In¯uence of the human endogenous retrovirus-like element HERV-E.PTN on the expression of growth factor pleiotrophin: a critical role of a retroviral Sp1-binding site Anke M Schulte* ,1 , Claudius Malerczyk 1 , Rafael Cabal-Manzano 1 , Jason J Gajarsa 1 , Heinz-Joachim List 1 , Anna Tate Riegel 1 and Anton Wellstein 1 1 Department of Oncology, Lombardi Cancer Center, Georgetown University, Washington DC, USA Germ line insertion of a human endogenous retrovirus- like element (HERV-E.PTN) into the growth factor pleiotrophin (PTN) gene generated a phylogenetically new promoter driving the expression of functional HERV-PTN fusion transcripts. Here we show by in situ hybridization, that HERV-PTN fusion transcripts are expressed in malignant trophoblasts (i.e. choriocarcino- ma) and in the proliferative and in the invasive trophoblasts of gestational trophoblastic tissue. Addi- tionally, a 1.9 kb fragment of the HERV-derived PTN promoter was analysed which has strong activity when transiently transfected into choriocarcinoma JEG-3 cells in contrast to HeLa cells. Deletion of the retrovirally- derived promoter portion abolished its activity and an enhancer (+443 to +486) was identi®ed in this region. Electrophoretic mobility shift and supershift experiments identi®ed a Sp1 binding site in this enhancer and site speci®c mutation of this site abolished its activity in choriocarcinoma cells. Sp1 overexpression in Drosophila SL2 cells showed that the enhancer activity is mediated via Sp1 binding in vivo. Furthermore, mutation of the Sp1 binding site reduced the activity of a promoter test fragment in choriocarcinoma cells by 80%. Our result shows that a retroviral Sp1 binding site in the PTN promoter is important for the expression of growth factor pleiotrophin in human choriocarcinoma cells. Oncogene (2000) 19, 3988 ± 3998. Keywords: pleiotrophin; HERV-E.PTN; promoter; Sp1; choriocarcinoma Introduction Pleiotrophin (PTN), is a secreted protein that belongs to a new family of highly conserved heparin-binding growth factors. PTN was originally described as a developmentally-regulated cytokine with neurite out- growth activity and later shown to play a role in human tumors (for review see, Schulte and Wellstein, 1997). In contrast to its limited expression in adult tissues (Bloch et al., 1992; Vanderwinden et al., 1992), PTN is upregulated in human cancer cell lines and tumor specimens of dierent origin (Wellstein et al., 1992; Fang et al., 1992; Vacherot et al., 1999). Beside its neurite-outgrowth activity (Boehlen et al., 1991), PTN has mitogenic and transforming activity on ®broblasts (Chauhan et al., 1993; Fang et al., 1992), growth promoting activity on epithelial and endothelial cells (Courty et al., 1991; Fang et al., 1992) and induces tube formation of endothelial cells in vitro and angio- genesis (Laaroubi et al., 1994; Choudhuri et al., 1997). Furthermore, PTN enhances plasminogen activator activity in endothelial cells (Boyd et al., 1993) and seems to play an essential role for the growth of models of human breast cancer (Zhang et al., 1997), choriocarcinoma (Schulte et al., 1996) and for the growth, angiogenesis and metastasis of human mela- noma (Czubayko et al., 1996). We recently de®ned the organization of the human PTN gene and identi®ed a novel human endogenous retrovirus-like element (HERV-E.PTN) inserted into the intron region immediately upstream of the ®rst coding exon. This insertion expands the region relative to the murine gene and phylogenetic studies suggest that its insertion into the PTN locus happened approximately 25 million years ago (Schulte et al., 1996; Schulte and Wellstein, 1998). Furthermore, a genomic fragment containing the retroviral insertion site showed strong transcriptional activity in tropho- blast-derived choriocarcinoma cells. Due to this phylogenetically novel promoter, fusion transcripts between HERV-E.PTN sequence and the open reading frame of PTN are expressed in normal trophoblast cells and in choriocarcinoma cell lines (Schulte et al., 1996). HERV elements are assumed to be prehistoric sequences from infective retroviruses which inserted into the germ-line of human progenitors millions of years ago (for reviews see Wilkinson et al., 1994; Loewer et al., 1996; Patience et al., 1997; Harris, 1998). Retroviral transcripts have been detected in dierent human tissues and cell lines, preferentially of placental, embryonic or neoplastic origin (Rabson et al., 1983; Gattoni-Celli et al., 1986; Herbst et al., 1996; Johansen et al., 1989), and some investigators reported the identi®cation of HERV fusion transcripts with en- dogenous genes (DiCristofano et al., 1995; Feuchter- Murthy et al., 1993; Liu and Abraham, 1991). However, to our knowledge, only two reports of a HERV germ-line insertion that generated changes in the expression pattern of a functional human gene product have been published to date: the phylogeneti- cally novel expression of human amylase in salivary gland (Samuelson et al., 1990; Ting et al., 1992), and the expression of the growth factor PTN in human trophoblasts and in choriocarcinoma cell lines (Schulte et al., 1996; Schulte and Wellstein, 1998). Oncogene (2000) 19, 3988 ± 3998 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc *Correspondence: AM Schulte, Lombardi Cancer Center, Research Building Room E312, Georgetown University, 3970 Reservoir Road N.W., Washington DC 20007, USA Received 10 March 2000; revised 8 June 2000; accepted 15 June 2000