Transcriptional regulation of the elastin gene by insulin-like growth factor-I involves disruption of Sp1 binding. Evidence for the role of Rb in mediating Sp1 binding in aortic smooth muscle cells.
We have recently identified a novel element (EFE 5/6) in the human elastin gene promoter that modulates the ability of insulin-like growth factor I (IGF-I) to up-regulate elastin gene transcription in aortic smooth muscle cells. In the present study, we have pursued the identification of those nuclear proteins binding to the EFE 5/6 element and affected by IGF-I treatment. Chelation inactivation and metal reactivation experiments together with supershift gel analyses demonstrated that Sp1 was one of the proteins affected by IGF-I. Southwestern and Western analyses showed that Sp1 was present in IGF-I nuclear extracts and capable of binding DNA after fractionation. Addition of retinoblastoma gene product (Rb) antibody mimicked the effect of IGF-I in gel shift analysis, suggesting that Sp1 binding may be regulated by an inhibitor normally associated with Rb. The fact that the phosphorylation state of Rb was affected by IGF-I was shown by Western blot analysis. The control smooth muscle cells transcribed the elastin gene at a high level without addition of IGF-I, so it is likely that disruption of Sp1 binding is the first step in allowing the binding of a more potent activating factor.
Jensen
D E
DE
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Rich
C B
CB
Terpstra
A J
AJ
Farmer
S R
SR
Foster
J A
JA
D000818:Animals; D001011:Aorta; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004549:Elastin / Q000235:genetics*; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D016160:Retinoblastoma Protein / Q000502:physiology*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*; D015032:Zinc / Q000494:pharmacology
D016160:Retinoblastoma Protein; D016329:Sp1 Transcription Factor; D007334:Insulin-Like Growth Factor I; D004247:DNA; D004549:Elastin; D015032:Zinc