Differential Fibronectin Expression in Activated C6 Glial Cells Treated with Ethanol

Abstract

The central nervous system is particularly susceptible to alcohol effects and toxicity. Glial cells constitute the most common cell type in the brain and play critical roles in normal brain function and during infection and injury. Astrocytes in particular seem to be important targets for alcohol neurotoxicity during both development and in adulthood. To gain more insight into alcohol-mediated effects on astrocytes at the molecular level, gene expression in rat C6 glial cells was studied in the presence or absence of ethanol. The differential display of mRNA technique was used to screen the expressed genes in ethanol-treated rat C6 cells before and after treatment with lipopolysaccharide (LPS) combined with phorbol-12-myristate-13-acetate (PMA), conditions that mimic an infectious inflammatory state and cause immunologic activation. The present data show that fibronectin appeared as a major gene whose expression is increased in C6 cells by LPS plus PMA stimulation and decreased by chronic ethanol exposure, both in mRNA and protein levels. Fibronectin is a dimeric glycoprotein found in the extracellular matrix of most tissues, in the blood, and on cell surfaces and is involved in many cellular processes. These results show that chronic exposure to ethanol is associated with changes in astrocyte properties during immunologic activation that reduce fibronectin expression. The discovery of astrocyte fibronectin expression as a potential regulated target for chronic alcohol abuse may be useful in understanding, preventing, and treating some brain disorders associated with alcohol abuse and alcoholism.

Dual Mechanisms for Ethanol-Induced Inhibition of Monocyte Chemotactic Protein-3 mRNA Expression in Activated Glial Cells

“…Cells were treated with ethanol as for previous studies (Syapin et al, 1999(Syapin et al, , 2001Ren et al, 2000). Concentrations used for acute exposure were from 50 to 300 mM.…”

“…The isolation of total cellular RNA, treatment with DNase I, and performance of the reverse transcription and polymerase chain reaction for differential display were performed exactly as described previously (Ren et al, 2000). Briefly, we used the HIEROGLYPH mRNA profile kit, the FluoroDD TMRfluorescent-anchored primer adapter kit for the HIEROGLYPH mRNA profile kit system, a Genomyx LR programmable DNA sequencer, and a Genomyx SC fluorescence scanner (Beckman Coulter, Inc., Fullerton, CA).…”

“…After electrophoretic separation on agarose, ethidium bromide-stained gels were photographed with a Polaroid camera system. Quantification was achieved by band densitometry of photographed gels using an Alpha imager (Alpha Innotech Corporation, San Leandro, CA) followed by normalization to the corresponding S12 ribosomal protein cDNA band density as reported previously (Ren et al, 2000). S12 mRNA served as an internal standard to verify whether equal quantities of jpet.aspetjournals.org cDNAs were amplified and allowed for an estimation of the integrity of the extracted RNA.…”

“…Briefly, we used the HIEROGLYPH mRNA profile kit, the FluoroDD TMRfluorescent-anchored primer adapter kit for the HIEROGLYPH mRNA profile kit system, a Genomyx LR programmable DNA sequencer, and a Genomyx SC fluorescence scanner (Beckman Coulter, Inc., Fullerton, CA). Conditions for electrophoresis and gel band reamplification were exactly as described previously (Ren et al, 2000). The reamplified PCR product was used as the template for direct DNA sequencing on an ABI prism genetic analyzer (PerkinElmer Life Sciences, Boston, MA).…”

“…To identify additional ethanol-responsive genes potentially related to host defense mechanisms and alcohol-related brain damage, we have used differential display of mRNA (Liang and Pardee, 1992) to screen the expressed genes in chronic ethanol-treated rat C6 glial cells after activation with bacterial LPS combined with PMA. With this technique we have detected several differentially regulated genes possibly involved in altered CNS host defense mechanisms or other responses to chronic ethanol (L. Ren and P. J. Syapin, unpublished data), including unsuspected genes such as that coding for fibronectin (Ren et al, 2000).…”