The long-term OBJECTIVE of the project is to determine the mechanism and consequences of ethanol effects on cell signaling mediated via tyrosine kinase (TK) and mitogen activated protein kinases (MAPK). Preliminary results suggest that ethanol modulated both kinases. In liver cells (BNLCL2) and rat hepatocytes, ethanol dramatically potentiates MAPK activation by serum. This is not due to upregulation of MAPK protein or physical interaction between ethanol and MAPK (p42 and p44) and therefore may be due to modulation on post-receptor signaling steps. The central hypothesis is that ethanol modulates serum-stimulated TK and Ras-Raf-MAPKK cascade leading to potentiation of MAPK and its translocation into the nucleus. Rat hepatocytes will be the major model of the proposal. There are three aims:
Aim 1. To determine pharmacology of serum stimulated tyrosine and MAP kinases in rat hepatocytes. Serum activated MAPK will be determined in ethanol (10-100 mM) treated (2-4 days) rat hepatocytes. Next these parameters will be tested in hepatocytes from control (CH) and ethanol-fed (EH) rats.
Aim II. To determine the mechanism of ethanol potentiation of serum stimulated MAPK. Involvement of Ras activation (active Ras GTP form) and Ras-Raf-interaction in this ethanol effect will be investigated.
Aim III. To determine nuclear translocation of MAPK by ethanol and its downstream relevance. MAPK levels in the nucleus of ethanol-treated hepatocytes (Aim1) will be determined. Next its level in the nuclei of CH and EH (+ serum stimulation) will be established. Consequences of the MAPK potentiation on c-PLA2 and of its nuclear translocation on the expression of acute phase proteins will be ascertained. Significance: This project will establish the mechanism and molecular target(s) involved in ethanol potentiation of serum stimulated MAPK. New knowledge on the nuclear effects of ethanol on MAPK will be gained. Ethanol potentiated MAP Kinase may emerge an as important link between cytosol and nucleus to elicit nuclear responses to ethanol. Such molecular elucidation of the ethanol actions offers the potential to design therapeutic tools to detect, treat, and prevent ethanol-induced liver injury.
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