Ubiquitin-dependent proteolysis of CXCL7 leads to posterior longitudinal ligament ossification
2018
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.
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