Regulation and Function of Mkp-1 During Sepsis.

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PROJECT SUMMARY Sepsis is common, has a high mortality rate, and is a very resource-intensive healthcare problem in the U.S.A. Sepsis is associated with cytokine storm, an exacerbated immune response, resulting in excessive vasodilation and severe vascular leakage leading to circulatory shock and multiple organ failure. As sepsis persists, patients often develop features of immunoparalysis. The majority of shock-related deaths occur during this secondary hypo-immune state, likely due to the failure to clear the initial infection and/or secondary nosocomial infection despite antibiotic treatment. We have found that mice deficient in mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1, an inducible negative regulator of the p38 and JNK MAPKs, exhibit exacerbated inflammation following bacterial infections. These mice experienced a substantially enhanced cytokine storm and showed markedly increased mortality associated with vascular leak and circulatory collapse. Given the enhanced inflammatory response, paradoxically, Mkp-1-/- mice exhibited defective bacterial clearance. This picture of an augmented inflammatory response, and yet an inability to clear bacteria, mimics what is often seen in septic patients that go on to develop shock and multi-organ failure. However, the mechanisms leading to the impaired bactericidal activity in Mkp-1-/- mice are not known. Additionally, using bone marrow reconstituted mice, we found that Mkp-1 in cells of non-bone marrow origin (such as endothelial cells) plays a pivotal role in maintaining vascular barrier function. Our central hypothesis is that adequately enhancing Mkp-1 expression may dampen inflammation, enhance anti-bacterial immune defense, and improve barrier function, ultimately leading to better outcomes for patients with sepsis. Although Mkp-1 is critically important in the innate immune response, its regulation remains poorly understood. In preliminary studies we found that mutation of two serine residues at the C-terminus of Mkp-1, which mimics phosphorylation by ERK, increased Mkp-1 stability 20-fold without affecting Mkp-1 ubiquitination. Computer-based analysis suggests that the C-terminus of Mkp-1, adjacent to these two serine residues, contains an intrinsically disordered domain and that phosphorylation of these two serine residues decreases the disorder of this domain. This suggests that the ERK pathway stabilizes Mkp-1 via increasing the order of an intrinsic disorderd domain. Therefore, we propose to elucidate the function of Mkp-1 in phagocytes and endothelial cells and to define the mechanism by which ERK stabilizes the Mkp-1 protein. We will also determine if enhancing ERK activity can be an effective strategy for sustaining Mkp-1 expression, thereby preventing the second phase of p38 activation, ultimately inhibiting cytokine production. The objective of this application is to understand the function and regulation of Mkp-1 in phagocytes and endothelial cells during bacterial sepsis and determine if selective ERK activation can be an effective means to attenuate inflammation.