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. 1998 Aug;9(8):2269–2285. doi: 10.1091/mbc.9.8.2269

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Copyright © 1998, The American Society for Cell Biology

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Identification of the FGF-2 promoter regions responsible for the activation by growth factors, PMA, and forskolin and for the maintenance of basal promoter activity. Astrocytes were transfected with FGF-2 promoter–luciferase constructs given in the figure. The numbers refer to the start and end points of FGF-2 promoter relative to the transcription start site (marked by the arrow). The luciferase activity for each plasmid is expressed relative to the activity in nonstimulated cells. (A) QG astrocytes: activation by growth factors. Bars show mean ± SEM from six to eight samples obtained in two independent experiments except (−255/+314)FGF-2Luc. Each treatment had a statistically significant effect on the expression of (−650/+314)FGF-2Luc (p < 0.05) or (−555/+314)FGF-2Luc (p < 0.01) but not on the expression of (−512/+314)FGF-2Luc or (−255/+314)FGF-2Luc. (B) QG astrocytes: basal promoter activity and activation by forskolin and PMA. Cells were transfected with the FGF-2 promoter–luciferase constructs and incubated in control serum-free medium (basal luciferase expression) or treated with 100 nM PMA or 5 μM forskolin as described in MATERIALS AND METHODS. Basal luciferase activity was calculated as light units per microgram of cellular protein per picogram of transfected DNA. In cells stimulated with forskolin or PMA, the luciferase activity for each plasmid is expressed relative to the activity in nonstimulated cells. Deletion from −555 to −513 bp significantly reduced basal promoter activity (p < 0.005). PMA had a statistically significant effect on the expression of (−650/+314)FGF-2Luc (p < 0.005) but not on the expression of the remaining constructs. Stimulation of (−650/+314)FGF-2Luc by forskolin did not attain a statistically significant level. FGF-2 promoter sequence from −650 to −512 bp is shown. The crossed bar below the sequence indicates GFRE (−555/−512 bp), and the solid bar indicates (−625/−556 bp) essential for PMA and forskolin stimulation. (C) NHA astrocytes: activation of the minimal (−103/+314 bp) FGF-2 promoter by upstream growth factor- and PKC-responsive elements. Astrocytes were transfected with the FGF-2 promoter-luciferase constructs and treated as in A or B. Bars show mean ± SEM of four (PMA) or eight (EGF) samples. EGF had a statistically significant effect on the expression of (−650/+314)FGF-2Luc, (−550/+314)FGF-2Luc, (−650/−453)(−103/+314)FGF-2Luc, and (−555/−453)(−103/+314)FGF-2Luc (p < 0.005). PMA had a statistically significant effect on the expression of (−650/+314)FGF-2Luc and (−650/−453)(−103/+314)FGF-2Luc (p < 0.0001). (D) FGF-2 promoter sequence from −624 to −513 bp is shown. The open bar below the sequence indicates growth the factor-responsive element (−555/−513 bp), and the solid bar indicates the region essential for PKC/cAMP stimulation (−624/−556 bp).

Identification of the FGF-2 promoter regions responsible for the activation by growth factors, PMA, and forskolin and for the maintenance of basal promoter activity. Astrocytes were transfected with FGF-2 promoter–luciferase constructs given in the figure. The numbers refer to the start and end points of FGF-2 promoter relative to the transcription start site (marked by the arrow). The luciferase activity for each plasmid is expressed relative to the activity in nonstimulated cells. (A) QG astrocytes: activation by growth factors. Bars show mean ± SEM from six to eight samples obtained in two independent experiments except (−255/+314)FGF-2Luc. Each treatment had a statistically significant effect on the expression of (−650/+314)FGF-2Luc (p < 0.05) or (−555/+314)FGF-2Luc (p < 0.01) but not on the expression of (−512/+314)FGF-2Luc or (−255/+314)FGF-2Luc. (B) QG astrocytes: basal promoter activity and activation by forskolin and PMA. Cells were transfected with the FGF-2 promoter–luciferase constructs and incubated in control serum-free medium (basal luciferase expression) or treated with 100 nM PMA or 5 μM forskolin as described in MATERIALS AND METHODS. Basal luciferase activity was calculated as light units per microgram of cellular protein per picogram of transfected DNA. In cells stimulated with forskolin or PMA, the luciferase activity for each plasmid is expressed relative to the activity in nonstimulated cells. Deletion from −555 to −513 bp significantly reduced basal promoter activity (p < 0.005). PMA had a statistically significant effect on the expression of (−650/+314)FGF-2Luc (p < 0.005) but not on the expression of the remaining constructs. Stimulation of (−650/+314)FGF-2Luc by forskolin did not attain a statistically significant level. FGF-2 promoter sequence from −650 to −512 bp is shown. The crossed bar below the sequence indicates GFRE (−555/−512 bp), and the solid bar indicates (−625/−556 bp) essential for PMA and forskolin stimulation. (C) NHA astrocytes: activation of the minimal (−103/+314 bp) FGF-2 promoter by upstream growth factor- and PKC-responsive elements. Astrocytes were transfected with the FGF-2 promoter-luciferase constructs and treated as in A or B. Bars show mean ± SEM of four (PMA) or eight (EGF) samples. EGF had a statistically significant effect on the expression of (−650/+314)FGF-2Luc, (−550/+314)FGF-2Luc, (−650/−453)(−103/+314)FGF-2Luc, and (−555/−453)(−103/+314)FGF-2Luc (p < 0.005). PMA had a statistically significant effect on the expression of (−650/+314)FGF-2Luc and (−650/−453)(−103/+314)FGF-2Luc (p < 0.0001). (D) FGF-2 promoter sequence from −624 to −513 bp is shown. The open bar below the sequence indicates growth the factor-responsive element (−555/−513 bp), and the solid bar indicates the region essential for PKC/cAMP stimulation (−624/−556 bp).