Ets transcription factors cooperate with Sp1 to activate the human tenascin-C promoter.

Tenascin-C (TN-C), an extracellular matrix glycoproteinis expressed during embryonic development, but ispresent only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, ﬁbrotic diseasesand in cancer. To better understand the mechanisms thatcontrol TN-C gene expression, we examined theregulation of the human TN-C promoter in humanﬁbroblasts. We demonstrate that a short segment of theTN-C promoter between bp 7133 and 727 containsthree evolutionarily conserved Ets binding sites (EBS).These three EBSs bind in vitro expressed Fli1 proteinand mediate transactivation of the TN-C gene by Fli1.Furthermore, two proximal EBSs contribute signiﬁcantlyto basal activity of the TN-C promoter. GABP, which ispresent in human ﬁbroblast nuclear extracts, interactswith the two proximal EBSs. In addition, several Sp1and Sp3 binding sites have been located in closeproximity to the EBSs within this promoter region.The studies performed in Drosophila cells demonstratethat either Fli1 or GABPa+b1 functionally interact withSp1 resulting in a synergistic stimulation of the TN-Cpromoter activity. In conclusion, this study shows for theﬁrst time that the TN-C gene is regulated by Etsproteins, which together with Sp1 act as potentactivators of TN-C expression.Keywords: Tenascin-C; transcriptional regulation; Fli1;GABP; Sp1IntroductionTenascin-C (TN-C) is a hexameric extracellular matrixglycoprotein, whose expression is precisely controlledtemporally and spatially during embryonic develop-ment (Erickson and Bourdon, 1989). It is expressed atsites where cells undergo extensive epithelial-mesench-ymal transformation such as the central and peripheralnervous system, lung, early heart, kidney and teeth(Erickson and Bourdon, 1989). TN-C is generallyfound at low levels in the normal adult tissues, but isupregulated in some physiological and pathologicalconditions including wound healing, inﬂammatorylesions, ﬁbrosis and in cancer tissues (Chiquet-Ehrismann et al., 1995; Crossin, 1996; Mackie, 1997).Studies have demonstrated elevated TN-C expressionin various human cancers, including breast (Shoji et al.,1992; Yoshida et al., 1997), lung (Kusagawa et al.,1998), colon (Sakai et al., 1993; Hanamura et al.,1997), and liver (Yamada et al., 1992). TN-C isexpressed in both cancer and stromal cells, suggestinga possible role in invasive properties of the cancer cellsthemselves as well as in the remodeling of cancerstroma. Strong positive TN-C staining is usuallyassociated with stromal ﬁbroblasts surrounding tumorcells, suggesting that TN-C is a stromal marker forepithelial malignancy. Furthermore, positive staining incancer cells has been associated with unfavorabledisease outcome (Yoshida et al., 1995). The transcrip-tional factors that regulate TN-C re-expression intumors are presently unknown.The transcriptional regulation of the TN-C gene hasbeen investigated in several species including human,mouse, and chicken (Jones et al., 1990; Copertino etal., 1995; Gherzi et al., 1995a). The nucleotide sequenceof the promoter region encompassing about 250 basepairs (bp) upstream of the transcriptional start site ishighly conserved among these three species. Thispromoter region also confers the highest promoteractivity in transient transfection assays. In contrast,human and mouse promoter regions upstream of 7250contain putative negative regulatory elements whichmay cause repression of TN transcription (Copertino etal., 1995; Gherzi et al., 1995a). In addition, the ﬁrstuntranslated exon of the human TN gene was shown tocontain additional positive and negative regulatoryelements (Gherzi et al., 1995b). Moreover, severalregulatory elements including a GCN4/AP-1 like site atposition 7146 and an NF-kb element at position7210 have been characterized by transfection of the7220 to +79 human promoter construct into primaryrat embryo ﬁbroblasts (Mettouchi et al., 1997). Recentstudies of the mouse TN promoter revealed thepresence of four distinct protein binding sites, includingsites for Krox24 at position 7257, octamer (N-Oct3,N-Oct5A and N-Oct5B) at position 7200, nuclearfactor one at position 7180, and an uncharacterizedprotein that binds to a site called the tenascin controlelement at position 7247 (Copertino et al., 1997).Interestingly, di•erent combinatorial e•ects of theseelements were found in cells of di•erent lineage(Copertino et al., 1997).