The CX3C chemokine fractalkine (CX3CL1) exists as a membrane-expressed protein promoting cell-cell adhesion and as a soluble molecule inducing chemotaxis. Transmembrane CX3CL1 is converted into its soluble form by defined proteolytic cleavage (shedding), which can be enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). PMA-induced CX3CL1 shedding has been shown to involve the tumor necrosis factor-α–converting enzyme (TACE), whereas the constitutive cleavage in unstimulated cells remains elusive. Here we demonstrate a role of the closely related disintegrin-like metalloproteinase 10 (ADAM10) in the constitutive CX3CL1 cleavage. The hydroxamate GW280264X, capable of blocking TACE as well as ADAM10, proved to be an effective inhibitor of the constitutive and the PMA-inducible CX3CL1 cleavage in CX3CL1-expressing ECV-304 cells (CX3CL1–ECV-304), whereas GI254023X, preferentially blocking ADAM10 but not TACE, reduced the constitutive cleavage only. Overexpression of ADAM10 in COS-7 cells enhanced constitutive cleavage of CX3CL1 and, more importantly, in murine fibroblasts deficient of ADAM10 constitutive CX3CL1 cleavage was markedly reduced. Thus, ADAM10 contributes to the constitutive shedding of CX3CL1 in unstimulated cells. Addressing the functional role of CX3CL1 shedding for the adhesion of monocytic cells via membrane-expressed CX3CL1, we found that THP-1 cells adhere to CX3CL1–ECV-304 cells but detach in the course of vigorous washing. Inhibition of ADAM10-mediated CX3CL1 shedding not only increased adhesive properties of CX3CL1–ECV-304 cells but also prevented de-adhesion of bound THP-1 cells. Our data demonstrate that ADAM10 is involved in the constitutive cleavage of CX3CL1 and thereby may regulate the recruitment of monocytic cells to CX3CL1-expressing cell layers.

E-cadherin controls a wide array of cellular behaviors, including cell-cell adhesion, differentiation, and tissue development. We show here that E-cadherin is cleaved specifically by ADAM (a disintegrin and metalloprotease) 10 in its ectodomain. Analysis of ADAM10-deficient fibroblasts, inhibitor studies, and RNA interference-mediated down-regulation of ADAM10 demonstrated that ADAM10 is responsible not only for the constitutive shedding but also for the regulated shedding of this adhesion molecule in fibroblasts and keratinocytes. ADAM10-mediated E-cadherin shedding affects epithelial cell-cell adhesion as well as cell migration. Furthermore, the shedding of E-cadherin by ADAM10 modulates the β-catenin subcellular localization and downstream signaling. ADAM10 overexpression in epithelial cells increased the expression of the β-catenin downstream gene cyclin D1 dose-dependently and enhanced cell proliferation. In ADAM10-deficient mouse embryos, the C-terminal E-cadherin fragment is not generated, and the full-length protein accumulates, highlighting the in vivo relevance for ADAM10 in E-cadherin shedding. Our data strongly suggest that this protease constitutes a major regulatory element for the multiple functions of E-cadherin under physiological as well as pathological conditions.

Cadherins are critically involved in tissue development and tissue homeostasis. We demonstrate here that neuronal cadherin (N‐cadherin) is cleaved specifically by the disintegrin and metalloproteinase ADAM10 in its ectodomain. ADAM10 is not only responsible for the constitutive, but also for the regulated, shedding of this adhesion molecule in fibroblasts and neuronal cells directly regulating the overall levels of N‐cadherin expression at the cell surface. The ADAM10‐induced N‐cadherin cleavage resulted in changes in the adhesive behaviour of cells and also in a dramatic redistribution of β‐catenin from the cell surface to the cytoplasmic pool, thereby influencing the expression of β‐catenin target genes. Our data therefore demonstrate a crucial role of ADAM10 in the regulation of cell–cell adhesion and on β‐catenin signalling, leading to the conclusion that this protease constitutes a central switch in the signalling pathway from N‐cadherin at the cell surface to β‐catenin/LEF‐1‐regulated gene expression in the nucleus.

The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumor initiation by activating natural killer cells and costimulating CD8 T cells. NKG2D-mediated recognition of malignant cells by cytotoxic lymphocytes is enabled through the tumor-associated expression of NKG2D ligands (NKG2DL) resulting from cellular or genotoxic stress. Shedding of NKG2DL is thought to constitute a major countermechanism of tumor cells to subvert NKG2D-mediated immunosurveillance. Here, we report that the prototypical NKG2DL MICA is released by proteolytic cleavage in the stalk of the MICA ectodomain, where deletions, but not alanine substitutions, impede MICA shedding. Small compound-mediated stimulation and inhibition of MICA shedding adduced characteristics that indicated an involvement of members of the “a disintegrin and metalloproteinase” (ADAM) family. Accordingly, MICA shedding by tumor cells was inhibited by silencing of the related ADAM10 and ADAM17 proteases, which are known to promote tumor growth by releasing epidermal growth factor receptor ligands. Collectively, our data show that ADAM10 and ADAM17 are critically involved in the tumor-associated proteolytic release of soluble MICA facilitating tumor immune escape. Hence, therapeutic blockade of ADAM10 and ADAM17 seems promising for cancer treatment by targeting both growth and immune escape of tumors. [Cancer Res 2008;68(15):6368–76]

Interleukin-6 (IL-6) activates cells by binding to the membrane-bound IL-6 receptor (IL-6R) and subsequent formation of a glycoprotein 130 homodimer. Cells that express glycoprotein 130, but not the IL-6R, can be activated by IL-6 and the soluble IL-6R which is generated by shedding from the cell surface or by alternative splicing. Here we show that cholesterol depletion of cells with methyl-β-cyclodextrin increases IL-6R shedding independent of protein kinase C activation and thus differs from phorbol ester-induced shedding. Contrary to cholesterol depletion, cholesterol enrichment did not increase IL-6R shedding. Shedding of the IL-6R because of cholesterol depletion is highly dependent on the metalloproteinase ADAM17 (tumor necrosis factor-α-converting enzyme), and the related ADAM10, which is identified here for the first time as an enzyme involved in constitutive and induced shedding of the human IL-6R. When combined with protein kinase C inhibition by staurosporine or rottlerin, breakdown of plasma membrane sphingomyelin or enrichment of the plasma membrane with ceramide also increased IL-6R shedding. The effect of cholesterol depletion was confirmed in human THP-1 and Hep3B cells and in primary human peripheral blood monocytes, which naturally express the IL-6R. For decades, high cholesterol levels have been considered harmful. This study indicates that low cholesterol levels may play a role in shedding of the membrane-bound IL-6R and thereby in the immunopathogenesis of human diseases.

The transmembrane metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various cell surface molecules including the IL6-receptor and the transmembrane chemokines CX3CL1 and CXCL16. These molecules are constitutively released from cultured cells, a process that can be rapidly enhanced by cell stimulation with phorbol esters such as PMA. Recent research supports the view that the constitutive cleavage predominantly involves ADAM10 while the inducible one is mediated to a large extent by ADAM17. We here describe the discovery of hydroxamate compounds with different potency against ADAM10 and ADAM17 and different ability to block constitutive and inducible cleavage of IL6R, CX3CL1 and CXCL16 by the two proteases. By screening a number of hydroxamate inhibitors for the inhibition of recombinant metalloproteinases, a compound was found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17, which may be explained by an improved fit of the compound to the S1 specificity pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage experiments this compound (GI254023X) potently blocked the constitutive release of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect the PMA-induced shedding, which was only blocked by GW280264X, a potent inhibitor of ADAM17. As expected, GI254023X did not further decrease the residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that the