Collagen alpha1(I) gene contains an element responsive to tumor necrosis factor-alpha located in the 5' untranslated region of its first exon.
The aims of the present study were to identify the cis-acting element through which tumor necrosis factor-alpha (TNFalpha) inhibits collagen alpha1(I) gene transcription and the trans-acting factors involved in this effect in cultured hepatic stellate cells. Deletion analysis of the collagen alpha1(I) promoter demonstrated that TNFalpha inhibited gene expression through an element located between -59 and + 116 bp relative to the transcription start site. DNase I protection assays revealed a footprint between +68 and +86 bp of the collagen first exon, the intensity of which decreased when the DNA probe was incubated with nuclear protein from TNFalpha-treated hepatic stellate cells. This footprint contained a G+C-rich box. Transfection experiments demonstrated that mutations in this G+C-rich element abrogated the inhibitory effect of TNFalpha on the collagen alpha1(I) promoter. Gel retardation experiments using a radiolabeled oligonucleotide containing sequences of this region confirmed that TNFalpha treatment decreased the formation of two complexes between nuclear proteins and DNA. These complexes were efficiently blocked with an oligonucleotide containing an Spl-binding site and were supershifted with specific Spl and Sp3 antibodies. These results suggest that TNFalpha inhibits collagen alpha1(I) gene expression by decreasing the binding of Spl to a G+C-rich box in the 5' untranslated region of its first exon.
Hernández
I
I
Centro de Investigación del Hospital 12 de Octubre, School of Medicine, Universidad Complutense, Madrid, Spain.
de la Torre
P
P
Rey-Campos
J
J
Garcia
I
I
Sánchez
J A
JA
Muñoz
R
R
Rippe
R A
RA
Muñoz-Yagüe
T
T
Solís-Herruzo
J A
JA
D020121:5' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003094:Collagen / Q000187:drug effects / Q000235:genetics*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005091:Exons; D020862:GC Rich Sequence; D005786:Gene Expression Regulation / Q000187:drug effects; D008099:Liver / Q000166:cytology; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D020218:Response Elements / Q000187:drug effects* / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D020121:5' Untranslated Regions; D004268:DNA-Binding Proteins; D016329:Sp1 Transcription Factor; C497123:Sp3 protein, rat; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D051705:Sp3 Transcription Factor; D003094:Collagen; D003850:Deoxyribonuclease I