Ethanol disrupts hepatic function with the eventual appearance of alcoholic liver disease (ALD). Kupffer cells, the resident macrophages in the liver, are critical to the onset of ethanol-induced liver injury. Activation of macrophages by endotoxin/lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, leads to the production of a variety of inflammatory cytokines, including tumor necrosis factor ? (TNF?). Increased production of TNF? contributes to the development of ethanol-induced liver injury. Using both an in vivo model of ethanol feeding and a cell culture model of ethanol exposure in vitro, we have discovered that chronic ethanol exposure results in a complex dysregulation of LPS-stimulated signaling pathways controlling TNF? production. These changes ultimately lead to increased production of TNF? by macrophages after chronic ethanol. These ethanol-induced changes influence both 1) the regulation of TNF? transcription and 2) TNF? mRNA stability. In brief, we discovered that chronic ethanol increases LPS-stimulated binding of Egr-1, a zinc finger transcription factor, to the TNF? promoter; this increase was dependent on increased LPS-stimulated ERK1/2 activation after chronic ethanol. Inhibition of ERK1/2 and/or Egr-1 activation normalized LPS-stimulated TNF? production after chronic ethanol. Despite these complex changes in regulation of TNF? transcription, we found that chronic ethanol exposure had no net effect on transcription of TNF? in response to LPS. This led us to study the effects of ethanol on the stability of TNF? mRNA. We discovered that chronic ethanol exposure resulted in a stabilization of the TNF? mRNA. This response was dependent on the activation of p38 MAPK, but was independent of ERKI/2 activation. We found that chronic ethanol increased LPS-stimulated activation of p38, contributing to the stabilization of the TNF? mRNA. Further, we found that PKC? is also required for ethanol-induced stabilization of the TNF?. Studies to investigate this novel mechanism for regulation of TNF? by chronic ethanol will be the focus of this competitive renewal.
Four specific aims will address 1) the mechanism for increased LPS-activation of p38 MAPK and PKC5 after chronic ethanol, 2) the downstream targets of p38 MAPK and PKC? involved in stabilization of TNF? mRNA, 3) determination of the relationship between TNF? mRNA translation and chronic ethanol-induced stabilization and 4) identification of cis- and trans-acting factors required for chronic ethanol-induced TNF? mRNA stabilization. These studies will define the molecular mechanisms of ethanol action leading to increased TNF? production and provide a foundation for the rational design of pharmacotherapeutic strategies to normalize TNF? production in patients with ALD.
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