The differential display of mRNA technique was used to screen the expressed genes in control and 50 mM chronic ethanoltreated rat C6 glial cells, with and without activation by lipopolysaccharide (LPS) combined with phorbol 12-myristate 13-acetate (PMA). One differentially expressed transcript was identified as that corresponding to the chemokine monocyte chemotactic protein (MCP)-3. MCP-3 is a broadly active chemokine that functions in chemoattraction and activation of monocytes, T lymphocytes, eosinophils, basophils, natural killer cells, and dendritic cells. Steady-state MCP-3 mRNA levels were elevated 6-fold after 24-h stimulation of control cells but less than 3-fold after stimulation of 9-day chronic ethanol-exposed cells. One-and 5-day exposures to 50 mM ethanol were not effective at reducing steady-state MCP-3 mRNA levels in stimulated cells, whereas 1-day exposure to Ͼ150 mM ethanol was effective. Stimulation with tumor necrosis factor-␣ elevated MCP-3 mRNA in C6 glial cells to a lesser extent than with LPS plus PMA, but the effects of ethanol were consistent. To gain insight into possible mechanisms for ethanol-induced reductions in steady-state MCP-3 mRNA, additional studies examined nuclear MCP-3 RNA levels and MCP-3 mRNA degradation. MCP-3 RNA content was greatly reduced in isolated nuclei from acute and chronic ethanol-exposed cells, suggesting transcriptional inhibition. On the other hand, acute ethanol exposure enhanced degradation of preexisting MCP-3 mRNA, indicating message destabilization. Thus, the results are consistent with a dual mechanism for ethanol-induced reductions in steady-state MCP-3 mRNA levels.
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