CD30 shedding from Karpas 299 lymphoma cells is mediated by TNF-alpha-converting enzyme

CD30 is a costimulatory receptor on activated lymphocytes and a number of h uman lymphoma cells. Specific ligation of membrane-bound CD30 or cellular s timulation by PMA results in a metalloproteinase-mediated down-regulation o f CD30 and release of its soluble ectodomain (sCD30), In this report, it is demonstrated that PMA-induced CD30 cleavage from Karpas 299 cells was medi ated by a membrane-anchored metalloproteinase which was active on intact ce lls following 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ext raction of membrane preparations. Moreover, CD30 shedding was blocked by th e synthetic hydroxamic acid-based metalloproteinase inhibitor BB-2116 (IC50 , 230 nM) and the natural tissue inhibitor of metalloproteinases (TIMP)-3 ( IC50, 30 nM), but not by the matrix metalloproteinase inhibitors TIMP-1 and TIMP-2, This inhibition profile is similar to that of the TNF-alpha- conve rting enzyme (TACE) and, indeed, mRNA transcripts of the membrane-bound met alloproteinase-disintegrin TACE could be detected in Karpas 299 cells. The ectodomain of TACE was expressed in bacteria as a GST fusion protein (GST-T ACE) which cleaved CD30 from the surface of Karpas 299 cells and concomitan tly increased the level of sCD30 in the cell supernatants. Hence, TACE does not only control the release of TNF-alpha, but also that of sCD30.