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

Autocrine TGF alpha is an important growth effector in the transformed phen otype. Growth stimulation of some colon cancer cells as well as other types of cancer cells is effected by activation of the epidermal growth factor r eceptor. Importantly, this receptor activation leads to further stimulation of TGF alpha transcription and increased peptide synthesis. However, the m olecular mechanism by which TGF alpha transcription is activated is poorly understood. In this paper, we describe the localization of a cis-sequence w ithin the TGF alpha promoter which mediates this stimulation. This region c ontains parallel cis-acting elements which interact to regulate both basal and EGF-induced TGF alpha expression. The well differentiated colon carcino ma cell line designated FET was employed in these studies. It produces auto crine TGF alpha 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 TGF alpha mRNA at 4 h. This addition of EGF also stimulated protein synthesis. The u se of deletion constructs of the TGF alpha promoter in chimeras with chlora mphenicol acetyl transferase localized EGF-responsiveness to between -247 a nd -201 within the TGF alpha promoter. A 25 bp sequence within this region conferred EGF-responsiveness to heterologous promoter constructs. Further u se of deletion/mutation chimeric constructs revealed the presence of at lea st two interacting cis-elements, one binding a repressor activity and the o ther, an activator. Gel shift studies indicate the presence of distinct com plexes representing activator and repressor binding, which are positively m odulated by EGF. The type and amount of complexes formed by these proteins interact to regulate both the basal activity and EGF-responsiveness of the TGF alpha promoter. The interaction of an activator protein with an EGF-res ponsive repressor may serve to regulate the level of this progression-assoc iated, transforming protein within tight limits.