Osteopontin (OPN), a secreted phosphoprotein, is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family of cell matrix proteins and participates in many biological activities. Studies have shown that OPN plays a role in bone metabolism and homeostasis. OPN not only is an important factor in neuronmediated and endocrine-regulated bone mass, but also is involved in biological activities such as proliferation, migration, and adhesion of several bone-related cells, including bone marrow mesenchymal stem cells, hematopoietic stem cells, osteoclasts, and osteoblasts. OPN has been demonstrated to be closely related to the occurrence and development of many bone-related diseases, such as osteoporosis, rheumatoid arthritis, and osteosarcoma. As expected, the functions of OPN in the bone have become a research hotspot. In this article, we try to decipher the mechanism of OPN-regulated bone metabolism and bone diseases.

Background and Purpose: Melatonin is a neurohormone involved in bone homeostasis. Melatonin directs bone remodelling and the role of bone marrow mesenchymal stem cells (BMMSCs) in the regulating melatonin-mediated bone formationresorption balance remains undefined. Experimental Approach: Osteoporosis models were established and bone tissue and serum were collected to test the effects of melatonin on bone homeostasis. Melatonin receptors were knocked down, the NF-κB signalling pathway and receptor activator of NF-κB ligand (RANKL) expression were investigated. Communication between bone marrow mesenchymal stem cells and osteoclasts was detected with directcontact or indirect-contact system. Key Results: Bone loss and microstructure disorder in mice were reversed after melatonin treatment, as a result of anabolic and anti-resorptive effects. In vitro, a physiological (low) concentration of melatonin promoted the bone marrow mesenchymal stem cells, osteogenic lineage commitment and extracellular mineralization but had no impact on extracellular matrix synthesis. After MT knockdown, especially MT 2 , the positive effects of melatonin on osteogenesis were attenuated. The canonical NF-κB signalling pathway was the first discovered downstream signalling pathway after MT receptor activation and was found to be down-regulated by melatonin during osteogenesis. Melatonin suppressed BMMSC-mediated osteoclastogenesis by inhibiting RANKL production in BMMSCs and this effect only occurred when BMMSCs and osteoclast precursors were co-cultured in an indirect-contact manner. Conclusion and Implications: Our work suggests that melatonin plays a crucial role in bone balance, significantly accelerates the osteogenic differentiation of bone marrow mesenchymal stem cells by suppressing the MT 2 -dependent NF-κB signalling pathway, and down-regulates osteoclastogenesis via RANKL paracrine secretion. Abbreviations: ALP, alkaline phosphatase; BMD, bone mineral density; BMMs, bone marrow monocytes; BMMSCs, bone marrow mesenchymal stem cells; BV/TV, trabecular bone volume per total volume; Col-I, collagen I; CTX-I, C-terminal telopeptide α1 chain of type I collagen; H&E, haematoxylin and eosin; Mel, melatonin; OCN, osteocalcin; OPG, osteoprotegerin; OVX, ovariectomy; PINP, N-terminal propeptide of type I procollagen; RANK, receptor activator of NF-κB; RANKL, receptor activator of NF-κB ligand; TRAP, tartrate-resistant acid phosphatase; Tb. N, trabecular bone number; Tb. Sp, trabecular bone separation; Tb. Th, trabecular bone thickness; μCT, micro-CT.