Differential implication of protein kinase C isoforms in cytotoxic T lymphocyte degranulation and TCR-induced Fas ligand expression.
CD8(+) cytotoxic T lymphocyte (CTL) clones are able to exert both perforin- and Fas-dependent cytotoxicity. We show in the present work that phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002 prevent TCR/CD3-induced functional Fas ligand (FasL) expression, but not perforin-dependent cytotoxicity. The specific inhibitor of classical protein kinase C (PKC) isoforms, Gö6976, completely inhibited perforin-dependent cytotoxicity and only affected slightly TCR/CD3-induced FasL expression, while the opposite was observed using rottlerin, an inhibitor with higher specificity for PKCtheta. To address further the dependence of FasL expression on PI3K, a luciferase reporter controlled by the FasL promoter was used. Reporter gene induction by anti-CD3 mAb was abolished in cells transfected with dominant-negative PI3K (PI3K-DN) and increased in cells transfected with constitutively active PI3K (PI3K*). Transfection with constitutively active mutants (A/E) of PKCepsilon, and especially of PKCtheta, improved anti-CD3 mAb-induced reporter expression and completely abolished inhibition by wortmannin, while transfection with dominant-negative (K/R) PKCtheta prevented the induction of the reporter. Finally, transfection with PKCalpha A/E, but not with PKCtheta A/E, cooperated with ionomycin to induce degranulation in the CTL line 1.3E6SN. Altogether, the results suggest that TCR/CD3-induced FasL gene transcription is controlled by PI3K and PKCtheta activation, while this signaling pathway is not implicated in CTL degranulation, which is rather dependent on the activation of classical PKC isoforms.
Pardo
Julián
J
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Buferne
Michel
M
Martínez-Lorenzo
María-José
MJ
Naval
Javier
J
Schmitt-Verhulst
Anne-Marie
AM
Boyer
Claude
C
Anel
Alberto
A
D000098:Acetophenones / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001578:Benzopyrans / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D015550:Cell Degranulation / Q000276:immunology / Q000502:physiology*; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D003609:Dactinomycin / Q000494:pharmacology; D053222:Fas Ligand Protein; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019169:Jurkat Cells; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D054353:Perforin; D010703:Phorbol Esters / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D052899:Pore Forming Cytotoxic Proteins; D011493:Protein Kinase C / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D000074121:Protein Kinase C-theta; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D014162:Transfection; D000077191:Wortmannin
D000098:Acetophenones; D000730:Androstadienes; D000911:Antibodies, Monoclonal; D001578:Benzopyrans; D002227:Carbazoles; D002867:Chromones; D003513:Cycloheximide; D003609:Dactinomycin; D053222:Fas Ligand Protein; D007211:Indoles; D015759:Ionomycin; D007527:Isoenzymes; D008156:Luciferases; D008562:Membrane Glycoproteins; D009025:Morpholines; D054353:Perforin; D010703:Phorbol Esters; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D052899:Pore Forming Cytotoxic Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D000074121:Protein Kinase C-theta; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; D000077191:Wortmannin; C505661:FASLG protein, human; C505658:Fasl protein, mouse; C081021:Go 6976; C119222:phorbol-12-myristate; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C085746:rottlerin; C497256:Prkce protein, mouse; C496628:PRKCA protein, human; C497255:PRKCE protein, human; C076233:PRKCQ protein, human; C496630:Prkca protein, mouse; C489593:Prkcq protein, mouse; C064758:Ro 31-8220