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 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-SPCdeltaDETCy), expressed in baculovirus could cleave preferentially (approximately 12-fold) the TNF-specific peptide over the matrix 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 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-alpha mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-alpha and IL-8 in ex vivo conditions. Addition of TNF-alphaR fused to IgG Fc fragment (TNF-alphaR: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-alpha in OA-affected cartilage that may depend, in part, on up-regulated levels of chondrocyte-derived TACE.
Patel
I R
IR
Department of Rheumatology and Medicine, Hospital for Joint Diseases, New York, NY 10003, USA.
Attur
M G
MG
Patel
R N
RN
Stuchin
S A
SA
Abagyan
R A
RA
Abramson
S B
SB
Amin
A R
AR
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D001172:Arthritis, Rheumatoid / Q000201:enzymology / Q000235:genetics*; D002356:Cartilage / Q000201:enzymology*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000302:isolation & purification; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D006801:Humans; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D017421:Sequence Analysis; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D018076:DNA, Complementary; D014409:Tumor Necrosis Factor-alpha; D051722:ADAM Proteins; D008666:Metalloendopeptidases; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human