No abstract

In articular chondrocytes, the inflammatory cytokine tumor necrosis factor-␣ (TNF-␣) induces the expression of bone morphogenetic protein-2 (BMP-2), a growth factor known to be involved in the induction of cartilage and bone. A study was performed to clarify the mechanism(s) underlying the induction of BMP-2 in chondrogenic ATDC5 cells and primary cultured adult human articular chondrocytes. In ATDC5 cells, the endogenous BMP-2 expression was consistently low throughout the process of chondrogenic differentiation, and TNF-␣ induced BMP-2 expression only after the cells acquired the chondrogenic phenotype. The results of nuclear run-off assay and cycloheximide treatment consistently indicated that ATDC5 cells acquire the capacity to synthesize BMP-2 mRNA in the nuclei during the differentiation process. In an attempt to explain the discrepancy between the active nuclear mRNA synthesis and the observed low expression level in differentiated ATDC5 cells, the stability of BMP-2 mRNA was evaluated, and the cells were found to regulate the expression of BMP-2 at the post-transcriptional level. Human chondrocytes were confirmed to have a similar post-transcriptional regulation. The result of 3-rapid amplification of cDNA end revealed that both human and mouse BMP-2 mRNAs contain multiple pentameric AUUUA motifs in a conserved manner in the 3-untranslated regions, and transient transfection experiments demonstrated that TNF-␣ increases the stability of BMP-2 mRNA through the pentameric motifs. Further experiments revealed that TNF-␣ modulates mRNA stability via p38 signal transduction pathway, whereas the cytokine also augmented the expression of BMP-2 through transcriptional up-regulation via the transcriptional factor NF-B.

Objective. To determine the change in metabolic activity of chondrocytes in osteoarthritic (OA) cartilage, considering regional difference and degree of cartilage degeneration.Methods. OA cartilage was obtained from knee joints with end-stage OA, at both macroscopically intact areas and areas with various degrees of cartilage degeneration. Control cartilage was obtained from agematched donors. Using laser capture microdissection, cartilage samples were separated into superficial, middle, and deep zones, and gene expression was compared quantitatively in the respective zones between OA and control cartilage.Results. In OA cartilage, gene expression changed markedly with the site. The expression of cartilage matrix genes was highly enhanced in macroscopically intact areas, but the enhancement was less obvious in the degenerated areas, especially in the upper regions. In contrast, in those regions, the expression of type III collagen and fibronectin was most enhanced, suggesting that chondrocytes underwent a phenotypic change there. Within OA cartilage, the expression of cartilage matrix genes was significantly correlated with SOX9 expression, but not with SOX5 or SOX6 expression. In OA cartilage, the strongest correlation was observed between the expression of type III collagen and fibronectin, suggesting the presence of a certain link(s) between their expression.Conclusion. The results of this study revealed a comprehensive view of the metabolic change of the chondrocytes in OA cartilage. The change of gene expression profile was most obvious in the upper region of the degenerated cartilage. The altered gene expression at that region may be responsible for the loss of cartilage matrix associated with OA.

Tumor necrosis factor-alpha (TNF-alpha) is initially synthesized as a membrane-bound protein and converted into a soluble form by proteolytic cleavage. Although a disintegrin and metalloproteinase 17 (ADAM17) is considered to be the primary sheddase for TNF-alpha, it is not known whether ADAM17 is solely responsible for that process in any type of cells. To identify the TNF-alpha sheddase(s) in varieties of cells, we performed experiments using a unique screening system and observed that ADAM9, ADAM10, ADAM17, and ADAM19 were capable of cleaving TNF-alpha. We then performed RNA interference experiments and confirmed that ADAM10 and ADAM17 were in fact involved in TNF-alpha shedding in 293A cells. In mouse macrophages, ADAM17 was confirmed to be the primary sheddase, but the involvement of ADAM10 was also demonstrated. In NIH3T3 cells, ADAM10 could be more important in the shedding than ADAM17. In mouse vascular endothelial cell line UVfemale2, ADAM10 and ADAM17 were equally involved in TNF-alpha shedding, whereas ADAM17 was a major sheddase in human osteoarthritic chondrocytes. From these observations and others, we concluded that both ADAM10 and ADAM17 can be a TNF-alpha sheddase and that their significance could be determined by their expression levels and the abundance of tissue inhibitor of metalloproteinases.