Diacylglycerol Kinase α Regulates the Secretion of Lethal Exosomes Bearing Fas Ligand during Activation-induced Cell Death of T Lymphocytes

Alonso, Roberto; Rodríguez, M. Carmen; Pindado, Jose; Merino, Ernesto; Mérida, Isabel; Izquierdo, Manuel

doi:10.1074/jbc.m501112200

Cited by 120 publications

(171 citation statements)

References 48 publications

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“…It has been demonstrated that FasL-mediated apoptosis can also be owing to the secretion of FasL-bearing exosomes/ microvesicles. 22 To exclude the detection of FasL released in exosomes into the culture medium, cell supernatants were subjected to ultracentrifugation before analyses. Although inhibitors of aspartyl and serine proteases (pepstatin A and AEBSF, respectively) did not show an obvious effect on FasL shedding, the wide-spectrum MP inhibitors GM6001 and TNF-a protease inhibitor (TAPI) led to a strong reduction of sFasL (Figure 1b).…”

Section: Resultsmentioning

confidence: 99%

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ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

et al. 2007

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The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.

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Section: Resultsmentioning

confidence: 99%

“…22 To exclude the detection of FasL released in exosomes into the culture medium, cell supernatants were subjected to ultracentrifugation before immunoblot or ELISA analyses.…”

Section: Methodsmentioning

confidence: 99%

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

et al. 2007

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“…The lipid mediator phosphatidic acid originating either from the activity of the diacylglycerol kinase [45] or from phospholipase D (PLD2) activities promotes exosome secretion [46].…”

Section: Regulated Secretionmentioning

confidence: 99%

“…Therefore understanding in depth the biogenesis and release mechanisms of exosomes in cells is essential. It has been shown that some lipid mediators could enhance their production [45,101]. On the reverse, decreasing the amount of tumor exosomes released by modifying the pH of MVB with amiloride enhances the efficacy of chemotherapeutic agents [102].…”

Section: E Pharmacology Of Exosomes: the Future Challengementioning

confidence: 99%

Exosomes as intercellular signalosomes and pharmacological effectors

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Silvente-Poirot

et al. 2011

Biochemical Pharmacology

483

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International audienceCell secretion is a general process involved in various biological responses. Exosomes are part of this process and have gained considerable scientific interest in the past five years. Several steps through investigations across the last 20years can explain this interest. First characterized during reticulocyte maturation, they were next characterized as a key player in the immune response and cancer immunotherapy. More recently they were characterized as vectors of mRNAs, miRNAs and also lipid mediators able to act on target cells. They are the only type of vesicles released from an intracellular compartment from cells in viable conditions. They appear as a vectorized signaling system operating from inside a donor cell towards either the periphery, the cytosol, or possibly to the nucleus of target cells. Exosomes from normal cells trigger positive effects, whereas those from pathological ones, such as tumor cells or infected ones may trigger non-positive health effects. Therefore regulating the biogenesis and secretion of exosomes appear as a pharmacological challenge to intervene in various pathophysiologies. Exosome biogenesis and molecular content, interaction with target cells, utilisation as biomarkers, and functional effects in various pathophysiologies are considered in this review

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“…[10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,[16][17][18][19][20][21] These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes.…”

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confidence: 99%

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

et al. 2015

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Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic. Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types. 1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process. 2,3 Two main pathways are involved in MVB maturation. 4,5 In addition to the ESCRT (endosomal complex required for traffic) proteins, 6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA), 7 ceramides 8 and diacylglycerol (DAG) 9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activationinduced cell death (AICD), antigen presentation and intercellular miRNA exchange. [10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,16-21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes. 22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG. 23,24 The secretory vesicle pathway involves several DAGc...

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Diacylglycerol Kinase α Regulates the Secretion of Lethal Exosomes Bearing Fas Ligand during Activation-induced Cell Death of T Lymphocytes

Cited by 120 publications

References 48 publications

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

ADAM10 regulates FasL cell surface expression and modulates FasL-induced cytotoxicity and activation-induced cell death

Exosomes as intercellular signalosomes and pharmacological effectors

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

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