Modulação gênica de RIPK3 e MLKL e seu impacto na sensibilidade a morte por necroptose

Abstract

Introduction: Necroptosis is a newly discovered biochemical pathway of cell death that is independent of caspases and with morphological aspects similar to necrosis. Activation of this pathway, which occurs through various stimuli, includes death receptors, and TRIFmediated signals involving a cascade of phosphorylation of the RIPK1 and RIPK3 kinases, and the MLKL pseudokinase. The relevance of cell death by necroptosis has already been demonstrated in several pathophysiological conditions such as bacterial and viral infections, ischemic processes, cancer and autoimmune diseases. In many of these models RIPK3 and MLKL expression levels have been reported to be modulated both positively and negatively. The expression intensity of these molecules are correlated directly with the sensitivity of the cell to induce death by necroptosis. So far few reports have been published in the literature about the mechanisms that regulate the modulation of these gene both regarding signaling pathways and transcription factors involved and the characterization of their promoter regions. Purpose: To elucidate the mechanisms of gene expression of RIPK3 and MLKL by studying their promoter regions and the transcription factors that regulate them. Methods: Cloning, luciferase assays, CRISPR-Cas9 screening, Western blot, polymerase chain reaction, in vitro assays. Results: To the murine MLKLpromoter, we defined a minimal region responsible for the basal promoter activity, which is also activated after the interferon gamma stimulation through luciferase assays.Additionally, we verified that MLKL expression by interferon gamma is mediated by the Janus Kinase pathway that indicates that the classic interferon-signaling pathway actively participates in this modulation. This suggestion was reinforced with CRISPR-Cas9 screening data showing that there is a region close to MLKL transcription start site that is rich in regulatory regions and with STAT1 binding sites. In these regions seems to be central for regulating the activation of MLKL transcription upon interferon gamma stimulation through the canonical JAK-STAT pathway. Conclusion: Important promoter regions were identified for the expression of murine MLKL both at baseline as well as after interferon gamma stimulation. The JAK-STAT pathway was also observed as participating in this process.