TNF-α Convertase Enzyme from Human Arthritis-Affected Cartilage: Isolation of cDNA by Differential Display, Expression of the Active Enzyme, and Regulation of TNF-α

Abstract

A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-α convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal + prodomain + catalytic region (TACE-SPCΔDETCy), expressed in baculovirus could cleave preferentially (∼12-fold) the TNF-specific peptide over the matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-α to TNF-α (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-α mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-α and IL-8 in ex vivo conditions. Addition of TNF-αR fused to IgG Fc fragment (TNF-αR:Fc) in the presence or absence of soluble IL-1R (with which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These experiments demonstrate a functional paracrine/autocrine role of TNF-α in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE.