KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration

Article https://doi.org/10.1038/s41467-023-37651-1 KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration Received: 2 June 2022 1 , Check for updates 1234567890():,; 1234567890():,; Accepted: 14 March 2023 Li Chen 1,5 , Kaikai Shi1, Nicholas Ditzel1, Weimin Qiu1, Florence Figeac Louise Himmelstrup Dreyer Nielsen1, Michaela Tencerova1, Justyna Magdalena Kowal 1, Ming Ding2, Christina Møller Andreasen 3, Thomas Levin Andersen 3 & Moustapha Kassem 1,4 Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cellsecreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions. Clinical trials employing bone marrow skeletal (also known as stromal or mesenchymal) stem cells (BMSC) for enhancing bone tissue regeneration, have revealed that the observed positive effects are majorly mediated by secreted factors1. Interestingly, the secretome of BMSC contains a large number of factors with unknown functions within bone biology, and with a possible therapeutic potential2–4. Also, these factors seem to be present at a biologically significant concentrations in the circulation, suggesting a systemic regulatory effect5. Another common feature of these novel factors is that their expression and secretion are regulated during BMSC lineage-specific differentiation, and in turn, they regulate at osteogenic differentiation, bone development, and bone mass6–8. We have previously described the composition of the secretome of human BMSC (hBMSC) during osteoblastic differentiation as revealed by quantitative mass-spectrometry-based proteomic analysis9. Among the identified factors is KIAA1199, which is a 150 KDa protein encoded by a gene located on chromosome 15q25.1, has a G8 domain, contains 8 conserved glycine residues, and consists of 5 beta- 1 Department of Endocrinology and Metabolism, Endocrine Research Laboratory (KMEB), Odense University Hospital & University of Southern Denmark, Odense, Denmark. 2Department of Orthopaedic Surgery and Traumatology, Odense University Hospital & University of Southern Denmark, Odense, Denmark. 3 Institute of Pathology, University of Southern Denmark, Odense, Denmark. 4Department of Cellular and Molecular Medicine (ICMM), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. 5Present address: Dept. of Pathology and Physiopathology, Guangxi Key Laboratory of Tumor e-mail: ; Immunology and Microenvironmental Regulation, Guilin Medical University, Guilin, Guangxi, China. Nature Communications | (2023)14:2016 1 Article chain pairs and 1 alpha helix10. Interestingly, mutation of the KIAA1199 causes human familial non-syndromic hearing loss11. The expression levels of KIAA1199 are increased in a number of cancer types12–15, and have been reported as a poor prognostic factor in patients with breast, colon cancer, and gastric cancer12,14,16,17, probably due to increased risk for metastasis15,18–20. Also, tissue expression of KIAA1199 is increased in the synovial tissues in patients with osteoarthritis (OA) and rheumatoid arthritis (RA)21–24. In addition, KIAA1199 binds and degrades hyaluronic acid (HA)21,25, a mechanism suggested to regulate endochondral ossification26. We have also reported that KIAA1199 regulates the motility and migration of hBMSC through changes in p38 MAPK kinase and Wnt signaling pathways27. According to these diverse biological functions, KIAA1199 is also called a ‘cell migration-inducing and hyaluronan-binding protein’ (CEMIP). In the present study, we investigated the biological role of KIAA1199 in hBMSC biology in vitro and its impact on bone formation in vivo. Our results demonstrate that KIAA1199 is a regulator of osteoblastic differentiation and bone mass, bone strength, bone fracture healing, as well as bone loss following estrogen deficiency. Moreover, the KIAA1199 expression levels of BMSC that obtained from patients are negatively correlated with their osteogenic potentials, and the plasma levels of KIAA1199 exhibit a positive correlation with the clinical estimate of fracture risk. Results KIAA1199 is highly expressed in bone, BMSC and in the circulation We have previously identified KIAA1199 as a protein secreted by cultured hBMSC9,28. To identify its in vivo cellular production, we performed in situ hybridization on human iliac crest bone biopsies. KIAA1199 is expressed in the ‘reversal cells’ and the ‘canopy cells’ i.e., osteoprogenitor cells present along the bone formation sites (Fig. 1A). In addition, KIAA1199 is expressed in a group of marrow cells surrounding marrow adipocytes (Fig. 1A). KIAA1199 expression was absent from osteocytes and osteoclasts. Gene expression analysis performed on murine tissues showed that KIAA1199 is expressed at high levels in brain, and in bones (skull and limbs), as well as in cultured BMSC (Fig. 1B). Considering the relative tissue volume, bone tissue is probably the main source of KIAA1199 production in the body. Also, KIAA1199 is detected in peripheral blood and bone marrow plasma by ELISA and its levels were highly correlated in the two compartments, the levels were also similar in men and women (Fig. 1C–E & Supplementary Fig. S1A, B). To test for clinical relevance, plasma levels of KIAA1199 were determined in a cohort of patients admitted to the hospital with bone fractures as described in a previous publication from our group29. We found that KIAA1199 levels in bone marrow plasma positively correlate with osteoporotic fracture risk as determined by FRAX score30 (Fig. 1F, G & Supplementary Fig. S1C–D). The data suggest that KIAA1199 is a potential biomarker to be included in the assessment of osteoporotic fracture risk. Additionally, we found that KIAA1199 expression levels in cultured hBMSC obtained from the same cohort29 negatively correlates with the number of CD146 + osteoprogenitor cells, and also with in vitro (...truncated)