The EGF/TGFalpha response element within the TGFalpha promoter consists of a multi-complex regulatory element

Abstract

Autocrine TGFalpha is an important growth effector in the transformed phenotype. Growth stimulation of some colon cancer cells as well as other types of cancer cells is effected by activation of the epidermal growth factor receptor. Importantly, this receptor activation leads to further stimulation of TGFalpha transcription and increased peptide synthesis. However, the molecular mechanism by which TGFalpha transcription is activated is poorly understood. In this paper, we describe the localization of a cis-sequence within the TGFalpha promoter which mediates this stimulation. This region contains parallel cis-acting elements which interact to regulate both basal and EGF-induced TGFalpha expression. The well differentiated colon carcinoma cell line designated FET was employed in these studies. It produces autocrine TGFalpha but requires exogenous EGF in the medium for optimal growth. Addition of EGF to FET cells maintained in the absence of EGF resulted in a 2 - 3-fold increase of both TGF promoter activity and endogenous TGFalpha mRNA at 4 h. This addition of EGF also stimulated protein synthesis. The use of deletion constructs of the TGFalpha promoter in chimeras with chloramphenicol acetyl transferase localized EGF-responsiveness to between -247 and -201 within the TGFalpha promoter. A 25 bp sequence within this region conferred EGF-responsiveness to heterologous promoter constructs. Further use of deletion/mutation chimeric constructs revealed the presence of at least two interacting cis-elements, one binding a repressor activity and the other, an activator. Gel shift studies indicate the presence of distinct complexes representing activator and repressor binding, which are positively modulated by EGF. The type and amount of complexes formed by these proteins interact to regulate both the basal activity and EGF-responsiveness of the TGFalpha promoter. The interaction of an activator protein with an EGF-responsive repressor may serve to regulate the level of this progression-associated, transforming protein within tight limits.