The decision between death or survival of lung epithelial cells exposed to oxidants is regulated by multiple signaling pathways. It is well known that the transcription factor, Nuclear Factor kappa B (NF-kappaB) plays an important role in ceII survival whereas c-Jun-N-terminal kinase (JNK) induces cell death. We have demonstrated that after exposure of lung epithelial cells to oxidants, a marked induction of JNK occurs in absence of activation of IKK, the upstream kinase in the NF-kappaB pathway, setting the stage for cell death. Importantly, NF-kappaB and JNK are coordinately activated in response to activation of the Tumor Necrosis Factor Receptor-1 (TNF-R1). The adapter proteins, TNF receptor associated factor 2 (TRAF2) and Receptor Interacting Protein (RIP), which are recruited to TNF-R1, direct signaling to either JNK or Inhibitory kappa B Kinase (IKK). The hypotheses to be tested in this proposal are that the imbalance in he activation of NF-kappaB and JNK by oxidants causes epithelial cell death, airway injury and remodeling in a well characterized model of nitrogen dioxide (NO2)-induced injury. We also hypothesize that oxidants promote signaling through JNK while preventing the activation of NF-kappaB, via stimulation of TNF-R1, and altered recruitment of the adapter proteins, TRAF2 and RIP. We will test our hypotheses in five Specific Aims. We will focus our efforts on NO2, an important inflammatory oxidant and air pollutant, which kills lung epithelial cells in a JNK-dependent manner. We will expose mouse lung epithelial cells and C57/B16 mice to NO2 and determine whether JNK and IKK activation occur in addition to recruitment of RIP and TRAF2 to TNF-R1. We will employ strategies aimed to prevent JNK activation and/or enhance IKK activation to determine whether patterns of cell death and survival can be altered. We will also test whether TNF-R1 dependent signaling via TRAF2 and RIP is required for JNK activation, injury or death in lung epithelial cells and mice and whether this occurs in a ligand-independent manner. Lastly, we will utilize transgenic mice that either are not capable of activating NF-kappaB or have constitutive activation of NF-kappaB in lung epithelium to determine whether the activation state of NF-kappaB affects survival, lung injury, repair, and development of lung fibrosis in response to NO2.
The aims of this proposal are crucial to the understanding of the regulation of survival and death responses of epithelial cells exposed to oxidants. The knowledge obtained from the combined in vitro and in vivo studies will be critical in the development of strategies aimed to minimize airway remodeling associated with inflammatory diseases where nitrating oxidants are present.
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