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

Ossification of 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 mouse model to study the molecular mechanisms underlying OPLL. CXCL7-null mice presented with a phenotype of OPLL, showing motor impairment, heterotopic ossification in the posterior ligament tissue, and osteoporosis in vertebrate tissue. To identify the mechanisms of CXCL7 deficiency 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 CXCL7 levels. 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 ubiquitin and ubiquitin-like modifiers, orchestrated by a cascade of specialized ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin ligase (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 cells expressed the antibody to ubiquitin (linkage-specific K48). Our data clearly show decreased CXCL7 levels 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 ligament ossified tissue and primary cells from CXCL7-null mice. We performed a phosphoproteomics analysis in CXCL7-deficient mice and identified increased phosphorylation of mitogen-activated protein kinase kinase (ME3K)15, ubiquitin protein ligase E3C (UBE3C) and protein kinase C (PKC) alpha, suggesting that ubiquitin-dependent degradation is involved in CXCL7 deficiency. Future studies in the CXCL7-null mouse model are, therefore, warranted to investigate the role of ubiquitination in the onset of OPLL. In conclusion, CXCL7 levels may be useful as a serum marker for the progression of OPLL. This study also suggests that increasing CXCL7 levels in patients can serve as an effective therapeutic strategy for the treatment of OPLL.


Immunostaining
We obtained 5-µm-thick sections of ossification ligament, pancreas, fat, and testis from the CXCL7 knockout mice and performed fixation with 4% paraformaldehyde.   Protein A-bound K48 antigen-antibody complex was finally extracted using the DynaMag™-2 magnet (Thermo Fisher Scientific), washed 3 times with wash buffer, and then analyzed by western blotting [3]. As a control, PBS was added to the mixture of Dynabeads Protein A magnetic beads and K48 antibodies.

Separation of white blood cells
Blood from healthy volunteers and OPLL patients was collected in PAXgene blood collection tubes (Becton Drive, Franklin Lakes, NJ, USA) and stored frozen at −80°C. Thawed samples were thawed at room temperature for 2 hours at room temperature and centrifuged at 4300 × g for 10 min in a swing rotor centrifuge (Kubota 5911, Tokyo, Japan; http://www.centrifuge.jp/products/model-5911/). The supernatant 6 / 14 was discarded, sterilized water was added to the pellets, and the erythrocytes were hemolyzed. The suspension was then centrifuged at 4300 g for 10 minutes, the supernatant was discarded, and white blood cells were extracted into the resuspension solution.

Western blotting
Cell lysates were subjected to SDS-PAGE and proteins were then transferred to a polyvinylidene difluoride membrane (0. The ChemiDoc Touch system (Bio-Rad) was used to visualize protein bands, which were then analyzed with imaging software (Bio-Rad).

Supplementary Discussion
Many OPLL patients develop diabetes and their blood sugar must be controlled prior to surgery. A lack of chemokines is associated with the development of OPLL, but the underlying mechanism is unclear, given that no abnormalities have been observed by exon sequencing or SNP analysis. Our microRNA analysis revealed that high miR-340 levels are present in OPLL patients compared to the levels in healthy subjects Furthermore, a proteome phosphorylation analysis was performed because of the inability to establish a direct correlation with the lack of the CXCL7 protein by SNP and microRNA. CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif are crucial mediators in neutrophil-dependent acute inflammation. NAP-2/CXCL7 is an NH2-terminally processed form of β-thromboglobulin (β-TG), which in turn is a cleavage product of the platelet basic protein (PBP) precursor derived from platelets, along with connective-tissue activating peptide-III (CTAP-III) and PBP [4]. We

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performed a kidney tissue RNA array analysis to determine the molecular mechanisms underlying OPLL caused by CXCL7 deficiency. Factors involved in gluconeogenesis, the acquisition of natural immunity, and MAPK and p53 signaling were highly represented in the DAVID pathway analysis v6.7 (https://david.ncifcrf.gov/) ( Table 3).
These results are consistent with the notion that induction of autophagy genes in response to p53 activation is associated with enhanced autophagy in diverse settings and depends on p53 transcriptional activity [5]. During chemokine degradation by the ubiquitin-proteasome system in CXCL7-null mice, the expression of Toll-like receptor 3, signaling through the downstream tumor necrosis factor receptor-associated factor (TRAF)3 and MAPK kinase kinase (MEKK)3, resulted in a modulation of E3 ubiquitin ligase activity. Posterior longitudinal ligament ossification observed in CXCL7-null mice is thought to be caused by osteoclast dysfunction owing to a decrease in TRAF6 [6][7][8]. Among the de-ubiquitinating (DUB) enzymes, DUB has been shown to removes the K63 polyubiquitin chain of TRAF6 and inhibits protein degradation from K48 polyubiquitination to suppress NF-κB activity [9][10][11]. This report suggests that the degradation of CXCL7 in this study is due to K48 polyubiquitination, which is a

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Our findings provide insight into the molecular basis of OPLL and suggest that increasing CXCL7 levels in patients could serve as an effective therapeutic strategy for the treatment of this disease.
DUB UCHL1 is strongly up-regulated, and inhibition of UCHL1 has been found to exacerbate rather than ameliorate the disease in a mouse model of spinal muscular atrophy (SMA) [12]. Although complex interactions of genetic and environmental factors underlie human neurodegenerative diseases, many of these conditions are suggested to share a common molecular signature: collapse of protein homeostasis [13,14]. Parameters for cortical bone were measured at the midpoint of the tibia. Data are expressed as the mean ± SEM of eight bones/group. Abbreviations related to osteocyte parameters in Table 2 are described in S1 Table. As a functional evaluation of the limbs in CXCL7 knockout mice, we present here videos of the animals walking and swimming. For the walking analysis, an 12 / 14 appropriately sized transparent cylinder was used, with both ends being sealed with a film containing air holes to prevent the animals from escaping. For the swimming analysis, it was necessary to evaluate limb function to detect any differences from a healthy mouse. A tank with an optimum depth and width so as not to stress the animals, was prepared. The water temperature was kept at 34°C, and the mouse was allowed to float for only 17 seconds. In addition, the laboratory room temperature was maintained at 27°C. After the experiment, a hot air dryer was used to quickly dry the animals to keep them in a good physical state.