title: PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence.
abstract: Transforming growth factor β1 (TGF-β1) induces Mv1Lu cell senescence through inactivating glycogen synthase kinase 3 (GSK3), thereby inactivating complex IV and increasing intracellular ROS. In the present study, we identified protein kinase C delta (PKCδ) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-β1-induced senescence. When Mv1Lu cells were exposed to TGF-β1, PKCδ phosphorylation simultaneously increased with GSK3 phosphorylation, and then AKT and ERK were phosphorylated. AKT phosphorylation and Smad signaling were independent of GSK3 phosphorylation, but ERK phosphorylation was downstream of GSK3 inactivation. TGF-β1-triggered GSK3 phosphorylation was blocked by inhibition of PKCδ, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKCδ mutant, but GSK3 inhibition with SB415286 did not alter PKCδ phosphorylation. Activation of PKCδ by PMA delayed cell growth and increased intracellular ROS level, but did not induce senescent phenotypes. In addition, overexpression of wild type or a constitutively active PKCδ mutant was enough to delay cell growth and decrease the mitochondrial oxygen consumption rate and complex IV activity, but weakly induce senescence. However, PMA treatment on Mv1Lu cells, which overexpress wild type and constitutively active PKCδ mutants, effectively induced senescence. These results indicate that PKCδ plays a key role in TGF-β1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.
authors.affiliation: Department of Biochemistry & Molecular Biology, Ajou University School of Medicine , Suwon , Republic of Korea.
authors.forename: H-O
authors.initials: HO
authors.lastname: Byun
authors.forename: H-J
authors.initials: HJ
authors.lastname: Jung
authors.forename: M-J
authors.initials: MJ
authors.lastname: Kim
authors.forename: G
authors.initials: G
authors.lastname: Yoon
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D016922:Cellular Senescence / Q000502:physiology*; D003576:Electron Transport Complex IV / Q000378:metabolism; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism*; D047468:Immunoprecipitation; D008928:Mitochondria / Q000378:metabolism*; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
chemical_list: D017382:Reactive Oxygen Species; D053773:Transforming Growth Factor beta1; D003576:Electron Transport Complex IV; D051745:Protein Kinase C-delta; D038362:Glycogen Synthase Kinase 3
keywords: GSK3; PKCδ; ROS; TGF-β1; senescence