Effects of acetaldehyde on nuclear protein binding to the nuclear factor I consensus sequence in the α 2(I) collagen promoter

Abstract

Acetaldehyde has been shown to increase collagen production in cultured rat myofibroblastlike cells and to activate the mouse α 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 α 1(I) and α 2(I) collagen genes. Activation of the α 2(I) collagen promoter was not observed when the NFI binding site of the promoter was deleted. In this study, we determined if acetaldehyde influences the binding of NF-I to the α 2(I) collagen promoter. Nuclear proteins extracted from NIH 3T3 cells, or myofibroblastlike cells, 36 hours after the addition of acetaldehyde (200 μmol/ 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 NFI oligo. Therefore, nuclear proteins, in addition to NF-I, bind to the NF-I consensus sequence and may have their binding altered by acetaldehyde. No supershift was obtained with antisera to Histone H1, which is known to also bind to the consensus sequence for NF-I in this promoter. This study suggests that the effect of acetaldehyde in enhancing transcription of the α 2(I) collagen promoter may be mediated by binding of NF-I or NFI-like proteins to the promoter, but may also include additional CCAAT binding proteins.