title: Gene targeting of the transcription factor Mohawk in rats causes heterotopic ossification of Achilles tendon via failed tenogenesis.
abstract: Cell-based or pharmacological approaches for promoting tendon repair are currently not available because the molecular mechanisms of tendon development and healing are not well understood. Although analysis of knockout mice provides many critical insights, small animals such as mice have some limitations. In particular, precise physiological examination for mechanical load and the ability to obtain a sufficient number of primary tendon cells for molecular biology studies are challenging using mice. Here, we generated Mohawk (Mkx)(-/-) rats by using CRISPR/Cas9, which showed not only systemic hypoplasia of tendons similar to Mkx(-/-) mice, but also earlier heterotopic ossification of the Achilles tendon compared with Mkx(-/-) mice. Analysis of tendon-derived cells (TDCs) revealed that Mkx deficiency accelerated chondrogenic and osteogenic differentiation, whereas Mkx overexpression suppressed chondrogenic, osteogenic, and adipogenic differentiation. Furthermore, mechanical stretch stimulation of Mkx(-/-) TDCs led to chondrogenic differentiation, whereas the same stimulation in Mkx(+/+) TDCs led to formation of tenocytes. ChIP-seq of Mkx overexpressing TDCs revealed significant peaks in tenogenic-related genes, such as collagen type (Col)1a1 and Col3a1, and chondrogenic differentiation-related genes, such as SRY-box (Sox)5, Sox6, and Sox9 Our results demonstrate that Mkx has a dual role, including accelerating tendon differentiation and preventing chondrogenic/osteogenic differentiation. This molecular network of Mkx provides a basis for tendon physiology and tissue engineering.
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Suzuki
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Ito
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan;
authors.forename: Masahiro
authors.initials: M
authors.lastname: Shinohara
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yamashita
authors.affiliation: Research Center for Medical and Dental Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Shizuko
authors.initials: S
authors.lastname: Ichinose
authors.affiliation: Department of Material-Based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan;
authors.forename: Akio
authors.initials: A
authors.lastname: Kishida
authors.affiliation: Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Takuya
authors.initials: T
authors.lastname: Oyaizu
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Kayama
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Ryo
authors.initials: R
authors.lastname: Nakamichi
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Naoki
authors.initials: N
authors.lastname: Koda
authors.affiliation: Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; Sports Science Organization, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Yagishita
authors.affiliation: Core Research for the Evolutionary Science and Technology, Japan Agency for Medical Research and Development, Chiyoda-ku, Tokyo 100-0004, Japan;
authors.forename: Martin K
authors.initials: MK
authors.lastname: Lotz
authors.affiliation: Department of Orthopaedic Surgery, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan;
authors.forename: Atsushi
authors.initials: A
authors.lastname: Okawa
authors.affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan; Core Research for the Evolutionary Science and Technology, Japan Agency for Medical Research and Development, Chiyoda-ku, Tokyo 100-0004, Japan; Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037 asahara@scripps.edu.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Asahara
mesh_terms: D000125:Achilles Tendon / Q000473:pathology; D050156:Adipogenesis; D000818:Animals; D020219:Chondrogenesis; D055786:Gene Knockout Techniques; D018398:Homeodomain Proteins / Q000502:physiology*; D008297:Male; D009999:Ossification, Heterotopic / Q000209:etiology* / Q000473:pathology; D010012:Osteogenesis; D017208:Rats, Wistar; D013314:Stress, Mechanical
chemical_list: D018398:Homeodomain Proteins; C000623076:Mkx protein, rat
keywords: Achilles tendon ossification; CRISPR-Cas9; Mkx; knockout rat; tendon development