Effects of acetaldehyde on nuclear protein binding to the nuclear factor I consensus sequence in the α2(I) collagen promoter

Abstract

Acetaldehyde has been shown to increase collagen production in cultured rat myofibroblastlike cells and to activate the mouse alpha 2(I) collagen promoter in transfected NIH 3T3 cells. Nuclear factor I (NF-I), a CCAAT binding transcription factor, is known to bind and activate the alpha 1(I) and alpha 2(I) collagen genes. Activation of the alpha 2(I) collagen promoter was not observed when the NF-I binding site of the promoter was deleted. In this study, we determined if acetaldehyde influences the binding of NF-I to the alpha 2(I) collagen promoter. Nuclear proteins extracted from NIH 3T3 cells, or myofibroblastlike cells, 36 hours after the addition of acetaldehyde (200 mumol/L) in serum-free media showed increased binding to the consensus sequence of the NF-I binding site by DNase I protection analysis and by electrophoretic mobility shift assay (EMSA) as compared with control nuclear extracts that were not exposed to acetaldehyde. Furthermore, nuclear proteins extracted from myofibroblastlike cells that had been previously exposed to acetaldehyde had a marked increase in NF-I protein, as shown by Western blot with NF-I antibodies. Antisera to NF-I resulted in a slow migrating DNA-protein-antibody complex (supershift) on EMSA. However, the NF-I antibody did not supershift all the DNA-protein complexes, and the supershift band was not increased with nuclear proteins from acetaldehyde-treated cells despite the increased binding of these nuclear protein preparations to the NF-I oligo. Therefore, nuclear proteins, in addition to NF-I, bind to the NF-I consensus sequence and may have their binding altered by acetaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)