The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3-portions of both 5-SHS strands in either singlestranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5-and 3-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (؊82 to ؉8). Activity of the negative regulatory region (؊1853 to ؊883), which contains the 5-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.The platelet-derived growth factor (PDGF) 1 family consists of three structurally similar glycoproteins (M r 30,000) that induce proliferation and other growth-related effects in cells of mesenchymal origin. These proteins arise from covalent dimerization of two PDGF subunits, designated the A-chain and B-chain, yielding the heterodimer PDGF-AB and two homodimers, PDGF-AA and PDGF-BB (1, 2). PDGF was implicated in tumorigenesis following the discovery of high sequence homology between the PDGF B-chain (PDGF-B) and the viral oncogene, v-sis (for a review, see Ref.3). Other studies suggest that both PDGF-A and PDGF-B may also mediate tumor progression to the metastatic phenotype (4, 5). Transcription of the PDGF-A gene is regulated by several enhancer and silencer elements that are poly-purine/pyrimidine-rich and possess a high degree of single-stranded, non-B DNA structure. Other laboratories (6, 7) as well as our own (8) have demonstrated that a highly GC-rich and nuclease-hypersensitive element (PDGF-A NHE) in the proximal 5Ј-flanking sequence of the PDGF-A promoter (Ϫ82 to Ϫ40) contributes most of the basal transcriptional activity of the gene. This activity is mediated by the binding of members of the Sp1 family of transcription factors and can be induced in vascular endothelial cells by phorbol ester treatment through displacement of Sp1 and Sp3 by the early growth response factor Egr-1 (7, 9) or repressed by binding of the Wilms' tumor gene product WT1 (10). More recently, we local...

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Transient transfection analysis of DNA subfragments from the distal 5-flanking region of the human plateletderived growth factor A-chain gene (؊18.3 to ؊1.8 kilobase pairs (kb)) revealed enhancer and silencer elements that contribute significantly to transcriptional regulation. Two adjacent regions (؊8.2 to ؊7.5 kb and ؊7.5 to ؊7.0 kb) enhanced transcription of both A-chain and heterologous thymidine kinase promoters, whereas repression was observed in two other nearby regions (؊9.9 to ؊8.2 kb and ؊7.0 to ؊5.9 kb). The ؊7.5 to ؊7.0-kb fragment, or J, was the strongest enhancer, and its activity was localized to a 66-base pair element (A-chain cell type-specific enhancer (ACE 66)). ACE66 activity was highly cell type-specific, with greatest activity seen in choriocarcinoma cell lines (4 -10-fold enhancement). Progressive 5-and 3-deletions of the ACE66 revealed distribution of activity across the element, with nucleotides 1-33 being critical for function. Electrophoretic mobility shift assays revealed cell type-specific patterns of high affinity protein binding to the element. Ethylation interference footprinting of JEG-3 extract localized guanine contacts on nucleotides 1-18 of both strands of the ACE element, whereas more extensive contacts were made with the phosphate backbone (nucleotides 1-32). The ACE66 element is a potent transcriptional regulator in placental cells and represents a valuable model of long distance regulation in a growth factor gene. Platelet-derived growth factor (PDGF)1 is a potent mitogen and chemoattractant for mesenchymal cells that was first purified from human platelet extracts but later shown to be expressed in a variety of normal and transformed cell types (1, 2). PDGF is a family of dimeric glycoproteins that arise from different combinations of two polypeptide chains, termed A and B, that are encoded by separate genes located on different chromosomes. The human A-chain gene contains 7 exons and is located on chromosome 7 (7pter-7q22), spanning approximately 24 kb (3, 4). Transcription of the A-chain gene is activated rapidly by a wide range of growth factors, cytokines and other mitogens (5-7), whereas complex cell type-specific patterns of A-chain transcription appear to underlie the regulation of cell proliferation during embryogenesis (8) and cellular differentiation (9). A-chain null mice exhibit abnormalities in early embryonic development and formation of lung alveolar myofibroblasts (10, 11), whereas expression of the A-chain and the PDGF ␣-receptor also appear to critical for development of the cardiovascular and nervous systems in mice (12)(13)(14). PDGF also appears to be critical for placental development, with high expression of A-chain observed in multiple cell types of the placenta including trophoblasts (2, 15), as well as extraembryonic membranes and uterine smooth muscle cells during pregnancy (15, 16). Inappropriate expression of the PDGF genes has also been implicated in many disease states involving abnormal cell proliferation, including atherosclerosis, pulmon...

Expression of the platelet-derived growth factor-A subunit (PDGF-A) is regulated to a significant degree by DNA elements located in the 5'-distal region of the gene. A potent basal enhancer (ACE66) located approximately 7 kb upstream of the transcription start site contains a number of half-sites for nuclear receptor binding, two of which are arranged in the form of direct repeat-3 motif that corresponds to a consensus vitamin D response element (VDRE). Electrophoretic mobility shift assays confirmed that the ACE66 sequence was recognized as a high affinity target for binding of heterodimers of recombinant vitamin D receptor (VDR) and its partner, retinoid-X receptor-alpha (RXRalpha). VDRE activity was localized by transient transfection analysis to a direct repeat-3 motif within the 5'-portion of the ACE66 element. Moreover, 1,25-(OH)2D3 was validated as a regulator of the endogenous PDGF-A gene by the vitamin D-stimulated upregulation of PDGF-A mRNA levels in a VDR-expressing clone of JEG-3 cells. Thus, PDGF-A represents a novel mitogenic target of 1,25-(OH)2D3 whose expression is induced via binding of hormone-activated VDR to a response element located far upstream of the transcription start site.

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