PKCδ phosphorylation is an upstream event of GSK3 inactivation-mediated ROS generation in TGF-β1-induced senescence

Transforming growth factor beta 1 (TGF-beta 1) 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 (PKC delta) as an upstream regulator of GSK3 inactivation in this mechanism of TGF-beta 1-induced senescence. When Mv1Lu cells were exposed to TGF-beta 1, PKC delta 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-beta 1-triggered GSK3 phosphorylation was blocked by inhibition of PKC delta, using its pharmacological inhibitor, Rottlerin, or overexpression of a dominant negative PKC delta mutant, but GSK3 inhibition with SB415286 did not alter PKC delta phosphorylation. Activation of PKC delta 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 PKC delta 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 PKC delta mutants, effectively induced senescence. These results indicate that PKC delta plays a key role in TGF-beta 1-induced senescence of Mv1Lu cells through the phosphorylation of GSK3, thereby triggering mitochondrial complex IV dysfunction and intracellular ROS generation.