Abstracts

RATIONALE: Experimentally induced epileptogenesis elicits profound changes in early response gene (ERG) expression in the brain. In these studies we describe in adult rat hippocampus, early (0-6 hr), medium (1-7 da) and long term (2-8 wk) changes in the DNA-binding of transcription factors (TFs) AP1, AP2, Egr1, STAT1, NF-kB, NFIL-6, SP1 and TFIID to target DNA sequences found in the immediate promoters of the cFOS and cyclooxygenase-2 (COX-2) ERGs after a single kainic acid (KA) injection). Using LAU8080, a specific intracellular platelet-activating factor (PAF) antagonist, we provide evidence that during experimental epileptogenesis this highly bioactive phospholipid is a key intermediate in triggering AP1-, NF-kB- and STAT1-DNA binding, elements known to drive the rapid induction of ERG expression. At the end of this activity participants should further understand the interrelationships between specific TF activation and cFOS and COX-2 gene expression.
METHODS: To trigger epileptogenesis, male albino Wistar rats were injected i.p. with 10mg/kg KA using saline as a vehicle. At indicated time points (0, 1, 3, 6 hours; 1, 3, 7 days and 2, 4, 8 weeks) rats were sacrificed and hippocampal and cortical cellular and nuclear proteins and total RNA were co-isolated. TF-DNA binding was studied using gel shift and super-shift assay; RNA message levels were determined using RT-PCR and cFOS and COX-2 protein levels were quantitated using Western immunochemistry.
RESULTS: AP1-, NF-[kappa]B- and STAT1-DNA binding were found to display phasic profiles over the short, medium and long term. AP1-DNA binding kinetics strongly correlated with COX-2 gene activation over the short term while NF-[kappa]B-DNA binding paralleled COX-2 gene expression at longer time points. Sustained increases in TFIID-DNA binding suggested prolonged de novo induction of transcription from TATA-containing brain genes. Both TF-DNA binding and cFOS and COX-2 ERG expression were significantly quenched using LAU8080 prior to epileptogenic stimuli.
CONCLUSIONS: Taken together, these results suggest that induction of the proinflammatory TF triad AP1, NF-[kappa]B and STAT1, and their binding to specific target DNAs in ERG promoters drives proinflammatory gene expression in the short term and re-programs hippocampal gene expression patterns long after the triggering of a single epileptogenic event.
[Supported in part by NIH NS23002 and AG18031]