Oncogene (1999) 18, 5923 ± 5935 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc The EGF/TGFa response element within the TGFa promoter consists of a multi-complex regulatory element Rana Awwad1,2,4, Lisa E Humphrey1,4,5, Baskar Periyasamy1, William Scovell Jr1, Wenhui Li1,5, Kevin Coleman3, Mark Lynch2, Joan Carboni2, Michael G Brattain1,5 and Gillian M Howell*,1,4,5 1Department of Biochemistry and Molecular Biology, Medical College of Ohio, PO Box 10008, Toledo, Ohio, OH 43699-0008, USA; 2Department of Molecular Genetics, Oncology Drug Discovery, Bristol Myers Squibb, PO Box 4000, Princeton, New Jersey, NJ 08543-4000, USA; 3Box 123, P®zer Central Research, Eastern Point Road, Groton, Connecticut, CT 06340, USA Autocrine TGFa is an important growth eector in the Introduction transformed phenotype. Growth stimulation of some colon cancer cells as well as other types of cancer cells TGFa (transforming growth factor a1) is a member of is eected by activation of the epidermal growth factor the family of mitogenic, autocrine growth factors receptor. Importantly, this receptor activation leads to which show homology to epidermal growth factor further stimulation of TGFa transcription and increased (EGF) and, like EGF, exert their biological eects via peptide synthesis. However, the molecular mechanism by the epidermal growth factor receptor (EGFr; Carpenter which TGFa transcription is activated is poorly under- and Cohen, 1990; Derynck, 1992; Todaro et al., 1990). stood. In this paper, we describe the localization of a cis- TGFa is secreted as a 50 amino acid peptide following sequence within the TGFa promoter which mediates this cleavage from a 160 (human) or 159 (rat) amino acid stimulation. This region contains parallel cis-acting precursor (Derynck, 1988) although secretion of larger elements which interact to regulate both basal and forms has been reported (Derynck, 1988; Watkins et EGF-induced TGFa expression. The well dierentiated al., 1988). The precursor molecule can also exist as a colon carcinoma cell line designated FET was employed transmembrane glycoprotein which has biological in these studies. It produces autocrine TGFa but requires activity (Brachman et al., 1989; Anklesaria et al., exogenous EGF in the medium for optimal growth. 1990). Addition of EGF to FET cells maintained in the absence The use of neutralizing antibodies to both TGFa of EGF resulted in a 2 ± 3-fold increase of both TGF and the EGFr, and TGFa antisense approaches have promoter activity and endogenous TGFa mRNA at 4 h. all demonstrated that TGFa is an autocrine growth- This addition of EGF also stimulated protein synthesis. stimulatory factor in colon carcinoma (Markowitz et The use of deletion constructs of the TGFa promoter in al., 1990; Sizeland and Burgess, 1991, 1992; Ziober et chimeras with chloramphenicol acetyl transferase loca- al., 1993) as well as other tumors (Derynck et al., 1987; lized EGF-responsiveness to between 7247 and 7201 Ennis et al., 1989; Imanishi et al., 1989; Malden et al., within the TGFa promoter. A 25 bp sequence within this 1989; Lee et al., 1991). TGFa has also been shown to region conferred EGF-responsiveness to heterologous be an autocrine growth factor in normal cells promoter constructs. Further use of deletion/mutation (Markowitz et al., 1990; Malden et al., 1989; Coey chimeric constructs revealed the presence of at least two et al., 1987; Madtes et al., 1988; Bates et al., 1990; interacting cis-elements, one binding a repressor activity Kudlow and Bjorge, 1990; Lewis et al., 1990) and and the other, an activator. Gel shift studies indicate the during development (Wilcox and Derynck, 1988). presence of distinct complexes representing activator and However, solid tumors generally produce higher levels repressor binding, which are positively modulated by of this growth factor than their normal counterparts EGF. The type and amount of complexes formed by (Derynck et al., 1987). these proteins interact to regulate both the basal activity Interestingly, activation of the EGFr by either and EGF-responsiveness of the TGFa promoter. The TGFa or EGF stimulates TGFa production (Coey interaction of an activator protein with an EGF- et al., 1987, 1992; Bjorge et al., 1989). Addition of responsive repressor may serve to regulate the level of TGFa or EGF to cultured cells including keratino- this progression-associated, transforming protein within cytes, breast and colon carcinoma cells results in a tight limits. time- and dose-dependent increase in TGFa mRNA production. Increased TGFa protein secretion into Keywords: colon carcinoma; EGF-response element; culture media is also observed with similar kinetics. TGFa promotor; transcription; transforming growth Activation of the EGFr has been implicated in this factora autoinduction as neutralizing antibodies to EGFr inhibit both TGFa accumulation into the media and the growth of many types of tumor cells in vitro (Markowitz et al., 1990; Ennis et al., 1989; Imanishi et *Correspondence: GM Howell al., 1989). Furthermore, overexpression of TGFa in 4 These authors contributed equally to this work the weakly tumorigenic colon carcinoma cell line GEO 5 Current address: Department of Surgery, University of Texas by stable transfection of a TGFa expression vector Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas, TX 78284, USA resulted in the isolation of clones with increased Received 30 June 1998; revised 10 May 1999; accepted 12 May 1999 tumorigenicity both in vitro and in vivo (Ziober et al., Multi-complex regulation of the TGFa promoter by EGF/TGFa RAwwadet al 5924 1993). Signi®cantly, the increased TGFa expression workers (Raja et al., 1991), using TGFa promoter- from the vector resulted in induction of endogenous luciferase constructs, reported evidence indicating that TGFa mRNA levels. EGF-responsiveness was located in the general region Despite the importance of TGFa autocrine activity of 7373 to 759 bp of the TGFa promoter, but and its consequent autoinduction as a growth speci®c elements were not identi®ed, and no known stimulant in cancer cells, little is known of the consensus motifs are present in this region. Further- molecular mechanisms controlling TGFa expression, more, as this area of the TGFa promoter shows no particularly with regard to control of transcription. homology to other known EGF-responsive consensus In order to study TGFa transcriptional regulation, sequences, it seemed likely that the EGF/TGFa we cloned a 2813 bp fragment of the human TGFa response element in this gene would represent a promoter (Saeki et al., 1991). This fragment extended novel response element. Therefore, starting with a the TGFa promoter characterization by 1673 bp in series of deletion TGFa-CAT constructs ranging from the 5' direction over that reported by a previous 7370 to 7201, we undertook studies to identify this group (Jakobovits et al., 1988). Where sequence element. overlap occurs, the sequence is identical. The rat The well dierentiated FET colon carcinoma cell TGFa promoter has also been cloned and is highly line was employed for these studies. The FET cell homologous to the human TGFa promoter (Blasband line was derived from a well dierentiated primary et al., 1990). The notable features of the TGFa tumor and has retained dierentiated characteristics promoter are that it is devoid of any CCAAT or in vitro (Brattain et al., 1984; Chantret et al., 1988; TATA box sequences. There are several Sp-1 sites Mulder and Brattain, 1989a,b; Wan et al., 1988). It located within the proximal 600 bp of DNA. There has been adapted to growth in a completely are several potential AP-2 sites located in the more chemically de®ned medium (Brattain et al., 1984). distal regions of the promoter between 7375 and This cell line expresses TGFa and EGFr and 7850 (Jakobovits et al., 1988). Since EGFr responds proliferatively to exogenous EGF (Mulder activation stimulates TGFa expression, the character- and Brattain, 1989a,b; Wan et al., 1988). While ization of the EGF/TGFa response element within exogenous EGF is not an absolute requirement for the human TGFa promoter is important in under- growth due to autocrine TGFa activity, optimal standing the control of this gene. Raja and co- growth is achieved only when EGF is present in the Figure 1 Induction of TGFa transcriptional activity and mRNA by EGF. (a) FET clone 3, stably transfected with p2813-CAT, was grown to 60% con¯uency in serum-free medium minus EGF. Cells were then changed to serum-free medium containing 10 ng/ ml EGF and harvested at 1, 4 and 24 h for assay of CAT activity as described in Materials and methods. (b) Parental FET cells adapted to growth in serum-free medium without EGF were switched to medium containing 10 ng/ml EGF and harvested at 1, 4 and 24 h later for RNA quantitation by RNase protection analysis as described in Materials and methods. (c) Quantitation of the CAT assay Multi-complex regulation of the TGFa promoter by EGF/TGFa RAwwadet al 5925 media (Mulder and Brittain, 1989b). Therefore the Results FET cell line represents an excellent model to study regulation of the TGFa promoter by activated Kinetics of induction of TGFa transcription by EGF EGFr. In this report, we describe the use of deletion Initial studies were performed to con®rm the EGF- fragments of the TGFa promoter in chloramphenicol transcriptional responsiveness of the TGFa promoter acetyltransferase (CAT) reporter-gene chimeras to in the FET cell line. The largest TGFa promoter localize the EGF/TGFa response element. Subse- construct p2813-CAT (containing the 2813 bp of quently, we de®ned a 47 bp region which conferred TGFa promoter 5' to the translation start site) was EGF-responsiveness to heterologous promoter reporter stably transfected into FET cells. Following Neo constructs. Further analysis of this sequence identi®ed selection and subsequent testing for CAT activity, 25 bp of promoter which was responsive to EGF.