EFFECTS OF ACETALDEHYDE ON NUCLEAR-PROTEIN BINDING TO THE NUCLEAR FACTOR-I CONSENSUS SEQUENCE IN THE ALPHA(2)(I) COLLAGEN PROMOTER

Acetaldehyde has been shown to increase collagen production in culture d rat myofibroblastlike cells and to activate the mouse alpha(2)(I) co llagen promoter in transfected NIH 3T3 cells. Nuclear factor I (NF-I), a CCAAT binding transcription factor, is known to bind and activate t he 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 o f the promoter was deleted. In this study, we determined if acetaldehy de influences the binding of NF-I to the alpha(2)(I) collagen promoter . Nuclear proteins extracted from NM 3T3 cells, or myofibroblastlike c ells, 36 hours after the addition of acetaldehyde (200 mu mol/ L) in s erum-free media showed increased binding to the consensus sequence of the NF-I binding site by DNase I protection analysis and by electropho retic mobility shift assay (EMSA) as compared with control nuclear ext racts that were not exposed to acetaldehyde. Furthermore, nuclear prot eins extracted from myofibroblastlike cells that had been previously e xposed to acetaldehyde had a marked increase in NF-I protein, as shown by Western blot with NP-I antibodies. Antisera to NP-I resulted in a slow migrating DNA-protein-antibody complex (supershift) on EMSA. Howe ver, the NF-I antibody did not supershift all the DNA-protein complexe s, and the supershift band was not increased with nuclear proteins hom acetaldehyde-treated cells despite the increased binding of these nuc lear protein preparations to the NF-I oligo. Therefore, nuclear protei ns, in addition to NF-I, bind to the NF-I consensus sequence and may h ave their binding altered by acetaldehyde. No supershift was obtained with antisera to Histone H1, which is known to also bind to the consen sus sequence for NF-I in this promoter. This study suggests that the e ffect of acetaldehyde in enhancing transcription of the alpha(2)(I) co llagen promoter may be mediated by binding of NF-I or NF-I-like protei ns to the promoter, but may also include additional CCAAT binding prot eins.