ABSTRACT

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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant HL13262. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Supported by Training Grant HL07429.

¶

To whom correspondence should addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 East Concord St., Boston, MA 02118. Tel.: 617-638-4361; Fax: 617-638-5339.

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The abbreviations used are: IGF-I, insulin-like growth factor I; bp, base pair(s); BSA, bovine serum albumin; EFE, elastin functional element (see Wolf et al., 1993); SMC, smooth muscle cell.

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A. J. Terpstra and J. A. Foster, manuscript in preparation.

()

C. B. Rich, H. D. Goud, and J. A. Foster, unpublished data.

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