Identification Of Novel Regulators Involved In Oxidative Stress-reprogrammed LPS Signaling Pathway By Comparative Phosphoprotein-affinity Profiling

Recent studies have suggested that oxidative stress generated during ischemia /reperfusion renders patients more susceptible to MODS (multiple organ dysfunction syndrome) by priming for an exaggerated response to a second inflammation stimulus (often bacteria LPS), the so-called "two-hit" hypothesis. Whereas oxidants-induced signaling cascade and LPS pathway in inflammation has been extensively studied, the synergistic effect of these two stimuli remains poorly elucidated. Previous works has shown that oxidants are able to prime macrophages for greatly increased activation of cytokines and chemoattractants, in response to LPS, whether in vitro or in vivo. Interestingly, this was reported to be attributed to largely augmented NF-kappaB translocation regulated by src/PI-3K, a manner different from that in the classical LPS pathway.These reports thus presented a theory that LPS pathway was actually reprogrammed by antecedent oxidative stress, diversifying from the traditional LPS pathway. However, limited signaling molecules known so far have prevented us from fully decoding its precise mechanism.To study the LPS pathway reprogrammed by antecedent oxidative stress more comprehensively and systematically, we demonstrated here an approach using PMA-induced human macrophage cell line Thp-1 as an in vitro model, trying to find differences of phosphoproteome between LPS stimulated Thp-1 cells with and without previous oxidative stress, aiming to reveal more potential regulators. This is the first comparative study between classical LPS pathway and the LPS pathway modified by oxidative stress, in a global scale. Utilizing PMAC columns from BD Biosciences, we could enrich up to 400μg suspected phosphoproteins. This larger loading amount than reported before, together with increased sensitivity introduced by a sequential staining method, makes less abundant signal-induced phosphoproteins more accessible. After 2-D electrophoresis, we resolved up to 1150 phosphoprotein spots on a single 2-D gel, in average. Compared to that of Thp-1 cells stimulated with LPS alone, 29 reproducibly changed spots on the 2-D map of LPS stimulated Thp-1 cells with previous oxidative stress were visualized and selected for MS analysis. Among these, 11 represented down-regulated spots (include those disappeared), 16 represented up-regulated (include those newly emerged) and the remain 2 represented spots with altered coordination.Up to now, 5 of these were identified as involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, proteasome beta-4 subunit, a major component of the proteasome complex, was dramatically down-regulated in LPS stimulated cells with oxidative stress, while proteasome has been reported to play important roles in traditional LPS pathway at various levels: 1, negatively regulate p38 activity; 2, negatively control IRAK activity by ubiquitin-proteasome pathway; 3, direct binding of LPS dictates its chymotrypsin activity to present the antigen of bacteria to MHC-1 class molecules. Whilst nuclear matrix protein NMP-238 was up-regulated significantly, which is recognized as a critical cofactor activating Wnt/beta-catenin controlled gene transcription. Although the role of Wnt/beta-catenin pathway hasn't been discovered in LPS pathway, it was recently reported to be involved in various inflammatory processes.Taken together, in addition to the previously reported src/PI-3K/NF-kappaB, we presented here a hypothesis that proteasome and NMP-238 might serve as novel regulators to diversify the LPS pathway reorganized by oxidant stimulation, at the following level: 1, dramatically repressed proteasome activity might lead to the massive induction of p38 activity, thus much more induction of some adhesive molecules and cytokines; 2, The negative control of proteasome on IRAK is probably minimized, consequently primes for massive induction of JNK/AP1 and NF-kappa B; 3, the antigen presentation ability of proteasome is also minimized, makes the body more susceptible to bacterial infection; 4, NMP-238 might induced additional inflammatory response via Wnt/oeta-catenin pathway. Collectively, these processes may work cooperatively and prime for an uncontrollable immune activation. To test this hypothesis, functional studies on whether and how proteasome and NMP-238 contributes to this diversification is in progress. Moreover, additional potential targets in the reprogrammed LPS pathway is being explored in detail by combined application of secretomic and membrane proteomic strategies.