The Design,Preparation,mechanism Research,and Application Of A Type Ⅰ Collagen Active Peptide Involved In Bone Remodeling Through OSCAR

PurposeInhibition of osteoclast bone resorption is the key to the prevention and treatment of osteoporosis.TypeⅠcollagen is the predominant organic component in bone tissue.However,whether its degradation fragments produced during bone resorption can regulate bone remodeling is unknown.This study aims to explore the typeⅠcollagen degradation active peptides（CAPs）that can bind to OSCAR in the bone resorption microenvironment,study its effect on osteoclast differentiation and bone resorption,and investigate its application in the diagnosis and treatment of osteoporosis.MethodsAccording to the known OSCAR core binding sites previously reported in collagen,the minimal putative OSCAR-binding amino sequence in typeⅠcollagen was analyzed,and possibly active peptides were designed.Then,we screened and verified the existence of these designed typeⅠcollagen peptides（CPs）in bone resorption conditioned medium by liquid chromatography-mass spectrometry/mass spectrometry（LC-MS/MS）and Western blot analysis.The verified CP were synthesized in vitro.The binding of CP1 to OSCAR was tested by solid-phase binding assay and immunofluorescence.Bone marrow macrophages（BMMs）from wild-type mice were treated with different concentrations（0,10,100,1000μg/m L）of CP1 to clarify the regulatory effect of CP1 on osteoclast differentiation and bone resorption in vitro.The bone-targeting peptide was constructed by conjugating CAP1 with（D-Asp）₈.Cy5 labeled bone-targeting CAP1（Cy5-DCAP1）was injected into wild-type mice via the tail vein.A biophotonic imaging system was used to verify the bone-targeting characteristic and half-life of Cy5-DCAP1.To explore the regulatory effect of CAP1 on bone resorption and bone mass in vivo and its therapeutic effect on osteoporosis,ovariectomized（OVX）wild-type mice were treated with high-dose（5mg/kg）bone-targeting CAP1,while wild-type mice were treated with low-dose（0.25 mg/kg）bone-targeting CAP1.The Oscar gene knockout mice were produced,and their phenotypic changes were observed.The regulatory effect of CAP1 on osteoclast differentiation and bone resorption through OSCAR in vivo and in vitro was demonstrated using Oscar knockout mice.By knocking down and overexpressing the nuclear factor of activated T cells c1（Nfatc1）and Oscar,the post-receptor mechanism of CAP1 regulating osteoclast differentiation through OSCAR was explored.CAP1 monoclonal antibody was prepared,and the CAP1 enzyme-linked immunosorbent assay（ELISA）kit was developed.The serum level of CAP1 was measured by the CAP1 ELISA kit in patients with normal bone mineral density（BMD）,osteopenia,and osteoporosis.The relationship between CAP1 and bone mass and its application in the diagnosis of osteoporosis was explored.ResultsThere are 13 putative OSCAR core binding sequences in typeⅠcollagen,among which the CP1 sequence was conserved in theα1 andα2chains of typeⅠcollagen and highly conserved in multiple species.LC-MS/MS and Western blot analysis showed that CP1 existed in the natural bone resorption microenvironment.CP1 bound firmly to His-tagged m OSCAR extracellular domain protein（OSCAR-His）fusion protein.Immunofluorescence showed that Cy5 labeled CP1（Cy5-CP1）co-localized with OSCAR on the cell membrane of osteoclasts.CP1 exerted dual effects on osteoclast differentiation:a low dose（10 and 100μg/m L）stimulated osteoclast differentiation,and a high dose high-dose（1000μg/m L）inhibited osteoclast differentiation.The bone-targeting CAP1 could specifically target bone tissue,especially the bone tissue around OSCAR positive cells.Its half-life in mouse bone tissue was more than 96 hours.High-dose（5 mg/kg）bone-targeting CAP1 could inhibit bone resorption in vivo,thus effectively treating OVX-induced bone mass loss.Low-dose（0.25 mg/kg）bone-targeting CAP1 could induce bone mass loss in mice by promoting bone resorption.Genetic deletion of Oscar resulted in decreased bone resorption and increased bone mass in both males and females.The osteoclast differentiation ability of BMMs from Oscar knockout(Oscar^-/-)mice was lower than that of wild-type control(Oscar^+/+)mice.Oscar deficiency not only abolished the effect of high-dose CAP1 on inhibiting osteoclast differentiation and improving OVX-induced bone mass loss but also eliminated the effect of low-dose CAP1 on promoting osteoclast differentiation and bone mass loss.The regulation of CAP1 on osteoclast differentiation was achieved by regulating NFATc1 expression through OSCAR-mediated changes in c-Fos and microphthalmia-associated transcription factor（MITF）.The antibody against CAP1 was prepared,and CAP1 could be detected in human peripheral blood.The results of ELISA showed that serum CAP1level was decreased in osteoporosis patients and was positively correlated with BMD and T-score at the femoral neck,hip,lumbar vertebrae,and overall（the lowest value in the femoral neck,hip,and lumbar vertebrae）.The Receiver Operating Characteristic curve analysis showed that CAP1had better performance in differentiating normal BMD from osteopenia,normal BMD from osteoporosis,osteoporosis from non-osteoporosis thanβisomer of C-terminal telopeptide of typeⅠcollagen,N-terminal propeptide of type I procollagen,and osteocalcin.Conclusion1.Type I collagen active peptide-CAP1 exists in the bone resorption microenvironment,which can stimulate osteoclast differentiation at low dose and inhibit osteoclast differentiation at high dose by binding to OSCAR on osteoclast precursor cells.The bone-targeting CAP1constructed by conjugating（D-Asp）₈ with CAP1 can promote bone resorption and lead to bone mass loss when given at a low dose,while at a high dose,it can inhibit bone resorption and improve the bone mass of OVX mice,which provides a novel approach for the treatment of osteoporosis.2.CAP1 can serve as a new bone turnover marker to reflect the state of bone metabolism,which will help to improve the early diagnosis of patients at a high risk of osteoporosis.