Annexin A2, up-regulated by IL-6, promotes the ossification of ligament fibroblasts from ankylosing spondylitis patients.

BACKGROUND: Annexin A2, a calcium-dependent phospholipid binding protein, is involved in osteogenesis. The objective of the present study was to explore the expression of Annexin A2 in spinal ligament tissues (LT) of ankylosing spondylitis (AS) patients and determine its pathological functions. METHODS: mRNA and protein expression of Annexin A2 was detected by real-time PCR and Western blotting, respectively. Interleukin-6 (IL-6) concentration in serum was assessed by enzyme linked immunosorbent assay. Alkaline phosphatase (ALP) activity was measured with ALP activity kit on a microplate reader. RESULTS: mRNA and protein expression of Annexin A2 in LT, and IL-6 concentration in serum were significantly increased in AS patients. Moreover, exogenous IL-6 treatment significantly up-regulated Annexin A2 expression and ALP activity. Silencing of Annexin A2 expression significantly ameliorated IL-6-induced ossification of fibroblasts from AS patients, as indicated by ALP activity, expression of proteins associated with osteogenic differentiation, including bone morphogenetic protein-2, osteocalcin and osterix, and the ratio of osteoprotegerin to receptor activator of NF-κB ligand. Further MEK inhibitor experiments suggested that Annexin A2 may exert its function through extracellular signal-related kinase pathway. CONCLUSIONS: Annexin A2, up-regulated by IL-6, may promote ligament ossification of AS patients.

Up-regulation of fatty acid oxidation in the ligament as a contributing factor of ankylosing spondylitis: A comparative proteomic study.

OBJECTIVES: The present study first utilized a standardized shotgun proteomic analysis method to determine differences in protein expression of fibroblasts in the ligament between AS patients and healthy controls. METHODS: Proteins extracted from primarily cultured FLLs from 35 AS patients and 10 normal subjects were analyzed by automated 2D-Nano-LC-ESI-MS/MS. Differentially expressed proteins were screened by 2-sample t-test and fold change. Bioinformatics analysis of differentially expressed proteins was based on the IPA. Fatty acid ß-oxidation-related proteins and INSR pathway-related proteins in the ligament were confirmed by real-time PCR and Western blot. RESULTS: A total of 556 differential proteins were screened in AS. Of them, 322 proteins were up-regulated and the remaining 234 proteins were down-regulated. GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in FLL cells, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. CONCLUSION: The lower body fat level in AS maybe due to up-regulation of fatty acid ß-oxidation-related enzymes regulated by INSR/PI3K/PKC pathway. BIOLOGICAL SIGNIFICANCE: Ankylosing spondylitis (AS), a common spondyloarthropathy, is an inflammatory rheumatic disease with a predilection for the axial skeleton. Clinical hallmarks of AS include sacroiliitis, uveitis, enthesitis and persistent spinal inflammation. The pathogenic mechanism of disease causation and perpetuation remains poorly understood. In this study, we primarily cultured fibroblast cells from ligament biopsies, knowing that fibroblast cells are dominant cells in the diseased ligament. One of the characteristic pathologic changes in AS is inflammation of the attachment points, including the muscle, ligament and bone or joint capsule. Inflammation of the tendon attachment point is usually non-bacterial and can lead to pain and swelling of the tendon ligament. To obtain more information, we used Shotgun proteomic analysis based on multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS). we firstly mixed the lysates of FLL cells derived from the ligaments of 35 AS patients and 10 normal subjects, identified proteins by automated 2D-Nano-LC-ESI-MS/MS method, GO and pathway analyses showed that six fatty acid ß-oxidation-related proteins (HADHB, ECHS1, ACSL4, ACADM, ACSL1 and HADH) were up-regulated in the ligament, which was consistent with the results obtained from real-time PCR, Western blot and MS, while INSR pathway-related proteins (INSR, IRS1, PI3K and PKC) was low in the ligament of AS as compared with that in healthy controls. We also find that AS subjects had significantly lower body mass index (BMI) and BMI Z-scores compared with that in healthy controls. The results remind us that up-regulation of fatty acid ß-oxidation-related proteins lower the body fat content, which is a new discovery contributing to the progression of AS. This is the first report on fatty acid oxidation in AS. It was found that the body fat level was low in AS due to high fatty acid oxidation, suggesting that insulin signaling may play an important role in the metabolic switch from predominant to fatty acid metabolism that characterizes the ligament of AS. One mechanism for this transition is increased expression of genes that regulate the rate of fatty acid oxidation. This effect may be mediated by PI3K, a downstream mediator of many receptor tyrosine kinases, including the INSR. This is a newly discovered factor contributing to the progression of AS.