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DECORIN INTERACTS WITH CTGF/CCN2 THROUGH LRR12

INHIBITING ITS BIOLOGICAL ACTIVITY

Cecilia Vial*, Jaime Gutiérrez*, Cristian Santander, Daniel Cabrera and Enrique Brandan1

Centro de Regulación Celular y Patología (CRCP), Centro de Regeneración y Envejecimiento

(CARE), Departamento de Biología Celular y Molecular, MIFAB, Pontificia Universidad Católica de Chile, Santiago, Chile

Running title: CTGF activity is regulated by the proteoglycan decorin * These authors contributed equally to this work 1To whom correspondence should be addressed: Enrique Brandan, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Casilla 114-D, Santiago, Chile. Fax 56 2 635 5395. Email: ebrandan@bio.puc.cl Fibrotic disorders are the endpoint of many chronic diseases in different tissues, where an accumulation of the extracellular matrix occurs, mainly because of the action of the connective tissue growth factor (CTGF/CCN2). Little is known about how this growth factor activity is regulated. We found that decorin null myoblasts are more sensitive to CTGF than wild type myoblasts, as evaluated by the accumulation of fibronectin or collagen III. Decorin added exogenously negatively regulated CTGF pro-fibrotic activity and the induction of actin stress fibers. Using co-immunoprecipitation and in vitro interaction assays, decorin and CTGF were shown to interact in a saturable manner with a Kd of 4.4 nM. This interaction requires decorin´s core protein. Experiments using decorin deletion mutants indicated that the leucine rich repeats (LRR) 10–12 are important for the interaction with CTGF and the negative regulation of the cytokine activity, and a peptide derived from the LRR12 was able to inhibit CTGF-decorin complex

formation and CTGF activity. Finally, we showed that CTGF specifically induced the synthesis of decorin, suggesting a mechanism of autoregulation. These results suggest that decorin interacts with CTGF and regulates its biological activity.

Fibrotic disorders are the endpoint of many chronic diseases in different tissues such as kidney, skin and skeletal muscle, among others (1-3). This complex biological process includes an inflammatory response and the activation of fibroblasts to myofibroblasts that have a highly contractile phenotype and are the principal source of connective tissue and extracellular matrix (ECM) proteins, such as collagen and fibronectin (4). The development of fibrosis is the consequence of the action of growth factors and cytokines that are overexpressed in fibrotic tissues. For years, transforming growth factor β (TGF-β) has been designated the principal inductor of fibrosis in different tissues (5), but now it is widely accepted that the fibrotic activity assigned to this growth factor was essentially because of another

http://www.jbc.org/cgi/doi/10.1074/jbc.M110.189365The latest version is at JBC Papers in Press. Published on March 23, 2011 as Manuscript M110.189365

Copyright 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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cytokine-denominated connective tissue growth factor (CTGF/CCN2), whose expression is stimulated by TGF-β and which is responsible for the fibrotic effects of TGF-β such as the accumulation of the ECM (6-9). CTGF is a growth factor that belongs to a highly conserved family called CCN (CTGF, CYR61, NOV-1 (10-12), and its over-expression is known to occur in a variety of fibrotic disorders such as skin fibrosis (13,14) renal fibrosis (15), hepatic fibrosis (16) and pulmonary fibrosis (17). CTGF´s function in fibrosis has gained interest in the past few years where it has been shown to be a strong mitogenic and chemotactic factor (18), which directly participates in the cell adhesion of fibroblasts (19) and the increase of ECM production, by inducing the synthesis collagen I and III, the integrin β1 subunit and fibronectin (20).

Duchenne muscular dystrophy (DMD) is a disease caused by the absence of the protein dystrophin, which produces muscle weakness that leads to cycles of degeneration and regeneration of the muscle fibers, resulting in a decrease of muscle mass and an increase of connective tissue (21-24). In this way, fibrotic scaring ensues and the regeneration of muscle is precluded by this fibrotic scar. Both TGF- and CTGF are known to be over-expressed in this disease (23,25,26). Moreover, we have previously shown that myoblasts are able to participate in this fibrotic process since they are able not only to produce CTGF but also to respond to the growth factor by increasing ECM production and actin stress fiber formation (27).

Since 45% of the deaths in developed countries are because of some type of fibroproliferative illness, finding a way to diminish fibrosis has become an important issue (3). Because CTGF is the principal molecule responsible for

fibrosis in many tissues it would be of great interest to find an inhibitor for its action. A good candidate to regulate CTGF action is decorin, which is a proteoglycan formed by a core protein with 12 leucine-rich repeats (LRRs) and a chondroitin/dermatan sulfate glycosaminoglycan (GAG) chain (28). Decorin is synthesized and secreted to the ECM were it binds and organizes the collagen fibers (29,30), and it interacts with other ECM proteins such as fibronectin (31). Decorin also interacts with some growth factors and growth factor transducing receptors, regulating their actions (32,33). The three isoforms of TGF- also interact with decorin (34-36), and this interaction reduces fibrosis because of the relocalization of TGF- to the ECM were it is sequestered by decorin (29,37). Since CTGF is responsible for TGF- fibrotic action, decorin could also be regulating this growth factor. In this study, we show that (i) decorin inhibits CTGF biological activity in myoblasts and fibroblasts, in which a peptide derived from LRR12 is sufficient to regulate CTGF action, (ii) both molecules interact in vitro and LRR12 is important for this interaction and (iii) CTGF induces the synthesis of its negative regulator decorin.

EXPERIMENTAL PROCEDURES

Cell Culture - The mouse skeletal cell line C2C12 (ATCC), the decorin null myoblasts (dcn null) (38) and the NIH-3T3 fibroblasts (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, CA) supplemented with fetal bovine serum (Hyclone/Thermo Fisher Sci, IL) as described previously (38,39). Cells were treated with recombinant purified CTGF containing a flag epitope (27) for 48 hours prior to

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starvation in DMEM for 18 hours. When CTGF was co-incubated with bovine cartilage decorin (Sigma Chemical, MO), WT and decorin deletion mutants or decorin-derived peptides (Peptide 2.0, VA), these molecules were preincubated with CTGF for 30 minutes at room temperature. Western Blotting - Cells were washed twice with PBS, lysed in lysis buffer (100 mM Tris pH 7.4, 150 mM NaCl, 0.1% Triton X-100) and scraped. Cell extracts were electrophoresed through SDS-polyacrylamide gels and electrotransferred onto PVDF filters. Filters were blocked for 1 hour at room temperature in Blotto (20 mM Tris–HCl, pH 7.4, 0.1 M NaCl, 5% nonfat dry milk) and incubated with polyclonal anti-fibronectin (Sigma Chemical, MO) (1/10000), anti-collagen III (Rockland-Inc, PA) (1/5000), anti-tubulin (Sigma Chemical, MO) (1/10000), anti-flag (1/10,000) (Sigma Chemical, MO), anti-rabbit HA-epitope (Santa cruz Biotechnology, Santa Cruz, CA.) and anti-mouse decorin antibody LF-136 (kindly donated by Dr. L. Fischer, NIDR, NIH, Bethesda, MD) at room temperature for 1 hour or at 4C overnight (38,40). The bound antibodies were visualized by enhanced chemiluminescence (Pierce, IL). Adenoviral Infection - Myoblasts were plated at a density of 20,000 cells/cm2 in 55 cm2 cell plates. After 6 hours cells were infected with an adenovirus that contained human decorin whole sequence (38) using 500 plaque-forming units/cell in 4 ml of DMEM supplemented with 2% of heat-inactivated fetal bovine serum (38). After 90 minutes of incubation, standard growing medium was added and the incubation continued for 24 hours. Cells were incubated with growing

factors or radiolabeled with H2[35S]SO4

(23). Decorin and Decorin´s deletion mutants - Either the wild type decorin and the mutants that lack different LRRs were generated by overlap PCR starting from human decorin (NM_133593) cloned in pcDNA 3.1 (kindly donated by Elke Schönherr, Institute of Physiological Chemistry and Pathobiochemistry, Münster, Germany). All decorins generated had an HA epitope in their C terminal. The constructs generated were sequenced and then stably transfected into CHO-K1 cells using media supplemented with G418 (400 µg/ml). After four weeks different clones were isolated. Decorin purification was performed from conditioned media of the CHO-K1 clones using a DEAE-Sephacel column (Bio-Rad Labs, CA) pre-equilibrated in 10 mM Tris–HCl, pH 7.4, 0.2 M NaCl and 0.1% Triton X-100. Sequential washes whit increasing NaCl concentrations were performed. All the decorins were eluted at 0,6 M NaCl. The purity was evaluated by 4–12% SDS-PAGE and Coomassie blue staining of samples digested or not with chondroitinase ABC (CABC) or heparitinase (Hase) (Seikagaku, Tokyo, Japan) (41). Decorin dimerization assay was performed as previously described (30). Briefly, decorin or its deletion mutants lacking LRR10-12 were maintained 1 hour at room temperature in PBS. Dithiobis (succinimidyl) propionate (DSS) was added at a final concentration of 0.15 mM for additional 30 minutes at room temperature. The reaction was stopped with a Tris-HCl pH 7.4 buffer at a final concentration of 30 mM for 15 min. Loading buffer was added and then decorin dimerization was evaluated by western blot using anti-HA antibodies.

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Design and synthesis of Leucine rich repeats peptides (LRR) - Peptides derived from the human decorin amino sequence (accession number: P07585, (accession number: P07585, http://www.uniprot.org/uniprot/P07585). LRR4 corresponds to the region between amino acid 142 to 162, LRR 5 to the region between 163 to186, LRR 6 to the region between 187 to 212, LRR 10 to the region between 282 to 304, LRR 11 to the region between 305 to 334 and LRR 12 to the region between amino acids 335 to 359. All peptides were obtained from Peptide 2.0, VA. Iodination of decorin and fibronectin -

Carrier-free bovine decorin (R&D, Minneapolis, MN) or fibronectin (Sigma Chemical, St.Louis, MO) was radiolabeled using Na[125I] and chloramine-T as previously described (30,42). Fibronectin degradation analysis – The degradation of fibronectin was evaluated as described with minor modifications (42). Briefly, 20 ng of radioactive 125I-fibronectin (1 X 106 cpm) were added to myoblast pre-treated for 6 hours with DMEM 1% FBS supplemented with CTGF, decorin and TGF-R&D, Minneapolis, MN) as indicated in the legend of the corresponding figure. After 42 hours the conditioned media were collected and fractionated by 7.5% SDS-PAGE. The radioactive bands were then visualized using a PerkinElmer Life Sciences phosphorimaging device (Cyclone model). CTGF – decorin binding assay – Recombinant CTGF (27) was immobilized in anti-flag M2 Affinity Gel (Sigma Chemical, MO) for 3 hours at 4 °C after 5 washes with PBS the gel was blocked with 5% BSA in PBS for 1 hour.

After 3 washes with PBS, 125I-decorin, decorin deletion mutants with or without the indicated competitors described in the corresponding figures, were incubated at room temperature for 3 hours. After 5 washes with PBS the bound material was eluted with protein loading buffer or was directly counted. Saturation isotherm and Scatchard analysis was performed whit GraphPad Prism software, version 5.03 adjusting the curve with non linear regression assuming one binding site. Co-immunoprecipitation - In vitro co-immunoprecipitation was performed as described (43). Briefly, purified recombinant CTGF was co-incubated with pure decorin or pure decorin core protein for 3 hours at room temperature. Then, the proteins were immunoprecipitated for 2 hours at 4C using an anti-mouse decorin antibody LF-136 that were previously attached to protein G beads (Pierce/Thermo Fisher Sci, IL). After washing, proteins were twice eluted in protein loading buffer, electrophoresed and analyzed either by western blot. Immunofluorescence Microscopy – The cells to be immunostained were grown on coverslips. The medium was removed, and the coverslips rinsed with PBS, fixed with 3% paraformaldehyde for 30 min at room temperature, then rinsed with blotto and further incubated for 1 hour in blotto. For actin filament staining, cells were incubated with 0.1 µM phalloidin conjugated with FITC (Sigma Chemical, MO) for 40 min and rinsed with PBS. For nuclear staining, cells were incubated with 1 g/ml Hoechst 33258 in PBS for 10 min. After rinsing, the coverslips were mounted and viewed under a Nikon Diaphot microscope, equipped for epifluorescence (27).

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RNA Isolation and Reverse transcription - Total RNA was isolated from cell cultures using TRIzolTM reagent according to the manufacturer’s instructions (Invitrogen, CA). Semi quantitative RT-PCR - Reverse transcriptase reaction was performed using Moloney murine leukemia virus reverse transcriptase according to the manufacturer’s instructions (Invitrogen, CA). PCR primers for TGF- mouse mRNA sequence. The primers and expression experiments for TGF- and fibronectin were done as published (42), (44).

RESULTS

Decorin null myoblasts are more sensitive to CTGF than wild type - Many cell processes including cell differentiation and fibrosis are regulated by proteoglycans. To study if the proteoglycan decorin could be regulating CTGF, we incubated a C2C12 myoblast or C2C12 myoblast cell line that does not express decorin (38) with different concentrations of CTGF and the amount of accumulated fibronectin was determined. Figure 1A shows that dcn null myoblasts presented an increased basal level of fibronectin and an augmented sensitivity to CTGF compared with WT myoblasts. The incubation of dcn null myoblasts with low CTGF concentrations resulted in a strong increase in fibronectin accumulation, whereas at higher concentrations of CTGF a reduction in fibronectin levels was observed. This reduction was also seen in wild type myoblasts incubated at even higher concentrations of CTGF (data not shown). To analyze if this CTGF effect is specific to decorin absence, we

re-expressed decorin in dcn null myoblasts using an adenovirus with the complete human decorin sequence (38). Figure 1B shows that wild type and dcn null myoblasts behaved as shown above, but when decorin is re-expressed in dcn null myoblasts, these cells behave more like wild type myoblasts, suggesting that dcn null sensitization to CTGF is specific to decorin absence. As a control, Figure 1C shows decorin levels determined by the autoradiographic analysis of incubation media from wild type, dcn null and dcn null infected with decorin adenovirus in the presence of H2[

35S]SO4. Altogether, these results show that in the absence of decorin, myoblasts are more responsive to CTGF. Decorin inhibits CTGF mediated induction of fibronectin, collagen III and actin stress fibers - To analyze the effect of decorin on wild type myoblasts, decorin and CTGF were preincubated for 30 minutes at room temperature and then added to myoblasts for 48 hours. Figure 2A shows that fibronectin and collagen III levels induced by CTGF are inhibited when decorin is present. Decorin inhibited fibronectin accumulation in a 60% and 80% at 1:1 and 1:4 CTGF: decorin molar ratios. Decorin inhibition of collagen III accumulation mediated by CTGF was even more pronounced. Another effect of CTGF on myoblasts is the induction of the formation of actin stress fibers (27). When myoblasts were incubated with CTGF, actin stress fiber formation was induced as previously reported (27), but when these myoblasts were co-incubated with CTGF and decorin the induction was inhibited (Figure 2B). To evaluate the mechanism of fibronectin accumulation induced by CTGF we analyzed if it was due to an inhibition of its degradation, and evaluate if the presence of CTGF and decorin

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affect the degradation of 125I-fibronectin. Figure 2C shows that CTGF and decorin have no effect on fibronectin degradation. To further analyze CTGF mediated fibronectin accumulation mechanism, we analyzed if the presence of CTGF and decorin affects fibronectin expression. Figure 2D shows that CTGF induces fibronectin mRNA and that this induction is inhibited in the presence of decorin. TGF-β1 was used as a positive control for the induction of fibronectin mRNA. So CTGF induction of fibronectin accumulation is due to an increase in fibronectin expression, since CTGF has no effect on fibronectin degradation. Next, we determine if the inhibition of CTGF by decorin induced, in a compensatory fashion, the synthesis of another pro-fibrotic factor, TGF-. Figure 2D shows that neither CTGF nor decorin have an effect on mRNA levels of TGF- The same figure shows that TGF- slightly induced the expression of TGF-, as it has been shown in other systems (45) Since fibroblasts are the major source of the ECM in fibrosis and are one of the main cell targets of CTGF, we analyzed if this decorin inhibitory effect on CTGF activity was the property of myoblasts or was a general mechanism involving these other cell types. 3T3 fibroblasts were incubated with CTGF in the presence of increasing concentrations of decorin. Figure 2E shows CTGF induced accumulation of fibronectin and decorin inhibition of 50% and 80% of fibronectin accumulation at a 1:1 and 1:4 CTGF:decorin molar ratio. Therefore, the decorin inhibition of CTGF could be a general mechanism of CTGF regulation. These results indicate that decorin is able to inhibit CTGF action inhibiting CTGF mediated fibronectin accumulation and expression and stress fiber formation.

CTGF and decorin interact in vitro - To analyze the mechanism by which decorin is able to inhibit the action of CTGF, we studied if these two molecules were able to interact. We analyzed if CTGF interacted with decorin by binding its core protein or the GAG chains to the cytokine. CTGF was co-incubated with the whole molecule of decorin or with the decorin core and immunoprecipitated with decorin antibodies. Figure 3A shows the absence of CTGF in both controls without antibody or without decorin. It also shows that CTGF immunoprecipitated with either the decorin whole molecule or just the protein core. Therefore, it binds at least to decorin´s core protein. To analyze CTGF and decorin interaction, and evaluate their affinity, we immobilized CTGF to beads and incubated it with different concentrations of 125I-decorin core protein. Figure 3B shows that decorin binds to CTGF in a dose-dependent manner. The specific binding is saturable with an estimate Kd of 4.4 nM. Altogether these results suggest that CTGF directly interacts with decorin core protein with high affinity.

LRR10–12 of decorin is important for decorin inhibition of CTGF - Decorin core protein has 12 LRRs that interact with various growth factors and in this way regulate their action (29,32,33,46). To analyze which of the LRRs was important to regulate CTGF we generated four deletion mutants of decorin that lack different LRRs (Figure 4A). These were purified by DEAE followed by western blot against its epitope HA as shown in Figure 4B. Some of the decorin deletions mutants show doublets which likely represent decorin with different degree of N-glycosilation (47,48). To evaluate which region(s) of decorin's core protein is/are important for the inhibitory effect

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on CTGF biological activity, C2C12 myoblasts were co-incubated with CTGF and the different decorins and the effect on the amount of fibronectin determined. Figure 4C (top) shows that fibronectin accumulation by CTGF was inhibited by wild type and all decorin mutants except for the one that lacks the LRR10–12. The Figure 4C (bottom) shows quantitative analysis of the western blot indicating that only decorin mutant lacking LRR10-12 was unable to inhibit the induction of fibronectin in response to CTGF. Since decorin lacking LRR10-12 was the only one that did not show inhibitory activity, we evaluated if this mutant decorin still has the capability to form dimers in solution as wild type decorin (49). Figure 4D shows that wild type decorin and decorin lacking LRR10-12 were able to form a dimer in solution, indicating that the absence of LRR10-12 would not affect the capability to form dimers. These results suggest that LRR10–12 is important for decorin inhibition of CTGF.

LRR10–12 of decorin is essential for the interaction with CTGF - As shown above, different deletion mutants of decorin had different effects on CTGF action, so we wanted to determine the ability of these deletion mutants to interact with CTGF. We co-incubated the different decorin core proteins with CTGF and immunoprecipitated CTGF with the anti-flag antibody. Figure 5 shows that wild type decorin as well as all the deletion mutants, except the one lacking LRR10–12, were able to interact with CTGF. The same figure shows as a control that the same amount of CTGF was immunoprecipitated and the input of the different core proteins used in this assay. To further evaluate if the LRR10-12 was critical to interact with CTGF, the later was immobilized to beads containing anti-flag antibodies and were incubated

with 125I-decorin and increasing concentrations of wild type decorin and decorin mutants lacking LRR4-6 and LRR10-12. Figure 5B shows that decorin lacking LRR10-12 was unable to displace bound 125I-decorin from CTGF. In contrast wild type and decorin lacking LRR4-6 substantially displaced bound decorin at a decorin:CTGF molar ratio of 2 and 4. These results suggest that LRR10–12 is critical for the interaction between CTGF and decorin.

Peptide derived from the decorin LRR10–12 region is able to inhibit decorin–CTGF interaction and repress biological effects mediated by CTGF -To further study the region of decorin responsible for the interaction with CTGF, we used different peptides derived from different regions of decorin, containing LRR4, 5 or 6 as controls, and LRR12 that showed to be important for the interaction and inhibition of CTGF biological activity. Figure 6A shows the specific amino-acid sequence of each peptide utilized. To analyze if any of these peptides affected the interaction of decorin and CTGF, the latter was immobilized to beads containing anti-flag antibodies, and incubated with decorin and the different peptides, molar ratio 2:1. Figure 6B shows the elute containing decorin, determined by western blot analysis of the precipitates using anti-HA antibodies. In the absence of peptides decorin interacts with the immobilized CTGF. Of all the peptides used, only the one derived from the LRR10–12 region, peptide LRR12, was able to inhibit the interaction between decorin and CTGF in about 50%. As a control of the assay figure 6B bottom panel shows that the same amount of CTGF was immobilized on the different conditions, and is observed in the elute with anti-flag antibodies. Next, we titrated the amount

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of LRR12 required to inhibit the interaction between decorin and CTGF. Figure 6C (left upper panel) shows that LRR12 inhibits the interaction between CTGF and decorin in a dose dependent manner. As a control the effect of LRR4 is show (left bottom panel). Figure 6C (right) shows a quantitative analysis of this experiment. These results indicate that a molar ratio of LRR12/decorin of around 2 is required to inhibit in a 50% the formation of the decorin-CTGF complex. In contrast, a small and rather erratic inhibition of the interaction between CTGF and decorin was observed when the LRR4 was used. These results strongly suggest that the LRR12 region of decorin's core protein is involved in the in vitro interaction between decorin and CTGF.

To analyze if the interaction of LRR12 with CTGF had any effect on the biological activity of the growth factor, we incubated C2C12 myoblasts with CTGF and the different peptides. Figure 7A shows fibronectin and collagen III accumulation by CTGF and how this is not affected when the control peptides were used. However, when the LRR12 was used along with CTGF, it inhibited CTGF action, as observed by the lack of fibronectin and collagen III accumulation in response to CTGF. The Figure also shows the values of induction of fibronectin and collagen III over tubulin and the exclusive inhibition exerted by LRR12. To evaluate the actin stress fiber formation induced by CTGF in C2C12 myoblasts, we co-incubated them with CTGF and the different LRR peptides. As seen in Figure 7B, when CTGF was co-incubated with the control peptides, CTGF induction of actin stress fibers was slightly increased when the peptides 4 or 5 were used. However, when LRR12 was used, actin stress fiber formation was

strongly inhibited. Finally, we also studied if the peptide could inhibit CTGF biological activity in fibroblasts. Figure 7C shows the CTGF accumulation of fibronectin and collagen III in 3T3 fibroblasts as seen for myoblasts. Control peptides had no effect on fibronectin accumulation and the LRR12 peptide was able to inhibit CTGF action on fibroblasts. To further evaluate peptides present in LRR10–12, myoblasts were incubated with peptides LRR10, 11 and 12 and the accumulation of fibronectin determined. Figure 7D shows that only LRR12 was able to inhibit the CTGF effect in the cells. These results suggest that LRR12 of decorin is necessary to inhibit CTGF action.

CTGF increases the amount of decorin - Since decorin specifically interacts and inhibits CTGF-mediated activity, we evaluated the levels of decorin in myoblasts treated with CTGF. To analyze this, C2C12 myoblasts were incubated with increasing concentrations of CTGF in the presence of H2[

35S]SO4. Figure 8A shows that CTGF induces the synthesis of a proteoglycan that migrated as smear of 120–80 kDa. To determine the GAG nature of this proteoglycan an aliquot of the radioactive medium from the myoblasts was incubated with two GAG-lyases CABC and Hase, that specifically degrade chondroitin/dermatan and heparan sulfate respectively. Figure 8B shows that most of the radioactive material induced by CTGF corresponds to proteoglycan containing the chondroitin/dermatan sulfate radioactive chain. The molecular weight as well as the GAG nature corresponds with the proteoglycan decorin previously described in the same types of cells (41). Interestingly, a proteoglycan of higher molecular weight also is induced by CTGF, its contains chondroitin /dermatan

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GAG chain and likely corresponds with the proteoglycan biglycan (50). To confirm that CTGF specifically induces decorin, aliquots from myoblast incubation media were incubated with CABC followed by western blot analyses with antibodies that specifically recognize decorin's core protein. Figure 8C shows that CTGF induces the synthesis of decorin in a dose-dependent manner as determined by western blot analyses. These results indicate that CTGF is able to induce the synthesis of decorin, which we have shown to be a specific inhibitor of CTGF-mediated biological activity.

DISCUSSION

Our results show that decorin can interact with CTGF and, moreover, can negatively regulate CTGF´s action. We also show that myoblasts that do not express decorin are more sensitive to the growth factor than are wild type ones. Finally, we show that CTGF is able to increase decorin accumulation in incubation media. This last result sheds light on how this could be a mechanism where CTGF autoregulates its own action by increasing the amount of its own inhibitor. In normal healing, CTGF is transiently increased and once the scar is formed is downregulated (51), but when this downregulation does not occur the tissue becomes fibrotic. One of the mechanisms that could be helping to regulate CTGF action so that the tissue does not become fibrotic could be decorin. In our experiments, when decorin null myoblasts were used, since CTGF induces decorin accumulation in wild type myoblasts, whereas in the decorin null myoblasts this is not possible, CTGF cannot negatively autoregulate itself and in this way these myoblasts were more sensitive to the growth factor. The finding that decorin interacts and inhibits CTGF action is critical since little is known about how

CTGF is regulated. Decorin is located in a cell´s ECM (30,52), so it could sequester the growth factor towards it as other growth factors such as TGF- (29,30). In this way, CTGF would not be available to interact with the transducing receptors. However, we have shown that decorin is able to bind to the endocytic LDL receptor-related protein receptor type-1 (LRP-1) and is endocyted and degraded as a consequence of this binding (53). Since CTGF is also able to bind LRP-1 (54), decorin could be regulating this binding and increasing CTGF´s degradation by LRP-1. In this way, it would decrease the time that CTGF is available to exert its biological functions. We determined that the binding of decorin to CTGF was saturable, with a half-saturation occurring at 4.4 nM. As mentioned CTGF is a multi-ligand protein and different values of interaction with receptors and growth factors have been described. Thus, it binds VEGF, BMP-7, -4 and TGF- with Kds of 26, 14, 15 and 30 nM respectively (55-57). Regarding the binding of CTGF to putative cells, cell surface receptors or molecules that mediate cell interaction, it has been described interaction with osteoblasts, integrins αIIbβ3 and fibronectin with Kds of 17, 15 and 64 nM respectively (58-60). As mentioned, particularly attractive is the binding of CTGF to LRP-1 with an observed Kd of 0.5–1 nM (54). All these interaction values are in general, in the same range of order of magnitude suggesting that competition between ligands and cell receptors for CTGF must results in variable biological effects, either during physiological as well as under pathogenic conditions. Whichever mechanism decorin is using to negatively regulate CTGF, it seems to be a general issue since the inhibition is seen in at least the

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two different cell types we used: myoblasts, which have been shown to participate in muscle fibrosis by responding to CTGF (27), and fibroblasts, which are another important cell target for this growth factor and mainly responsible for ECM production in many fibrotic disorders (3,61).

In this paper, we also show that mutant decorins that lack different LRRs are all able to inhibit CTGF except for the one lacking LRR10–12. The models that predict decorin structure show that it has the shape of a horseshoe where the inner part would be formed by sheets composed of the different LRRs (49,62,63). The crystal structure of the dimeric protein core of decorin has been elucidated (49). It is suggested that decorin dimerizes through the concave surfaces of the LRR domains, which have been implicated previously in protein-ligand interactions (49). Through cross-link assay, we demonstrated that decorin and the deletion mutant lacking LRR10-12 are able to dimerize in vitro, indicating that the ability of deletion mutant lacking LRR10-12 to form a dimer is not lost by the absence of this LRR. If the dimeric or monomeric form of decorin is required to interact and inhibit CTGF biological activities require further investigation. It has been shown that the majority of the growth factors, collagen and transducing receptors which interact with decorin´s core protein do so through LRR4–8 (36,64,65). Therefore, the finding that the important region for CTGF´s inhibition is not located within this region would help develop therapies that will not interfere with the decorin regulation of other growth factors. In this work, we used a peptide derived from the LRR10–12 region that was able to inhibit CTGF by itself. Several investigators have used decorin as a therapeutic inhibitor of TGF-

(37,66-70), a strong inductor of fibrosis (3,71,72). Since TGF- has pleiotropic effects this type of therapeutic approach is not very promising. Previously, we have shown that decorin interacts with TGF- through a sequence present in LRR3–5 (36). Interestingly, some partial effects on myoblast fibronectin accumulation and actin stress fiber formation were observed when CTGF was co-incubated with LRR4 and 5. Previously, it has been suggested that CTGF could be an agonist of TGF- (57). The role of these LRRs regions on TGF-β activity deserves further evaluation. The finding that LRR12 interacts and inhibits CTGF is attractive. These are potentially important findings since although fibrotic disorders are responsible for 45% of human deaths an effective therapy for these fibrotic diseases does not exist (3). In DMD, all the therapies being developed try to either restore dystrophin expression or inject dystrophin-expressing cells that repair the injured muscle. However, these have not been effective and one of the reasons is because the muscle tissue is already fibrotic (73,74). In vivo experiments have shown that myostatin knock-out mice developed significantly less fibrosis and displayed better skeletal muscle regeneration compared with wild type mice at two and four weeks following gastrocnemius muscle laceration injury (75). The fibrotic scar in DMD muscles and other skeletal muscular dystrophies is large, and it would be of great help to decrease it before cell therapies are used to restore defective protein expression (74). In this way, muscle regeneration could be more effective. The LRR12 peptide could be of great help on this matter, and we are developing in vivo experiments to assess the effectiveness of this peptide in animal models of muscle fibrosis. Finally, we propose a model

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summarized in Figure 9 in which as previously shown, CTGF induces the accumulation of ECM proteins and actin

stress fiber formation, but we now show that these are inhibited by decorin. This

molecule is also induced by CTGF, and this could be an autoregulatory inhibitory.

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FOOTNOTES

This study was supported by research grants from FONDAP-Biomedicine # 13980001, CARE PFB12/2007, FONDECYT (103193 and 21050032 to CV and JG respectively) and MDA 89419 and Fundación Chilena para Biología Celular Proyecto MF-100. Abbreviations. CTGF: connective tissue growth factor; dcn, decorin; LRR: leucine-rich repeat; ECM, extracellular matrix; TGF-, transforming growth factor β; DMD: Duchenne muscular dystrophy; LRP, LDL receptor-related protein

LEGENDS TO FIGURES

FIGURE 1: Decorin absence increases myoblast sensitivity to CTGF. A. Wild type C2C12 and decorin null myoblasts (Dcn null) were serum starved for 18 hours and then incubated with CTGF for 48 hours. Fibronectin (FN) levels were determined by western blot from the cell extracts. Tubulin (tub) was used as a loading control. B. Wild type, decorin null and decorin null myoblasts that re-express decorin by infection with an adenovirus (Adv-dcn) were treated as in A. Fibronectin levels were determined by western blot from the cell extracts, and tubulin was used as a loading control. C. Autoradiography of the [35S]-H2SO4 radiolabeled conditioned medium from cells infected as in B. The figure shows the wild type, Dcn null and Dcn null myoblasts that re-express decorin. The brackets indicate Dcn migration. FIGURE 2: Exogenously added decorin inhibits the fibrotic effect of CTGF on myoblasts and fibroblasts. A. C2C12 myoblasts were serum starved for 18 hours and then incubated with the indicated concentrations of CTGF and decorin (Dcn). When both molecules were added, they were preincubated for 30 min. After 48 hours the fibronectin (FN, left panel) and Collagen III (Col III, right panel) levels were determined by western blot from the cell extracts. Tubulin levels were used as a loading control in both cases. FN/tub and Col-III/tub correspond to a quantification of FN and Col-III levels normalized to tub levels of this representative experiment, of three independent experiments. B. C2C12 myoblasts were grown on coverslips and serum starved for 18 hours and then incubated with CTGF preincubated with or without decorin at the indicated concentrations. After 48 hours, actin stress fibers were observed by immunofluorescence using phalloidin coupled to FITC.

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Bar = 50 µm. C. C2C12 myoblast were serum starved and treated as indicated, 6 hours later 20 ng of 125I-FN (1X106 cpm) were added to each well. After 42 hours the conditioned media were collected, subjected to electrophoresis and analyzed by phosphor imaging. Medium alone, correspond to a well with culture medium in the absence of cells. D. C2C12 cells were treated as indicated for 24 hours. Total RNA was extracted and semi quantitative RT-PCR were carried out to determine the expression levels of FN and TGF-rRNA 28S were used as loading control. A control without reverse transcriptase was performed (-). E. 3T3 fibroblasts were serum starved for 18 hours and then incubated with the indicated concentrations of CTGF and Dcn. When both molecules were added, they were pre-incubated for 30 min. After 48 hours the FN levels were determined by western blot. Tub levels were used as a loading control. Fn/tub corresponds to a quantification of FN levels normalized to tub levels of this representative experiment. FIGURE 3: CTGF interacts with decorin’s core protein. A. CTGF was incubated with complete decorin or with decorin´s core protein (Dcn core) and then immunoprecipitated with an anti-decorin (anti-Dcn) antibody. CTGF presence on the immunoprecipitate was determined by an anti-flag western blot. B. Binding isotherm of increasing concentration of 125I-Dcn to immobilized CTGF. The specific binding derived from the subtraction of non-specific binding to the total binding in each point. The dissociation constant (Kd: 4.4 nM) is indicated in the graph. The values correspond to one experiment performed in triplicate. Scatchard analysis of the presented data is shown. FIGURE 4: Decorin´s leucine rich repeats 10 to 12 are required to inhibit the CTGF-induced accumulation of ECM molecules. A. Diagram of wild type decorin (WT) with its 12 leucine rich repeats (LRRs) and the four deletion mutants generated in which the core protein lacks the (i) LRRs 4, 5 and 6 (Δ 4–6), (ii) LRRs 4 and 5 (Δ 4–5), (iii) LRRs 5 and 6 (Δ 5–6) and (iv) LRR 10 and 12 (Δ 10–12). All of these construction contains an HA epitope in its C-terminal. B. Dcn and its deletion mutants were purified through DEAE-Sephacel. Their core proteins, after CABC digestion, were visualized by a western blot using anti-HA antibodies. C. C2C12 myoblasts were serum starved for 18 hours and then incubated with CTGF 0.8 nM and increasing concentrations of Dcn or its deletion mutants for 48 hours (1.6 and 3.2 nM). Fibronectin levels were determined by a western blot of the cell extracts, using tubulin as a loading control. At the bottom a quantification of this representative experiment is shown. Two independent experiments were done. D. Dcn and the mutant Δ 10–12 were incubated 30 minutes at room temperature with Dithiobis (succinimidyl) propionate (DSS) as cross linking agent. The decorin´s monomers and dimers were detected by western blot using anti-HA antibodies. FIGURE 5: CTGF interacts with decorin's core protein through the LRRs 10-12. A. CTGF (0.4 nM) was preincubated with Dcn or its deletion mutants Δ 4–5, Δ 4–6, Δ 5–6 or Δ 10–12. The proteins were immunoprecipitated with an anti-flag antibody and further analyzed by western blot using an anti-HA antibody that recognizes the epitope present in all decorin constructions. Before loading the gel the samples were treated with CABC to detect only decorin core protein for a more accurate quantification. The bottom panel shows the levels of the different decorin´s constructions that were incubated with CTGF as an input

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control (each input was diluted 1:20 before being loaded onto the gel) and analyzed by western blot with an anti-HA antibody. A quantification of the bound Dcn or deletion mutant normalized to the corresponding input is presented as the percentage of the specific binding of Dcn (% bound Dcn) B. 125I-Dcn was incubated whit immobilized CTGF in the absence or presence of increasing amount of cold Dcn, Δ 4–6 or Δ 10–12 as competitors, at the indicated competitor/125I-Dcn molar ratios. The bound 125I-Dcn was determined in a gamma counter, and presented as the percentage of the specific binding in the absence of competitors. Results are from one experiment done in triplicate. Values represent mean with standard error. FIGURE 6: Peptide derived from the LRR 10-12 compete with WT decorin for CTGF. A. Amino-sequence of the leucine rich repeats peptides (LRR) derived from human decorin (Dcn). B. Inmobilized CTGF on anti-FLAG M2 Affinity Gel was incubated with Dcn in the presence of different LRR peptides (molar ratio LRR:Dcn = 2). Since all the LRR stocks are in DMSO, a DMSO control was executed. The bound material was eluted with loading buffer and then analyzed by western blot with antibodies against HA to detect decorin or anti-FLAG for CTGF. A quantification of the bounded Dcn is shown normalized to the total CTGF and presented as the percentage of the specific binding in the absence of competitors (% Control/CTGF) C. Immobilized CTGF as in B, was incubated with Dcn and increasing amount of LRR12 or LRR4 at the indicated LRR:Dcn molar ratios. Nonspecific corresponds to incubation in the absence of CTGF. The bound material was analyzed as in B. A quantification of this experiment is shown on the right, in this case the nonspecific binding was subtracted to the bound material. FIGURE 7: Peptide derived from the LRR12 region of decorin inhibits CTGF action. A. C2C12 myoblasts were serum starved for 18 hours and then incubated with CTGF 0.8 nM and increasing concentrations of different LRR peptides for 48 hours (1.6 and 3.2 nM), preincubated for 30 minutes at room temperature. FN and Col-III levels were determined by western blot of the cell extracts, using tubulin as a loading control. The normalized relation between FN or Collagen III levels to tubulin levels is shown under each blot. These esperiments were realized twice. B. C2C12 myoblasts were grown on coverslips, serum starved for 18 hours and then incubated for 48 hours with CTGF 0.8 nM and different LRR peptides at 3.2 nM, preincubated as in A. Actin stress fibers were observed by immunofluorescence using phalloidin coupled to FITC. Bar=100 µm. C. 3T3 fibroblasts were treated as in A. FN and Col-III levels were determined by a western blot of the cell extracts using tubulin as a loading control. D. C2C12 myoblasts were serum starved for 18 hours as in A, and incubated with CTGF and the indicated LRR peptides (3.2 nM). FN was determined by a western blot of the cell extracts using GADPH as a loading control. FIGURE 8: CTGF induces decorin in C2C12 myoblasts. A. C2C12 myoblasts were serum starved for 18 hours, then incubated with different concentrations of CTGF for 48 hours. The last 18 hours was in the presence of [35S]-H2SO4. The cell media were concentrated by DEAE-Sephacel, electrophoresed and the gel autoradiographed. B. C2C12 myoblasts were treated as in A, the conditioned mediums were incubated with the indicated GAG-lyases, electrophoresed and autoradiographed. The bracket in A indicates Dcn migration, and in B

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indicate Dcn and biglycan (Bgn) migration. C. C2C12 myoblasts were serum starved for 18 hours and then incubated with different concentrations of CTGF for 48 hours. Dcn levels from the media were determined after Cabc digestion by western blot using antibodies against mouse decorin that recognize the Dcn core protein. Tubulin (tub) levels were used as a loading control. Hase and Cabc enzyme that hydrolyzes heparan sulfate and chondroitin/dermatan sulfate GAG chains respectively. FIGURE 9: Proposed model for CTGF regulation by decorin. CTGF is responsible for ECM accumulation and actin stress fiber formation. CTGF is also able to induce decorin accumulation, which would be a mechanism of autoregulation since decorin is able to inhibit CTGF action through its LRR12.

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47

118

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KDaKDa

Dcn118

47

KDa

FN

tub

CTGF nM

A

FN

tub

CTGF nM

WT Dcn null WT Dcn nullDcn nullAdv-Dcn

B C

FIGURE 1

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GF

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55

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tub

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55

1.0 2.0 1.6 1.2 1.1 1.0

FNkDa

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Col- III

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170

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1.0 4.0 2.6 1.5

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Fn/tub Col-III/tub

Fn/tub

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FN

TGF-β

Dcn- - + + -CTGF - + - + -TGF-- - - - +

28S

bp

FIGURE 2

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WB: anti-FLAG47

kDa

Dcn-+ + - -

Dcn core- +- + -

anti-Dcn-+ + - +

CTGF+ + + + +

B

FIGURE 3

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i) ii) iii) iv)

FN

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CTGF+ ++- + + + + + + + +

170

55

FIGURE 4

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WB: anti-FLAG

WB: anti-HA

A

CTGF+- + + + +Dcn++ - - - -Dcn ∆ 4-5-- + - - -Dcn ∆ 4-6-- - + - -Dcn ∆ 5-6-- - - -+Dcn ∆10-12-- - - - +

EL

UT

ED

INP

UT

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43

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43

55

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100 65 72 100 20 % Bound Dcn

B

FIGURE 5

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100 103 99 86 106 % Control/CTGF47

LRR4: KELPEKMPKTLQELRAHENEILRR5: TKVRKVTFNGLNQMIVIELGTNPLLRR6: KSSGIENGAFQGMKKLSYIRIADTNILRR12: QYWEIQPSTFRCVYVRSAIQLGNYK

A

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0,1 1 10 100

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FIGURE 6

LRR 12 / Dcn WTMolar Ratio

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CTGF + LRR 12

CTGF + Vehicle

CTGF + LRR 6

Control

CTGF + LRR 5

HA-Dcn WTCTGF

FN

Col-III

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kDa

55

CTGF++

++ ++

FN

GAPDH

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34

kDa

C D

A

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tub

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KDaCTGF+ ++- + + + + + +

Col-III

1.0 1.8 1.8 1.7 1.8 1.8 1.9 1.7 1.4 0.9

1.0 5.9 6.1 6.0 6.0 6.0 4.6 5.1 1.3 0.1

1.0 1.7 0.7 1.6 2.6 1.0

170

FIGURE 7

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0 0.1 0.2

CTGF

Dcn118

85

kDa(nM)

B

Dcn

118

85

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Hase

Cabc

CTGF (nM)0 0.2 0 0.2 0 0.2

+- - + - --- - - + +

C

0 0.1 0.20 0.1 0.2

tub

Dcn

Control Cabc

CTGF (nM)

47

55

kDa

1.0 1.2 2.0

FIGURE 8

Bgn

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CTGF

DCN

ECMFibronectinCollagen III

ACTIN STRESS FIBERS

FIGURE 9

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Cecilia Vial, Jaime Gutierrez, Cristian Santander, Daniel Cabrera and Enrique BrandanDecorin interacts with CTGF/CCN2 through LRR12 inhibiting its biological activity

published online March 23, 2011J. Biol. Chem. 

  10.1074/jbc.M110.189365Access the most updated version of this article at doi:

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