Cutting Edge: The Metalloproteinase ADAM17/TNF-α-Converting Enzyme Regulates Proteolytic Shedding of the MHC Class I-Related Chain B Protein

Abstract

MHC class I-related chain (MIC)AT he MHC class I-related chain (MIC) 4 A and B proteins are polymorphic MHC-related molecules that bind the activating receptor NKG2D. Engagement of NKG2D by these ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells and thus plays a central role in immune system activation (for review, see Refs. 1 and 2). The vast majority of healthy cells do not express MIC molecules and instead their expression at the cell surface is up-regulated in pathological situations like cancer and autoimmunity; so, broadly, MICA/B expression is related to stress, although much remains unclear about the molecular mechanisms regulating their expression (3).The interaction between NKG2D and its ligands plays an important role in the immunosurveillance of cancer and tumor control. Thus, it is not surprising that tumor cells have evolved mechanisms to inhibit this system. Cytokines such as TGF-␤ can provoke down-regulation of NKG2D and its ligands (4, 5). Moreover, NKG2D ligands can be shed from tumor cells and have been detected in sera from patients with various types of cancer. Soluble NKG2D ligands appears to impair immune surveillance by promoting down-regulation of NKG2D (6), and high levels of soluble MIC (sMIC) molecules in sera correlate strongly with poor clinical outcome in patients suffering from various types of cancer, including colon (7) and prostate cancers (8). The mechanisms that regulate shedding of MI-CA/B are not well understood, but inhibition of cellular metalloproteinase activity markedly interferes with their release (9 -12), and recently shedding of MICA has also been shown to depend on ERp5, a thiol isomerase (13).In this study, we report that the ADAM (a disintegrin and metalloproteinase) family member ADAM17, also known as TNF-␣-converting enzyme (TACE), is involved in the shedding of soluble MICB and that regions of the membrane enriched in cholesterol and sphingolipids, also known as detergent-resistant membrane microdomains (DRMs), are an important site for this proteolysis. Materials and Methods Experimental proceduresA detailed description of the experimental procedures is provided in the supplemental data online. 5ELISA sMICB was detected using a sandwich ELISA procedure. Plates were coated with an anti-MICB mAb from R&D Systems (5 g/ml) and blocked with 2% BSA-PBS. Tissue culture supernatant was added for 1 h at 37°C. Bound MICB protein was detected using biotinylated goat anti-MICB (R&D Systems) followed by streptavidin-HRP (Amersham) and developed using the peroxidase substrate system (ABTS; Roche). The absorbance was measured at 410 nm with a reference wavelength of 490 nm. Samples were analyzed in duplicates. Under these conditions, the cutoff for detection of recombinant sMICB (BioSupply

Genotoxic Stress Induces Senescence-Associated ADAM10-Dependent Release of NKG2D MIC Ligands in Multiple Myeloma Cells

“…Several studies, primarily performed in different types of tumor cell lines or transfected cells in steady-state conditions, indicate the direct involvement of both ADAM10 and ADAM17 enzymes in MICA and MICB release, but the relative contribution of these enzymes is still controversial. Boutet et al (32) found that ADAM17 mediates MICB proteolytic cleavage in CV1 epithelial transfectants; however, recent evidence highlights a cell type-specific role for these metalloproteinases (33). Our results also reveal high levels of ADAM10 expression in several MM cell lines and in primary malignant PCs, with enzyme expression not correlating with the disease stage.…”

“…To investigate whether drug-induced MICB release involves metalloproteinase activity (32,33), we evaluated MICB levels in the supernatants of DOX-and MEL-treated SKO-007(J3) cells in the presence of the metalloproteinase inhibitor marimastat. As MICA gene typing was performed as described in Materials and Methods.…”

“…The relative roles for ADAM10 and ADAM17 metalloproteinases on MIC shedding were primarily investigated in steady-state conditions in a variety of tumor cell lines or transfected cells (9,(32)(33)(34). To identify the ADAM enzymes responsible for the druginduced MICB release, we examined whether the inhibition of ADAM10 and ADAM17 expression on drug-treated MM cells interfered with MICB shedding.…”

Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands

“…Recently, members of the ADAM (a disintegrin and metalloproteinase) family have been identified as key proteases involved in the shedding of some alleles of MICA and MICB (21,22), and a member of the disulfide isomerase family, ERp5, has also been proposed to play a role in the shedding of MICA (23). ADAMs 10 and 17 have been shown to be involved in proteolytic cleavage of ULBP2 (22), but nothing else is known about the shedding of other ULBPs.…”

Shaping of NK Cell Responses by the Tumor Microenvironment

“…Many ADAMs are overexpressed in tumors and affect different steps of tumor progression [117]. NK cell recognition of target cells is hampered by ADAM10 and ADAM17 activation, which were shown to be involved in the proteolytic release of soluble MICA and MICB from tumor cells [118,119]. Grzywacz et al suggested that NK cell encounter of target cells led to activation of MMPs that subsequently shed CD16 from the surface of NK cells [120].…”