Regulation of gene expression at the translational level (i.e., mRNA-protein) is poorly understood in normal megakaryocytes, and it is virtually unknown if inflammatory diseases alter translational responses in megakaryocytes and platelets. With that said, inflammation contributes to the pathogenesis of numerous diseases such as sepsis, and inflammatory agonists are considered potent triggers of mRNA translation. This grant proposal will utilize human and mouse model systems (in vitro and in vivo) and state-of-the-art sequencing (ribosomal footprinting and eIF4E-enrichment) to determine how translational control pathways regulate megakaryocyte and platelet gene expression ? in the setting of health and septic situations. Preliminary data supporting this project demonstrates that the mammalian target of rapamycin (mTOR) and the MAP kinase-interacting serine/threonine-protein kinase 1 (MNK1), two translational control pathways that converge on the initiation step of translation, regulate protein synthetic events and functions in megakaryocytes and platelets. These preliminary results have led to the hypothesis that the septic milieu will significantly modify mTOR and/or MNK1-dependent translation in megakaryocytes and platelets, resulting in inappropriate formation and function of platelets during the course of sepsis.
Specific Aim1 will test the hypothesis that inflammatory agonists generated during sepsis will alter protein synthetic events in megakaryocytes and the formation of platelets in an mTOR and MNK1-dependent manner.
Specific Aim 2 will determine how genetic deletion of mTOR or MNK1 regulates the ex vivo and in vivo function of platelets in the presence or absence of inflammation.
Specific Aim 3 will test the hypothesis that the septic milieu activates the mTOR and/or MNK1 signaling pathways and thereby increases protein synthesis by platelets. It will also determine if deletion of mTor and/or Mnk1 in mouse platelets leads to adverse outcomes in murine models of sepsis. Successful completion of these aims will 1) identify translated mRNAs in megakaryocytes that are under mTOR and/or MNK1-dependent control and whether these translational signaling pathways regulate the final steps of platelet production, 2) delineate the effects of mTOR and MNK1 on functions of megakaryocytes and platelets in health and inflammation, 3) characterize inadvertent side- effects of clinically-used mTOR and MNK1 inhibitors on megakaryocytes and platelets, and 4) determine if mTOR or MNK1 in megakaryocytes and platelets regulate outcomes in models of sepsis. Data generated will immediately increase our understanding of how translational control pathways alter megakaryocyte and platelet functions in the setting of sepsis.
Megakaryocytes and platelets are critical effectors of acute and infectious inflammatory diseases such as sepsis. Results generated from this proposal will advance our understanding of how gene expression pathways in megakaryocytes (i.e., platelet precursors) and platelets regulate outcomes in sepsis.
