TNF-ALPHA CONVERTASE ENZYME FROM HUMAN ARTHRITIS-AFFECTED CARTILAGE -ISOLATION OF CDNA BY DIFFERENTIAL DISPLAY, EXPRESSION OF THE ACTIVE ENZYME, AND REGULATION OF TNF-ALPHA

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-alpha convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine sw itch, 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-SPC Delta DETCy), expressed in baculovirus could cleave p referentially (similar to 12-fold) the TNF-specific peptide over the m atrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-alpha to TNF-alpha (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differen tially expressed in a variety of human tissues, is up-regulated in art hritis-affected cartilage, but not normal cartilage. OA-affected carti lage also expressed TNF-alpha U mRNA that was not detected in normal c artilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alp ha R fused to IgG Fc fragment (TNF-alpha R:Fc) in the presence or abse nce of soluble IL-1R (with which it acted additively) significantly at tenuated the spontaneous/autocrine release of articular IL-8 in this a ssay. These experiments demonstrate a functional paracrine/autocrine r ole of TNF-alpha in OA-affected cartilage that may depend, in part, on upregulated levels of chondrocyte-derived TACE.