Sp family of transcription factors is involved in iron-induced collagen alpha1(I) gene expression.
The purpose of this study was to identify the cis-acting elements and the trans-acting factors involved in the iron-induced expression of the collagen alpha1(I) (COL1aI) gene. Rat hepatic stellate cells were cultured in the presence of 50 microM ferric chloride, 50 microM ascorbic acid, and 250 microM citric acid (Fe/AA/CA), and the effects on collagen gene expression and the binding of nuclear proteins to the COL1aI promoter were measured. The Fe/AA/CA treatment induced a time- and dose-dependent increase in the cellular levels of COL1aI mRNA that was abrogate by pretreating cells with cycloheximide, antioxidants, and inhibitors of aldehyde-protein adduct formation. Transient transfection experiments showed that Fe/AA/CA exerted its effect through regulatory elements located between -220 and -110 bp of the COL1aI promoter. Gel retardation assays showed that Fe/AA/CA increased the binding of nuclear proteins to two elements located between -161 and -110 bp of the COL1aI promoter. These bindings were blocked by unlabeled consensus Sp1 oligonucleotide and supershifted with Sp1 and Sp3 antibodies. Finally, Fe/AA/CA increased cellular levels of the Sp1 and Sp3 proteins and Sp1 mRNA. Treatment with Fe/AA/CA stimulates COL1aI gene expression by inducing the synthesis of Sp1 and Sp3 and their binding to two regulatory elements located between -161 and -110 bp of the COL1aI promoter.
Ruiz
I G
IG
Department of Gastroenterology, Research Center, Hospital Universitario 12 de Octubre, Madrid, Spain.
de la Torre
P
P
Diaz
T
T
Esteban
E
E
Morillas
J D
JD
Muñoz-Yagüe
T
T
Solís-Herruzo
J A
JA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides; D019343:Citric Acid / Q000494:pharmacology; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005290:Ferric Compounds / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D009687:Nuclear Proteins / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D017392:Thiobarbituric Acid Reactive Substances / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
D000975:Antioxidants; D002712:Chlorides; D004268:DNA-Binding Proteins; D005290:Ferric Compounds; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D017392:Thiobarbituric Acid Reactive Substances; D014157:Transcription Factors; D051705:Sp3 Transcription Factor; D019343:Citric Acid; D003094:Collagen; D003513:Cycloheximide; D011392:Proline; D001205:Ascorbic Acid; C024555:ferric chloride