Sclerostin modulates mineralization degree and stiffness profile in the fibrocartilaginous enthesis for mechanical tissue integrity.

Fibrocartilaginous entheses consist of tendons, unmineralized and mineralized fibrocartilage, and subchondral bone, each exhibiting varying stiffness. Here we examined the functional role of sclerostin, expressed in mature mineralized fibrochondrocytes. Following rapid mineralization of unmineralized fibrocartilage and concurrent replacement of epiphyseal hyaline cartilage by bone, unmineralized fibrocartilage reexpanded after a decline in alkaline phosphatase activity at the mineralization front. Sclerostin was co-expressed with osteocalcin at the base of mineralized fibrocartilage adjacent to subchondral bone. In Scx-deficient mice with less mechanical loading due to defects of the Achilles tendon, sclerostin+ fibrochondrocyte count significantly decreased in the defective enthesis where chondrocyte maturation was markedly impaired in both fibrocartilage and hyaline cartilage. Loss of the Sost gene, encoding sclerostin, elevated mineral density in mineralized zones of fibrocartilaginous entheses. Atomic force microscopy analysis revealed increased fibrocartilage stiffness. These lines of evidence suggest that sclerostin in mature mineralized fibrochondrocytes acts as a modulator for mechanical tissue integrity of fibrocartilaginous entheses.

Scleraxis-lineage cells are required for correct muscle patterning.

Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scx-lineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue-tissue interaction in musculoskeletal morphogenesis.

Tenogenic Induction From Induced Pluripotent Stem Cells Unveils the Trajectory Towards Tenocyte Differentiation.

The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-ß2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-ß2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.

Deficiency of TMEM53 causes a previously unknown sclerosing bone disorder by dysregulation of BMP-SMAD signaling.

Bone formation represents a heritable trait regulated by many signals and complex mechanisms. Its abnormalities manifest themselves in various diseases, including sclerosing bone disorder (SBD). Exploration of genes that cause SBD has significantly improved our understanding of the mechanisms that regulate bone formation. Here, we discover a previously unknown type of SBD in four independent families caused by bi-allelic loss-of-function pathogenic variants in TMEM53, which encodes a nuclear envelope transmembrane protein. Tmem53-/- mice recapitulate the human skeletal phenotypes. Analyses of the molecular pathophysiology using the primary cells from the Tmem53-/- mice and the TMEM53 knock-out cell lines indicates that TMEM53 inhibits BMP signaling in osteoblast lineage cells by blocking cytoplasm-nucleus translocation of BMP2-activated Smad proteins. Pathogenic variants in the patients impair the TMEM53-mediated blocking effect, thus leading to overactivated BMP signaling that promotes bone formation and contributes to the SBD phenotype. Our results establish a previously unreported SBD entity (craniotubular dysplasia, Ikegawa type) and contribute to a better understanding of the regulation of BMP signaling and bone formation.