Regulation of TNF biosynthesis by MK2/3: Role of MK2/3-dependent expression and modification of TTP and its interplay with further ARE-binding proteins and co-factors

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TNF is a master cytokine of inflammatory signaling of macrophages. Its biosynthesis is tightly controlled to allow rapid secretion but also to avoid delay or leakiness in its down-regulation, which could result in exaggerated or persistent inflammation. The levels of regulation comprise transcription, processing, nuclear export and stability of the TNF mRNA, translation of pro-TNF and shedding of TNF at the cell membrane. The p38 MAPK/MK2/3 pathway regulates TNF-biosynthesis mainly at the translational level depending on the AU-rich element (ARE) in the 3’-UTR of TNF mRNA, which binds to p38- and MK2/3-substrates such as hnRNP A0, tristetraprolin (TTP), and KSRP. So far the molecular mechanisms regulating ARE-dependent translation of pro-TNF via phosphorylation are not understood. The ARE-binding MK2/3-substrate TTP is mainly held responsible for regulation of the stability of specific mRNAs. Its role in translational regulation and a possible switch in its function between regulation of stability and translation of mRNA have remained uncharacterized to date.We have generated macrophage cell lines representing the MK2/3-deficient and MK2-rescued genotype and have obtained evidence that TTP is involved in MK2-dependent translational regulation of TNF mRNA and also of TTP’s own mRNA. We have successfully demonstrated transcriptional regulation of the TTP gene by SRF and identified the SRF-cofactor MRTF-A (MAL, MKL1) as a substrate of MK2. Furthermore, we have characterized components of the proteasome as substrates of MK2 and interaction partners of TTP. Based on this preliminary work, the scientific objective of the project proposed is the elucidation of the molecular mechanisms of p38/MK2/3-dependent translational regulation of TNF biosynthesis with specific regard to the role of TTP and other ARE-binding proteins and regulation of their expression and activity. In addition, the role of TTP in regulated mRNA decay will be further analyzed.The identification and characterization of components influencing MK2- and TTP-dependent translation in macrophages will comprise sub-cellular fractionation and polysome profiling in combination with the analysis of the distribution of relevant mRNA-ARE-binding proteins and the effects of their knockdown on the translation of TNF- as well as TTP-mRNA. The effects of these proteins on cap-dependent translation and on ARE-binding of TTP will be analyzed by reporter and in vitro systems. The role of the specific phosphorylations of TTP by MK2 will be characterized by rescue of TTP-deficient macrophages with phosphorylation-site mutants of TTP. In addition, further biochemical and genetic analysis of the regulation of TTP expression, modification and degradation will be performed.Fulfilling the aims of this project will contribute to the understanding of major principles of post-transcriptional regulation of gene expression in inflammation.