Bone remodeling is characterized by the sequential, local tethering of osteoclasts and osteoblasts and is key to the maintenance of bone integrity. While bone matrix-mobilized growth factors, such as TGF-β, are proposed to regulate remodeling, no in vivo evidence exists that an osteoclast-produced molecule serves as a coupling factor for bone resorption to formation. We found that CTHRC1, a protein secreted by mature boneresorbing osteoclasts, targets stromal cells to stimulate osteogenesis. Cthrc1 expression was robustly induced when mature osteoclasts were placed on dentin or hydroxyapatite, and also by increasing extracellular calcium. Cthrc1 expression in bone increased in a high-turnover state (such as that induced by RANKL injections in vivo), but decreased in conditions associated with suppressed bone turnover (such as with aging and after alendronate treatment). Targeted deletion of Cthrc1 in mice eliminated Cthrc1 expression in bone, whereas its deficiency in osteoblasts did not exert any significant effect. Osteoclast-specific deletion of Cthrc1 resulted in osteopenia due to reduced bone formation and impaired the coupling process after resorption induced by RANKL injections, impairing bone mass recovery. These data demonstrate that CTHRC1 is an osteoclastsecreted coupling factor that regulates bone remodeling. IntroductionBone is constantly remodeled through the removal of old bone (resorption) and the replacement of new bone (formation) by hematopoietic-derived osteoclasts and mesenchymal-derived osteoblasts, respectively, to meet structural and metabolic demands (1, 2). It has long been believed that a preceding resorption phase is a prerequisite for the initiation of subsequent bone formation (3), which is also supported by recent clinical observations that treatment of osteoporotic patients with the potent antiresorptive drug alendronate blunts the anabolic action of parathyroid hormone (PTH) (4). Although the coupling of bone formation to resorption has been long recognized, the mechanism and the factors mediating this fundamental process in skeletal homeostasis are not fully understood (5).Recently, it was suggested that active TGF-β1 released from bone matrix during bone resorption is coupled to bone formation by inducing migration of marrow stromal cells to resorption sites (6). Bidirectional signaling between EPHRINB2 on osteoclasts and the receptor EPHB4 on osteoblasts has also been proposed to link bone resorption and formation through direct cell-cell contact (7). In addition, mature osteoclasts produce and secrete factors, such as WNT10B, BMP6, and the lipid mediator sphingosine-1-phosphate (S1P), that have been shown to stimulate osteoblast recruitment and survival (8-10). However, the in vivo function of these factors in the coupling process remains to be elucidated.We reasoned that a factor mediating the coupling reaction linking bone resorption to formation would have to meet the following criteria: (a) it should be produced and secreted/presented by osteoclasts; (b) it should exert a...

Bone remodeling is regulated by a coupling of resorption to subsequent formation; however, the "coupling factor" and underlying mechanism are not fully understood. Here, we found that the condition medium (CM) of mature osteoclasts contains a humoral factor that stimulates the differentiation of primary osteoblasts, as determined by alkaline phosphatase (ALP) activity. We purified osteoblastogenesis-stimulating activity from 3 L of osteoclast CM through successive ion exchange chromatographies by monitoring the ALP activity of osteoblasts and identified complement component 3 (C3). Expression of the C3 gene increased during osteoclastogenesis, and the cleavage product C3a was detected by ELISA in the CM of osteoclasts but not in that of bone marrow macrophages. The osteoblastogenesis-stimulating activity present in osteoclast CM was inhibited by a specific antagonist of the C3a receptor (C3aR), SB290157. Conversely, the retroviral expression of C3a as well as treatment with the C3aR agonist, benzeneacetamide, stimulated osteoblast differentiation. C3 gene expression in bone was increased in the high bone turnover states of ovariectomy (OVX) or a receptor activator of NF-kB ligand (RANKL) injection, and blocking the action of C3a with the daily administration of SB290157 resulted in the attenuation of bone formation elevated by OVX and the exacerbation of bone loss. These results suggest that osteoclast-derived C3a functions in the relay from bone resorption to formation and may be a candidate for a coupling factor.

Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm2 for 265 nm, 3.0 mJ/cm2 for 280 nm, and 23 mJ/cm2 for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.

To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.