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.Achilles tendon ossification T endons play a critical role in the musculoskeletal system by connecting muscle to bone to transmit mechanical loads and enable movement. Tendon injuries and damage are repaired slowly and incompletely because of poor intrinsic healing capacity, which in part results from tissue hypocellularity and hypovascularity (1). Even after surgical tendon repair, a standard treatment for tendon rupture, clinical outcomes are not satisfactory because of recurrent rupture or adhesions (2). To develop cell-based or pharmacological approaches for promoting tendon repair, the molecular mechanism of tendon development and regeneration must be determined; however, the key genome network for tendon differentiation and homeostasis has not been well characterized.We, along with other researchers, recently reported the tendon-specific expression and functions of the transcription factor Mohawk (Mkx), which regulates tendon-related gene expression (3, 4). Mkx knockout mice showed general tendon hypoplasia (5, 6), suggesting that Mkx plays an important role during tendon development. Moreover, overexpression of Mkx in mesenchymal stem cells (MSC) elevates tendon-related markers, and transplantation of these cells increases the diameter of collagen fibers in tendons (7,8), suggesting the potential application of Mkx in cell therapy for tendon injury.Although the results from analysis of Mkx knockout mice have provided critical informatio...

The main pathogenesis of intervertebral disc (IVD) herniation involves disruption of the annulus fibrosus (AF) caused by ageing or excessive mechanical stress and the resulting prolapse of the nucleus pulposus. Owing to the avascular nature of the IVD and lack of understanding the mechanisms that maintain the IVD, current therapies do not lead to tissue regeneration. Here we show that homeobox protein Mohawk (Mkx) is a key transcription factor that regulates AF development, maintenance and regeneration. Mkx is mainly expressed in the outer AF (OAF) of humans and mice. In Mkx−/− mice, the OAF displays a deficiency of multiple tendon/ligament-related genes, a smaller OAF collagen fibril diameter and a more rapid progression of IVD degeneration compared with the wild type. Mesenchymal stem cells overexpressing Mkx promote functional AF regeneration in a mouse AF defect model, with abundant collagen fibril formation. Our results indicate a therapeutic strategy for AF regeneration.

Mechanoforces experienced by an organ are translated into biological information for cellular sensing and response. In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover. We show that Mohawk (Mkx), a tendon-specific transcription factor, is essential in mechanoresponsive tenogenesis through regulation of its downstream ECM genes such as type I collagens and proteoglycans such as fibromodulin both in vivo and in vitro. Wild-type (WT) mice demonstrated an increase in collagen fiber diameter and density in response to physical treadmill exercise, whereas in Mkx−/− mice, tendons failed to respond to the same mechanical stimulation. Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx. In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation. Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

The periodontal ligament (PDL), which connects the teeth to the alveolar bone, is essential for periodontal tissue homeostasis. Although the significance of the PDL is recognized, molecular mechanisms underlying PDL function are not well known. We report that mohawk homeobox (Mkx), a tendon-specific transcription factor, regulates PDL homeostasis by preventing its degeneration. Mkx is expressed in the mouse PDL at the age of 10 weeks and expression remained at similar levels at 12 months. In Mkx −/− mice, agedependent expansion of the PDL at the maxillary first molar (M1) furcation area was observed. Transmission electron microscopy (TEM) revealed that Mkx −/− mice presented collagen fibril degeneration in PDL with age, while the collagen fibril diameter gradually increased in Mkx +/+ mice. PDL cells lost their shape in Mkx −/− mice, suggesting changes in PDL properties. Microarray and quantitative polymerase chain reaction (qPCR) analyses of Mkx −/− PDL revealed an increase in osteogenic gene expression and no change in PDL-and inflammatory-related gene expression. Additionally, COL1A1 and COL1A2 were upregulated in Mkxoverexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.

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