Tumor necrosis factor alpha-dependent aggrecan cleavage and release of glycosaminoglycans in the meniscus is mediated by nitrous oxide-independent aggrecanase activity in vitro

INTRODUCTION Little is known about factors that induce meniscus damage. Since joint inflammation appears to be a causative factor for meniscal destruction, we investigated the influence of tumor necrosis factor (TNFalpha) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model. METHODS Meniscal explant disks (3 mm diameter x 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFalpha-treatment GAG release (DMMB assay), biosynthetic activity (sulfate incorporation), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR, zymography), and immunostaining of the aggrecan fragment NITEGE were determined. RESULTS TNFalpha induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFalpha increased matrix metalloproteinase (MMP)-3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 mRNA expression, whereas collagen type I was decreased, and aggrecan, collagen type II as well as MMP-1, -2, -13 and ADAMTS-5 were variably affected. Zymography also showed a TNFalpha-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFalpha-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFalpha-dependent GAG release and NITEGE production, whereas inhibition of TNFalpha-dependent NO generation with the NO-synthetase inhibitor L-NMMA failed to inhibit GAG release and NITEGE production. CONCLUSIONS Our study shows that aggrecanase activity (a) is responsible for early TNFalpha-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFalpha-dependent source for MMP-3. However, the TNFalpha-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.