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.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...

Modeling human somite development and fibrodysplasia ossificans progressiva with induced pluripotent stem cells

“…The results showed that iPSC-derived SYN and human primary tenocytes upregulated SYN-related markers in response to mechanical stress, consistent with previous reports (Fig. 4H) (Suzuki et al, 2016; Yang et al, 2004). These data demonstrate one aspect of the function of iPSC-derived SYNs.…”

Novel Model of Tendon Regeneration Reveals Distinct Cell Mechanisms Underlying Regenerative and Fibrotic Tendon Healing

“…Inflammation can also trigger heterotopic endochondral ossification, as established by several studies of traumatic or neurogenic injuries and congenital diseases383940. In addition, a recent study showed that the tendon transcription factor Mkx may also function to inhibit chondrogenic differentiation in tenocytes;41 our study showed downregulation of Mkx expression after adult tendon injury, which may permit cartilage differentiation at the expense of tenogenesis. Although our lineage tracing results showed a clear contribution of Scx lin tenocytes to ectopic cartilage, we also observed a number of non-labeled cells.…”

Mohawk transcription factor regulates homeostasis of the periodontal ligament

“…The transcription factor mohawk homeobox (Mkx), a member of the three-amino-acid loop extension (TALE) superclass of atypical homeobox genes, is expressed in the embryonic progenitor cell populations of the cartilage, skeletal muscle, tendons and bones, in addition to the tips of the ureteric buds in the metanephric kidneys and the testis cords of the male gonad testis in the mouse embryo (Anderson et al, 2006). Additionally, Mkx plays a crucial role in tendon maturation by regulating the expression of type I collagen (Ito et al, 2010;Liu et al, 2010;Nakamichi et al, 2016;Onizuka et al, 2014;Suzuki et al, 2016). However, the significance of Mkx in the PDL has never been explored.…”