Research ArticleOsteoarthritis

ITGBL1 modulates integrin activity to promote cartilage formation and protect against arthritis

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Science Translational Medicine  10 Oct 2018:
Vol. 10, Issue 462, eaam7486
DOI: 10.1126/scitranslmed.aam7486
  • Fig. 1 ITGBL1 is involved in facial cartilage development in X. laevis.

    (A) Alcian blue–stained images of craniofacial cartilages in control, Itgbl1-depleted (MO), and Itgbl1-rescued embryos of X. laevis. Upper panels show the ethmoidal plates and lower panels show the ceratohyal cartilages. Scale bars, 500 μm. (B) Quantification of craniofacial cartilages in (A). n = 33, control; n = 42, Itgbl1-MO; n = 18, Itgbl1-MO + 300; n = 38, Itgbl1-MO + 500; n = 41, Itgbl1-MO + 1000. (C) Alcian blue–stained images of craniofacial cartilages of control and Itgbl1-overexpressing embryos. Scale bars, 500 μm. (D) Quantification of facial cartilages in (C). n = 10, control; n = 20, Itgbl1; n = 20, Itgbl1-Flag + 300. (E) Expression of Sox9 and Col2a1 in control and Itgbl1-overexpressing embryos. Scale bars, 100 μm. (F) Schematic illustrating Itgbl1-MO unilateral injection [contralateral region injected with mGFP (membrane green fluorescent protein)] into two-cell-stage Xenopus embryos to examine the Itgbl1 loss-of-function phenotype in a mosaic animal. (G) Immunostaining for COL2A1 (green) in facial cartilages in unilaterally injected embryos (50-μm sections). (G′) mGFP pseudo-colored blue. Histone-red fluorescent protein (H-RFP; G″-G‴) was co-injected as a tracer. Scale bars, 100 μm. (H and I) High-magnification images of the ethmoidal plate (Eth in G‴) in control and Itgbl1-MO–injected embryos. The dashed lines indicate cell boundaries. mGFP was pseudo-colored as blue. Scale bars, 20 μm. (J) ITGBL1-FLAG fusion proteins expressed in craniofacial prechondrocytes. Immunofluorescent staining was performed with anti-FLAG antibody (green). mGFP was pseudo-colored as red. Scale bars, 20 μm. (K) Control and Itgbl1-overexpressing Xenopus embryo (stage 37) sections stained for COL2A1 (green). Scale bars, 100 μm. Data are shown as means ± SEM. Statistical significances in (B) and (D) were determined using an unpaired two-tailed Student’s t test. A.U., arbitrary units.

  • Fig. 2 Functions of ITGBL1 during chondrogenesis in mouse and human cells.

    (A) Itgbl1 and Aggrecan (Acan) expression quantified by quantitative PCR (qPCR) analysis during chondrogenesis. (B) Itgbl1 and alkaline phosphatase (Alpl) expression quantified by qPCR analysis during osteogenesis. (C and D) Frozen sections (10 μm) of control or ITGBL1-depleted hBMSC-derived chondrogenic pellets stained with anti-COL2A1 antibody (C) or Alcian blue (D). Scale bars, 10 μm (C) and 100 μm (D). DAPI, 4′,6-diamidino-2-phenylindole. (E) Chondrogenic gene expression upon Itgbl1 overexpression with increasing doses of transfecting DNA in the absence of differentiation medium analyzed by RT-PCR. (F) Chondrogenic gene expression shown in (E) quantified by qPCR. Data are shown as means ± SEM from three biological replicate experiments. Statistical significances in (F) were determined using an unpaired two-tailed Student’s t test. (G) Mouse limb-bud mesenchyme isolated and induced for chondrogenesis by serum (S+) or by overexpressing Itgbl1 without serum (S−), stained with Alcian blue. Scale bars, 1 mm. (H) Quantification of glycosaminoglycan (GAG) expression in the micromasses shown in (G). Data are shown as means ± SEM from three replicate experiments. Statistical significance in (H) was determined using an unpaired two-tailed Student’s t test.

  • Fig. 3 ITGBL1 inhibits integrin-ECM complex formation in PC3 cells.

    (A) Immunofluorescent staining of FAK-positive focal adhesion complexes in control or ITGBL1-depleted PC3 cells. (B) Expression intensity and number of FAK-positive focal adhesion complexes in (A). FAK intensity: n = 18, control (CTL); n = 21, Itgbl1-siRNA. FAK puncta: n = 12, control; n = 19, Itgbl1-siRNA. (C) Immunofluorescent staining of integrin-β1–positive FA complexes in control and ITGBL1-depleted PC3 cells. (D) Intensity of integrin-β1 expression and size of the cells in (C). Scale bars, 10 μm. ITG-β1 intensity: n = 20, control; n = 14, Itgbl1-siRNA. Cell size: n = 19, control; n = 13, Itgbl1-siRNA. Statistical significances in (B) and (D) were determined using an unpaired two-tailed Student’s t test. (E) FACS analysis using active integrin-β1–specific antibodies (HUTS-4) in control, ITGBL1-depleted, or Itgbl1-overexpressing PC3 cells. (F) Active integrin-β1–specific antibody staining in control or ITGBL1-depleted PC3 cells. (G) Co-immunoprecipitation (IP) of ITGBL1-HA and integrin-β–Flag in the presence or absence of Ca2+. The red box indicates co-immunoprecipitated ITGBL1-HA, and the red asterisks indicate the absence of the ITGBL1-HA signals. Transfection of C-terminal hemagglutinin (HA)–tag fusion construct of ITGBL1 yielded two major products of 63 and 48 kDa in human embryonic kidney (HEK) 293T cells, which were ITGBL1-L and ITGBL1-S, respectively. Scale bars, 10 μm (A, C, and F).

  • Fig. 4 ITGBL1 inhibits integrin-ECM complex formation in various cell types including chondrocytes.

    (A and B) Phase-contrast images of cell adhesion and spreading and quantification of cell size in control, ITGBL1-depleted, or Itgbl1-overexpressing PC3 cells. Increasing doses of Mn2+ were added to the Itgbl1-overexpressing cells to activate integrins. n = 54, control; n = 60, siRNA; n = 51, Itgbl1-OE; n = 45, Itgbl1-OE + 0.1 mM; n = 43, Itgbl1-OE + 0.2 mM; n = 37, Itgbl1-OE + 0.4 mM. Scale bars, 20 μm. (C and D) Immunofluorescent images using anti-FAK antibody (C) and the quantification of FAK intensity (D) in control, ITGBL1-depleted, or Itgbl1-overexpressing human chondrocytes. n = 13, control; n = 8, siRNA; n = 14, Itgbl1-OE. Scale bars, 10 μm. (E and F) Images of cell adhesion and spreading and quantification of cell size in control, ITGBL1-depleted, or Itgbl1-overexpressing human articular chondrocytes. Increasing doses of Mn2+ were added to the Itgbl1-overexpressing cells to activate integrins. n = 70, control; n = 48, siRNA; n = 81, Itgbl1-OE; n = 53, Itgbl1-OE + 0.1 mM; n = 54, Itgbl1-OE + 0.2 mM; n = 31, Itgbl1-OE + 0.4 mM. Scale bars, 20 μm. Data are shown as means ± SEM. Statistical significances in (B), (D), and (F) were determined using one-way analysis of variance (ANOVA) with Bonferroni post hoc analysis (*P < 0.05, ***P < 0.0005).

  • Fig. 5 ITGBL1 promotes chondrogenesis via integrin inhibition.

    (A and B) Chondrogenic protein expression in Itgbl1-overexpressing ATDC5 cells treated with Mn2+ or DTT. (B) Quantification of (A); three replicate experiments. Statistical significances in (B) were determined using an unpaired two-tailed Student’s t test. (C) Alcian blue staining in micromass cultures of control and Itgbl1-overexpressing ATDC5 cells in the presence of Mn2+ or DTT. Scale bars, 1 mm. (D and E) Average size of micromasses and GAG/total protein ratio in (C). n = 6 for each experimental sample. Statistical significances in (D) and (E) were determined using one-way ANOVA with Bonferroni post hoc analysis (**P < 0.005, ***P < 0.0005). (F) Sox9 expression in human chondrocytes upon depletion of integrin1, integrin3, integrin5, or integrin10 in combination with β1 subunits analyzed by qPCR. Data are shown as means ± SEM from three replicate experiments. (G) Sox9 expression in human chondrocytes upon depletion of integrin1β1, integrin3β1, integrin5β1, or integrin10β1 in combination with Itgbl1 overexpression analyzed by qPCR. Data are shown as means ± SEM from six replicate experiments. Statistical significances in (F) and (G) were determined using an unpaired two-tailed Student’s t test (*P < 0.05, **P < 0.005, ***P < 0.0005).

  • Fig. 6 ITGBL1 protects chondrocytes from catabolic gene expression.

    (A) Mmp3 and Mmp13 expression analyzed by RT-PCR after treatment of control or Itgbl1-overexpressing human chondrocytes with 29-kDa Fn-f. Itgbl1-overexpressing cells were treated with Mn2+ or DTT to activate integrins. (B and C) Quantification of Mmp3 (B) or Mmp13 (C) expression shown in (A). Data are shown as means ± SEM from three replicate experiments. (D) N-terminal 29-kDa Fn-f expression in control, Itgbl1-knockdown, and Itgbl1-overexpressing human chondrocytes. Itgbl1-overexpressing chondrocytes were treated with Mn2+ or DTT to activate integrins. Scale bars, 10 μm. (E) Quantification of fluorescence intensity in (D). n = 14, control; n = 16, Itgbl1-siRNA; n = 17, Itgbl1-OE; n = 16, Itgbl1-OE+Mn2+; n = 14, Itgbl1-OE+DTT. (F) Mmp3 and Mmp13 expression analyzed by RT-PCR in control and ITGBL1-depleted human chondrocytes treated with various integrin inactivators. (G and H) Quantification of (F). Data are shown as means ± SEM from three replicate experiments. Statistical significances in (B), (C), (E), (G), and (H) were determined using one-way ANOVA with Bonferroni post hoc analysis (*P < 0.05, **P < 0.005, ***P < 0.0005).

  • Fig. 7 ITGBL1 depletion causes OA-like cartilage damage in chondrocytes and in a mouse model.

    (A) Itgbl1 expression in chondrocytes infected with Ad-Itgbl1 shRNA. (B) Col2a1 and Sox9 expression in chondrocytes infected with Ad-Itgbl1 shRNA. (C) Sox9 activity measured by luciferase assay in chondrocytes infected with Ad-Itgbl1 shRNA. (D) Itgbl1 expression in chondrocytes infected with Ad-Itgbl1. (E) Col2a1 and Sox9 expression in chondrocytes infected with Ad-Itgbl1. (F) Sox9 activity measured by luciferase assay in chondrocytes infected with Ad-Itgbl1. Data in (A) to (F) are shown as means ± SEM from three replicate experiments. (G) Safranin-O staining and immunohistochemistry of mouse knees intra-articularly injected with Ad-control or Ad-Itgbl1 shRNA with or without ATN-161, an integrin-α5β1 inhibitor (n = 9 per experimental condition). Scale bars, 100 μm. (H) Scoring of cartilage destruction, osteophyte maturity, and subchondral bone sclerosis was quantified in injected knee joints in (G). Statistical significances in (A) to (F) and (H) were determined using an unpaired two-tailed Student’s t test. MOI, multiplicity of infection; PBS, phosphate-buffered saline.

  • Fig. 8 ITGBL1 expression protects cartilage tissues from OA development.

    (A) Col2a1 and Itgbl1expression in mouse chondrocytes treated with IL-1β for 0 to 36 hours. (B and C) Expression of Itgbl1 (B) or Col2a and Sox9 (C) in IL-1β–treated chondrocytes with or without Ad-Itgbl1 infection. (D) Sox9 activity measured by luciferase assay in IL-1β–treated chondrocytes with or without Ad-Itgbl1 infection. (E and F) Mmp3 (E) and Mmp13 (F) expression in IL-1β–treated chondrocytes with or without Ad-Itgbl1 infection. Data in (A) to (F) are shown as means ± SEM from three replicate experiments. (G) Alcian blue staining and ITGBL1 protein expression (upper panel) and mRNA (lower plot) in cartilage samples from human patients with OA and undamaged controls (n = 10). Scale bars, 100 μm. IHC, immunohistochemistry. (H) Safranin-O staining and immunohistochemistry of DMM-induced OA mouse knees injected with Ad-Itgbl1 or control. Scale bars, 100 μm. (I) Scoring of cartilage destruction (upper panel), osteophyte maturity (middle panel), and subchondral bone sclerosis (lower panel) from (H). n = 12, Osteoarthritis Research Society International (OARSI) score; n = 9, osteophyte maturity; n = 12, subchondral bone plate thickness. Statistical significances in (A) to (F), (G), and (I) were determined using an unpaired two-tailed Student’s t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/462/eaam7486/DC1

    Materials and Methods

    Fig. S1. Itgbl1 expression pattern in facial cartilage tissues and homology of ITGBL1 across vertebrates.

    Fig. S2. Neural crest cell migration, hypertrophic chondrocyte development, and osteogenic differentiation after ITGBL1 depletion.

    Fig. S3. Fibronectin deposition after ITGBL1 depletion.

    Fig. S4. Itgbl1 expression during chondrogenesis analyzed after siRNA transfection.

    Fig. S5. ITGBL1 promotes chondrogenesis.

    Fig. S6. Gene expression analysis during chondrogenesis of Itgbl1-overexpressing hBMSCs.

    Fig. S7. ITGBL1 reduces cell-ECM adhesion.

    Fig. S8. ITGBL1-induced integrin inactivation is not recovered by integrin-activating antibody.

    Fig. S9. ITGBL1 inhibits focal adhesion complex formation.

    Fig. S10. Relative expression of integrin subunits in human primary chondrocytes and efficacy of siRNAs used in Fig. 5 (F and G).

    Fig. S11. Itgbl1 expression pattern in embryonic mouse limbs and subcellular localization in ATDC5 cells.

    Fig. S12. Cell viability and efficiency of adenovirus delivery in Ad-Itgbl1–infected chondrocytes and cartilage in mice.

    Fig. S13. ITGBL1 expression protects cartilage tissue against OA development in mice.

    Fig. S14. Scoring of synovial inflammation in the OA mouse model.

    Fig. S15. Human OA cartilage information.

    Table S1. List of the top 100 putative secreted proteins identified by transcriptome analysis.

    Table S2. Individual subject-level data presented in the figures.

    References (74, 75)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Itgbl1 expression pattern in facial cartilage tissues and homology of ITGBL1 across vertebrates.
    • Fig. S2. Neural crest cell migration, hypertrophic chondrocyte development, and osteogenic differentiation after ITGBL1 depletion.
    • Fig. S3. Fibronectin deposition after ITGBL1 depletion.
    • Fig. S4. Itgbl1 expression during chondrogenesis analyzed after siRNA transfection.
    • Fig. S5. ITGBL1 promotes chondrogenesis.
    • Fig. S6. Gene expression analysis during chondrogenesis of Itgbl1-overexpressing hBMSCs.
    • Fig. S7. ITGBL1 reduces cell-ECM adhesion.
    • Fig. S8. ITGBL1-induced integrin inactivation is not recovered by integrin-activating antibody.
    • Fig. S9. ITGBL1 inhibits focal adhesion complex formation.
    • Fig. S10. Relative expression of integrin subunits in human primary chondrocytes and efficacy of siRNAs used in Fig. 5 (F and G).
    • Fig. S11. Itgbl1 expression pattern in embryonic mouse limbs and subcellular localization in ATDC5 cells.
    • Fig. S12. Cell viability and efficiency of adenovirus delivery in Ad-Itgbl1–infected chondrocytes and cartilage in mice.
    • Fig. S13. ITGBL1 expression protects cartilage tissue against OA development in mice.
    • Fig. S14. Scoring of synovial inflammation in the OA mouse model.
    • Fig. S15. Human OA cartilage information.
    • Table S1. List of the top 100 putative secreted proteins identified by transcriptome analysis.
    • References (74, 75)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Individual subject-level data presented in the figures.

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