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Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

ABSTRACT: Cleavage and release (shedding) of membrane proteins is a critical regulatory step in many normal and pathological processes. Evidence suggests that the antiaging transmembrane protein Klotho (KL) is shed from the cell surface by proteolytic cleavage. In this study, we attempted to identify the enzymes responsible for the shedding of KL by treating KL-transfected COS-7 cells with a panel of proteinase inhibitors and measuring cleavage products by Western blot. We report that metalloproteinase inhibitors, including EDTA, EGTA, and TAPI-1, inhibit the shedding of KL, whereas insulin increases shedding. The effects of the inhibitors in KL-transfected COS-7 cells were repeated in studies on rat kidney slices ex vivo, which validates the use of COS-7 cells as our model system. Tissue inhibitor of metalloproteinase (Timp)-3 effectively inhibits KL cleavage, whereas Timp-1 and Timp-2 do not, a profile that indicates the involvement of members of the A Desintegrin and Metalloproteinase (ADAM) family. Cotransfection of KL with either ADAM10 or ADAM17 enhances KL cleavage, whereas cotransfection of KL with small interference RNAs specific to ADAM10 and ADAM17 inhibits KL secretion. These results indicate that KL shedding is mediated mainly by ADAM10 and ADAM17 in KL-transfected COS-7 cells. The effect of insulin is abolished when ADAM10 or ADAM17 are silenced. Furthermore, we demonstrate that the effect of insulin on KL shedding is inhibited by wortmannin, showing that insulin acts through a PI3K-dependent pathway. Insulin enhances KL shedding without increasing ADAM10 and ADAM17 mRNA and protein levels, suggesting that it acts by stimulating their proteolytic activities.

SUBMITTER: Chen CD

PROVIDER: S-EPMC2148378 | biostudies-literature | 2007 Dec

REPOSITORIES: biostudies-literature

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Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Chen Ci-Di CD Podvin Sonia S Gillespie Earl E Leeman Susan E SE Abraham Carmela R CR

Proceedings of the National Academy of Sciences of the United States of America 20071203 50

Cleavage and release (shedding) of membrane proteins is a critical regulatory step in many normal and pathological processes. Evidence suggests that the antiaging transmembrane protein Klotho (KL) is shed from the cell surface by proteolytic cleavage. In this study, we attempted to identify the enzymes responsible for the shedding of KL by treating KL-transfected COS-7 cells with a panel of proteinase inhibitors and measuring cleavage products by Western blot. We report that metalloproteinase in ...[more]

PMID: 18056631

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Project description:Reports of the stimulated release of extracellular vesicles (EVs) are few, and the mechanisms incompletely understood. To our knowledge, the possibility that the activation of any one of the multitudes of G-protein-coupled receptors (GPCRs) expressed by a single cell-type might increase EV release has not been explored. Recently, we identified the expression of cholecystokinin (CCK), gastrin, gastrin/cholecystokinin types A and/or B receptors (CCKAR and/or -BR), and the bitter taste receptor, TAS2R14 in the human and mouse placenta. specifically, trophoblast. These GPCR(s) were also expressed in four different human trophoblast cell lines. The current objective was to employ two of these cell lines-JAR choriocarcinoma cells and HTR-8/SVneo cells derived from first-trimester human villous trophoblast-to investigate whether CCK, TAS2R14 agonists, and other GPCR ligands would each augment EV release. EVs were isolated from the cell-culture medium by filtration and ultracentrifugation. The preparations were enriched in small EVs (<200 nm) as determined by syntenin western blot before and after sucrose gradient purification, phycoerythrin (PE)-ADAM10 antibody labeling, and electron microscopy. Activation of TAS2R14, CCKBR, cholinergic muscarinic 1 & 3, and angiotensin II receptors, each increased EV release by 4.91-, 2.79-, 1.87-, and 3.11-fold, respectively (all p < .05 versus vehicle controls), without significantly changing EV diameter. A progressive increase of EV concentration in conditioned medium was observed over 24 hr consistent with the release of preformed EVs and de novo biogenesis. Compared to receptor-mediated stimulation, EV release by the calcium ionophore, A23187, was less robust (1.63-fold, p = .08). Diphenhydramine, a TAS2R14 agonist, enhanced EV release in JAR cells at a concentration 10-fold below that required to increase intracellular calcium. CCK activation of HTR-8/SVneo cells, which did not raise intracellular calcium, increased EV release by 2.06-fold (p < .05). Taken together, these results suggested that other signaling pathways may underlie receptor-stimulated EV release besides, or in addition to, calcium. To our knowledge, the finding that the activation of multiple GPCRs can stimulate EV release from a single cell-type is unprecedented and engenders a novel thesis that each receptor may orchestrate intercellular communication through the release of EVs containing a subset of unique cargo, thus mobilizing a specific integrated physiological response by a network of neighboring and distant cells.

Dataset Information

Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

Publications

Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17.

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