Acute exposure of cerebellar granule neurons to ethanol suppresses stress-activated protein kinase-1 and concomitantly induces AP-1.
The current studies were designed to examine the mechanisms of acute effects of ethanol on cerebellar granule neurons (CGNs) during neurodevelopment, with specific reference to activator protein-1 (AP-1). CGNs, isolated from 3-day-old Sprague-Dawley rats and cultured for 3 days, were exposed to 0, 22.5, and 100 mM ethanol for 1 h. Gel shift assays performed on the nuclear protein extracts showed increased AP-1 and heat shock factor-1 (HSF-1) transcriptional activation in response to ethanol. Western blots and RT-PCR showed increased c-JUN and phosphorylated c-JUN (serine 73) protein, as well as c-jun mRNA. Ethanol paradoxically decreased the activity of stress-activated protein kinase-1 (SAPK-1) while increasing p44 and p42 mitogen-activated protein kinase (MAPK) activity. The protein synthesis-inhibiting and SAPK-1 activity-inducing antibiotic, anisomycin (30 and 500 microM) decreased AP-1 transcriptional activation to 47 and 23% of control values, respectively. The anisomycin effect was enhanced in the presence of 100 mM ethanol. Similarly, cycloheximide decreased ethanol-induced AP-1 transcriptional activation. Pretreatment with the MAPK kinase (MEK) pathway inhibitor PD98059 resulted in decreases in both ethanol-induced and control AP-1 DNA binding. Thus this acute ethanol-induced increased AP-1 transcriptional activation requires protein synthesis and involves MEK-independent increased MAPK phosphorylation, on the one hand, and decreased SAPK-1 activity on the other. The ethanol effect is thus ascribed to the activities of alternate kinase pathways and/or the inhibition of (a) protein phosphatase(s). Exposure of CGNs to ethanol for 24 h resulted in decreased AP-1 DNA binding, an observation that could have consequences for overall neuronal function under chronic exposure conditions.
Acquaah-Mensah
G K
GK
Division of Pharmacology and Toxicology, College of Pharmacy, Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, 78712, USA. gacqmens@mail.utexas.edu
Leslie
S W
SW
Kehrer
J P
JP
D000818:Animals; D002492:Central Nervous System Depressants / Q000494:pharmacology*; D002531:Cerebellum / Q000187:drug effects* / Q000201:enzymology; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D000431:Ethanol / Q000494:pharmacology*; D000076249:Heat Shock Transcription Factors; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D051381:Rats; D017207:Rats, Sprague-Dawley; D018808:Transcription Factor AP-1 / Q000096:biosynthesis*; D014157:Transcription Factors
D002492:Central Nervous System Depressants; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D000431:Ethanol; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases