Evaluation of the Contribution of Different ADAMs to Tumor Necrosis Factor α (TNFα) Shedding and of the Function of the TNFα Ectodomain in Ensuring Selective Stimulated Shedding by the TNFα Convertase (TACE/ADAM17)

Abstract

Tumor necrosis factor-␣ (TNF␣), a potent proinflammatory cytokine, is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-␣ converting enzyme (TACE; a disintegrin and metalloprotease17; ADAM17) is known to have a key role in the ectodomain shedding of TNF␣ in several cell types. However, because purified ADAMs 9, 10, and 19 can also cleave a peptide corresponding to the TNF␣ cleavage site in vitro, these enzymes are considered to be candidate TNF␣ sheddases as well. In this study we used cells lacking ADAMs 9, 10, 17 (TACE), or 19 to address the relative contribution of these ADAMs to TNF␣ shedding in cell-based assays. Our results corroborate that ADAM17, but not ADAM9, -10, or -19, is critical for phorbol ester-and pervanadate-stimulated release of TNF␣ in mouse embryonic fibroblasts. However, overexpression of ADAM19 increased the constitutive release of TNF␣, whereas overexpression of ADAM9 or ADAM10 did not. This suggests that ADAM19 may contribute to TNF␣ shedding, especially in cells or tissues where it is highly expressed. Furthermore, we used mutagenesis of TNF␣ to explore which domains are important for its stimulated processing by ADAM17. We found that the cleavage site of TNF␣ is necessary and sufficient for cleavage by ADAM17. In addition, the ectodomain of TNF␣ makes an unexpected contribution to the selective cleavage of TNF␣ by ADAM17: it prevents one or more other enzymes from cleaving TNF␣ following PMA stimulation. Thus, selective stimulated processing of TNF␣ by ADAM17 in cells depends on the presence of an appropriate cleavage site as well as the inhibitory role of the TNF ectodomain toward other enzymes that can process this site. TNF␣1 is a pro-inflammatory cytokine that has a critical role in autoimmune disorders such as rheumatoid arthritis and Crohn's disease (1, 2). TNF␣ is synthesized as a trimeric type II membrane-anchored precursor referred to as pro-TNF␣ (3). Upon cleavage in the juxtamembrane domain, the mature form of TNF␣ is released from the cell and can enter the blood stream (4, 5). This proteolytic release of TNF␣ from the membrane is referred to as "protein ectodomain shedding" (6, 7). Protein ectodomain shedding also affects the function of a variety of other structurally and functionally diverse molecules on the cell surface, including cytokines and growth factors, their receptors, adhesion proteins, and other molecules, such as the amyloid precursor protein, Notch and Delta (6 -9).Because of the critical role of TNF␣ in rheumatoid arthritis, considerable efforts have been made to identify the TNF␣ convertase. ADAM17 (a disintegrin and metalloprotease 17, also referred to as TNF␣ converting enzyme or TACE) is considered to be an important, if not the major, sheddase for TNF␣ (10, 11). ADAM17 was initially purified based on its ability to process a peptide, which mimics the physiological cleavage site of TNF␣, in exactly the same position that is used by the TNF␣ converting activity in cells (see Table I and Refs. 10 and 11). A targeted de...

The Fas ligand intracellular domain is released by ADAM10 and SPPL2a cleavage in T-cells

“…1 Matrix metalloproteinases have been reported to be involved in the release of sFasL, and several matrix metalloprotease (MMP) 7 (matrilysin) cleavage sites have been identified in the human and murine FasL sequences ( 20 and references therein). However, as the ADAM family members ADAM10 and ADAM17 have been implicated in TNFa processing, 21 we examined a potential role for both proteases in FasL shedding and production of APL in 293 cells. In the first experiment, we used the metalloproteinase inhibitor GI254023X, which preferentially blocks ADAM10, 22 on stable 293 transfectants overexpressing hFasL, and analyzed FasL processing by immunoblotting.…”

Expression of ADAMs (“a disintegrin and metalloprotease”) in the human lung

“…This is exemplified by both ADAM17 and ADAM19, which are both capable of shedding TNF-alpha. ADAM17 plays a key role in this release of soluble TNF-alpha by cleavage of the membraneanchored precursor of TNF-alpha [16][17][18]. TNF-alpha is a proinflammatory cytokine and a key mediator in immune defense with a role in induction and amplification of inflammation, as a response to external, potential threatening stimuli.…”

Overexpression of sigma-1 receptor inhibits ADAM10 and ADAM17 mediated shedding in vitro

“…PMA (phorbol 12-myristate 13-acetate) is a known activator of ADAM17 through protein kinase C (PKC) activation (Zheng et al, 2002(Zheng et al, , 2004Sahin et al, 2004). Therefore, we used PMA induced HB-EGF shedding to check whether ADAM17-dependent shedding is affected by sigma-1 receptors or not.…”

“…Since ADAM10 function is induced by free Ca 2+ in cell (Sanderson et al, 2005;Horiuchi et al, 2007), such inhibition could thus inhibit ADAM10-dependent BTC shedding. On the other hand, although sigma-1 receptor agonists could activate PKC (Fu et al, 2010;Yoon et al, 2010) which can in turn activate ADAM17-dependent shedding (Zheng et al, 2002(Zheng et al, , 2004Sahin et al, 2004), such an effect might be covered by the Figure 3. ADAM10-depedent BTC shedding is more sensitive to membrane lipid change while ADAM17-dependent HB-EGF shedding was not.…”

“…The alkaline phosphatase tagged substrate shedding assay has been described previously (Zheng et al, 2002(Zheng et al, , 2004Sahin et al, 2004). In brief, cells seeded in six-well tissue culture plates (Corning) were transfected with the appropriate expression plasmids using Fugene6 reagent (Roche).…”