Dual Mechanisms for Ethanol-Induced Inhibition of Monocyte Chemotactic Protein-3 mRNA Expression in Activated Glial Cells
- Departments of 1Pharmacology (L.R., P.J.S.) and 2Anesthesiology (P.J.S.), Alcohol and Brain Research Laboratory, Texas Tech University Health Sciences Center, Lubbock, Texas
- Dr. Peter J. Syapin, Department of Pharmacology, Texas Tech University Health Sciences Center, 3601 4th St., Lubbock, TX 79430-0001. E-mail: peter.syapin{at}ttmc.ttuhsc.edu
Abstract
The differential display of mRNA technique was used to screen the expressed genes in control and 50 mM chronic ethanol-treated 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 >150 mM ethanol was effective. Stimulation with tumor necrosis factor-α 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.
Footnotes
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This work was supported by National Institutes of Health Grant AA11643 and the Texas Advanced Research Program under Grant 010674-011.
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DOI: 10.1124/jpet.102.035253
- Abbreviations:
- CNS
- central nervous system
- PMA
- phorbol 12-myristate 13-acetate
- MCP
- monocyte chemotactic protein
- LPS
- lipopolysaccharide
- TNF
- tumor necrosis factor
- MIP
- macrophage inflammatory protein
- PCR
- polymerase chain reaction
- RT-PCR
- reverse transcription-polymerase chain reaction
- DBR
- 5,6-dichlorobenzimidazole riboside
- RANTES
- regulated upon activation normal T cell expressed and secreted
- IDV
- integrated density value
- iNOS
- inducible nitric-oxide synthase
- ARE
- A+U-rich element
- ANOVA
- analysis of variance
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- Received February 26, 2002.
- Accepted June 14, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
