Reverse Phase Protein Array Used to Elucidate Pro-inflammatory Signalomes Provides an Excellent Guide for RPPA Methodologies and Techniques

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Application of Reverse-Phase Protein Arrays (RPPA) has primarily centered on the discovery and identification of cell signaling pathways involved in cancer development and progression. The RPPA approach and technology has found expanded utility in other disciplines such as in the study of inflammatory diseases.Recently Negm, et al. employed RPPA to study the signaling pathways downstream of the mutated TNF receptor 1 (TNFR1) which causes the autosomal dominant TNF receptor-associated periodic syndrome (TRAPS) (1). This systemic auto-inflammatory disorder is manageable with variable efficacy using corticosteroids and by the blocking of some pro-inflammatory cytokines. Many patients fail to respond to such therapy indicating that multiple inflammatory pathways may be involved. These investigators used RPPA to elucidate the variety and complexity in the intracellular signaling pathways affected by a TRAPS-associated TNFR1 mutation and created a cell model using transfected SK-Hep-1 cells with C33Y or WT TNFR1 constructs and compared to C33Y TRAPS patients’ peripheral blood mononuclear cells (PBMCs). By using RPPA, this study was able to provide the most comprehensive report to date of the pro-inflammatory signalome which is constitutively activated by mutant TNFR1. The findings showed that in both the model system and in patient PBMCs, cellular homeostasis was shifted to a hypersensitive state which was more reactive to, not only TNFα, but to a variety of stimuli. Data linked multiple signaling pathways including NF-κB, p38, Jnk and Erk, MAPK, Akt, Jak2, and STAT3. The results provided an explanation for why cytokine-blocking treatments were effective for some TRAPS patients but not for others due to the cells’ adaptability using alternative pathways and also provided data explaining the diversity in the pathophysiology of TRAPS. Future studies include similar analysis using other TNFR1 mutants to further characterize the disease states with the goal of expanding the target pool for future, more effective treatments. This study also serves as an excellent example of how to adopt RPPA technology for the novice user. The optimized methods described for these experiments are clearly outlined and described. Important RPPA parameters included are optimal cell lysis, lysate printing (optimal buffer and PNC substrate), and assay conditions such as blocking and, most importantly, detection antibody validation. These conditions are critical to for optimal RPPA performance and should be established prior to the initiation of any RPPA studies.

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(1)Negm, Ola H., et al. “A pro‐inflammatory signalome is constitutively activated by C33Y mutant TNF receptor 1 in TNF receptor‐associated periodic syndrome (TRAPS).” European journal of immunology 44.7 (2014): 2096-2110.