Research ArticleOsteoarthritis

FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis

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Science Translational Medicine  14 Feb 2018:
Vol. 10, Issue 428, eaan0746
DOI: 10.1126/scitranslmed.aan0746
  • Fig. 1 Body size, growth plate, and articular cartilage in Col2Cre-FoxO KO mice.

    (A) Photograph and quantification of body length and tail length in 1-month-old mice. KO, knockout; TKO, triple KO. (B) Histological sections of proximal tibial growth plate from postnatal day 7 (P7) and 1-month-old mice. Scale bar, 200 μm. Red bar indicates proliferative zone and blue bar indicates hypertrophic zone. (C) Quantification of lengths of proliferative zone and (D) hypertrophic zone. (E) Histological sections of articular cartilage from 1- and 2-month-old mice. Scale bar, 200 μm. (F) Articular cartilage thickness measured as the distance between the articular surface and the subchondral bone interface across three points in each medial tibial plateau of the knee joint in mice at 1 and 2 months. (G) Ratios of height of cartilage above tidemark measured at 2 months. (H) Cell density above the tidemark and (I) cell size in the cartilage of tibial plateau measured at 2 months. Numbers of mice are listed in table S1. Data are means ± SD. *P < 0.05, **P < 0.01 [one-way analysis of variance (ANOVA) and Bonferroni’s post test].

  • Fig. 2 FoxO effect on cell proliferation and gene expression in chondrocytes.

    (A) Immunohistochemistry and quantification of 5-bromo-2′-deoxyuridine (BrdU) in control (n = 5) and Col2Cre-TKO mouse (n = 8) femur and tibia sections. Scale bars, 100 μm. (B) Fluorescence images and quantification of BrdU-positive cells in immature mouse articular chondrocytes (IMACs) from P6 control (n = 4) and Col2Cre-TKO mice (n = 3) in culture (×40 magnification). DAPI, 4′,6-diamidino-2-phenylindole. (C) Quantification of proliferation (MTT assay) in IMACs from P6 control (n = 4) and Col2Cre-TKO mice (n = 5). (D) Flow cytometry cell cycle analysis of IMACs from P6 control and Col2Cre-TKO mice. The percentages of cells in G1, S, and G2-M phase are indicated (n = 5 each). (E) Real-time polymerase chain reaction (PCR) analysis for chondrogenic markers and cell cycle genes using RNA from knee joint cartilage from 1-month-old Col2Cre-TKO mice and control mice (n = 4 each). Expression values are relative to Gapdh. (F) Real-time PCR analysis for chondrogenic markers and cell cycle genes in adenoviral FoxO1-AAA–transduced human chondrocytes (n = 6). Values are relative to Actb, and expression values are normalized to Ad-GFP (green fluorescent protein)–transfected cells. Data are means ± SD. *P < 0.05, **P < 0.01 (Mann-Whitney test).

  • Fig. 3 Spontaneous development of OA-like changes in joint tissues of Col2Cre-FoxO KO mice.

    (A) Summed Osteoarthritis Research Society International (OARSI) scores for the medial femoral condyle and tibial plateau from 2-, 4-, and 6-month-old mice. (B) Histological sections of knee joints from 4- and 6-month-old mice. Scale bar, 200 μm. (C) Synovium and bone scores obtained from the same sections from 6-month-old mice. (D) Histological sections of knee joints and summed OARSI scores for the medial femoral condyle and tibial plateau from 18-month-old control, Col2Cre-FoxO3 KO, and Col2Cre-FoxO4 KO mice. Numbers of mice are listed in table S1. Data are means ± SD. *P < 0.05, **P < 0.01 (one-way ANOVA and Bonferroni’s post test).

  • Fig. 4 Cell viability and cellular homeostasis gene expression.

    (A) TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) staining (scale bars, 100 μm) and quantitative analysis of the TUNEL-positive cell counts per field in the central weight-bearing region of the medial tibial plateau in 2-month-old mice. Cell viability under 500 μM H2O2 was measured by resazurin assay (n = 4 each). Results are shown as % viability compared to cells not treated with H2O2. (C) Real-time PCR analysis using RNA from knee joint cartilage from 2-month-old Col2Cre-TKO, Col2Cre-FoxO1 KO, and control mice (control, n = 4; Col2Cre-TKO, n = 4; Col2Cre-FoxO1 KO, n = 4; Col2Cre-FoxO3 KO, n = 3; Col2Cre-FoxO4 KO, n = 3). Expression values were normalized to Gapdh as control. (D) Real-time PCR analysis from adenoviral FoxO1-AAA–transduced healthy human chondrocytes (n = 4) and C57BL6/J wild-type mouse IMACs (n = 6). The values are relative to Actb, and expression values are normalized to Ad-GFP–transfected cells. Data are means ± SD. *P < 0.05, **P < 0.01 (Mann-Whitney test for two groups and one-way ANOVA and Bonferroni’s post test for multiple groups).

  • Fig. 5 Cartilage superficial zone changes in Col2Cre-FoxO KO mice.

    (A) Images and quantification of superficial zone cells in the cartilage on the tibial plateau per superficial zone area (10 μm from the surface) measured in 1-month-old mice. Scale bar, 50 μm. Yellow arrows indicate superficial zone cells in healthy cartilage. Numbers of mice are listed in table S1. (B) Images of Picrosirius red–stained knee joint sections from 1-month-old mice under standard light (left) or polarized light (right). Intensity of stained area in superficial zone of cartilage on tibial plateau (10 μm from the surface) was measured by ImageJ software (n = 4 each). Scale bar, 100 μm. (C) Immunohistochemistry and quantification for superficial zone cell markers superficial zone protein (SZP) and vascular cell adhesion molecule 1 (VCAM1) in the cartilage on the tibial plateau from 1-month-old mice (n = 4 each). Scale bar, 100 μm. (D) Real-time PCR analysis for Prg4 using RNA isolated from cartilage from P1, 1-month-old, and 2-month-old mice (P1: n = 7 each; 1-month-old: control, n = 4; Col2Cre-TKO, n = 4; Col2Cre-FoxO1 KO, n = 3; Col2Cre-FoxO3 KO, n = 3; and Col2Cre-FoxO4 KO, n = 3; 2-month-old: control, n = 4; Col2Cre-TKO, n = 4; Col2Cre-FoxO1 KO, n = 4; Col2Cre-FoxO3 KO, n = 3; and Col2Cre-FoxO4 KO, n = 3). The expression values are relative to Gapdh, and each value is compared to control. Data are means ± SD. *P < 0.05, **P < 0.01 (Mann-Whitney test for two groups and one-way ANOVA and Bonferroni’s post test for multiple groups).

  • Fig. 6 Phenotype of postnatal deletion of FoxO in cartilage using Aggrecan-CreERT knock-in mice.

    (A) Real-time PCR analysis using RNA from knee joint cartilage in control and Aggrecan-CreERT2 TKO (AcanCreERT-TKO) mice 2 weeks after tamoxifen (TMX) injection (n = 4 each). The expression values are relative to Gapdh. (B) Images and quantification of the thickness of articular cartilage measured as the distance between the articular surface and the subchondral bone interface (scale bar, 100 μm) across three points in each medial tibial plateau of the knee joint in mice at 2 months after tamoxifen injection (control, n = 10; AcanCreERT-TKO, n = 8). (C) Quantification of the numbers of superficial zone cells of cartilage on the tibial plateau per superficial zone area (10 μm from the surface) measured in mice 2 months after tamoxifen injection (control, n = 10; AcanCreERT-TKO, n = 8). (D) Immunohistochemistry for SZP and quantification of percent SZP-positive cells in the cartilage on the tibial plateau of mice 2 months after tamoxifen injection (n = 4 each). Scale bar, 100 μm. (E) Histological sections and OARSI scores of knee joints from mice 2 and (F) 5 months after tamoxifen injection, (G) 4 weeks after surgical osteoarthritis (OA) model (scale bar, 200 μm), and (H) 6 weeks after treadmill-induced OA model (coronal section; scale bar, 200 μm). DMM, destabilization of the medial meniscus. OARSI scores were obtained by summing the scores for the medial femoral condyle and tibial plateau. Numbers of mice are listed in table S1. Data are means ± SD. *P < 0.05, **P < 0.01 (Mann-Whitney test).

  • Fig. 7 FoxO and Prg4 expression in mouse articular chondrocytes and chondrogenic cells.

    (A) Real-time PCR analysis for Prg4 in adenoviral FoxO1-AAA– or Ad-GFP–transduced IMACs (n = 6) and plasmid FoxO1-AAA– or plasmid GFP control–transfected ATDC5 cells (n = 3). The values are relative to Actb, and expression values are normalized to Ad-GFP– or plasmid GFP–transduced cells. (B) Real-time PCR for Prg4 expression in cultured adipose tissue–derived mesenchymal stem cells from FoxO triple floxed mice with or without adenovirus-Cre infection (n = 6 each). Day 0 was defined as the start date 72 hours after transfection. Values are shown relative to those from day 0 cells not infected with adenovirus. (C) Real-time PCR analysis of Prg4 in IMACs from control and Col2Cre-TKO mice treated with TGFβ1 (transforming growth factor–β; 10 ng/ml) or vehicle for 6 hours (control, n = 5; Col2Cre-TKO, n = 4). The expression values are relative to Gapdh. (D) Western blotting for secreted PRG4 protein. IMACs from control and Col2Cre-TKO mice (n = 3 each) were treated with TGFβ1 (10 ng/ml) or vehicle for 24 hours. Secreted PRG4 protein in the culture supernatants was measured by Western blotting. Equal amounts of concentrated supernatants from identical cell numbers were loaded on the gels. Band intensity was quantified by ImageJ. (E) Real-time PCR analysis of Prg4 expression in ATDC5 cells transfected with GFP or FoxO1-AAA and treated with TGFβ1 or vehicle for 24 hours. The expression values are relative to Gapdh. (F) Real-time PCR analysis of Prg4 in ATDC5 cells transfected with GFP, FoxO1H2015RAAA, and treated with TGFβ1 (10 ng/ml) or vehicle for 24 hours. The expression values are relative to Gapdh. All experiments were performed in duplicate. Data are means ± SD. P values were calculated with Mann-Whitney test (A to D) and Student’s t test (E and F). *P < 0.05, **P < 0.01.

  • Fig. 8 Overexpression of FoxO1 in human OA chondrocytes and effects on IL-1β stimulation.

    (A) Real-time PCR analysis for inflammatory and cartilage catabolic genes and (B) FoxO expression and autophagic genes using RNA from healthy human (n = 4) and OA chondrocytes (n = 4) with basal medium. (C) Real-time PCR analysis of autophagic genes in OA chondrocytes (n = 3) transfected with Ad-GFP or FoxO1-AAA. (D) Real-time PCR analysis for inflammatory and cartilage catabolic genes in Ad-GFP control or FoxO1-AAA–transduced human OA chondrocytes (n = 3) with or without interleukin-1β (IL-1β). The expression values in all experiments are relative to B2M. Data are means ± SD. P values were calculated with Mann-Whitney test (A and B) and Student’s t test (C and D). *P < 0.05, **P < 0.01.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/428/eaan0746/DC1

    Fig. S1. Skeletal and growth plate changes in Col2Cre-FoxO KO mice.

    Fig. S2. Real-time PCR of cartilage from 1- and 2-month-old Col2Cre KO mice.

    Fig. S3. Histological features in the growth plate in 4- and 6-month-old Col2Cre KO mice.

    Fig. S4. Real-time PCR of cartilage from AcanCreERT-TKO mice.

    Fig. S5. Synovial and bone histological scores in AcanCreERT-TKO mice.

    Table S1. Numbers of mice used for each experiment.

    Table S2. TaqMan probes used for real-time PCR analysis.

    Table S3. Subjective-level data.

  • Supplementary Material for:

    FoxO transcription factors modulate autophagy and proteoglycan 4 in cartilage homeostasis and osteoarthritis

    Tokio Matsuzaki, Oscar Alvarez-Garcia, Sho Mokuda, Keita Nagira, Merissa Olmer, Ramya Gamini, Kohei Miyata, Yukio Akasaki, Andrew I. Su, Hiroshi Asahara, Martin K. Lotz*

    *Corresponding author. Email: mlotz{at}scripps.edu

    Published 14 February 2018, Sci. Transl. Med. 10, eaan0746 (2018)
    DOI: 10.1126/scitranslmed.aan0746

    This PDF file includes:

    • Fig. S1. Skeletal and growth plate changes in Col2Cre-FoxO KO mice.
    • Fig. S2. Real-time PCR of cartilage from 1- and 2-month-old Col2Cre KO mice.
    • Fig. S3. Histological features in the growth plate in 4- and 6-month-old Col2Cre KO mice.
    • Fig. S4. Real-time PCR of cartilage from AcanCreERT-TKO mice.
    • Fig. S5. Synovial and bone histological scores in AcanCreERT-TKO mice.
    • Table S1. Numbers of mice used for each experiment.
    • Table S2. TaqMan probes used for real-time PCR analysis.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3 (Microsoft Excel format). Subjective-level data.

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