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TAp63? influences mouse cartilage development.

ABSTRACT: Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63?. To investigate the role of TAp63? in chondrocyte differentiation and maturation, we developed stable TAp63? expressing ATDC5 cells. Compared to control cells, TAp63? cells showed significant upregulation of Col10a1 after 4 and 7 days in culture. Moreover, alkaline phosphatase, Alizarin red, and Alcian blue staining were stronger in TAp63? cells, suggesting that TAp63? promotes chondrocyte proliferation, hypertrophic differentiation, and possibly matrix mineralization. To investigate the in vivo function of TAp63? during endochondral bone formation, we established transgenic mice that express flag-tagged TAp63? driven by Col10a1 regulatory elements. Skeletal staining of transgenic mice at postnatal day 1 showed accelerated ossification in long bone, tail, and digit bones compared to wild-type littermates. Furthermore, Sox9 expression was reduced and Runx2 expression was increased in the proliferative and/or hypertrophic zones of these mice. Altogether, these results suggest that TAp63? promotes endochondral ossification and skeletal development, at least partially via controlling chondrocyte differentiation and maturation.

SUBMITTER: Wang Q

PROVIDER: S-EPMC7244026 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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TAp63γ influences mouse cartilage development.

Wang Qian Q Li Na N Chen Fangzhou F Hei Ruoxuan R Gu Junxia J Lu Yaojuan Y Sun Lichun L Zheng Qiping Q

Aging 20200511 9

Depletion of tumor protein p63 results in severe epithelial as well as limb defects in mice, suggesting that p63 is also required for endochondral ossification during long bone development. A key stage in endochondral ossification is chondrocyte hypertrophy, which has been associated with elevated levels of the p63 variant TAp63γ. To investigate the role of TAp63γ in chondrocyte differentiation and maturation, we developed stable TAp63γ expressing ATDC5 cells. Compared to control cells, <i>TAp63 ...[more]

PMID: 32392534

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Project description:Skeletal growth by endochondral ossification involves tightly coordinated chondrocyte differentiation that creates reserve, proliferating, prehypertrophic, and hypertrophic cartilage zones in the growth plate. Many human skeletal disorders result from mutations in cartilage extracellular matrix (ECM) components that compromise both ECM architecture and chondrocyte function. Understanding normal cartilage development, composition, and structure is therefore vital to unravel these disease mechanisms. To study this intricate process in vivo by proteomics, we analyzed mouse femoral head cartilage at developmental stages enriched in either immature chondrocytes or maturing/hypertrophic chondrocytes (post-natal days 3 and 21, respectively). Using LTQ-Orbitrap tandem mass spectrometry, we identified 703 cartilage proteins. Differentially abundant proteins (q < 0.01) included prototypic markers for both early and late chondrocyte differentiation (epiphycan and collagen X, respectively) and novel ECM and cell adhesion proteins with no previously described roles in cartilage development (tenascin X, vitrin, Urb, emilin-1, and the sushi repeat-containing proteins SRPX and SRPX2). Meta-analysis of cartilage development in vivo and an in vitro chondrocyte culture model (Wilson, R., Diseberg, A. F., Gordon, L., Zivkovic, S., Tatarczuch, L., Mackie, E. J., Gorman, J. J., and Bateman, J. F. (2010) Comprehensive profiling of cartilage extracellular matrix formation and maturation using sequential extraction and label-free quantitative proteomics. Mol. Cell. Proteomics 9, 1296-1313) identified components involved in both systems, such as Urb, and components with specific roles in vivo, including vitrin and CILP-2 (cartilage intermediate layer protein-2). Immunolocalization of Urb, vitrin, and CILP-2 indicated specific roles at different maturation stages. In addition to ECM-related changes, we provide the first biochemical evidence of changing endoplasmic reticulum function during cartilage development. Although the multifunctional chaperone BiP was not differentially expressed, enzymes and chaperones required specifically for collagen biosynthesis, such as the prolyl 3-hydroxylase 1, cartilage-associated protein, and peptidyl prolyl cis-trans isomerase B complex, were down-regulated during maturation. Conversely, the lumenal proteins calumenin, reticulocalbin-1, and reticulocalbin-2 were significantly increased, signifying a shift toward calcium binding functions. This first proteomic analysis of cartilage development in vivo reveals the breadth of protein expression changes during chondrocyte maturation and ECM remodeling in the mouse femoral head.

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TAp63? influences mouse cartilage development.

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TAp63γ influences mouse cartilage development.

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