IL- 1? and LPS stimulate related receptors (IL-1R1 and TLR-4, respectively) from the Toll-like receptor family. Each of these receptors play an important role in either initiating or propagating signaling events that promote inflammation following exposure to bacterial endotoxin. IL-1R1 and TLR-4 share many similar effectors (MyD88, IRAK, TRAF6), but also have unique effectors (TIRAP/Mal, TRAM, TRIP), that specify unique biological properties of each receptor pathway. However, both receptors can stimulate NF-KB in a redox-dependent manner that involves NADPH oxidases (Nox). Our laboratory has recently described a unique mechanism whereby NADPH oxidases spatially regulate the redox-dependent activation of receptors at the level of the endosome. Our data suggests that LPS, IL- 1?, and TNFa all signal via this mechanism to activate NF-KB. The goal of this proposal is to further define how redox-active endosomes facilitate the IL- 1? and LPS signaling pathways. I propose to study these mechanisms in cell culture and mouse models through the use of high throughput proteomics and by focusing on how Toll-like receptors activate Nox2 in the endosomal compartment.
In Aim1, IL- 1? will be used as the model system to test the hypotheses that Nox2 enters redox-active endosomes from the plasma membrane (as opposed to early endosome fusion), that endocytosis is caveolin-1 mediated, and that Rac1 is activated by one of four candidate GEFs: ?-Pix, Sos, Vav1, or P-Rex1. Experiments will utilize siRNA, chemical inhibitors and dominant negative mutants to dissect these pathways.
In Aim 2, proteomic analyses of redox-active endosomes will be performed using both 2D gel / MOLDI TOF experiments and the use of tandem mass spectrometry. These studies will identify effector proteins in-common and unique to redox-active endosomes harboring ligand activated Toll- like receptors. Identifying unique proteins in the LPS pathway is of particular interest, as this could lead to key therapeutic targets for the treatment of sepsis. Further proteomic work will include the use of floxed Rac1, MyD88 KO, and Nox2 KO mice to generate mechanistic pathways of LPS and IL- 1? redox signaling. Statement of Relevance: This study will investigate novel aspects of inflammation caused by a common bacterial toxin called LPS. Results of this research will also translate into practical knowledge for many other types of environmental injuries that involve inflammation and reactive oxygen species. Eventually, this research could allow for the development of medical treatments to attenuate inflammation caused by LPS and other diseases involving inflammation. ? ? ?
| Oakley, Fredrick D; Abbott, Duane; Li, Qiang et al. (2009) Signaling components of redox active endosomes: the redoxosomes. Antioxid Redox Signal 11:1313-33 |
| Oakley, Fredrick D; Smith, Rachel L; Engelhardt, John F (2009) Lipid rafts and caveolin-1 coordinate interleukin-1beta (IL-1beta)-dependent activation of NFkappaB by controlling endocytosis of Nox2 and IL-1beta receptor 1 from the plasma membrane. J Biol Chem 284:33255-64 |
