Reactive oxygen species (ROS) produced in the Gl tract by bacteria and host cells participate in the signal transduction of stresses and stimuli in normal tissue homeostasis, and also contribute to barrier disruption, ulcer formation, and malabsorption associated with intestinal disease. The mechanisms whereby ROS participate in biological responses including survival, proliferation, and programmed cell death remain enigmatic. The long-term objective of this proposal is to elucidate signal transduction pathways and assess the consequences of ROS-induced signals on the fate of Gl epithelial cells. Protein kinase D (PKD1), recently identified as a novel downstream effector protein kinase of protein kinase C (PKC), is involved in multiple biological processes including mitogenesis and apoptosis. PKD1 is dramatically activated in response to oxidative stress. We describe a model that predicts a role for PKC/PKD cascades in signal transduction mediating cellular responses to oxidative stress in Gl epithelial cells. Multiple stresses and stimuli including oxidative stress are linked to coordinated changes in gene expression and apoptosis via the JNK-c-Jun pathway. Activated PKD1 attenuates JNK-mediated c-Jun phosphorylation. Further, PKD1 directly phosphorylates the amino-terminal portion of c-Jun at sites distinct from those modified by JNK. These findings have led to two central hypotheses: 1. ROS signaling via Src and PKC potently activates PKD1, and induces subcellular redistribution of the activated kinases in intestinal epithelial cells; 2. In response to ROS signaling, PKD1 exert a protective effect to modulate apoptosis in Gl epithelial cells. The research objectives in this proposal are organized into three specific aims: 1. Determine the extent of PKD1 and JNK activation and apoptosis induction by oxidant signaling in intestinal epithelial cells. 2. Examine the roles of PKD1 activity and JNK-c-Jun signaling to modulate oxidant-induced apoptosis in intestinal epithelial cells. 3. Examine PKD1 inhibition of JNK activation and identify direct PKD1 phosphorylation sites in c-Jun as possible mechanisms mediating overall attenuation of JNK-c-Jun signaling. Successful completion of these aims will improve our understanding of normal and pathological signaling mechanisms in the Gl tract, allowing us to pinpoint new targets for therapy. ? ? ?
