Identification of novel bone-resorption markers by osteoclasts in vitro and in vivo

Bone resorption involves dissolution of mineral and enzymatic degradation of bone matrix. The primary enzyme is cathepsin K but other proteases including matrix metalloproteinases are involved. Some cleavage products of cathepsin K have been partially identified, including crossed-linked telopeptides of type I collagen. However, the pathway of type I bone collagen degradation has not been fully elucidated. The aim of this study was to comprehensively characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in vitro. Complete characterisation of these breakdown products will advance understanding of osteoclast biology and has the potential to reveal new biomarkers of bone resorption. We analysed extracellular media from osteoclasts cultured on dentine substrates, using untargeted liquid chromatography mass spectrometry. We discovered 22 breakdown products resulting from osteoclastic action. These products were peptide fragment sequences that mapped to various collagen proteins present in bone and dentine matrix. Nine peptide fragments mapped exclusively to collagen I alpha-1 chain (COL1A1), the most abundant protein in bone. Analysis of the reported cleavage sites in the COL1A1 protein sequence indicated 7/9 COL1A1-specific fragments not explained by known proteolytic events. We subsequently showed that 14 of the fragment products were present in human serum and/or urine from metabolomic datasets obtained from patients with the inherited metabolic disease alkaptonuria (serum) and lung cancer (urine). Two products were at higher concentration (P <0.05, fold change >2) in urine from patients with bone metastasis (29/112) from the lung cancer cohort. The range of collagen peptide fragments we discovered as a direct result of osteoclast activity indicates a complexity of bone resorption pathways not previously known. Monitoring the concentrations of these novel bone markers in biofluids has the potential to capture multiple pathways of bone resorption activity beyond the existing assays based on Cathepsin K. Lay summaryBreakdown of bone tissue is performed by specialised bone cells called osteoclasts in a process known as bone resorption. Knowledge of specific molecules produced from osteoclasts acting on bone is important for a) understanding bone resorption in health and disease, and b) clinical tests of bone resorption from measurement of these breakdown products. Here we aimed to characterise the entire complement of bone breakdown products resulting from osteoclast action under controlled conditions in the laboratory. We found a total of 22 breakdown products produced from osteoclasts cultured on wafers of dentine, a tissue with almost identical composition to bone. Analysis of the structures of these products revealed fragments of varying size produced from digestion of specific proteins present in bone. We then showed that 14/22 bone protein fragments observed in our cell culture experiments were also present in human blood and/or urine. In our analysis of urine from patients with lung cancer, we found that two of the protein fragments we identified were higher in a subset (29/112) of patients with bone metastasis. Our findings provide new insights into the mechanisms of bone resorption and show the potential for monitoring the range of urine bone protein fragments in bone disorders.