 (Control) 10, 20, or 40 ng/ml of CTGF for 24 h at 37 °C in 40 ␮l of Dulbecco’s modified Eagle’s medium. Then the samples were separated by SDS-PAGE. The gel was exposed in a photo-sensitive film for the visualization of FN. To analyze the degradation of FN by cells, 12 ng of radioactive FN (1 ⫻ 106 cpm) were added to the indicated cells and incubated in the presence of CTGF (0, 10 and 20 ng/ml) in minimum medium VOLUME 284 • NUMBER 20 • MAY 15, 2009.

(3) FN Excess Affects the CTGF Fibrotic Response Statistical Methods—The results are given as the means ⫾ S.E. for the indicated number of experiments. Comparisons among the different experimental groups were carried out using one-way analysis of variance test utilizing SigmaPlot 10 with SigmaStat software. Differences were considered statistically significant at p ⬍ 0.05.. RESULTS CTGF Increases FN Amount, MMP-2 Expression, and Gelatinase Activity in 3T3 Fibroblasts—To evaluate the fibrotic effect of CTGF in 3T3 fibroblasts, we determined the FN amount in response to recombinant CTGF. We found that FN increases in the presence of CTGF in a dose-dependent manner as we described previously (40) (Fig. 1, A and B). These results demonstrate the fibrotic effect of CTGF in 3T3 fibroblasts. To determine whether the effect of CTGF on the accumulation of FN in 3T3 fibroblasts is due to variations in MMP-2 expression and in consequence its activity, we evaluated the MMP-2 expression and amount and gelatinase activity in response to recombinant CTGF in these cells. The rqPCR shows that CTGF increases MMP-2 mRNA levels at 5.0 ng/ml of the factor (Fig. 1C). The expression of metalloproteinase type 9 (MMP-9 or gelatinase B), TIMP-1, and TIMP-2 was not FIGURE 1. Fibronectin amount, MMP-2 expression, and gelatinase activity in conditioned medium altered by CTGF (data not shown). increases in response to CTGF in 3T3 fibroblasts. A, fibroblasts were incubated for 24 h with increasing concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% A similar effect of CTGF on MMP-2 SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using amount was observed by gelatin anti-FN and anti-tubulin antibodies, as explained under “Experimental Procedures.” Tubulin immunostaining zymography (Fig. 1D). Next, the is shown as a loading control. B, the graph shows the quantification of FN bands as a ratio to the tubulin bands intensity; the mean and standard error (n ⫽ 3) are represented. The p value between control condition and cells effect of CTGF on the levels of treated with 5.0 and 10 ng/ml CTGF was ⬍0.05 (*). C, fibroblasts were incubated for 24 h with different con- active enzyme determined by gelacentrations of CTGF in serum-free medium. Then rqPCR was carried out. The graph shows the quantification of MMP-2 bands as a ratio to the 18 S bands intensity; the mean and standard error are represented. All of the tinase activity was evaluated in conconditions show statistically significant difference (n ⫽ 3, p ⬍ 0.05). D, fibroblasts were incubated for 48 h with ditioned medium. We observed that different concentrations of CTGF in serum-free medium. After that, the conditioned medium was collected, gelatinase activity increased greatly and gelatin zymography was carried out. E, serum-free conditioned medium of cells treated for 48 h with different concentrations of CTGF was used in the gelatinase activity assay (n ⫽ 3). The enzymatic activity was from 5.0 ng/ml CTGF (Fig. 1E). This represented as (A30 ⫺ A0/mg) ⫻ fV, where A30 is the absorbance of the sample after 30 min of incubation; A0 is indicates that the increase in MMP-2 the absorbance of the sample at time 0; mg is the amount of protein/well; and fV is the ratio between the total expression and protein levels is volume of conditioned medium in the well and the volume used in the assay. The p value between control condition and 5.0 ng/ml CTGF was ⬍0.05 (*). The gelatinase activity between 5.0 and 10 ng/ml of CTGF did not reflected in an increase of the gelatinshow statistically significant difference. ase activity. These results show a positive regulation of MMP-2 expression during 24 h. After that, the cell extracts were prepared and and in consequence of the gelatinase activity by CTGF. CTGF Decreases the Amount of FN in Fibroblasts Treated with fractionated by 7.5% SDS-PAGE. The radioactive bands were then visualized and quantified using a PerkinElmer Life Sci- the Metalloproteinase Inhibitor GM-6001—Fibrotic disorders are characterized by FN accumulation. This effect can be ences phosphorimaging device (Cyclone model). MAY 15, 2009 • VOLUME 284 • NUMBER 20. JOURNAL OF BIOLOGICAL CHEMISTRY. 13553.

(4) FN Excess Affects the CTGF Fibrotic Response. FIGURE 2. CTGF decreases the amount of fibronectin in 3T3 cells treated with GM-6001. A, fibroblasts were incubated for 24 h with different concentrations of CTGF in serum-free medium in the presence of 25 ␮M GM-6001. After that, the cell extracts were prepared, fractionated by 7.5% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control. B, the graph shows the quantification of FN bands as a ratio to the tubulin bands intensity; the mean and standard error (n ⫽ 3) are represented. The p value between the control condition (without GM-6001 and CTGF) and cells treated with CTGF in the presence of GM-6001 was ⬍0.05 (*). The p value between cells treated with GM-6001 without CTGF and cells treated with 5.0 and 10 ng/ml CTGF in the presence of GM-6001 was ⬍0.001 (**).. explained by an increase in the synthesis or decrease in the degradation of this substrate. To evaluate whether FN accumulation in fibrotic tissues involved MMP activity inhibition, 3T3 fibroblasts were incubated with the cytokine in the presence of the metalloproteinase inhibitor GM-6001 (Ilomastat). We observed that in the presence of GM-6001, the basal amount of FN was higher and decreased after CTGF addition in a dose-dependent manner (Fig. 2). These results show that FN levels in the extracellular space are regulated by MMP activity and suggest that they are important to regulate the cellular response to CTGF. FN Is Increased in MMP-2-deficient 3T3 Cells—We observed that CTGF increases MMP-2 expression. Although FN is a target of this enzyme and cells with inhibited MMP activity show FN accumulation, we hypothesized that in fibrotic tissues, MMP-2 can be inhibited by a mechanism that does not involve inhibition of expression but implicates the direct inhibition of its enzymatic activity. Then to evaluate the specific role of MMP-2 in the FN amount regulation and to emulate a specific enzymatic inhibition, a stable clone that expresses shRNA against MMP-2 was produced using lentiviral vectors. This clone was called 3T3-M2(⫺). As control, we made a cell line called 3T3-C (that expresses a scramble sequence). We observed an inhibition greater than 4.3-fold in the expression of MMP-2 in 3T3-M2(⫺) cells (Fig. 3A). The levels of expression. 13554 JOURNAL OF BIOLOGICAL CHEMISTRY. of MMP-2 were equal in wild type and control cells (data not shown). The gelatin zymography shows a great decrease in the MMP-2 amount observed, whereas the levels of MMP-9 (gelatinase B) are unaltered (Fig. 3B). Subsequently, the levels of FN in 3T3-M2(⫺) cells were observed through immunofluorescence (Fig. 3C) and Western blot (Fig. 3D). The levels of FN were equal in wild type and control cells (for comparison observe Fig. 4A, left bar). In 3T3-shM2(⫺) we observed a clear increase in FN amount relative to control cells (Fig. 3D). These results support the ideas that FN is a major substrate for MMP-2 and that its enzymatic activity is critical to regulate the FN levels in the extracellular space. CTGF Decreases the Amount of FN in MMP-2-deficient 3T3 Cells—The above results indicate that MMP-2 activity is critical for the regulation of FN levels. Therefore, we evaluated the effect of CTGF on FN levels in 3T3-M2(⫺) cells. We observed that the basal amount of FN is higher, as expected, and decreases in a dose-dependent manner in response to CTGF (Fig. 4, A and B). This paradoxical behavior is similar to that observed in the presence of the metalloproteinase inhibitor GM-6001 (Fig. 2). Similar effect was observed for collagen III, another important ECM protein (Fig. 4C). MMP-9 was not responsible for the different behavior of FN in 3T3-M2(⫺) cells because its amount did not increase in the presence of CTGF (data not shown). These results suggest that accumulation of FN generated by MMP-2 deficiency notably alters the response to CTGF, which translates into a paradoxical response to the cytokine. Interaction between CTGF and FN Does Not Have a Direct Effect in the Paradoxical Response to the Growth Factor— The paradoxical effect observed in the MMP-2-deficient cells could be explained as a consequence of the interaction between FN and CTGF. We analyzed this interaction through solid phase binding assay, and then we determined whether FN could be degraded by CTGF. We observed that CTGF clearly interacts with FN (Fig. 5A) with a KD for FN of 81.2 ␮g/ml (360.8 nM) at 100 nM CTGF. We then analyzed whether CTGF was able to degrade FN and observed that CTGF did not degrade FN in the range of concentrations used for the experiments in cells (Fig. 5B). These results suggest that the decrease in the amount of FN in response to CTGF cannot be explained by the degradative activity of CTGF. CTGF Decreases the Amount of Endogenous FN in 3T3 Cells Preincubated with an Excess of Exogenous FN—To evaluate whether the paradoxical response to CTGF in MMP-2-deficient fibroblasts is due to a FN excess, 3T3 cells (wild type) were incubated with 200 nM FN for 24 h prior to the treatment with CTGF. We observed that cells incubated with FN have a different response to CTGF than the control. The FN-treated cells show a decrease in the amount of FN in the conditioned medium in a dose-dependent manner in response to CTGF (Fig. 6, A and C) in contrast with the control condition (Fig. 6, B and D). The contribution of the FN used for the treatment in relation to the one produced by the cells was negligible (Fig. 6A, lane C). This behavior is analogous to that observed in MMP2-deficient cells (Fig. 4, A and B). These results clearly indicate that FN excess alters the effect of CTGF over endogenous FN amount. VOLUME 284 • NUMBER 20 • MAY 15, 2009.

(5) FN Excess Affects the CTGF Fibrotic Response the access of CTGF to integrins, altering its signaling through these putative receptors. Formation of Stress Fibers and Focal Adhesion Sites Is Altered in MMP-2-deficient Cells—To evaluate whether the integrin signaling pathway is altered in the presence of high levels of FN, we evaluated the formation of stress fibers and focal adhesion sites, two classical integrin-mediated effects (58), in MMP2-deficient cells in response to CTGF. We observed that control cells form stress fibers and focal adhesion sites in response to 20 ng/ml CTGF (Fig. 7). On the other hand, MMP-2-deficient cells were unable to generate stress fibers and focal adhesion sites in response to CTGF (Fig. 7). These results support the idea that integrin signaling is altered when high levels of FN are present, thus affecting the cellular response to CTGF. Phosphorylation of ERK Proteins in Response to CTGF Is Altered in MMP-2-deficient Cells and in Cells Treated with Antibodies against Integrin ␣V Subunit—Recently, we have shown that CTGF induces ERK phosphorylation in C2C12 cells (40). In fibroblasts we analyzed ERK phosphorylation in response to FIGURE 3. Fibronectin is increased in MMP-2-deficient cells. A stable clone of 3T3 cells expressing shRNA CTGF and to an excess of FN. We against MMP-2 was produced using lentiviral vectors. This clone was called 3T3-M2(⫺). A, RNA from control observed an increase in ERK phos3T3-C (control cells) and 3T3-M2(⫺) was isolated. rqPCR was carried out to evaluate the expression of MMP-2 phorylation in control fibroblasts mRNA. The graph shows the quantification of MMP-2 bands as a ratio to the 18 S bands intensity; the mean and standard error (n ⫽ 3) are represented. The clone 3T3-M2(⫺) shows an inhibition of 4.3-fold in MMP-2 expres- cells in response to CTGF (Fig. 8A, sion relative to control cells (p ⬍ 0.001). B, conditioned medium from 3T3-C and 3T3-M2(⫺) cells was collected left panel). In the MMP-2-deficient and gelatin zymography was carried out. The zymogram shows the amount of MMP-2 and MMP-9 present in the conditioned medium of each cell line. C, wild type (WT) and 3T3-M2(⫺) cells were cultured for 48 h in cells that have a FN excess, we do control conditions and then processed for immunofluorescence, using anti-FN antibodies (green). The nuclei not observe any effect of CTGF over were stained with Hoechst (blue). Scale bar, 10 ␮m. D, wild type and 3T3-M2(⫺) cells were incubated for 48 h ERK phosphorylation (Fig. 8A, midunder control conditions. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN and anti-tubulin dle panel). Quantification analyses antibodies. Tubulin immunostaining is shown as a loading control. of these experiments (Fig. 8B) indicate a significant difference in CTGF Decreases the Amount of FN in 3T3 Cells Incubated the amount of phosphorylation of ERK-2 in response to CTGF with RGDS Peptide—Because FN has been described to bind in control but not in MMP-2-deficient cells. It has been described that integrins containing the ␣V subto integrins (48 –51), which are putative CTGF receptors (52–56), we evaluated whether the altered response to CTGF unit act as putative CTGF receptors (55). Because these parby the excess of FN is due to the blockage of integrins by this ticular integrins are expressed in 3T3 cells (59) and bind FN FN excess. 3T3 cells incubated with CTGF were treated with and RGDS peptide, it is tempting to speculate that the use of 0.2 mg/ml of RGDS peptide, a specific peptide contained in blocking antibodies against this subunit would generate the the FN sequence (57) that binds integrins (49, 51) and blocks same effect as the excess of FN over CTGF response in fibrothe interaction between integrins and CTGF (53). We blasts. We clearly observed that pretreated cells with the observed that cells incubated with RGDS peptide show a blocking antibody anti-integrin ␣V subunit were unable to decrease in the amount of FN in response to CTGF (Fig. 6, E phosphorylate ERK in response to CTGF (Fig. 8A, right and G) in contrast to control conditions (Fig. 6, F and H). panel). This is analogous to cells that present high concenThese results suggest that the increased FN levels can block trations of FN (MMP-2-deficient cells) (Fig. 8A, middle MAY 15, 2009 • VOLUME 284 • NUMBER 20. JOURNAL OF BIOLOGICAL CHEMISTRY. 13555.

(6) FN Excess Affects the CTGF Fibrotic Response. FIGURE 4. CTGF decreases the amount of fibronectin in MMP-2-deficient cells. A, 3T3-C and 3T3-M2(⫺) cells were incubated for 24 h with increasing concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control. B, the graph shows the quantification of FN bands as a ratio to the tubulin bands intensity; the mean and standard error (n ⫽ 3) are represented. The black bars show the effect of CTGF in 3T3-C cells, and the gray bars show the effect of CTGF in 3T3-M2(⫺) cells. The p value between control cells without CTGF and control cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*). The p value between control cells without CTGF and 3T3-M2(⫺) cells without CTGF was ⬍0.001 (**). The p value between 3T3-M2(⫺) cells without CTGF and 3T3-M2(⫺) cells treated with CTGF was ⬍0.05 (*) and ⬍0.001(**). C, 3T3-C and 3T3-M2(⫺) cells were incubated for 24 h with different concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-collagen III and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control.. panel). These results strongly suggest that integrins are involved in ERK phosphorylation in response to CTGF. Thus, blockage of integrins by a FN excess or by anti-integrin ␣V antibodies could explain the paradoxical effect observed in the MMP-2-deficient cells. CTGF Decreases the Amount of FN in Cells Treated with Antibodies against Integrin ␣V Subunit—To evaluate whether blockage of integrins with anti ␣V antibodies in control cells abolishes the induction of FN in response to CTGF, fibroblasts were preincubated with anti-integrin ␣V antibodies and then with increasing concentrations of CTGF, and the amount of FN was determined. Pretreatment of the cells shows a decrease in the amount of FN in response to CTGF in contrast to control conditions (Fig. 9). These results clearly show that the blockage of integrin ␣V is capable of emulating the effect of FN excess on fibroblasts in their paradoxical response to CTGF and support. 13556 JOURNAL OF BIOLOGICAL CHEMISTRY. FIGURE 5. CTGF interacts with fibronectin without degrading it. A, the interaction between CTGF and FN was evaluated using solid phase binding assay as explained under “Experimental Procedures.” The binding analysis was carried out using three concentrations of CTGF (1, 10, and 100 nM). The bound FN was determined as A405 as explained under “Experimental Procedures.” The KD for FN calculated at 100 nM CTGF was 81.2 ␮g/ml (360.8 nM). B, the putative degradation of FN by CTGF was determined incubating 6 ng of 125 I-FN with 0 (control), 10, 20, or 40 ng/ml of CTGF by 24 h at 37 °C. Then the samples were separated by SDS-PAGE. The gel was exposed in a photo-sensitive film.. the idea that at least integrins with ␣V subunit participate as a receptors for CTGF. CTGF Does Not Affect the mRNA Expression of FN in MMP-2-deficient Cells—Previously we showed that the excess of FN in MMP-2-deficient cells produces a reduction of FN amount in response to CTGF. One possible explanation is that CTGF down-regulates the FN mRNA expression when the extracellular levels of FN are increased. We observed that in MMP-2-deficient cells the basal expression of FN is increased and that these cells do not respond to CTGF like the control cells (Fig. 10A). These results suggest that high levels of FN stimulate the endogenous FN expression and that the paradoxical response to CTGF observed in MMP-2-deficient cells or cells treated with exogenous FN does not involve changes in FN expression. MMP-2-deficient Cells Show a Greater Degree of Degradation of FN in Response to CTGF—Because FN mRNA expression is not affected by CTGF when FN concentration is high, we evaluated whether the decrease in FN in response to CTGF would be caused by an increase in FN degradation. We incubated 3T3-C and 3T3-M2(⫺) with 125I-FN in the presence or absence of CTGF for 24 h. We observed that both cell lines degraded FN, but in MMP-2-deficient cells the degree of proteolysis was higher than in control (5% of degradation in control cells versus 15% of degradation in 3T3-M2(⫺) cells at 20 ng/ml of CTGF) (Fig. 10, B and C). These results VOLUME 284 • NUMBER 20 • MAY 15, 2009.

(7) FN Excess Affects the CTGF Fibrotic Response suggest that the paradoxical effect in response to CTGF observed in cells with high amounts of FN could be explained by an increase in FN degradation.. FIGURE 6. CTGF decreases the amount of fibronectin in 3T3 cells treated with exogenous fibronectin and RGDS peptide. A, 3T3 wild type cells were incubated for 24 h in the presence of 25 ␮g/ml FN, then were washed, and treated with CTGF for 24 h in serum-free medium. After that, conditioned medium was prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN antibody. As a control for the FN treatment, the wells were incubated with FN in the absence of cells. These wells were subjected to the same treatment that wells with cells. Then the serum-free medium of these control wells was collected to observe the contribution of the FN incubation to the FN observed by Western blot. This condition was called control (lane C). B, 3T3 wild type cells were incubated for 24 h with different concentrations of CTGF in serum-free medium. After that, conditioned medium was prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN antibody. C, the graph shows the quantification of FN bands in conditioned medium from FN-treated cells, as a ratio to the tubulin bands intensity in Western blots from the protein extracts (not shown), used to normalize the loading of the conditioned medium. The mean and standard error (n ⫽ 3) are represented. The p value between cells without CTGF and cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*). D, the graph shows the quantification of FN bands (control of A) intensity in conditioned medium as a ratio to the tubulin bands intensity in Western blots from the protein extracts (not shown), used to normalize the loading of the conditioned medium. The mean and standard error (n ⫽ 3) are represented. The p value between cells without CTGF and cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*). E, 3T3 wild type cells were incubated for 24 h with CTGF in the presence of 0.2 mg/ml RGDS peptide in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control. F, 3T3 wild type cells were incubated for 24 h with different concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and transferred onto nitrocellulose membranes. Western blot analyses were carried out using anti-FN and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control. G, the graph shows the quantification of FN bands from RGDStreated cells as a ratio to the tubulin bands intensity, The mean and standard error (n ⫽ 3) are represented. The p value between cells without CTGF and cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*). H, the graph shows the quantification of FN bands (control of E) intensity as a ratio to the tubulin bands intensity; the mean and standard error (n ⫽ 3) are represented. The p value between cells without CTGF and cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*).. MAY 15, 2009 • VOLUME 284 • NUMBER 20. DISCUSSION MMPs, together with cytokines, have become a fundamental element of study for the understanding of fibrosis. First, was reported that pro-fibrotic cytokines, such as CTGF, activate signal transduction pathways that in consequence increase the synthesis of ECM proteins and produce its accumulation (14, 16). Then an increase in the amount of MMPs related to the degradation of basal lamina was observed, an event connected to a high rate of regeneration of tissues in early stages of fibrosis, after inflammation (60). This point is very interesting because fibrosis is considered irreversible when the appropriate cellular source of MMPs is no longer present, and in consequence it is impossible to revert the accumulation of connective tissue proteins (61). Furthermore, an increase in the expression of TIMPs in fibrosis has been described, which can be associated with ECM accumulation because of MMP inhibition (62). Then it appears that in fibrosis coexists an increase in MMP expression with MMP inhibition, which will facilitate the ECM accumulation that would turn the pathological state into an irreversible state. CTGF has been involved in the regulation of ECM proteins production and thus in wound repair and fibrosis (16). In consequence, elucidating its effect on MMP expression is very important to understand the processes involved in fibrosis. Previously, an increase in the MMP-2 expression caused by CTGF in vascular smooth muscle cells (63) and renal interstitial fibroblasts was reported (36). Those studies analyzed the mRNA expression and the protein amount, but they did not deepen in the variation of the real enzymatic activity. In our study we observed an increase in the MMP-2. JOURNAL OF BIOLOGICAL CHEMISTRY. 13557.

(8) FN Excess Affects the CTGF Fibrotic Response. FIGURE 7. The formation of stress fibers and focal adhesion sites in response to CTGF is altered in MMP-2-deficient cells. Control cells and 3T3-M2(⫺) cells were cultured for 24 h under control conditions or in the presence of 20 ng/ml CTGF and then processed for immunofluorescence, using anti-vinculin antibodies (red) and fluorescein-labeled phalloidin (green) to observe actin fibers. The nuclei were stained with Hoechst (blue). The stress fibers can be observed as several actin fibers (green) across the cell. The focal adhesion sites can be observed as encounter points at the end of actin fibers with vinculin staining (red). A few focal adhesion sites are marked with white arrows. Stress fibers and focal adhesion sites were not observed in response to CTGF in MMP-2-deficient cells. Scale bar, 10 ␮m.. FIGURE 8. Phosphorylation of ERK proteins is diminished in MMP-2-deficient cells and in cells treated with antibodies against integrin ␣V. A, 3T3-C, 3T3-M2(⫺), and 3T3-C ⫹ Ab␣V-pretreated cells (incubation with 5 ␮g/ml of antibody for 48 h) were serum-starved for 18 h and then incubated for 15 min with increasing concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Western blot analyses were carried out using anti-phospho-ERK and anti-ERK antibodies. ERK immunostaining is shown as a loading control. B, the graph shows the quantification of phosphor-ERK-2 bands as a ratio to the ERK-2 bands intensity; the mean and standard error (n ⫽ 3) are represented. The p value between 3T3-C cells without CTGF and 3T3-C cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*).. 13558 JOURNAL OF BIOLOGICAL CHEMISTRY. FIGURE 9. CTGF decreases the amount of FN in cells treated with blocking antibodies against integrin ␣V. A, wild type fibroblasts under control conditions or pretreated with anti-integrin ␣V antibodies were incubated for 24 h with the indicated concentrations of CTGF in serum-free medium. After that, cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes. Western blot analyses were carried out using anti-FN and anti-tubulin antibodies. Tubulin immunostaining is shown as a loading control. B, the graph shows the quantification of FN bands intensity as a ratio to the tubulin bands intensity, the mean and standard error (n ⫽ 3) are represented. The p value between control cells without CTGF and control cells treated with 10 and 20 ng/ml CTGF was ⬍0.05 (*). The p value between control cells without CTGF and pretreated cells without CTGF was ⬍0.001 (**). The p value between pretreated cells without CTGF and pretreated cells with 10 and 20 ng/ml CTGF was ⬍0.05 (*).. expression caused by CTGF. We observed saturation of the expression at 5.0 ng/ml of CTGF (Fig. 1C). These results are in complete agreement with the gelatinase activity in response to CTGF (Fig. 1E). This contrasts with the results of Yang et al. (36), which showed an increase in MMP-2 caused by CTGF, but the concentrations used of the factor are very far from the physiological range (64). Zymography (65, 66) is not properly an enzymatic assay; rather it is an assay that shows the amount of active and inactive forms of gelatinases (in the case of gelatin zymography) utilizing its catalytic activity recovered after SDS treatment. For this reason, we mounted a gelatinase activity assay and analyzed the effect of CTGF. Because we observed that MMP-9 expression was not significantly altered by CTGF, any change in the gelatinase activity in the conditioned medium was due to variations in the levels of MMP-2. The real variation in the gelatinase activity in response to CTGF was measured. It showed a great increase in the enzymatic activity caused by the factor (Fig. 1E). In this way CTGF together with the increase in the ECM production contribute to the pathogenesis of fibrosis altering the MMP-2 levels. The apparent paradox observed in fibrosis in which there is an increase in the MMP production and at the same time a great accumulation of ECM such as the observed in liver fibrosis (38, 67– 69) is reinforced by these results. Here, we observed that CTGF increases the gelatinase activity, but when the general MMP or MMP-2 activity is diminished (inhibiting MMPs VOLUME 284 • NUMBER 20 • MAY 15, 2009.

(9) FN Excess Affects the CTGF Fibrotic Response. FIGURE 10. CTGF does not affect mRNA expression of FN, but it does affect the amount of FN in MMP-2-deficient cells. A, control and 3T3-M2(⫺) cells were incubated for 24 h with the indicated concentrations of CTGF in serumfree medium. Then rqPCR was carried out as explained under “Experimental Procedures.” The graph shows the quantification of FN bands as a ratio to the 18 S bands intensity; the mean and standard error are represented (n ⫽ 3). The p value between control cells in the absence of CTGF and control cells treated with 20 ng/ml CTGF was ⬍0.05 (*). The p value between control cells without CTGF and 3T3-M2(⫺) cells in the absence of CTGF was ⬍0.005 (*). B, control and 3T3-M2(⫺) cells were incubated with 125I-FN in the presence of 0, 10, and 20 ng/ml CTGF for 24 h in minimum medium. After that, the cell extracts were prepared, fractionated by 7.5% SDS-PAGE, and then the radioactive bands were visualized using phosphorimaging. C, the graph shows the quantification of 125I-FN bands intensity relative to control (3T3-C cells without CTGF); the mean and standard error (n ⫽ 3) are represented. The p value between control cells without CTGF and treated with 20 ng/ml CTGF was ⬍0.05 (*). The p value between 3T3-M2(⫺) cells without CTGF and treated with 20 ng/ml CTGF was ⬍0.05 (*).. chemically or specifically decreasing the MMP-2 mass), the cells develop a phenotype with an important FN accumulation. To this point, although FN is a known MMP-2 substrate (70), this protein is also substrate for MMP-1, -3, -7, -10, -12, -13, -14, -16, -19, and -26 (for review see Ref. 71). Therefore, we did not expect the great effect on FN accumulation when we specifically decreased the MMP-2 amount. Our results support the great importance of MMP-2 activity in the maintenance of FN levels. Surprisingly, we observed that MMP-2-deficient cells present an opposite response to CTGF showing a decrease in the FN protein levels in response to the factor (Fig. 4). These results are analogous to our previous findings (72) in fibroblasts from dystrophic mice (73) obtained from fibrotic muscles that showed a MAY 15, 2009 • VOLUME 284 • NUMBER 20. significant increase in FN relative to fibroblasts from normal mice (C57) in basal conditions and a decrease in the protein in the presence of CTGF (72). We could explain the CTGF effect in both cases as a consequence of FN excess, which can affect the response of the cells to cytokine. For this reason, we evaluated the effect of incubation with exogenous FN excess previous to the treatment with CTGF on FN amount produced by 3T3 cells. Here, we observed that cells treated with exogenous FN show the same behavior as MMP-2-deficient cells, a decrease in the FN amount in response to CTGF (Fig. 6, A and C). Apparently, the interaction observed between CTGF and FN is not a crucial factor in this effect because CTGF does not have a proteolytic effect over FN (Fig. 5). A possible trapping of the factor by FN does not explain the decrease in the amount of FN and the dose response observed in the paradoxical effect. A more plausible explanation would be that FN excess could alter the preferential signaling pathway of CTGF by blockage of its receptors. Previously, integrins have been described to bind CTGF (52– 56), and it has been demonstrated that FN binds with high affinity to integrins (48 –51). For these reasons, we speculate that integrins could be the receptors involved in this phenomenon, explaining the different cellular response to CTGF in MMP-2deficient and FN-treated cells, where the excess of FN would compete with CTGF for the binding to integrins. To evaluate this idea, we blocked integrins with an excess of FN (Fig. 6, A and C), with RGDS peptide (Fig. 6, E and G) and the putative CTGF receptor (55) using blocking integrin ␣V antibodies (Fig. 9). In these experiments we clearly observed a decrease in FN amount in response to CTGF. Moreover, we observed that cells in the presence of high concentrations of FN (MMP-2-deficient cells) or when the integrin ␣V subunit was blocked by antibodies were unable to phosphorylate ERK in response to CTGF (Fig. 8). Furthermore, MMP-2-deficient cells apparently did not show some integrin-mediated effects, such as generate stress fibers or focal adhesion sites (58), in response to CTGF (Fig. 7). Together, all of these results support the hypothesis that integrins (at least integrins with ␣V subunit) are involved in the paradoxical response to CTGF observed in MMP-2-deficient cells. In this context, these integrins can act like CTGF receptors when the amount of FN is low, but when the cells develop a phenotype with high accumulation of FN, these receptors could be blocked by this protein causing a different cellular response to CTGF, possibly because of the binding of the factor to a different kind of receptor. To date, other nonintegrin proteins that bind CTGF have been described, like LRP (74), syndecan-4 (75), and perlecan (76), but further studies are necessary to evaluate the participation of these receptors in the paradoxical effect observed. The altered response to CTGF observed in the presence of high levels of FN does not involve changes in the FN mRNA expression (Fig. 10A) but implicates changes in the proteolytic capacity of the cells because MMP-2-deficient cells show a greater degradation of FN than control cells (Fig. 10, B and C). It is possible that under these conditions (great amount of FN and the presence of CTGF) other proteases would be activated, explaining the decrease of endogenous FN amounts in the presence of high basal concentrations of FN. This paradoxical JOURNAL OF BIOLOGICAL CHEMISTRY. 13559.

(10) FN Excess Affects the CTGF Fibrotic Response mechanism could be a compensatory mechanism that protects against a major damage in the fibrotic tissues, when the ECM accumulation goes from a normal process to a pathologic course. Three aspects are clear in this study. First, the regulation of MMP-2 activity can be important in the genesis of fibrosis, because MMP-2-deficient cells show FN accumulation in the conditioned medium and a response to CTGF similar to that of fibroblasts obtained from fibrotic tissues (72). Second, the amount of FN can regulate the cellular response to CTGF caused by the blockage of integrins (at least integrins with ␣V subunit) that act like primary CTGF receptors in fibroblasts. Third, under these conditions, CTGF regulates the endogenous amount of FN by a mechanism that does not involve changes in the expression of FN but might involve changes in the proteolytic activity of the cell. 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