Cyclin‑dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice

Chinzei,Nobuaki; Hayashi,Shinya; Hashimoto,Shingo; Kanzaki,Noriyuki; Iwasa,Kenjiro; Sakata,Shuhei; Kihara,Shinsuke; Fujishiro,Takaaki; Kuroda,Ryosuke; Kurosaka,Masahiro

doi:10.3892/mmr.2014.2889

Cyclin‑dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice

Authors:
- Nobuaki Chinzei
- Shinya Hayashi
- Shingo Hashimoto
- Noriyuki Kanzaki
- Kenjiro Iwasa
- Shuhei Sakata
- Shinsuke Kihara
- Takaaki Fujishiro
- Ryosuke Kuroda
- Masahiro Kurosaka
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Affiliations: Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo 650‑0017, Japan
Published online on: November 6, 2014 https://doi.org/10.3892/mmr.2014.2889
Pages: 1601-1608
Copyright: © Chinzei et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].

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Abstract

Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin‑dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild‑type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5‑bromo‑2'‑deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion.

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