Research ArticleFibrosis

Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites

See allHide authors and affiliations

Science Signaling  29 May 2018:
Vol. 11, Issue 532, eaar2566
DOI: 10.1126/scisignal.aar2566
  • Fig. 1 Fibrillar collagens and LOX in skin samples from wild-type and Thbs1−/− mice.

    (A) Immunoblots showing TSP1 in spleen samples from wild-type (WT) and Thbs1−/− mice (four mice of each genotype, numbered 1 to 4). Ponceau staining of the gel used for immunoblotting is shown as a loading control. (B) Hydroxyproline content of HAc extracts from WT and Thbs1−/− skin samples. n = 4 for each genotype. (C) SDS-PAGE analysis of acid-extracted collagens from the skin of WT and Thbs1−/− mice under reducing conditions. The samples were equalized for hydroxyproline content. Bands corresponding to various forms of collagen trimers, dimers, and monomers are noted. n = 4 for each genotype. (D) Quantification of collagen α2(I) in WT and Thbs1−/− samples. n = 4 for each genotype. (E) Proportions of different collagen forms extracted from the skin of WT and Thbs1−/− mice, given as mean ± SD. n = 4 animals per genotype. (F) Immunoblot showing proLOX and mLOX in total skin extracts from WT and Thbs1−/− mice. Ponceau staining of the gel used for immunoblotting is shown as a loading control. n = 4 for each genotype. (G and H) Quantification of proLOX (G) and mLOX (H) from immunoblots, normalized to the major loading control band. n = 4 for each genotype. a.u., arbitrary units. (I) mLOX/proLOX ratio from the quantitations in (G) and (H) (P = 0.057). In each scatter plot, the bar indicates the mean. Data were analyzed by Mann-Whitney U test. Each dot in (B) and (D) represents the value from an individual animal. Each dot in (G) to (I) represents the mean of four separate measurements from each animal.

  • Fig. 2 TSP1 binds to proLOX and fibrillar collagens I, II, and III in vitro.

    (A) Representative immunoblots of assays for cleavage of recombinant human proLOX by BMP-1 in the absence or presence of increasing concentrations of TSP1, as indicated. BSA was used as a negative control. n = 3 independent experiments. (B) Quantification of the concentration-dependent inhibition of BMP-1–mediated proLOX cleavage by TSP1. Each data point represents the mean from three independent experiments, and the error bars indicate SEM. (C) Schematic diagram of the domains of a fibrillar procollagen molecule. (D) Binding of TSP1 to immobilized collagens in solid-phase binding assays. Collagen I (Col I) (denatured), Col I, Col II, and Col III samples were derived from pepsin-digested collagen preparations. GPP10 and BSA were included as negative controls. One-way analysis of variance (ANOVA) and Bonferroni’s multiple comparison test were performed against GPP10. n = 4 independent experiments. (E) Concentration dependence of TSP1 binding to the indicated immobilized proteins (ligands). The ligands (CPI or CPIII) were immobilized and incubated with or without TSP1 (T). A mouse antibody recognizing TSP1 (C9) and goat anti-mouse antibody (GAM) were included in each reaction to quantify binding. n = 4 independent experiments. (F) Solid-phase binding assays testing TSP1 (T) binding to immobilized CPI or BSA in the presence of either Ca2+ or EDTA and quantified with the antibodies C9 and GAM. n = 4 independent experiments. One-way ANOVA and Bonferroni’s multiple comparison test were performed against BSA. (G) Concentration dependence of TSP1 (T) binding to the indicated immobilized proteins (ligands), rhProCOL1A1, Col I, and CPI. Antibodies C9 and GAM were used to quantify binding. n = 4 independent experiments. Dotted lines in (E) and (G) indicate negative control assays without TSP1. Data points in (D) to (G) indicate the mean, and error bars indicate the SEM.

  • Fig. 3 Identification of specific TSP1-binding peptides within the triple-helical region of collagen II.

    (A) Quantification of the binding to TSP1 to the 56 peptides (numbered for every fifth peptide as II1, etc.) of the Collagen Toolkit II in the presence of Ca2+ and Zn2+. The horizontal line represents the background binding to GPP10. Each bar represents the mean of duplicate samples. (B) Binding of TSP1 (T) to specific TSP1-binding peptides as identified from the initial hits in Toolkit II in the presence of Ca2+ and Zn2+. Binding was quantified by indirect colorimetric assay using the antibodies C9 and GAM. n = 4 independent experiments. (C) Quantification of the binding of TSP1 (T) to the indicated peptides in the presence of the Ca2+ chelator EDTA. n = 4 independent experiments. (D) Binding of proLOX to immobilized TSP1 and the indicated proteins and peptides derived from fibrillar collagens. n = 4 independent experiments. In all panels, GPP10 and BSA were included as negative controls. Pepsin-digested collagens I and III were used as positive controls in (B) and (C). In (B) to (D), one-way ANOVA and Bonferroni’s multiple comparison tests were performed against GPP10. Each bar indicates the mean, and error bars indicate the SEM.

  • Fig. 4 KGHR is a minimal and conserved motif for TSP1 binding.

    (A) Binding of TSP1 (T) to derivatives of TSP1-binding peptides from the Collagen Toolkit II, truncated or mutated as indicated (guest peptides). Binding was quantified by indirect colorimetric assay using the antibodies C9 and GAM. Data are from five independent experiments. (B) Alignment showing the KGHR motifs (red text) in the N-terminal and C-terminal triple-helical regions of the indicated collagen chains of Homo sapiens (Hs). The numbers 87 and 930 refer to the amino acid position in the triple-helical domain of collagen α1(I). Black shading, identical residues; gray shading, conservative substitutions; no shading, no conservation. (C) Concentration-dependent inhibition of TSP1 binding to collagen I fibrils by Collagen Toolkit peptide II-52. Each data point represents the mean, and bars represent the SEM of three independent experiments. (D to F) Merged phase-contrast and immunofluorescence images of TSP1 binding to collagen I fibrils isolated from tendons (D); TSP1 binding to collagen I fibrils in the presence of the indicated Collagen Toolkit II peptides (E) or in the presence of peptides of the indicated sequence (F). Images are representative of four independent experiments. Scale bar, 15 μm. (G) Quantification of immunofluorescence staining for TSP1 bound to collagen fibrils, calculated as total fluorescence intensity within fibrils normalized on pixel count within fibrils. Each dot represents the value from an individual experiment, each bar indicates the mean, and the error bars indicate the SEM from four independent experiments. Data were analyzed by Kruskal-Wallis test and pairwise tests against BSA with Holm’s correction.

  • Fig. 5 Localization of TSP1, fibrillar collagen, and LOX in human dermal fibroblast cultures.

    (A) Dual indirect immunofluorescence staining of the indicated pairs of proteins in permeabilized or nonpermeabilized HDFs cultured for 72 hours. The regions shown at higher magnification in the insets are boxed by dotted lines in the main panels. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Images are representative of four independent experiments. (B) Quantification by Pearson correlation (per cell) of colocalization of the indicated pairs of proteins in permeabilized (P) HDF cultures at the indicated times. C, collagen I; T, TSP1; L, LOX. n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. Data were analyzed by Kruskal-Wallis test and Dunn’s comparison. (C) Quantification by Pearson correlation (per field) of colocalization of the indicated pairs of proteins in nonpermeabilized (NP) HDF cultures at the indicated times. n = 4 independent experiments with at least six fields analyzed per condition per experiment. (D) Dual indirect immunofluorescence staining of the indicated pairs of proteins in permeabilized and nonpermeabilized HDFs after 10 days of culture. Images are representative of four independent experiments. The regions shown at higher magnification in the insets are boxed by dotted lines in the main panels. (E) Quantification by Pearson correlation (per field) of colocalization of the indicated pairs of proteins in HDFs after 10 days of culture. n = 4 independent experiments with at least six fields analyzed per condition per experiment. (F) In situ proximity ligation assay for the indicated pairs of proteins in HDFs cultured for 72 hours. Images are representative of four independent experiments. (G to L) Quantification of in situ proximity ligation signals for the indicated pairs of proteins over time in permeabilized (P) or nonpermeabilized (NP) HDFs. 2Ab, secondary antibodies only. Each data point represents the mean from one experiment, and the bars indicate the mean. n = 4 (G to J) and 3 (K to L) independent experiments. At least 36 cells were analyzed per condition for each experiment. (A), (D), and (F) show results for HDFs at passage 7 and are representative of four experiments on HDFs between passages 4 and 8. Scale bars, 50 μm (main images) and 10 μm (insets).

  • Fig. 6 Effects of LOX inhibition or KGHR-containing peptides on the localization of TSP1 and fibrillar collagen in human dermal fibroblast cultures.

    (A) HDFs were cultured for 10 days in the absence or presence of the LOX inhibitor βAPN. The soluble and insoluble fractions of cell extracts and CM from each treatment group were analyzed by immunoblotting for collagen I α(l), TSP1, and proLOX. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a loading control (CTRL). Molecular markers are indicated to the left of the blots in kilodaltons. (B) Quantification of the indicated proteins from the immunoblots in (A). Protein abundances were normalized to GAPDH and expressed as a ratio versus control cells. Each bar represents the mean of three independent experiments, and the error bars indicate SEM. Mann-Whitney U tests were performed for each pair (not significant). (C) Indirect immunofluorescence images showing TSP1 and collagen I in HDFs cultured for 10 days in the absence or presence of βAPN. Nuclei were stained with DAPI (blue). Images are representative of four independent experiments. The regions shown at higher magnification in the insets are boxed by dotted lines in the main panels. (D) Quantitation by Pearson correlation (per field) of colocalization of TSP1 and collagen I under the indicated conditions. n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. (E) In situ proximity ligation assays in permeabilized HDFs after 10 days culture in the absence or presence of βAPN. Images are representative of four independent experiments. (F) Quantitation of proximity ligation signals under the indicated conditions. n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. (G) Immunofluorescence showing collagen and TSP1 in HDFs cultured and treated for 48 hours with the indicated peptides, then fixed, and stained by indirect immunofluorescence (IF) or in situ proximity ligation (Prox). In the top row, the regions shown at higher magnification in the insets are boxed by dotted lines in the main panels. Each row is representative of four independent experiments. (H) Quantification by Pearson correlation of immunofluorescence colocalizations as in (G). n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. (I) Quantification of proximity ligation signals for conditions as in (G). n = 4 independent experiments with at least 36 cells analyzed per condition per experiment. Data in (H) and (I) were analyzed by Kruskal-Wallis test and pairwise tests against HAc with Holm’s correction. Scale bars, 50 μm (main images) and 10 μm (insets).

  • Fig. 7 KGHR-containing, TSP1-binding collagen triple-helical peptides increase myofibroblast differentiation through a TGF-βR1–dependent mechanism.

    (A) Merged immunofluorescence images of HDFs cultured for 96 hours in the presence of HAc, TGF-β1, collagen peptide II-8, or collagen peptide II-52 and stained for αSMA (green) and nuclei (DAPI; blue). Images are representative of three independent experiments. (B) Quantification of the percentage of αSMA-positive cells under the indicated conditions from three independent experiments. (C) Effects of the various treatments on cell numbers over time; n = 3 independent experiments. (D) Merged immunofluorescence images showing αSMA (green) and DNA (blue) in HDFs cultured for 96 hours in the presence of HAc, TGF-β1, or the indicated peptides. Images are representative of three independent experiments. (E) Quantification of αSMA-positive cells per field after treatment with the indicated peptides. n = 3 independent experiments. (F) Effects of the indicated peptides on cell numbers at 96 hours. n = 3 independent experiments. (G) Merged immunofluorescence images showing αSMA (green) and DNA (blue) in HDFs cultured for 96 hours in the presence of the inhibitor of TGF-βR1 kinase activity SB-431542 (SB) or the β-catenin signaling inhibitor PNU-74654 (PNU). Images are representative of three independent experiments. (H) Quantification of αSMA-positive cells after 96 hours in the presence of the indicated inhibitors. n = 3 independent experiments. (I) Quantification of bioactive and total (determined after acid activation) active TGF-β1 in media from HDFs cultured under the indicated conditions. UT, untreated. n = 3 independent experiments with duplicate samples per condition per experiment. In all graphs, each data point indicates the mean, and error bars indicate the SEM. In (B), (C), (E), (F), and (H), at least 30 cells were scored per condition per experiment. Data were analyzed by Dunnett’s multiple comparison test (one-way ANOVA) against HAc (B) or by Kruskal-Wallis test and pairwise tests against HAc (E and F) or against dimethyl sulfoxide (DMSO) (H). Scale bars, 50 μm.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/532/eaar2566/DC1

    Fig. S1. Identification of specific TSP1-binding peptides within the triple-helical region of collagen III.

    Fig. S2. Detection of collagen I, TSP1, FN, LOX and collagen I C-propeptide in cell fractions, CM, and ECM isolated from HDFs.

    Fig. S3. Detection of the association of TSP1 with collagens I and II and LOX in HDFs by in situ proximity ligation.

    Fig. S4. TGF-β1–mediated induction of αSMA in HDFs.

    Table S1. Primary antibodies and the dilutions used in this study.

    Table S2. Secondary antibodies and the dilutions used in this study.

    Table S3. Chemicals used in this study.

    Table S4. Chemical inhibitors used in this study.

    Table S5. Proteins used in this study.

    Table S6. Buffers used in this study.

  • Supplementary Materials for:

    Thrombospondin-1 promotes matrix homeostasis by interacting with collagen and lysyl oxidase precursors and collagen cross-linking sites

    Silvia Rosini, Nicholas Pugh, Arkadiusz M. Bonna, David J. S. Hulmes, Richard W. Farndale, Josephine C. Adams*

    *Corresponding author. Email: jo.adams{at}bristol.ac.uk

    This PDF file includes:

    • Fig. S1. Identification of specific TSP1-binding peptides within the triple-helical region of collagen III.
    • Fig. S2. Detection of collagen I, TSP1, FN, LOX and collagen I C-propeptide in cell fractions, CM, and ECM isolated from HDFs.
    • Fig. S3. Detection of the association of TSP1 with collagens I and II and LOX in HDFs by in situ proximity ligation.
    • Fig. S4. TGF-β1–mediated induction of αSMA in HDFs.
    • Table S1. Primary antibodies and the dilutions used in this study.
    • Table S2. Secondary antibodies and the dilutions used in this study.
    • Table S3. Chemicals used in this study.
    • Table S4. Chemical inhibitors used in this study.
    • Table S5. Proteins used in this study.
    • Table S6. Buffers used in this study.

    [Download PDF]


    © 2018 American Association for the Advancement of Science

Stay Connected to Science Signaling

Navigate This Article