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Platelet-derived growth factor A-chain is a potent mitogen expressed in a restricted number of normal and transformed cells. Transient transfection and deletion analysis in BSC-1 (African green monkey, renal epithelial) cells revealed that the -1680 to -1374 region of the A-chain gene repressed homologous and heterologous promoter activities by 60-80%. An S1 nuclease-hypersensitive region (5'SHS) was identified within this region (-1418 to -1388) that exhibited transcriptional silencer activity in BSC-1 and a variety of human tumor cell lines (U87, HepG2, and HeLa). Electrophoretic mobility shift assays conducted with 5'SHS oligodeoxynucleotide probes revealed several binding protein complexes that displayed unique preferences for binding to sense, antisense, and double-stranded forms of the element. Southwestern blot analysis revealed that the antisense strand of 5'SHS binds to nuclear proteins of molecular mass 97, 87, 44, and 17 kDa, whereas the double-stranded form of 5'SHS is recognized by a 70-kDa factor. Mutations within 5'SHS element indicated the necessity of a central 5'-GGGGAGGGGG-3' motif for protein binding and silencer function, while nucleotides flanking both sides of the motif were also critical for repression. These results support a model in which silencer function of 5'SHS is mediated by antisense strand binding proteins, possibly by stabilizing single-stranded DNA conformations required for interaction with enhancer sequences in the proximal promoter region of the A-chain gene.

Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.

Experiments were performed to ascertain whether circulating levels of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 varied in a cyclic manner during the 24-hr ovulatory period in Japanese quail hens and to assess the effects of dietary calcium restriction upon levels of the plasma vitamin D3 metabolites during that period. Birds fed a marginally-deficient calcium diet (1.7%) had significantly elevated plasma 1,25-dihydroxyvitamin D3 (.71 +/- .11 ng/ml, mean +/- SEM) compared with those fed high (3.5%) calcium (.35 +/- .04; P less than .01), although these values did not reveal any significant cyclic variation or correlation with known diurnal pulses in circulating 17 beta-estradiol. The magnitude of the plasma 1,25-dihydroxyvitamin D3 response to dietary calcium stress in some individuals was as great as 2.2 ng/ml, which are certainly among the highest values reported in any avian or mammalian species to date. These observations suggest that the total circulating level of plasma 1,25-dihydroxyvitamin D3 is not important in the direct cyclic regulation of laying hen calcium metabolism. The levels of free plasma hormone, target cell membrane permeability of the hormone, or regulation of cytosolic/nuclear 1,25-dihydroxyvitamin D3 receptor activity are potential aternate levels of the control of vitamin D3 mediated calcium metabolism in the quail hen.