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. 2012 Feb 24;287(9):6773-87.
doi: 10.1074/jbc.M111.312488. Epub 2011 Dec 27.

The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles

Claudio Cabello-Verrugio  1 , Cristian Santander, Catalina Cofré, Maria José Acuña, Francisco Melo, Enrique Brandan
Affiliations

The internal region leucine-rich repeat 6 of decorin interacts with low density lipoprotein receptor-related protein-1, modulates transforming growth factor (TGF)-β-dependent signaling, and inhibits TGF-β-dependent fibrotic response in skeletal muscles

Claudio Cabello-Verrugio et al. J Biol Chem. .

Abstract

Decorin is a small proteoglycan, composed of 12 leucine-rich repeats (LRRs) that modulates the activity of transforming growth factor type β (TGF-β) and other growth factors, and thereby influences proliferation and differentiation in a wide array of physiological and pathological processes, such as fibrosis, in several tissues and organs. Previously we described two novel modulators of the TGF-β-dependent signaling pathway: LDL receptor-related protein (LRP-1) and decorin. Here we have determined the regions in decorin that are responsible for interaction with LRP-1 and are involved in TGF-β-dependent binding and signaling. Specifically, we used decorin deletion mutants, as well as peptides derived from internal LRR regions, to determine the LRRs responsible for these decorin functions. Our results indicate that LRR6 and LRR5 participate in the interaction with LRP-1 and TGF-β as well as in its dependent signaling. Furthermore, the internal region (LRR6i), composed of 11 amino acids, is responsible for decorin binding to LRP-1 and subsequent TGF-β-dependent signaling. Furthermore, using an in vivo approach, we also demonstrate that the LRR6 region of decorin can inhibit TGF-β mediated action in response to skeletal muscle injury.

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Figures

FIGURE 1.
FIGURE 1.
The LRR4–6 decorin region is necessary for LRP-1 binding and LRP-1-mediated decorin endocytosis. A, schematic showing decorin deletion mutants. B, deletion mutants generated in CHO-K1, which lack LRR4 and 5 (Δ4–5); 4, 5, and 6 (Δ4–6); 5 and 6 (Δ5–6); or 10, 11, and 12 (Δ10–12); were purified by DEAE-Sephacel and separated in a 4–12% SDS-PAGE gradient for detection of glycanated forms (upper) or protein-core after treatment with chondroitinase ABC (lower). Decorins were visualized by Western blot using anti-HA antibodies. C, C2C12 cells were incubated with 35S-labeled decorin mutants at 4 °C for 3 h. Extracts were immunoprecipitated with anti-LRP-1 antibodies and the presence of decorin deletion mutants in the immunoprecipitate (IP) was evaluated by autoradiography (18). Protein levels were detected in the immunoprecipitate with an anti-LRP-1 antibody by Western blot. D, C2C12 myoblasts were incubated with 35S-labeled decorin mutants at 37 °C for 3 h in the absence (control) or presence of 1 μm receptor-associated protein (RAP), then cells were analyzed to determine the level of endocytosis as described before (5). Values correspond to the mean ± S.D. from three independent experiments (*, #, and &, p < 0.001; †, p < 0.05).
FIGURE 2.
FIGURE 2.
Decorin-mediated LRP-1-dependent TGF-β signaling requires the LRR4–6 region of decorin (Dcn). A, wild type and decorin-deficient myoblasts were transiently transfected with plasmids containing p3TP-lux and pRL-SV40 sequences and incubated with 75 nm or without complete (Full) human decorin (hDcnHA) or deletion mutants lacking LRR (Δ4–5), (Δ4–6), (Δ5–6), or (Δ10–12) as described in the legend to Fig. 1. After 6 h of TGF-β1 (1.0 ng/ml) treatment, cells were lysed and reporter activities were determined. Values correspond to the mean ± S.D. from three independent experiments (*, #, &, and ##, p < 0.05). B, decorin-deficient myoblasts were incubated with or without TGF-β1 (5.0 ng/ml) in the absence or presence of 75 nm hDcnHA or deletion mutants as described for A. Total RNA was isolated and CTGF and GAPDH expression was determined by qRT-PCR according to “Experimental Procedures.” Values correspond to the mean of dCT value ± S.D. of three independent experiments (* and #, p < 0.05). C, wild type and decorin-deficient myoblasts were transfected with control siRNA or LRP-1 siRNA, together with the TGF-β responding p3TP-lux reporter, and pRL-SV40 as a control for transfection efficiency (6). Cells were incubated with TGF-β1 in the absence or presence of 75 nm hDcnHA or deletion mutants as described in A. After 6 h, luciferase activity was determined. Values for wild type cells treated with Lipofectamine (control) and TGF-β1 (1.0 ng/ml) correspond to 100%. Values correspond to the mean ± S.D. from three independent experiments (*, p < 0.001).
FIGURE 3.
FIGURE 3.
Two regions contained in decorin LRR4–6 are directly involved in decorin (Dcn) endocytosis and TGF-β-mediated signaling mediated by LRP-1. A, protein cores after treatment with chondroitinase ABC, corresponding to hDcnHA or deletion mutants generated in CHO-K1, which lack LRR4 and -5 (Δ4–5), LRR4, -5, and 6 (Δ4–6), LRR5 and -6 (Δ5–6), LRR5 (Δ5), LRR6 (Δ6) or LRR10, -11, and -12 (Δ10–12). Decorins were visualized by Western blot using anti-HA antibodies. B, wild type and decorin-deficient myoblasts were transfected with control siRNA or LRP-1 siRNA, together with the TGF-β responding p3TP-lux reporter, and pRL-SV40 as a control for transfection efficiency (6). Cells were incubated with TGF-β1 in the absence or presence of decorins or deletion mutants as described in the legend of Fig. 2A. After 6 h, luciferase activity was determined. Values for wild type cells treated with Lipofectamine (control) and TGF-β1 (1.0 ng/ml) correspond to 100%. Values correspond to the mean ± S.D. from three independent experiments (*, p < 0.001). C, decorin-deficient myoblasts were incubated with or without TGF-β1 (5.0 ng/ml) in the absence or presence of 75 nm hDcnHA or deletion mutants as described for B. CTGF and GAPDH expression was determined by qRT-PCR as described in the legend to “Experimental Procedures.” Values correspond to the mean of dCt value ± S.D. of three independent experiments. (* and #, p < 0.05).
FIGURE 4.
FIGURE 4.
Two synthetic peptides derived from the LRR4–6 region of decorin (Dcn) decreases LRP-1-mediated decorin endocytosis and TGF-β-mediated signaling. A, C2C12 myoblasts were incubated with 35S-decorin mutants at 37 °C for 3 h in the absence or presence of 225 nm synthetic peptides derived from the LRR4, LRR5, LRR6, or LRR12 regions of decorin. The cells were then analyzed to determine levels of decorin endocytosis as described in the legend to Fig. 1D. Values correspond to the mean ± S.D. from three independent experiments (* and &, p < 0.05; #, p < 0.001). B, wild type and decorin-deficient myoblasts were transiently transfected with plasmids containing p3TP-lux and pRL-SV40 sequences and incubated with 75 nm hDcnHA in the absence or presence of 225 nm synthetic peptides derived from LRR4, LRR5, LRR6, or LRR12 regions of decorin. After 6 h of TGF-β1 (1.0 ng/ml) treatment, cells were lysed and reporter activities were determined. Values correspond to the mean ± S.D. from three independent experiments (* and &, p < 0.05; #, p < 0.001). C, wild type and decorin-deficient myoblasts were transiently transfected with p3TP-lux and pRL-SV40, and incubated with hDcnHA in the absence or presence of the indicated amounts of synthetic LRR5 and LRR6 peptides. After 6 h of TGF-β1 (1.0 ng/ml) treatment, cells were lysed and reporter activities were determined. 100% correspond to p3TP-lux induction in WT cells. Values correspond to the mean ± S.D. from three independent experiments (*, #, and &, p < 0.05). D, C2C12 myoblasts were incubated with 75 nm hDcnHA at 4 °C for 3 h in the absence or presence of the 75 or 225 nm LRR6 peptides. Lactoferrin was used as a protein that displaces the binding of ligands to LRP-1. Cell extracts were obtained and the immunoprecipitated anti-LRP-1 antibody was evaluated by Western blot using an anti-HA and anti-LRP-1 antibodies, respectively.
FIGURE 5.
FIGURE 5.
A peptide containing the internal region of LRR6 decorin competes with full-length decorin for binding to LRP-1 and reduces LRP-1-mediated decorin endocytosis. A, sequence of peptides from the LRR6 region of decorin, LRR6 (Full), LRR6i (In), and LRR6e (Ext), are depicted and their potential locations in the three-dimensional decorin molecule are shown based on the structural model described by Scott et al. (44). B, C2C12 myoblasts were incubated with 75 nm hDcnHA at 4 °C for 3 h in the absence or presence of 225 nm of the synthetic peptides derived from LRR6 as described in A, cell extracts were immunoprecipitated with anti-LRP-1 antibody and the presence of hDcnHA and LRP-1 in the immunoprecipitate were evaluated by Western blot using anti-HA and anti-LRP-1 antibodies, respectively. C, C2C12 myoblasts were incubated with 35S-decorin mutants at 37 °C for 3 h in the absence or presence of synthetic peptides from the LRR6 region of decorin as described in A and B, or scramble peptide. The cells were then analyzed to determinate decorin endocytosis levels as described in the legend to Fig. 1C. Values correspond to the mean ± S.D. from three independent experiments (* and #, p < 0.001).
FIGURE 6.
FIGURE 6.
TGF-β activity is decreased by a synthetic peptide derived from the LRR6 internal region of decorin (Dcn). A, wild type and decorin-deficient myoblasts were transiently transfected with plasmids containing p3TP-lux and pRL-SV40 sequences and incubated with 75 nm hDcnHA in the absence or presence of 225 nm synthetic peptides from the LRR6 region of decorin, LRR6 (Full), LRR6i (In), or LRR6e (Ext). After 6 h of TGF-β1 (1.0 ng/ml) treatment, cells were lysed and reporter activities were determined. Values correspond to the mean ± S.D. from three independent experiments (*, #, and &, p < 0.05). B, wild type and decorin-deficient myoblasts were transiently transfected with p3TP-lux and pRL-SV40 plasmids and incubated with 75 nm hDcnHA-Full in the absence or presence of different amounts of LRR6 (Full, In, or Ext) or scramble peptide (Scramble). Treatment with TGF-β1 (1.0 ng/ml) and reporter activities were determined as in A. Values correspond to the mean ± S.D. from three independent experiments (*, p < 0.05; # and &, p < 0.001). C, decorin-deficient myoblasts were incubated with TGF-β1 (5.0 ng/ml) plus 75 nm hDcnHA in the absence or presence of 225 nm synthetic peptides LRR6: Full, In, or Ext, or scramble (Scr) peptide. Total RNA was isolated and CTGF and GAPDH expression was determined by qRT-PCR according to “Experimental Procedures.” Values correspond to the mean of dCT value ± S.D. of three independent experiments (* and #, p < 0.05).
FIGURE 7.
FIGURE 7.
The region contained in LRR4–6 of decorin (Dcn) contains the binding site for TGF-β1. A, C2C12 myoblasts were incubated with the indicated amount of 125I-TGF-β1 at 4 °C for 3 h. Extracts were immunoprecipitated with anti-LRP-1 antibodies and the presence of 125I-TGF-β1 in the immunoprecipitate was evaluated by autoradiography. Levels of LRP-1 were detected by Western blot assay. B, C2C12 myoblasts were incubated with 125I-TGF-β1 at 4 °C for 3 h in the absence or presence of RAP (1 μm). Extracts were immunoprecipitated with anti-LRP-1 antibodies. The presence of 125I-TGF-β1 in the immunoprecipitate was evaluated by autoradiography and the protein levels of LRP-1 by Western blot. C, C2C12 myoblasts were incubated with 125I-TGF-β1 as in A in the absence or presence of 75 nm hDcnHA or deletion mutants lacking LRR (Δ4–6), (Δ4–5), (Δ5–6), (Δ5), (Δ6), or (Δ10–12). Extracts were immunoprecipitated with anti-LRP-1 antibodies as in A. The presence of hDcnHA or the corresponding deletion mutants in the immunoprecipitate was evaluated by Western blot using an anti-HA antibodies. 125I-TGF-β1 in the immunoprecipitate (IP) was evaluated by autoradiography. LRP-1 levels were detected by Western blot analysis. D, C2C12 myoblasts were incubated with 75 nm hDcnHA in the absence or presence of 125I-TGF-β1 as in A. In some cases different LRR-derived peptides (LRR5, LRR6, LRR12) were added to the cells. Extracts were immunoprecipitated with anti-LRP-1 antibodies as in A. The presence of hDcnHA and LRP-1 in the immunoprecipitate was evaluated by Western blot using anti-HA and LRP-1 antibodies, respectively. 125I-TGF-β1 in the immunoprecipitate was evaluated by autoradiography.
FIGURE 8.
FIGURE 8.
A synthetic peptide from the LRR6 region of decorin decreases TGF-β activity in an injured skeletal muscle mouse model. A, tibialis anterior muscles of C57BL10 mice were injured by BaCl2 injection (36). Tibialis anterior muscles were removed at different times post-injury and protein extracts were separated by SDS-PAGE. Levels of LRP-1, CTGF, and embryonic myosin were determined by Western blot analysis. GAPDH levels are shown as a loading control. B, tibialis anterior muscles of C57BL10 mice were injured as in A. At day 3 post-injury, tibialis anterior muscles were injected with TGF-β1 (20 ng) in the absence or presence of complete synthetic LRR6 peptide (10 μg). Tibialis anterior muscles were removed on day 4 post-injury and protein extracts were separated by SDS-PAGE. Levels of CTGF and fibronectin (FN) were determined by Western blot analysis. GAPDH levels are shown as a loading control. C, quantification of three independent experiments as shown in B. Values correspond to the mean ± S.D. for fold-induction relative to control cells without TGF-β1 and LRR6 (* and #, p < 0.05). D, tibialis anterior muscles of C57BL10 mice were injured as in B. TGF-β1 (20 ng) was injected in the absence or presence of complete synthetic LRR6i peptide (10 μg) or scramble peptide (Scr, 10 μg). Tibialis anterior muscles were removed on day 4 post-injury and protein extracts were separated by SDS-PAGE. Levels of phospho-Smad-2 and total Smad-2 were determined by Western blot analysis.
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