ObjectivesThe aim of this study was to identify the role of tenascin-C (TNC) in entheseal new bone formation and to explore the underlying molecular mechanism.MethodsLigament tissue samples were obtained from patients with ankylosing spondylitis (AS) during surgery. Collagen antibody-induced arthritis and DBA/1 models were established to observe entheseal new bone formation. TNC expression was determined by immunohistochemistry staining. Systemic inhibition or genetic ablation of TNC was performed in animal models. Mechanical properties of extracellular matrix (ECM) were measured by atomic force microscopy. Downstream pathway of TNC was analysed by RNA sequencing and confirmed with pharmacological modulation both in vitro and in vivo. Cellular source of TNC was analysed by single-cell RNA sequencing (scRNA-seq) and confirmed by immunofluorescence staining.ResultsTNC was aberrantly upregulated in ligament and entheseal tissues from patients with AS and animal models. TNC inhibition significantly suppressed entheseal new bone formation. Functional assays revealed that TNC promoted new bone formation by enhancing chondrogenic differentiation during endochondral ossification. Mechanistically, TNC suppressed the adhesion force of ECM, resulting in the activation of downstream Hippo/yes-associated protein signalling, which in turn increased the expression of chondrogenic genes. scRNA-seq and immunofluorescence staining further revealed that TNC was majorly secreted by fibroblast-specific protein-1 (FSP1)+fibroblasts in the entheseal inflammatory microenvironment.ConclusionInflammation-induced aberrant expression of TNC by FSP1+fibroblasts promotes entheseal new bone formation by suppressing ECM adhesion forces and activating Hippo signalling.

Recent cancer studies have found that the netrin family of proteins plays vital roles in the development of some cancers. However, the functions of the many variants of these proteins in cancer remain incompletely understood. In this work, we used the most comprehensive database available, including more than 10000 samples across more than 30 tumor types, to analyze the six members of the netrin family. We performed comprehensive analysis of genetic change and expression of the netrin genes and analyzed epigenetic and pathway relationships, as well as the correlation of expression of these proteins with drug sensitivity. Although the mutation rate of the netrin family is low in pan-cancer, among the tumor patients with netrin mutations, the highest number are Uterine Corpus Endometrial Carcinoma patients, accounting for 13.6% of cases (54 of 397). Interestingly, the highest mutation rate of a netrin family member is 38% for NTNG1 (152 of 397). Netrin proteins may participate in the development of endocrine-related tumors and sex hormone-targeting organ tumors. Additionally, the participation of NTNG1 and NTNG2 in various cancers shows their potential for use as new tumor markers and therapeutic targets. this analysis provides a broad molecular perspective of this protein family and suggests some new directions for the treatment of cancer. Cytokines, growth factors, angiogenic factors, and extracellular proteases are secreted to the outside of the cell to produce biological functions 1. These secreted proteins participate in the immune regulation of chronic inflammation 2,3 and the occurrence of lipid metabolism diseases 4 , but also play important roles in tumor invasion, metastasis, immunity, and drug resistance 5-8. For example, CD317 and EGFR are secreted proteins that are potential diagnostic markers for non-small cell lung cancer 9. CD63 is secreted by melanoma into the blood and can be used as a protein marker 10. HGF inhibits the treatment of RAF inhibitors of BRAF mutant melanoma 11. The netrins are neuroguiding factors with axonal guiding function, and are similar to laminin in structure. Netrins were first discovered in C. elegans in 1990 12 , and this family of proteins includes the secreted proteins Netrin-1 (NTN1), Netrin-3 (NTN3), Netrin-4 (NTN4), and Netrin-5 (NTN5). The secreted proteins have a common domain structure, with an N-terminal laminin repeat (Laminin N-terminal), three cysteine-rich EGF modules (V-1, V-2, and V-3), and a positively charged C-terminal domain (NTR) 13. The netrin family also includes two membrane-binding proteins, Netrin-G1 (NTNG1) and Netrin-G2 (NTNG2) 14. Although these proteins also have Laminin N-terminal and Laminin EGF domains, their ends have different functions due to GPI 15. The major binding receptors of the secreted netrin proteins are DCC and UNC5 homologue family UNC5A-D, which are both dependent receptors. Netrin binding to a receptor promotes cell survival, proliferation, and differentiation, and without netrin binding, the receptor induces apoptosis 16,17....

No abstract