Glucocorticoids and Tumor Necrosis Factor α Increase Oxidative Stress and Suppress Wnt Protein Signaling in Osteoblasts

Endogenous glucocorticoids (GCs) and inflammatory cytokines contribute to the age-associated loss of bone mass and strength, but the molecular mechanisms responsible for their deleterious effects on the aging skeleton are unclear. Based on evidence that oxidative stress is a causal mechanism of the insulin resistance produced by either one of these two agents, we tested the hypothesis that their adverse skeletal effects also result from increased oxidative stress. We report that administration of prednisolone to mice increased reactive oxygen species (ROS) and the phosphorylation of p66(shc) (an amplifier of H2O2 generation in mitochondria) in bone. Dexamethasone (Dex) and TNF alpha had a similar effect on osteoblastic cells in vitro. The generation of ROS by Dex and TNF alpha required PKC beta/p66(shc) signaling and was responsible for the activation of JNK and induction of apoptosis by both agents. The activity of Forkhead box O (FoxO) transcription factors was also increased in response to ROS; however, FoxO activation opposed apoptosis induced by Dex and TNF alpha. In addition, both agents suppressed Akt phosphorylation as well as Wnt-induced proliferation and osteoblast differentiation. However, the inhibitory actions on Wnt signaling were independent of PKC beta/p66(shc). Instead, they were mediated by inhibition of Akt and stimulation of FoxOs. These results demonstrate that ROS-induced activation of a PKC beta/p66(shc)/JNK signaling cascade is responsible for the pro-apoptotic effects of Dex and TNF alpha on osteoblastic cells. Moreover, modulation of Akt and FoxOs by GCs and TNF alpha are cell-autonomous mechanisms of Wnt/beta-catenin antagonism contributing to the adverse effects of GC excess and inflammatory cytokines on bone alike.