Transcriptional Regulation of the SIS/PDGF-B Gene in Human Osteosarcoma Cells by the Sp Family of Transcription Factors

Abstract

Expression of PDGF-B, the gene encoding the plateletderived growth factor B chain, has been implicated as a participant in an autocrine growth loop in the human osteosarcoma cell line U2-OS. In previous work, we identified a primary site in the PDGF-B promoter, the SIS proximal element (SPE), which is critical for transcription of the PDGF-B gene in U2-OS cells. We also identified Sp1 as one of the SPE-binding proteins in U2-OS nuclear extracts. In the present work, we have identified another SPE-binding protein to be Sp3. Gel mobility shift assays showed that both Sp1 and Sp3 require the CACCC motif within the SPE for binding. In vitro transcription assays showed that Sp1 or/and Sp3 is necessary for transcription of the PDGF-B gene. Cotransfection experiments functionally demonstrated that Sp1 and Sp3 can independently or additively activate the PDGF-B promoter through the SPE as well as a synthetic promoter. However, the CACCC motif within the SPE is not the only site within the minimal PDGF-B promoter through which Sp1/Sp3 acts; additional nested deletion analyses showed that multiple cis-acting elements within the minimal promoter are required for full level transcription of the PDGF-B gene in U2-OS cells. Platelet-derived growth factor (PDGF)1 is a potent mitogen and chemotactant for cells that express functional PDGF receptors, typically cells of mesenchymal origin (Antoniades, 1991;Deuel et al., 1982). It is a homo-or heterodimeric protein consisting of two related polypeptide chains, A and B (Johnsson et al., 1982). The three isoforms of PDGF (AA, AB, and BB) stimulate biological responses by binding to two cell surface PDGF receptors, ␣ and ␤ (Claesson-Welsh, 1994).A functional, contributory role for PDGF-B in the development and maintenance of cancer cells is supported by several observations: (i) the B chain gene of PDGF was identified as the cellular homolog of the simian sarcoma virus oncogene, v-sis (Doolittle et al., 1983;Waterfield et al., 1983), (ii) when under the control of a strong promoter, both v-sis and its cellular homolog PDGF-B caused the transformation of mouse 3T3 cells or human fibroblasts Clarke et al., 1984;Gazit et al., 1984;Stevens et al., 1988), (iii) the cellular PDGF-B gene has been shown to be constitutively expressed in a large percentage of human tumor cells, cells of both mesenchymal and epithelial origin, whereas it was not detectably expressed in the normal counterparts of these cells (Eva et al., 1982;Peres et al., 1987;Maxwell et al., 1990), and (iv) expression of a recombinant, dominant-negative mutant of PDGF-B in PDGF-B-expressing astrocytoma cells resulted in a substantial reduction in the tumorigenic growth of the cells (Shamah, 1993). The above evidence, especially the fact that high level expression of the cellular form of the sis oncogene is sufficient for oncogenesis, implies that loss of its transcriptional regulation provides a contributory step in neoplastic transformation.In U2-OS, a human osteosarcoma cell line, coexpression of the PDGF-B gene as well as ...

Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene

“…The transcriptional start site of L-CPT I conforms well with a consensus initiator sequence Py\Py\A (j1)\N\(A\T)\Py\Py in which the A is the first transcribed nucleotide [28]. The first 140 bp of the promoter are extremely GC rich with 77 % of the nucleotides being G or C. There is no apparent TATA box but there are several consensus Sp1 binding sites at k21 to k27 and k100 to k107 [29,30]. Within this region, there are several AP-2 binding sites and binding sites for the CCAAT enhancer binding protein (C\EBP) [23,31].…”

“…Sp1 in particular has been shown to tether the TATA factors and participate in transcriptional initiation [33]. Since there are multiple members of the Sp family of transcription factors, others may also be involved in L-CPT I expression [29,30]. AP-2 has also been reported to be involved in the transcriptional regulation of TATA-less genes [31,35].…”

Transcriptional regulation of the hepatocyte growth factor (HGF) gene by the Sp family of transcription factors

“…It is known that the major regulatory function of Sp1 is upregulation of its target genes. In contrast, the major function of Sp3 is down regulation (although exceptions exist; for example, the PDGF-B promoter was reported to be upregulated by Sp3 (Hagen et al, 1994;Liang et al, 1996), presumably by competing with Sp1 for the same binding site). Because Sp1 and Sp3 bind to the same DNA sequence, it is di cult to di erentiate their functions by changing the binding site.…”

Identification of regulatory elements in the human adipose most abundant gene transcript-1 (apM-1) promoter: role of SP1/SP3 and TNF-? as regulatory pathways

“…SP1 and SP3 contain a similar DNA-binding domain with three zinc fingers and two glutamine-and serine/threonin-rich trans-activation domains [33,40]. SP3 comprises an additional inhibitory domain, leading to either activation or repression depending on the promoter and cellular context [40,41,42,43,44,45,46]. In this context, we reported, that the apM-1 promoter lacks a classical TATAA box, but contains a classical CCAAT box and has several transcription start sites [22], thus belonging to the group of class II promoters.…”