Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification

Ossificationof the posterior longitudinal ligament(OPLL), a spinal ligament, reduces the range of motion in limbs. No treatment is currently available for OPLL, which is why therapies are urgently needed. OPLL occurs in obesity, is more common in men, and has an onset after 40 years of age. The mechanisms underlying OPLL remain unclear. In this study, we performed a serum proteomic analysis in both OPLL patients and healthy subjects to identify factors potentially involved in the development of OPLL, and found reduced levels of a protein that might underlie the pathology of OPLL. We isolated the protein, determined its amino acid sequence, and identified it as chemokine (C-X-C motif) ligand 7 ( CXCL7). Based on these proteomics findings, we generated a CXCL7 knockout mousemodel to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossificationin the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7deficiency in OPLL, we searched for single nucleotide polymorphisms and altered DNA exons, but no abnormalities were found. Although miR-340 levels were found to be high in an miRNA array, they were insufficient to reduce CXCL7levels. Ubiquitin C-terminal hydrolase1 (UCHL1) was found to be overexpressed in CXCL7-null mice and in the sera of patients with OPLL, and was correlated with OPLL severity. Post-translational modifications of proteins with ubiquitinand ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme(E1), ubiquitin conjugating enzyme(E2), and ubiquitinligase (E3) enzymes, are thought to control a wide range of cellular processes, and alterations in the ubiquitin–proteasome system have been associated with several degenerative disorders. In addition, the OPLL tissue of CXCL7-null mouse and its primary cellsexpressed the antibody to ubiquitin(linkage-specific K48). Our data clearly show decreased CXCL7levels in patients with OPLL, and that OPLL developed in mice lacking CXCL7. Tumor necrosis factor receptor-associated factor (TRAF)6 expression was decreased in CXCL7-null mouse primary cells. Furthermore, K48 polyubiquitination was found in posterior longitudinal ligamentossified tissue and primary cellsfrom CXCL7-null mice. We performed a phosphoproteomicsanalysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase(ME3K)15, ubiquitinprotein ligase E3C (UBE3C) and protein kinase C ( PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitinationin the onset of OPLL. In conclusion, CXCL7levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.