Differential gene expression and regulation of the bone morphogenetic protein antagonists follistatin and gremlin in normal and osteoarthritic human chondrocytes and synovial fibroblasts

Abstract

Objective. To compare gene expression in normal and osteoarthritic (OA) human chondrocytes using microarray technology. Of the novel genes identified, we selected follistatin, a bone morphogenetic protein (BMP) antagonist, and investigated its expression/regulation as well as that of 3 other antagonists, gremlin, chordin, and noggin, in normal and OA chondrocytes and synovial fibroblasts.Methods. Basal and induced gene expression were determined using real-time polymerase chain reaction. Gene regulation was monitored following treatment with inflammatory, antiinflammatory, growth, and developmental factors. Follistatin protein production was measured using a specific enzyme-linked immunosorbent assay, and localization of follistatin and gremlin in cartilage was determined by immunohistochemical analysis.Results. All BMP antagonists except noggin were expressed in chondrocytes and synovial fibroblasts. Follistatin and gremlin were significantly up-regulated in OA chondrocytes but not in OA synovial fibroblasts. Chordin was weakly expressed in normal and OA cells. Production of follistatin protein paralleled the gene expression pattern. Follistatin and gremlin were expressed preferentially by the chondrocytes at the superficial layers of cartilage. Tumor necrosis factor ␣ and interferon-␥ significantly stimulated follistatin expression but down-regulated expression of gremlin. Interleukin-1␤ (IL-1␤) had no effect on follistatin but reduced gremlin expression. Conversely, BMP-2 and BMP-4 significantly stimulated expression of gremlin but down-regulated that of follistatin. IL-13, dexamethasone, transforming growth factor ␤1, basic fibroblast growth factor, platelet-derived growth factor type BB, and endothelial cell growth factor down-regulated the expression of both antagonists.Conclusion. This study is the first to show the possible involvement of follistatin and gremlin in OA pathophysiology. The increased activin/BMP-binding activities of these antagonists could affect tissue remodeling. The data suggest that follistatin and gremlin might appear at different stages during the OA process, making them interesting targets for the treatment of this disease.Osteoarthritis (OA) is a progressive, debilitating disease of the joints characterized by the erosion of articular cartilage. Although much is known about the expression and regulation of genes associated with OA (e.g., metalloprotease, tissue inhibitor of metalloproteases, inflammatory cytokines, and extracellular matrix proteins such as collagens and proteoglycans), it is likely that the expression of many other genes is also affected during this pathologic process, and more is yet to be learned in order to gain a comprehensive understanding of this disease.

A sodium dodecyl sulfate–polyacrylamide gel electrophoresis–liquid chromatography tandem mass spectrometry analysis of bovine cartilage tissue response to mechanical compression injury and the inflammatory cytokines tumor necrosis factor α and interleukin‐1β

“…We also observed proteins recently described in cartilage, including cytokine-like protein C17, connective tissue growth factor, IL-17B, scrapie-responsive protein 1 (SCRG-1), and follistatin-like protein (18,25,26). Other proteins, such as CD109, plateletderived growth factor receptor-like (PDGFR-like), angiopoietin-like 7, and peptidoglycan-recognition protein long (PGRP-L), represent novel cartilage proteins.…”

Differential modulation of RANKL isoforms by human osteoarthritic subchondral bone osteoblasts: Influence of osteotropic factors

“…The RNA was quantitated using the RiboGreen RNA quantitation kit. The RT reactions were primed with random hexamers as described previously [29]. According to the published RANKL sequence (hRANKL1, gi: 18143618; hRANKL2, gi: 16610212; hRANKL3, gi: 21536432), primers were designed to amplify specifically RANKL1 and RANKL3.…”

Activation of the receptor EphB4 by its specific ligand ephrin B2 in human osteoarthritic subchondral bone osteoblasts

“…RNA was quantified using a RiboGreen RNA quantification kit. The reverse transcription reactions were primed with random hexamers as previously described (30).…”

“…The CASE assay includes a complete antibody-based detection system for colorimetric quantification of the relative amount of phosphorylated protein. Briefly, following treatment with ephrin B2 ligand at 100 ng/ml for different time periods (5,15,30, and 60 minutes), the cells were fixed to preserve any activation-specific protein modification. The procedure was performed according to the manufacturer's specifications (SuperArray Bioscience), and the amount of bound antibody was determined using a developing solution and an ELISA plate reader.…”