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. 2008 Sep;22(9):2049–2060. doi: 10.1210/me.2007-0489

Fig. 3.

Fig. 3.

Dexamethasone Induction of the P2 Promoter of 11β-HSD1 Requires C/EBP-Binding Sites FP3 and FP4

A, In transiently transfected A549 cells, dexamethasone dose-dependently increased activity of p11β1(−1799/+49)-LUC, a luciferase reporter construct encoding −1799 to +49 of the 11β-HSD1 P2 promoter. Data are expressed as fold induction relative to vehicle-treated cells and are mean ± sem of at least two experiments, each carried out in triplicate. B, Effect of 5′ deletion or internal deletion of the 11β-HSD1 P2 promoter upon dexamethasone response. Data are expressed relative to vehicle-treated p11β1(−1799/+ 49) (arbitrarily set to 1) and are mean ± sem of at least three experiments, each carried out in triplicate. C, Effect of mutation of FP3 and/or FP4 upon the glucocorticoid response of the proximal 11β-HSD1 P2 promoter, encoded by p11β1(−196/+49)-LUC. Data are expressed relative to vehicle-treated p11β1(−196/+49)-LUC (arbitrarily set to 1) and are mean ± sem of at least three experiments, each carried out in triplicate. D, The fragment encoding −196 to −88 of the 11β-HSD1 P2 promoter in one [P(−196/−88)1], two [P(−196/−88)2], or three [P(−196/−88)3] copies confers a glucocorticoid response upon the heterologous promoter present in the vector pGL2-promoter. Data are expressed relative to vehicle-treated vector (arbitrarily set to 1) and are mean ± sem of at least three experiments, each carried out in triplicate. E, No effect of mutation of the putative GRE (indicated between FP3 and FP4 by an X) upon the glucocorticoid response of the proximal 11β-HSD1 P2 promoter, encoded by p11β1(−196/+49)-LUC, either alone or in combination with either FP3 or FP4 mutation. Data are expressed relative to vehicle-treated p11β1(−196/+49)-LUC (arbitrarily set to 1) and are mean ± sem of at least five experiments, each carried out in triplicate. In each transfection, 1 × 105 cells were seeded per well in six-well plates and transfected with 250 ng test plasmid and 250 ng pRSV-lacZ (internal control). Promoter activity is expressed as luciferase/β-galactosidase (internal control) activity. Black bars represent activity in vehicle-treated cells; white bars represent activity in dexamethasone-treated cells (10−6 m unless stated otherwise). *, P < 0.05; **, P < 0.001; ***, P < 0.0001.