Osteopetrosis-like disorders induced by osteoblast-specific retinoic acid signaling inhibition in mice

Abstract

Osteopetrosis is an inherited metabolic disease, characterized by increased bone density and narrow marrow cavity. Patients with severe osteopetrosis exhibit abnormal bone brittleness, anemia, and infection complications, which commonly cause death within the first decade of life. Pathologically, osteopetrosis impairs not only the skeletal system, but also the hemopoietic and immune systems during development, while the underlying osteoimmunological mechanisms remain unclear. Osteoclastic mutations are regarded as the major causes of osteopetrosis, while osteoclast non-autonomous theories have been proposed in recent years with unclear underlying mechanisms. Retinoic acid (RA), the metabolite of Vitamin A, is an essential requirement for skeletal and hematopoietic development, through the activation of retinoic acid signaling. RA can relieve osteopetrosis symptoms in some animal models, while its effect on bone health is still controversial and the underlying mechanisms remain unclear. In this study, we constructed an osteoblast-specific inhibitory retinoic acid signaling mouse model and surprisingly found it mimicked the symptoms of osteopetrosis found in clinical cases: dwarfism, increased imperfectly-formed trabecular bone deposition with a reduced marrow cavity, thin cortical bone with a brittle skeleton, and hematopoietic and immune dysfunction. Micro-CT, the three-point bending test, and histological analysis drew a landscape of poor bone quality. Single-cell RNA sequencing (scRNA-seq) of the femur and RNA-seq of osteoblasts uncovered an atlas of pathological skeletal metabolism dysfunction in the mutant mice showing that osteogenesis was impaired in a cell-autonomous manner and osteoclastogenesis was impaired via osteoblast-osteoclast crosstalk. Moreover, scRNA-seq of bone marrow and flow cytometry of peripheral blood, spleen, and bone marrow uncovered pathology in the hematopoietic and immune systems in the mutant mice, mimicking human osteopetrosis. Results showed that hematopoietic progenitors and B lymphocyte differentiation were affected and the osteoblast-dominated cell crosstalk was impaired, which may result from transcriptional impairment of the ligands Pdgfd and Sema4d. In summary, we uncovered previously unreported pathogenesis of osteopetrosis-like disorder in mice with skeletal, hematopoietic, and immune system dysfunction, which was induced by the inhibition of retinoic acid signaling in osteoblasts, and sheds new insights into a potential treatment for osteopetrosis.