Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development Yoko Hosakaa,1, Taku Saitoa,b,1, Shurei Sugitaa, Tomohiro Hikatac, Hiroshi Kobayashia, Atsushi Fukaia, Yuki Taniguchia, Makoto Hirataa,d, Haruhiko Akiyamae, Ung-il Chungd, and Hiroshi Kawaguchia,2 aSensory and Motor System Medicine, bBone and Cartilage Regenerative Medicine, and dCenter for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan; cDepartment of Orthopaedic Surgery, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-8582, Japan; and eDepartment of Orthopaedic Surgery, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Edited by Marc Feldmann, Imperial College London, London, United Kingdom, and approved December 19, 2012 (received for review May 6, 2012) Here we examined the involvement of Notch signaling in the endo- hairy and enhancer of split (Hes) / hairy/enhancer-of-split related chondral ossification process, which is crucial for osteoarthritis (OA) with YRPW motif (Hey) family of basic helix–loop–helix tran- development. Intracellular domains of Notch1 and -2 were trans- scription factors: Hes1, Hes5, Hes7, Hey1, Hey2, and HeyL (10, located into the nucleus of chondrocytes with their differentiation 11). Accumulating evidence has shown that these Notch family in mouse limb cartilage and in mouse and human OA articular car- members are highly expressed in chondrocytes of developmental tilage. A tissue-specific inactivation of the Notch transcriptional ef- skeletal cartilage (12–14). Furthermore, a recent report disclosed κ fector recombination signal binding protein for Ig kappa J (RBPjκ)in that chondrocyte differentiation is regulated via the RBPj -de- chondroprogenitor cells of SRY-box containing gene 9 (Sox9)-Cre; pendent Notch signaling in chondrocytes (15). The Notch family fl/fl Rbpj mouse embryos caused an impaired terminal stage of endo- members are also expressed in adult articular cartilage (16, 17), fi κ and over 70% of chondrocytes on the surface zone of articular chondral ossi cation in the limb cartilage. The RBPj inactivation in fi adult articular cartilage after normal skeletal growth using type II cartilage express Notch1 receptor (18), suggesting a signi cant collagen (Col2a1)-CreERT;Rbpjfl/fl mice by tamoxifen injection caused role of the Notch signaling in regulating articular cartilage ho- resistance to OA development in the knee joint. Notch intracellular meostasis during adult life (16, 19). A recent study demonstrated that the Notch pathway is activated in OA cartilage because domain with the effector RBPjκ stimulated endochondral ossifica- several Notch signaling molecules are much more abundant than tion through induction of the target gene Hes1 in chondrocytes. in healthy cartilage (14, 16). Here we examined the involvement Among the Notch ligands, Jagged1 was strongly induced during κ N N fl of the RBPj -dependent Notch signaling in the endochondral OA development. Finally, intraarticular injection of -[ -(3,5-di ur- ossification process during skeletal growth and OA development. ophenylacetate)-L-alanyl]-(S)-phenylglycine t-butyl ester (DAPT), We further investigated the underlying mechanism and the pos- a small compound Notch inhibitor, to the mouse knee joint pre- sibility of clinical application of the signaling as a therapeutic κ vented OA development. The RBPj -dependent Notch signaling in target of OA. chondrocytes modulates the terminal stage of endochondral ossifi- cation and OA development, representing an extracellular thera- Results peutic target of OA. Notch Signaling Molecules Are Expressed and Activated During Chondrocyte Differentiation. We initially looked at the in vitro skeletal development | cartilage degradation and in vivo expression patterns of the Notch family members during chondrocyte differentiation. In cultures of mouse chon- ndochondral ossification is an essential process not only for drogenic cell line ATDC5 and primary chondrocytes, Notch1,-2, physiological skeletal growth (1) but also for development of Rbpj,andHes1 were strongly expressed during the differentiation, E MEDICAL SCIENCES osteoarthritis (OA), which is the most prevalent form of arthritis whereas Notch3 and -4 and other Hes/Hey family members were with articular cartilage degradation (2, 3). In the process, after little expressed (Fig. 1 A and B). In the mouse limb cartilage and chondrocytes undergo hypertrophic differentiation characterized knee articular cartilage, ICDs of Notch1 and Notch2 remained in by secretion of type X collagen (COL10A1), the avascular car- the plasma membrane of less differentiated chondrocytes of the tilage tissue is converted into highly vascularized bone tissue via proliferative zone and undegraded normal cartilage; however, degradation of the cartilage matrix and vascular invasion (1, 4). both were translocated into the nucleus of highly differentiated The matrix degradation requires proteinases, among which ma- chondrocytes in the hypertrophic zone and in the degraded OA trix metalloproteinase-13 (MMP13) plays a major role (4, 5), and cartilage (Fig. 1C). The translocation of the ICDs into the nucleus the vascular invasion depends on an angiogenic switch by vas- of chondrocytes during OA development was reproducible in the cular endothelial growth factor A (VEGFA) (6). human knee OA cartilage (Fig. 1D). Contrarily, RBPjκ remained Notch is a single-pass transmembrane cell surface receptor, in the nucleus without being affected by the differentiation stages which plays a crucial role in cell fate assignment by regulating or OA induction in both mouse and human samples (Fig. 1 C differentiation and apoptosis during embryogenesis as well as and D). various developmental systems like neurogenesis and hemato- poiesis (7, 8). In mammals, the signaling is initiated by binding of a membrane ligand (Delta-like 1, 3, 4, or Jagged1 and -2) to the Author contributions: Y.H., T.S., T.H., and H. Kawaguchi designed research; Y.H., T.S., S.S., Notch receptor (Notch1–4) on the adjacent cell (9). Upon bind- T.H., H. Kobayashi, and A.F. performed research; Y.H., T.S., T.H., H. Kobayashi, and H.A. ing, the Notch receptor is cleaved by proteinases like a disintegrin contributed new reagents/analytic tools; Y.H., T.S., Y.T., M.H., U.-i.C., and H. Kawaguchi and metalloproteinase domain-containing proteins, and sub- analyzed data; and Y.H., T.S., and H. Kawaguchi wrote the paper. sequently by the γ-secretase complex, and the Notch intracellular The authors declare no conflict of interest. domain (ICD) is released in the cytoplasm. Then, the Notch ICD This article is a PNAS Direct Submission. translocates to the nucleus and binds to the transcriptional ef- 1Y.H. and T.S. contributed equally to this work. fector recombination signal binding protein for Ig kappa J 2To whom correspondence should be addressed. E-mail: [email protected]. κ (RBPj ), converting it into an activator and ultimately inducing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the expression of downstream target genes like members of the 1073/pnas.1207458110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1207458110 PNAS | January 29, 2013 | vol. 110 | no. 5 | 1875–1880 Downloaded by guest on October 2, 2021 two genotypes, suggesting unaffected proliferation and hyper- trophic differentiation of chondrocytes by the RBPjκ knockout (Fig. 2C). However, the percentage of the hypertrophic zone relative to the limb length was much increased, whereas that of fl fl the bone area was considerably decreased in the Sox9-Cre;Rbpj / limbs, indicating that the RBPjκ knockout impaired the terminal differentiation stage in such aspects as matrix degradation and vascular invasion (Fig. 2C). Immunofluorescence showed that intensities of the proliferating cell nuclear antigen (Pcna) and Col10a1 were comparable, whereas those of Mmp13 and Vegfa, as well as Hes1, were decreased by the RBPjκ knockout (Fig. 2D). These were confirmed by the expressions of these factors in the pellet cultures of primary chondrocytes derived from the two genotypes (Fig. 2E). RBPjκ-Dependent Notch Signaling Modulates OA Development. We next examined the contribution of RBPjκ in cartilage to OA development in which pathological endochondral ossification in the adult articular cartilage is known to play a crucial role (2). fl fl Because the Sox9-Cre;Rbpj / mice died shortly after birth, we sought to create RBPjκ knockout mice that would undergo normal skeletal growth and joint formation under physiological conditions. To inactivate RBPjκ in later stages of chondrocyte fl fl differentiation than Sox9-Cre;Rbpj / mice, we generated condi- tional knockout mice by mating type II collagen (Col2a1)pro- moter-driven Cre-transgenic mice (Col2a1-Cre)(22)withthe fl fl fl fl Rbpj / mice. Among three lines of the Col2a1-Cre;Rbpj / mice obtained, two were perinatal lethal as previously reported (23); however, one line survived even after birth and grew normally without skeletal abnormality (Fig. S1A). This may be because the RBPjκ inactivation in cartilage was not complete but partial in this line (Fig. S1B). Our further analysis of the knee joints showed that joint morphology as well as phenotypes of articular cartilage, fl fl meniscus, and subchondral bone were comparable to the Rbpj / Fig. 1. In vitro and in vivo expression patterns of the Notch signaling littermates under physiological conditions (Fig. S1C). However, molecules during chondrocyte differentiation