Cyclin-dependent Kinase Inhibitor, P21 Is Not Essential ForEndochondral OssificationNobuaki Chinzei, Shinya Hayashi, Shingo Hashimoto, Noriyuki Kanzaki, Kenjiro Iwasa, Shuhei Sakata, Shinsuke Kihara, TakaakiFujishiro, Ryosuke Kuroda, Masahiro Kurosaka.Kobe University Graduate School of Medicine, Kobe, Japan.

Disclosures:N. Chinzei: None. S. Hayashi: None. S. Hashimoto: None. N. Kanzaki: None. K. Iwasa: None. S. Sakata: None. S. Kihara: None. T.Fujishiro: None. R. Kuroda: None. M. Kurosaka: None.

Introduction: At growth plate, chondrogenic differentiation occurs from diaphyseal side toward metaphyseal side, forming alayered structure. These processes with continuing chondrocyte proliferation, differentiation and growth arrest is regulated bymany factors and hormones. Cyclin-dependent kinases CDKs) are recognized as a regulator of cell cycle progression, and CDKactivation is regulated by CDK inhibitors (CKIs). Two families of CKIs are famous, the Cip/Kip and the Ink4 family. In Cip/Kipfamily, p21 was identified as a cell cycle regulator which its induction by p53 during a DNA damage-induced G (1)-phasecheckpoint response inhibits both CDK 4 and CDK 2 activities. Several papers reported that the expression of p21 is essential forchondrogenesis in vitro [1, 2]. However, they did not show any evidence in vivo. Therefore, the aim of this study is to clarify thep21 function in endochondral ossification.

Methods: P21 knockout mice (B6.129S6 (Cg)-Cdkn1atm1Led/J) were obtained from The Jackson Laboratory (Bar Harbor, Maine).To make heterozygous mice, we backbred the mice gainst a C57BL/6 background. Then, heterozygous mice were mated eachother to obtain 2 groups of embryo, p21 knockout (KO) and Wild type (WT) mice. Pregnant mice were sacrificed at E13.5, E15.5and E18.5 days respectively. After obtaining the embryos, forearms of embryos were dissected and embedded in paraffin.Sagittal histological sections were stained with Safranin O and BrdU. Additionally, we examined the expression of Cyclin D1, TypeII collagen, Type X collagen, SOX9, and p16 which is known as the one of the INK4 family. These sections were analyzed byimmunohistochemistry. Other Cip/Kip families, p27 and p57, were evaluated by immunofluorescence. For western blottinganalysis, chondrocytes were isolated from the ribs of mouse embryos at E18.5 days according to the previous report [3]. Eachsample was electrophoresed on polyacrylamide gradient gel and transblotted electrically onto the blotting membrane. Theexpressions of Cyclin D1, p16, p27, and p57 were evaluated.

Results: Safranin O staining showed that there was no structural change in each time point between WT and p21KO mice (Fig.1). Type II collagen was expressed ubiquitously(Fig. 2a), and Type X collagen was expressed hypertrophic zone in cartilagetissues. However, we did not find any changes in each time point between 2 groups (Fig. 2b). Sox9 showed that there was nochange in each time point between 2 groups (Fig. 2c). The expression levels of Cyclin D1 were higher in E13.5 cartilagetissues(Fig.3a), and the uptake of BrdU seemed to be much higher in the earlier time point (Fig.3b), . However, we did not findany differences in 2 groups. For CKIs analysis, p16 was expressed ubiquitously. However, we did not find any differences in 2groups (Fig. 4). P27 was expressed ubiquitously and p57 was much expressed in in hypertrophic zone adjacent to metaphysis. Inrelation to the expression periods, the expression levels of p27 were increased in E18.5, and the expression levels of p57 weredecreased. However, we did not find any differences in 2 groups (Figs. 5a-e). Western blotting analysis of cartilage proteinobtained from E18.5 mice of the two groups showed that the expression levels of p27, p57, p16, and Cyclin D1 were to find anydifferences in each protein. However, the expression level of p27 was much higher than that of p57 (Fig. 6).

Discussion: Although the Cip/Kip family has similar functions respectively, they also have different functions according to thedifference of the patterns of the expression and the structures of the protein. Previous studies reported that p27 and p57 playan important role in endochondral ossification. In p27 KO mice, although multiple organ overgrowth was observed, theexpression levels of p27 mRNA was similar in the hypertrophic zone and the resting/proliferative zone in adult mice [4]. In p57KO mice, they have altered cell proliferation and differentiation, leading to endochondral bone ossification defects withincomplete differentiation of hypertrophic chondrocytes [5]. With regard to the expression periods of these two CKIs, asprevious reports said in another tissues [6, 7], our results showed similar tendencies that p27 may play an important role in thelatter period of endochondral ossification. Additionally, p57 may play an important role in the middle period, especially at thehypertrophic zone. In contrast, we showed that p21 deficiency did not impact the morphology of cartilage tissue in mouseembryo. Further, p21 deficiency affected neither ECM productions nor SOX 9 expressions. The equivalence of expressions ofCyclin D1 and p16 indicated that there were also no differences in the remaining parts of cell cycle progression that Cip/Kipfamily involved in. The uptake of BrdU seemed to be much higher in the earlier time point. However, we did not find any

differences in 2 groups, revealing that the developmental speed was equivalent. Taken these results into consideration, p21deficiency also did not affect chondrocytes proliferation. Therefore, our defining finding was that p21 may not be essential forthe endochondral ossification of mice embryo and may be compensated by complicate other networks including Cip/Kip family.

Significance: Our findings showed that p21 may not be essential for the endochondral ossification of mice embryo. P27 and p57in itself may play an important role in endochondral ossification of mice embryo.

Acknowledgments: We thank Ms. Kyoko Tanaka, Ms.Minako Nagata, Ms. Maya Yasuda, and Mr. Takeshi Ueha for technicaladvice and assistance.

References: 1. Aikawa T, et al., J Biol Chem, 20012. Nakajima M, et al., Biochem Biophys Res Commun, 20043. Gosset M, et al., Nat Protoc, 20084. Kiyokawa H, et al., Cell, 19965. Zhang P, et al., Nature, 19976. Kim ST, et al., Placenta, 20057. Ilyin GP, et al., Am J Physiol Gastrointest Liver Physiol, 2003

ORS 2014 Annual MeetingPoster No: 1322