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. 2013 Jan 29;110(5):1875-80.
doi: 10.1073/pnas.1207458110. Epub 2013 Jan 14.

Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development

Yoko Hosaka  1 , Taku Saito, Shurei Sugita, Tomohiro Hikata, Hiroshi Kobayashi, Atsushi Fukai, Yuki Taniguchi, Makoto Hirata, Haruhiko Akiyama, Ung-il Chung, Hiroshi Kawaguchi
Affiliations

Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development

Yoko Hosaka et al. Proc Natl Acad Sci U S A. .

Abstract

Here we examined the involvement of Notch signaling in the endochondral ossification process, which is crucial for osteoarthritis (OA) development. Intracellular domains of Notch1 and -2 were translocated into the nucleus of chondrocytes with their differentiation in mouse limb cartilage and in mouse and human OA articular cartilage. A tissue-specific inactivation of the Notch transcriptional effector recombination signal binding protein for Ig kappa J (RBPjκ) in chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre;Rbpj(fl/fl) mouse embryos caused an impaired terminal stage of endochondral ossification in the limb cartilage. The RBPjκ inactivation in adult articular cartilage after normal skeletal growth using type II collagen (Col2a1)-Cre(ERT);Rbpj(fl/fl) mice by tamoxifen injection caused resistance to OA development in the knee joint. Notch intracellular domain with the effector RBPjκ stimulated endochondral ossification through induction of the target gene Hes1 in chondrocytes. Among the Notch ligands, Jagged1 was strongly induced during OA development. Finally, intraarticular injection of N-[N-(3,5-diflurophenylacetate)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), a small compound Notch inhibitor, to the mouse knee joint prevented OA development. The RBPjκ-dependent Notch signaling in chondrocytes modulates the terminal stage of endochondral ossification and OA development, representing an extracellular therapeutic target of OA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
In vitro and in vivo expression patterns of the Notch signaling molecules during chondrocyte differentiation and OA development. (A) Time course of mRNA levels of Notch signaling molecules, and Col2a1, Col10a1, Mmp13, and Vegfa during differentiation of mouse chondrogenic ATDC5 cells cultured with insulin, transferrin, and sodium selenite (ITS) for 3 wk and for 2 d more with inorganic phosphate (Pi). Data are expressed as means ± SD. (B) mRNA levels of the Notch signaling molecules above in mouse primary costal chondrocytes and articular chondrocytes cultured for 5 and 7 d, respectively. (C) Immunostaining with antibodies to ICDs of Notch1 and Notch2 (NICD1 and NICD2), and RBPjκ in the mouse proximal tibia (E18.5) (Left; red, blue, and green bars to the Top indicate layers of proliferative, hypertrophic zones, and bone area, respectively) and in the mouse knee articular cartilage with or without surgical OA induction for 8 wk (16 wk old) (Right). Insets indicate the regions shown in the enlarged images immediately below. (Scale bars, 100 μm and 10 μm for low and high magnification images, respectively.) (D) Safranin-O staining and immunofluorescence with antibodies to NICD1, NICD2, and RBPjκ in the normal and OA cartilage of human knee joints. Insets in the safranin O staining indicate the regions shown in the enlarged immunofluorescence images. (Scale bars, 200 μm and 50 μm for low and high magnification images, respectively.)
Fig. 2.
Fig. 2.
Skeletal abnormality in Sox9-Cre;Rbpjfl/fl mouse embryos. (A) Double staining with Alizarin red and Alcian blue of the whole skeleton (Top), upper extremities (Middle), and lower extremities (Bottom) of Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos (E17.5). (Scale bars, 1 mm.) (B) Length of long bones and vertebra (first to fifth lumbar spines) of Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos. Data are expressed as means ± SD of six mice per group. *P < 0.05 versus Rbpjfl/fl. (C) H&E staining of whole femurs of the Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos. (Scale bars, 200 μm.) Lower graph indicates percentage of the length of proliferative zone (red), hypertrophic zone (blue), and bone area (green) over the total femoral length of the Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos. (D) H&E staining and immunofluorescence with antibodies to Pcna, Col10a1, Mmp13, Vegfa, Hes1, and RBPjκ of distal femurs of the Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos. Red, blue, and green bars (Upper) indicate layers of proliferative, hypertrophic zones, and bone area, respectively. (Scale bars, 200 μm.) (E) mRNA levels of Pcna, Col10a1, Mmp13, Vegfa, Hes1, and Rbpj in the pellet cultures of primary costal chondrocytes derived from Rbpjfl/fl and Sox9-Cre;Rbpjfl/fl littermate embryos. Data are expressed as means ± SD *P < 0.01 versus Rbpjfl/fl.
Fig. 3.
Fig. 3.
OA development in Col2a1-CreERT;Rbpjfl/fl mice. (A) Beta-galactosidase (LacZ) staining of knee joints of Col2a1-CreERT;R26Rf/+ mice and the Cre negative control littermates (R26Rf/+) 8 wk after tamoxifen induction (16 wk old). (Scale bars, 100 μm.) Haematoxylin staining was performed as a counterstain. (B) Plain radiographs of the entire bodies of Rbpjfl/fl and Col2a1-CreERT;Rbpjfl/fl littermates (8 wk old) after tamoxifen injection for 5 d. (Scale bars, 10 mm.) (C) Safranin O staining of mouse knee joints in 8-wk-old Rbpjfl/fl and Col2a1-Cre;Rbpjfl/fl littermates above under physiological conditions. Insets in the Upper safranin O-stained image indicate the regions shown in the enlarged images (Lower). (Scale bars, 400 μm and 100 μm for low and high magnification images, respectively.) (D) mRNA levels of Rbpj in articular chondrocytes from Rbpjfl/fl and Col2a1-CreERT;Rbpjfl/fl littermates above (8 wk old). Data are expressed as means ± SD *P < 0.01 versus Rbpjfl/fl. (E) Cartilage degradation assessed by safranin O staining and immunofluorescence with antibodies to Col10a1, Mmp13, Vegfa, Hes1, and RBPjκ in mouse knee joints 8 wk after creating a surgical OA model in 8-wk-old Rbpjfl/fl and Col2a1-CreERT;Rbpjfl/fl littermates above. Insets in the Upper safranin O-stained images indicate the regions shown in the enlarged safranin O-stained or immunofluorescence images (Lower). (Scale bars, 400 μm and 100 μm for low and high magnification images, respectively.) (F) Quantification of OA development by Osteoarthritis Research Society International (OARSI) grading systems. Data are expressed as means ± SD of nine mice per group. *P < 0.05 versus Rbpjfl/fl.
Fig. 4.
Fig. 4.
Regulation of Col10a1, Mmp13, Vegfa, and Hes/Hey family members in cultures of chondrocytes. (A) mRNA levels of the factors above, and alkaline phosphatase (ALP) and Alizarin red stainings in stable lines of ATDC5 cells retrovirally transfected with GFP, RBPjκ, and Notch1–ICD (NICD1) after culture for 3 wk with ITS and 2 d more with Pi. *P < 0.01 versus GFP. (B) mRNA levels of the factors above, Pcna, and ALP staining in primary articular chondrocytes adenovirally transfected with GFP and NICD1 after culture for 5 d with differentiation medium. *P < 0.01 versus GFP. (C) Luciferase activities by the transfections of GFP, RBPjκ, NICD1, and HES1 into HeLa, ATDC5, and human chondrogenic SW1353 cells with a reporter construct containing a fragment of the MMP13 gene (Upper) or the VEGFA gene (Lower) (−1000 bp to transcriptional start site). *P < 0.01 versus GFP. (D) mRNA levels of Mmp13, Vegfa, and Hes1 in ATDC5 cells that were transfected with GFP and NICD1, and further cotransfected with siRNA for GFP (si-GFP) or Hes1 (si-Hes1). Data are expressed as means ± SD #P < 0.05, *P < 0.01.
Fig. 5.
Fig. 5.
Effects of a small compound DAPT, a Notch inhibitor, on endochondral ossification and OA development. (A) mRNA levels of Col10a1, Mmp13, Vegfa, Hes1, and ALP and Alizarin red stainings in mouse articular chondrocytes cultured for 10 d with and without DAPT (1, 10, 100 nM in DMSO) or DMSO alone (vehicle). Data are expressed as means ± SD *P < 0.05 versus vehicle. (B) Cartilage degradation assessed by safranin O staining and immunofluorescence with antibodies to Col10a1, Mmp13, Vegfa, and Hes1 in the mouse surgical knee OA joints with intraarticular injection (two times per week) of 10 μL of 2.5 μM DAPT solution (25 mM DAPT/DMSO in PBS, 1:10,000) or DMSO alone in PBS (1:10,000; vehicle) for 10 wk. Insets in the Upper safranin O-stained image indicate the regions shown in the enlarged safranin O-stained or immunofluorescence images (Lower). (Scale bars, 400 μm and 100 μm for low and high magnification images, respectively.) (C) Quantification of OA development by OARSI grading systems. Data are expressed as means ± SD of seven mice per group. *P < 0.05 versus vehicle.
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