The L1 adhesion molecule plays an important role in axon guidance and cell migration in the nervous system. L1 is also expressed by many human carcinomas. In addition to cell surface expression, the L1 ectodomain can be released by a metalloproteinase, but the biological function of this process is unknown. Here we demonstrate that membrane-proximal cleavage of L1 can be detected in tumors and in the developing mouse brain. The shedding of L1 involved a disintegrin and metalloproteinase (ADAM)10, as transfection with dominant-negative ADAM10 completely abolishes L1 release. L1-transfected CHO cells (L1-CHO) showed enhanced haptotactic migration on fibronectin and laminin, which was blocked by antibodies to αvβ5 and L1. Migration of L1-CHO cells, but not the basal migration of CHO cells, was blocked by a metalloproteinase inhibitor, indicating a role for L1 shedding in the migration process. CHO and metalloproteinase-inhibited L1-CHO cells were stimulated to migrate by soluble L1-Fc protein. The induction of migration was blocked by αvβ5-specific antibodies and required Arg-Gly-Asp sites in L1. A 150-kD L1 fragment released by plasmin could also stimulate CHO cell migration. We propose that ectodomain-released L1 promotes migration by autocrine/paracrine stimulation via αvβ5. This regulatory loop could be relevant for migratory processes under physiological and pathophysiological conditions.

Exosomes are small membrane vesicles that are secreted from a variety of cell types into various body fluids including the blood and urine. These vesicles are thought to play a role in cell-cell interactions. CD24 is a small but extensively glycosylated protein linked to the cell surface by means of a glycosyl-phosphatidylinositol anchor. In this study we found that CD24 is present in membrane vesicles characterized as exosomes that were isolated from the urine of normal individuals. CD24 was expressed by both tubule cells and podocytes and treatment of the latter with a cholesterol-extracting agent, but not with a calcium ionophore, caused the release of CD24-containing exosomes. Using CD24 as a marker, we found exosomes in the urine of newborn infants and in the amniotic fluid of pregnant women with similar findings made in mice. Interestingly, studies with CD24 knockout mice showed that the exosomes are released from the fetus but not from the mother; however, exosome release was similar from both the knockout and the wild-type mice. This indicates that CD24 is not essential for exosome formation or release but may be a convenient exosome marker. Our studies suggest that exosomal secretion from the embryonic kidney could play a biological role at the fetal-maternal interphase.

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Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.

Cells can release membrane components in a soluble form and as membrane vesicles. L1, an important molecule for cell migration of neural and tumor cells, is released by membrane-proximal cleavage, and soluble L1 promotes cell migration. Release of L1 is enhanced by shedding inducers such as phorbol ester and pervanadate, but it is also enhanced by depletion of cellular cholesterol with methyl-beta-cyclodextrin (MCD). How such different compounds can induce shedding is presently unknown. We show here that ADAM10 is involved in L1 cleavage, which occurs at the cell surface and in the Golgi apparatus. MCD and pervanadate treatment induced the release of microvesicles containing full-length L1 and the active form of ADAM10. L1 cleavage occurred in isolated vesicles. L1-containing microvesicles could trigger haptotactic cell migration. Only the neural L1 form carrying the RSLE signal for clathrin-dependent endocytosis was recruited and cleaved in vesicles. Phorbol ester treatment activated L1 cleavage predominantly at the cell surface. Our results provide evidence for two pathways of L1 cleavage, based on ADAM10 localization, that can be activated differentially: 1) direct cleavage at the cell surface, and 2) release and cleavage in secretory vesicles most likely derived from the Golgi apparatus. The findings establish a novel role for ADAM10 as a vesicle-based protease.

Purpose: The L1adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1is released from carcinoma cells in a soluble form. Soluble L1is present in serum and ascites of ovarian carcinoma patients.We investigated the mode of L1cleavage and the function of soluble L1. Experimental Design: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1and L1cleavage byWestern blot analysis and ELISA. Results: We find that in ovarian carcinoma cells the constitutive cleavage of L1proceeds in secretory vesicles. We show that apoptotic stimuli like C 2 -ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. Conclusions: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.Many transmembrane proteins can undergo cleavage and release of their ectodomain into the medium (for review see Refs. 1-3). These proteins are diverse in structure and function and comprise molecules such as TNF-␣ 1 (4 -8), FasL (9), interleukin-6 receptor (10, 11), L-selectin (12-14), pro-TGF-␣, and the ␤-amyloid precursor protein (15-17). Although there is evidence for a physiological function of many released molecules, a general significance for ectodomain shedding is still disputed (2). Recently, members of the ADAM metalloproteinase family, which are membrane proteins composed of a disintegrin and metalloproteinase domain, were shown to be important in ectodomain release (for review see Refs. 3 and 18). TACE/ADAM17 mediates the membrane release of TNF-␣, Lselectin, TGF-␣ (8), and TRANCE (tumor necrosis factor-related activation-induced cytokine), a TNF family member involved in osteoclastogenesis and dendritic cell survival (19). ADAM10/Kuz has been described as an ␣-secretase for the cleavage of ␤-amyloid precursor protein (20), and ADAM9 is involved in the phorbol ester-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) (21, 22). Certain released molecules can be cleaved by more than one enzyme, and some enzymes can cleave more than one substrate. For example, TNF-␣ cleavage can also be mediated by ADAM10 (23), and ␣-secretase activity for ␤-amyloid precursor protein has been attributed to TACE/ADAM17 (24) and ADAM9 (25). It is not known how the protease(s) select their substrate, because consensus cleavage sites have not been identified. In addition to the proteolytic function, some members of the ADAM famil...

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of Factin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of ␣ v ␤ 5 to focal contact sites, but not of ␣ v ␤ 3 or ␣ 5 ␤ 1 , was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with ␣ v ␤ 5 and paxillin but not with ␣ v ␤ 3 that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the ␣ v ␤ 5 integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-␣ v ␤ 5 antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the ␣ v ␤ 5 integrin that supports tumor cell invasion.The ability of tumor cells to invade beyond controlled hemostatic boundaries and re-emerge from blood vessels to establish new metastatic colonies continuous to present a major obstacle in cancer cure. It is well known that in tumor invasion and metastasis, the pericellular proteolytic systems, consisting of proteases and their specific cell surface receptors, are tightly regulated to modulate cellular functions and degrade selective matrix barriers (1, 2). We have previously demonstrated that the proteolytically activated receptor 1 (PAR1, 1 thrombin receptor) plays a central role in the malignant and physiological invasion processes of both breast carcinoma metastasis and placental implantation (3). At the molecular level, tumor invasion is mediated via the combined interactions of the host cell signaling machinery and the regulation of the stromal extracellular matrix (ECM). Extensive proteolysis in the tumor microenvironment is also responsible for the activation of several enzymatic precursors, like plasminogen, pro-matrix metalloproteinase, and prothrombin (4 -6). In addition, the extravascular deposition of fibrin within the tumor microenvironment...