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 (TNFα) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model. METHODS: Meniscal explant disks (3 mm diameter × 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFα-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: TNFα induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFα 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 TNFα-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFα-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFα-dependent GAG release and NITEGE production, whereas inhibition of TNFα-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 TNFα-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFα-dependent source for MMP-3. However, the TNFα-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.