Signaling pathway components such as Ihh/Pthrp, TGF?, BMPs, Wnt/?-catenin, FGFs, and Sox-related proteins represent important regulators of cartilage formation and development. These same signaling pathways and related molecules are being targeted for clinical treatment of cartilage injuries and diseases (osteoarthritis, rheumatoid arthritis, and fracture repair) and cartilage tissue engineering applications (maintenance and manipulation of mesenchymal progenitor cells (MPCs)). We provide original, unpublished data indicating that the Notch signaling pathway is another important regulator of MPC differentiation and chondrocyte maturation, leading to questions regarding the mechanisms by which Notch controls these processes. To begin addressing these questions, we propose to test the novel hypothesis that RBPj?-dependent Notch signaling suppresses MPC differentiation and chondrogenesis by interacting with stabilized ?-catenin to regulate Hes1 and ultimately Sox9, and is later required to promote chondrocyte maturation via cartilage specific regulation of Hes1 and Runx2 activities. To test these hypotheses, we will address two Specific Aims. Experiments in Specific Aim 1 will investigate whether Hes1 is required for MPC differentiation and chondrogenesis or the Notch-mediated suppression of MPC differentiation using limb mesenchyme specific conditional loss-of-function and genetic rescue mouse models. Secondly, we will determine whether the Wnt/beta-catenin signaling pathway is necessary and sufficient for Notch mediated induction of Hes1 and suppression of MPC differentiation. Finally, we will determine whether Hes1 suppresses MPC differentiation by directly regulating Sox9 expression.
Specific Aim 2 will first examine the potential roles for both RBPJ?- dependent and -independent Notch signaling in promoting chondrocyte maturation using various tissue specific Notch gain- and loss-of-function mouse models. Additionally, we will perform in vitro experiments using primary chondrocyte cultures to identify RBPJ?-dependent Notch targets that regulate chondrocyte maturation and examine whether Notch mediates Wnt/beta-catenin or BMP signaling during this process. Secondly, we will use two different conditional Hes1 mutant mouse models to determine whether Hes1 is the primary RBPJk-dependent Notch regulator of chondrocyte maturation. Finally, in vitro studies will be performed to determine whether Hes1 promotes Runx2 activity and chondrocyte maturation via competitive interactions with the mutual co-repressor, Groucho related gene (Grg1). Completion of these aims will identify the Notch signaling mechanisms important in regulating MPC maintenance and expansion, as well as, chondrocyte maturation. These molecules will likely serve as therapeutic targets for cartilage injuries or diseases and provide us with potential tools for use in cartilage tissue engineering applications.

Public Health Relevance

We have identified the RBPJ?-dependent Notch pathway as an important regulator of MPC differentiation and chondrocyte maturation. Our proposal will determine the exact Notch signaling mechanisms responsible for suppressing chondrogenic commitment from mesenchymal progenitor cells (MPCs) and for promoting chondrocyte maturation. Data generated by this proposal will likely implicate specific Notch signaling molecules as potential therapeutic targets for cartilage related injuries and diseases, as well as, provide potential tools in MPC maintenance and expansion for use in tissue engineering applications.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Biology Development and Disease Study Section (SBDD)
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Tyree, Bernadette
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University of Rochester
Schools of Dentistry
United States
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Rutkowski, Timothy P; Kohn, Anat; Sharma, Deepika et al. (2016) HES factors regulate specific aspects of chondrogenesis and chondrocyte hypertrophy during cartilage development. J Cell Sci 129:2145-55
Liu, Z; Ren, Y; Mirando, A J et al. (2016) Notch signaling in postnatal joint chondrocytes, but not subchondral osteoblasts, is required for articular cartilage and joint maintenance. Osteoarthritis Cartilage 24:740-51
Shang, Xifu; Wang, Jinwu; Luo, Zhengliang et al. (2016) Notch signaling indirectly promotes chondrocyte hypertrophy via regulation of BMP signaling and cell cycle arrest. Sci Rep 6:25594
Wang, Cuicui; Inzana, Jason A; Mirando, Anthony J et al. (2016) NOTCH signaling in skeletal progenitors is critical for fracture repair. J Clin Invest 126:1471-81
Zhang, Yongchun; Sheu, Tzong-jen; Hoak, Donna et al. (2016) CCN1 Regulates Chondrocyte Maturation and Cartilage Development. J Bone Miner Res 31:549-59
Zhang, Hengwei; Sun, Wen; Li, Xing et al. (2016) Use of Hes1-GFP reporter mice to assess activity of the Hes1 promoter in bone cells under chronic inflammation. Bone 90:80-9
Qiu, Tao; Xian, Lingling; Crane, Janet et al. (2015) PTH receptor signaling in osteoblasts regulates endochondral vascularization in maintenance of postnatal growth plate. J Bone Miner Res 30:309-17
Wang, Cuicui; Shen, Jie; Yukata, Kiminori et al. (2015) Transient gamma-secretase inhibition accelerates and enhances fracture repair likely via Notch signaling modulation. Bone 73:77-89
Liu, Zhaoyang; Chen, Jianquan; Mirando, Anthony J et al. (2015) A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis. Sci Signal 8:ra71
Rutkowsky, Timothy; Sharma, Deepika; Hilton, Matthew J (2014) Whole-mount in situ hybridization on murine skeletogenic tissues. Methods Mol Biol 1130:193-201

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