Sufficiency of the CRE box for MAPK pathway-mediated signaling to chromogranin A gene expression in pheochromocytoma cells
| Plasmid | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| pXP-1133 | mCgA-CRE-TK | Perfect-CRE-TK | Mutant-CRE-TK | pTK-LUC (vector) | |||||
| Promoter CRE box | 1133 bp mCgA TGACGTAA | TK (110 bp) TGACGTAA | TK (110 bp) TGACGTCA | TK (110 bp) TGA-GTAA | TK (110 bp) None | ||||
| Pathway | |||||||||
| Stimulus | |||||||||
| None | |||||||||
| Mock | 318 ± 12.4 | 407 ± 17.7 | 513 ± 20.1 | 154 ± 10.0 | 128 ± 11.5 | ||||
| Nicotinic cholinergic | |||||||||
| Nicotine (1 mm, 48 hr) | 948 ± 47.63-a | 1650 ± 67.83-a | 2580 ± 98.73-a | 208 ± 12.1 | 157 ± 9.23 | ||||
| Ratio (nicotine/mock) | 2.98 | 4.06 | 5.03 | 1.35 | 1.23 | ||||
| cAMP | |||||||||
| cAMP (0.1 mm, 48 hr) | 1130 ± 52.83-a | 1600 ± 74.53-a | 4160 ± 2513-a | 172 ± 10.2 | 135 ± 8.67 | ||||
| Ratio (cAMP/mock) | 3.55 | 3.92 | 8.11 | 1.11 | 1.05 | ||||
| MAPK pathway activators | |||||||||
| ATA (100 μm, 6 hr) | 1370 ± 65.53-a | 1290 ± 60.23-a | 2130 ± 77.83-a | 194 ± 11.5 | 127 ± 8.56 | ||||
| Ratio (ATA/mock) | 4.31 | 3.16 | 4.16 | 1.26 | 0.99 | ||||
| PAF (C16) (100 nm, 6 hr) | 935 ± 60.23-a | 924 ± 52.83-a | 2020 ± 1243-a | 186 ± 9.78 | 163 ± 7.79 | ||||
| Ratio (PAF/mock) | 2.94 | 2.27 | 3.93 | 1.21 | 1.27 | ||||
| C6 ceramide (10 μm, 6 hr) | 1080 ± 40.33-a | 1140 ± 43.93-a | 1990 ± 1113-a | 241 ± 12.33-a | 148 ± 6.62 | ||||
| Ratio (C6/mock) | 3.39 | 2.79 | 3.87 | 1.56 | 1.16 | ||||
PC12 cells were transfected with the indicated promoter/luciferase reporter plasmid (top row) and in the presence or absence of secretagogues (left column), and cells were harvested for reporter assay after the times noted. In the TK (thymidine kinase) plasmids, the herpes simplex virus 110-bp TK promoter drives expression of a luciferase reporter; isolated CRE (cAMP response element) boxes have been fused just 5′ (upstream) of the TK promoter. Both the mouse chromogranin A (mCgA) promoter and the herpes simplex virus TK promoter contain TATA boxes. Units are luciferase activity/mg protein. In the wild-type mouse Cg A promoter, the functional CRE box (TGACGTAA) is located at position −71 to −64 bp upstream of the cap site.
↵3-a Results in a treatment (secretagogue) group significantly (p < 0.05) different from the mock group in the same column. Results shown are mean ± standard error for four replicates/condition. mCgA-CRE, form in which the functional CRE box occurs in the mouse chromogranin A promoter (TGACGTAA); Perfect-CRE, consensus 8 bp CRE motif (TGACGTCA), originally described in the somatostatin promoter; Mutant-CRE, single point-gap mutation in the mCgA CRE box (TGA-GTAA). The effect of nicotine or cAMP on gene expression of these plasmids has been reported previously (Tang et al., 1996; Wu et al., 1995).