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Transcriptional Regulation of the Human Osteopontin Promoter: Functional Analysis and DNA-Protein Interactions

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Transcriptional Regulation of the Human Osteopontin Promoter: Functional Analysis and DNA-Protein Interactions Oncogene (2000) 19, 5801 ± 5809 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions Dongyan Wang1, Shunsuke Yamamoto3, Naoki Hijiya3, Etty N Benveniste2 and Candece L Gladson*,1 1Department of Pathology, Division of Neuropathology, The University of Alabama at Birmingham, LHRB 567, 701 19th Street South, Birmingham, Alabama, AL 35294, USA; 2Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama, AL 35294, USA; 3Department of Pathology, Oita Medical University, Hasama-machi, Oita 879-55, Japan Synthesis of cell attachment proteins and cytokines, such requires integrin-mediated attachment and migration as osteopontin (OPN), can promote tumor cell remodel- of the tumor cells to the extracellular matrix. Although ing of the extracellular matrix into an environment that migration can occur on the existing matrix of the promotes tumor cell attachment and migration. We normal brain (reviewed in Gladson, 1999), the tumor investigated the transcriptional regulation of OPN in the cells can actively remodel their extracellular matrix U-251MG and U-87MG human malignant astrocytoma through the synthesis of matrix proteins and cell cell lines. Deletion and mutagenesis analyses of the OPN attachment proteins that promote cell attachment and promoter region identi®ed a proximal promoter element migration. Malignant astrocytoma cells in vivo exhibit (724 to 794 relative to the transcription initiation site) upregulation of at least two such proteins, vitronectin that is essential for maintaining high levels of OPN (Gladson and Cheresh, 1991; Gladson et al., 1995) and expression in the tumor cells. This element, designated osteopontin (OPN) (Saitoh et al., 1995). OPN is not RE-1, consists of two cis-acting elements, RE-1a (755 expressed in the normal matrix of most tissues, other to 786) and RE-1b (722 to 745), which act than mineralized tissues, but it is synthesized by cells in synergistically to regulate the activity of the OPN response to various stimuli, and thus is considered to promoter. Gel shift assays using nuclear extracts of U- be a cytokine (Denhardt and Noda, 1998). OPN also 251MG cells demonstrated that RE-1a contains binding promotes cell attachment and migration through its sites for transcription factors Sp1, the glucocorticoid recognition as a ligand by several cell adhesion receptor, and the E-box-binding factors, whereas RE-1b receptors, e.g., integrins avb3 and avb5; thus, OPN also contains a binding site for the octamer motif-binding is considered to be a cell attachment protein (Liaw et protein (OCT-1/OCT-2). Inclusion of antibodies directed al., 1995; Gladson et al., 2000). Integrins avb3 and avb5 toward Myc and OCT-1 in the gel shift assays indicated are upregulated on malignant astrocytoma cells in vivo that Myc and OCT-1 participate in forming DNA- (Gladson et al., 1995), therefore, OPN could poten- protein complexes on the RE-1a and RE-1b elements, tially promote malignant astrocytoma cell attachment respectively. Our results identify two previously un- and migration. recognized elements in the OPN promoter that act The elevated levels of OPN in a wide variety of synergistically to promote upregulation of OPN synth- malignant cells (Denhardt and Guo, 1993; Brown et esis by tumor cells but are regulated by dierent al., 1994) suggests that it may play a key role in transcription factors. Oncogene (2000) 19, 5801 ± 5809. tumorigenesis; indeed, OPN was originally called transformation-associated protein. Several lines of Keywords: osteopontin; promoter; transcriptional reg- evidence support this concept. The concentration of ulation; malignant astrocytoma; astrocytes; glioma OPN is substantially elevated in the blood of patients with metastatic cancer (Senger et al., 1998). Increased expression of OPN is associated with ras transforma- Introduction tion of NIH3T3 ®broblasts (Chambers et al., 1992), and the ras-transformed ®broblasts require OPN for Accumulating evidence indicates that tumors can their growth (Gardner et al., 1994; Su et al., 1995). In actively promote their own expansion by manipulating murine cells, the v-src transforming oncogene stimu- their microenvironment. We are particularly interested lates OPN expression (Tezuka et al., 1996) and cells in the ability of malignant astrocytomas to remodel homozygous for c-src disruption exhibit suppression of their extracellular matrix by secreting matrix proteins, OPN synthesis (Chackalaparampil et al., 1996). and proteins that promote cell attachment (Gladson Finally, transformation of mouse epidermal cells by and Cheresh, 1991; Gladson et al., 1995; Pijuan- the tumor promoter, TPA, is associated with the Thompson et al., 1999). The signi®cant mortality induction of OPN (Smith and Denhardt, 1987; Chang associated with malignant astrocytomas is directly and Prince, 1993). attributable to the invasion of the normal brain by Although these studies implicate the regulation of the tumor (Russell and Rubenstein, 1989), which OPN expression in the promotion of tumor cell proliferation and attachment, as well as migration, the mechanisms that regulate the expression of OPN are still only poorly understood. Expression of OPN is *Correspondence: CL Gladson Received 19 June 2000; revised 5 September 2000; accepted 12 known to be in¯uenced by a number of dierent September 2000 factors, including hormones, growth factors, tumor Transcriptional regulation of the human osteopontin promoter D Wang et al 5802 promoters, and oncogenes (Denhardt and Guo, 1993; Denhardt and Noda, 1998; Manji et al., 1998; Sato et al., 1998; Vanacker et al., 1998). The regulation of OPN expression appears to be exerted largely at the level of transcription (Denhardt and Noda, 1998) and the OPN promoter region, speci®cally the 5' upstream sequence of the human OPN gene (up to base pair 7250), is highly conserved among the human, murine, and porcine genes (Hijiya et al., 1994). In osteosarcoma cells, transforming growth factor-b has been shown to stimulate OPN transcription as well as protein expression (Noda et al., 1988). Similarly, OPN mRNA is increased in ras-transformed NIH3T3 cells in proportion to the level of Ras expression (Chambers et al., 1992). The transactivating eect of ras on OPN expression has been attributed to a DNA regulatory element in the OPN promoter (Guo et al., 1995) and the v-src transforming oncogene has been shown to stimulate mouse OPN expression through a CCAAT box-like sequence in the OPN promoter (Tezuka et al., 1996). A previous study of the human OPN promoter demonstrated binding of nuclear proteins to several DNA elements within a proximal sequence of the promoter (Hijiya et al., 1994). Here, we report the identi®cation of two regulatory sequences in the proximal region of the promoter that are essential for OPN expression in astrocytic cells and identify Figure 1 A proximal segment in the human OPN promoter transcription factors that bind to these sequences (RE-1) is essential for maximal expression of the gene. (a) and (b) 5'-deletion analysis of a region of the OPN promoter. Various speci®cally. These two sequences appear to function portions of the human OPN promoter were cloned upstream of synergistically in maintaining the high level of OPN the luciferase gene and transfected into U-251MG and U-87MG expression in malignant astrocytic cells. Using muta- cells (a) and primary rat astrocytes (b). The names of the 5' genesis analysis, we also located elements within these deletion constructs indicate the OPN promoter sequences that two sequences that are important for the function of were placed upstream of the luciferase gene. Luciferase activities were assayed 30 h after transfection, with that of the 1.3 kb the entire sequence. promoter construct (OPN71206/+90) standardized to 100%. Luciferase activity from the wild type promoter ranged between 10 000 and 20 000 light units, while the background activity of the assay was between 100 and 200 light units. The relative position Results of RE-1 is indicated. (c) RE-1 is able to activate transcription from a heterologous promoter. The RE-1 OPN promoter Deletion analysis of an OPN promoter sequence fragment was cloned upstream of the herpes simplex virus TK promoter in the pT81Luc plasmid to create pRE-1-TK-Luc. Sequential 5'-deletion mutant constructs were prepared pT81Luc and pRE-1-TK-Luc were transiently transfected into U- 251MG, U-87MG, and U-373MG cells. Luciferase activities were as described by Hijiya et al. (1994). A region of the assayed 25 h after transfection, with that of the pT81Luc promotor sequence shown to confer ecient transcrip- construct standardized to 100%. These experiments were repeated tion of the OPN gene and the 5' deletion sequences twice and the results analysed and presented as the mean+ (Figure 1) were cloned into the pGLG2-basic reporter s.e.mean plasmid, and the luciferase activity was assayed in transfected primary rat astrocytes and two representa- tive malignant astrocytoma cell lines (U-251MG and had either no eect or only a minimal eect on the U-87MG). promoter activity (Figure 1a). Also, deletion of the In both the human astrocytoma cell lines and the sequence between 7439 and 7124 caused a slight normal rat astrocytes, deletion of a 71 bp fragment, increase (*20%) in OPN transcription in the U- from 794 to 724, resulted in a dramatic loss of OPN 251MG cells, suggesting that a negative regulatory promoter activity (490% reduction) (Figure 1a,b, element could be present in the sequence between respectively), pinpointing this proximal region (794 7439 and 7124. to 724) of the OPN promoter as the region that To determine the functional importance of the RE-1 contains the essential regulatory sites for basal sequence (794 to 724) in OPN transcription, we expression of OPN transcription. We named this placed this fragment upstream of the herpes simplex proximal 71 bp fragment RE-1 and focused our studies TK promoter in the pT81Luc plasmid and evaluated on this region.
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