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.