Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation

Ibaraki,Kazuyuki; Hayashi,Shinya; Kanzaki,Noriyuki; Hashimoto,Shingo; Kihara,Shinsuke; Haneda,Masahiko; Takeuchi,Kazuhiro; Niikura,Takahiro; Kuroda,Ryosuke

doi:10.3892/mmr.2020.11028

Deletion of p21 expression accelerates cartilage tissue repair via chondrocyte proliferation

Authors:
- Kazuyuki Ibaraki
- Shinya Hayashi
- Noriyuki Kanzaki
- Shingo Hashimoto
- Shinsuke Kihara
- Masahiko Haneda
- Kazuhiro Takeuchi
- Takahiro Niikura
- Ryosuke Kuroda
View Affiliations

Affiliations: Department of Orthopedic Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo 650‑0017, Japan
Published online on: March 13, 2020 https://doi.org/10.3892/mmr.2020.11028
Pages: 2236-2242

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Articular cartilage tissue has a poor healing potential, and when subjected to traumatic damage this tissue undergoes cartilage degeneration and osteoarthritis. The association between the regulation of cell cycle checkpoints and tissue regeneration has been previously investigated, and p21 was initially identified as a potent inhibitor of cell cycle progression. However, the effects of p21 defects on damaged tissue remain controversial. Therefore, the aim of the present study was to evaluate the effects of p21 deficiency on cartilage repair. A mouse model of articular cartilage repair was generated by inducing a patellar groove scratch in 8‑week‑old p21‑knockout (KO) mice and C57Bl/6 wild‑type (WT) mice. Mice were sacrificed at 4 and 8 weeks post‑surgery. The present study also investigated the effect of p21 deficiency on cartilage differentiation in ATDC5 cells in vitro. Safranin O staining results indicated that cartilage repair initially occurred in p21 KO mice. In addition, immunohistochemical analysis demonstrated that p21 KO upregulated proliferating cell nuclear antigen and increased cell proliferation. However, type II collagen and Sox9 expression levels remained unchanged in p21 KO and WT mice. Moreover, it was identified that p21 downregulation did not affect Sox9 and type II collagen expression levels in vitro. Furthermore, p21 deficiency promoted healing of articular cartilage damage, which was associated with cell proliferation in vivo, and increased chondrocyte proliferation but not differentiation in vitro. Therefore, the present results suggested that p21 does not affect Sox9 or type II collagen expression levels during cartilage differentiation in the repair of cartilage defects.

View Figures

View References

1	Rai MF and Sandell LJ: Regeneration of articular cartilage in healer and non-healer mice. Matrix Biol. 39:50–55. 2014. View Article : Google Scholar : PubMed/NCBI
2	Buckwalter JA, Rosenberg LC and Huniker EB: Articular cartilage: composition, structure, response to injury, and methods of facilitating repair. Articular Cartilage and Knee Joint Function: Basic Science and Arthroscopy. Ewing JW: Raven Press Ltd.; New York, NY: 1990
3	Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS and Little CB: Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage. 16:1319–1326. 2008. View Article : Google Scholar : PubMed/NCBI
4	Eltawil NM, De Bari C, Achan P, Pitzalis C and Dell'accio F: A novel in vivo murine model of cartilage regeneration. Age and strain-dependent outcome after joint surface injury. Osteoarthritis Cartilage. 17:695–704. 2009. View Article : Google Scholar : PubMed/NCBI
5	Bedelbaeva K, Snyder A, Gourevitch D, Clark L, Zhang XM, Leferovich J, Cheverud JM, Lieberman P and Heber-Katz E: Lack of p21 expression links cell cycle control and appendage regeneration in mice. Proc Natl Acad Sci USA. 107:5845–5850. 2010. View Article : Google Scholar : PubMed/NCBI
6	Arthur LM and Heber-Katz E: The role of p21 in regulating mammalian regeneration. Stem Cell Res Ther. 2:302011. View Article : Google Scholar : PubMed/NCBI
7	Harper JW, Adami GR, Wei N, Keyomarsi K and Elledge SJ: The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 75:805–816. 1993. View Article : Google Scholar : PubMed/NCBI
8	Suzuki A, Tsutomi Y, Akahane K, Araki T and Miura M: Resistance to Fas-mediated apoptosis: Activation of caspase 3 is regulated by cell cycle regulator p21WAF1 and IAP gene family ILP. Oncogene. 17:931–939. 1998. View Article : Google Scholar : PubMed/NCBI
9	Hayashi S, Fujishiro T, Hashimoto S, Kanzaki N, Chinzei N, Kihara S, Takayama K, Matsumoto T, Nishida K, Kurosaka M, et al: p21 deficiency is susceptible to osteoarthritis through STAT3 phosphorylation. Arthritis Res Ther. 17:3142015. View Article : Google Scholar : PubMed/NCBI
10	Seoane J, Le HV and Massagué J: Myc suppression of the p21(Cip1) Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature. 419:729–734. 2002. View Article : Google Scholar : PubMed/NCBI
11	Kondo S, Nakagawa Y, Mizuno M, Katagiri K, Tsuji K, Kiuchi S, Ono H, Muneta T, Koga H and Sekiya I: Transplantation of aggregates of autologous synovial mesenchymal stem cells for treatment of cartilage defects in the femoral condyle and the femoral groove in microminipigs. Am J Sports Med. 47:2338–2347. 2019. View Article : Google Scholar : PubMed/NCBI
12	Qasim M, Chae DS and Lee NY: Bioengineering strategies for bone and cartilage tissue regeneration using growth factors and stem cells. J Biomed Mater Res A. 108:394–411. 2020. View Article : Google Scholar : PubMed/NCBI
13	Nakagawa Y, Fortier LA, Mao JJ, Lee CH, Goodale MB, Koff MF, Uppstrom TJ, Croen B, Wada S, Carballo CB, et al: Long-term evaluation of meniscal tissue formation in 3-dimensional-printed scaffolds with sequential release of connective tissue growth factor and TGF-β3 in an ovine model. Am J Sports Med. 47:2596–2607. 2019. View Article : Google Scholar : PubMed/NCBI
14	Premnath P, Jorgenson B, Hess R, Tailor P, Louie D, Taiani J, Boyd S and Krawetz R: p21-/- mice exhibit enhanced bone regeneration after injury. BMC Musculoskelet Disord. 18:4352017. View Article : Google Scholar : PubMed/NCBI
15	Kihara S, et al: Cyclin-dependent kinase inhibitor-1-deficient mice are susceptible to osteoarthritis associated with enhanced inflammation. JMBR. 32:991–1001. 2017.
16	Matsuoka M, Onodera T, Sasazawa F, Momma D, Baba R, Hontani K and Iwasaki N: An articular cartilage repair model in common C57Bl/6 Mice. Tissue Eng Part C Methods. 21:767–772. 2015. View Article : Google Scholar : PubMed/NCBI
17	Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI and Goldberg VM: Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am. 76:579–592. 1994. View Article : Google Scholar : PubMed/NCBI
18	Shukunami C, Shigeno C, Atsumi T, Ishizeki K, Suzuki F and Hiraki Y: Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: Differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor. J Cell Biol. 133:457–468. 1996. View Article : Google Scholar : PubMed/NCBI
19	Shukunami C, Ishizeki K, Atsumi T, Ohta Y, Suzuki F and Hiraki Y: Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro. J Bone Miner Res. 12:1174–1188. 1997. View Article : Google Scholar : PubMed/NCBI
20	Atsumi T, Miwa Y, Kimata K and Ikawa Y: A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. Cell Differ Dev. 30:109–116. 1990. View Article : Google Scholar : PubMed/NCBI
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
22	Schönenberger F, Deutzmann A, Ferrando-May E and Merhof D: Discrimination of cell cycle phases in PCNA-immunolabeled cells. BMC Bioinformatics. 16:1802015. View Article : Google Scholar : PubMed/NCBI
23	Bi W, Deng JM, Zhang Z, Behringer RR and de Crombrugghe B: Sox9 is required for cartilage formation. Nat Genet. 22:85–89. 1999. View Article : Google Scholar : PubMed/NCBI
24	Akiyama H, Chaboissier MC, Martin JF, Schedl A and de Crombrugghe B: The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev. 16:2813–2828. 2002. View Article : Google Scholar : PubMed/NCBI
25	Ng LJ, Wheatley S, Muscat GE, Conway-Campbell J, Bowles J, Wright E, Bell DM, Tam PP, Cheah KS and Koopman P: SOX9 binds DNA, activates transcription, and coexpresses with type II collagen during chondrogenesis in the mouse. Dev Biol. 183:108–121. 1997. View Article : Google Scholar : PubMed/NCBI
26	Chinzei N, Hayashi S, Hashimoto S, Kanzaki N, Iwasa K, Sakata S, Kihara S, Fujishiro T, Kuroda R and Kurosaka M: Cyclin dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice. Mol Med Rep. 11:1601–1608. 2015. View Article : Google Scholar : PubMed/NCBI