ABSTRACT
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 10â weeks and expression remained at similar levels at 12â months. In Mkx-/- mice, age-dependent 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 Mkx-overexpressing human PDL fibroblasts, whereas osteogenic genes were downregulated. Our results indicate that Mkx prevents PDL degeneration by regulating osteogenesis.
Subject(s)
Homeodomain Proteins/physiology , Homeostasis/genetics , Periodontal Ligament/physiology , Alveolar Bone Loss/genetics , Alveolar Bone Loss/pathology , Animals , Cell Differentiation/genetics , Cells, Cultured , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Fibroblasts/physiology , Gene Expression Regulation , HEK293 Cells , Humans , Mice, Inbred C57BL , Mice, Knockout , Osteogenesis/geneticsABSTRACT
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.
