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. 2011 Jan 7;286(11):9049–9062. doi: 10.1074/jbc.M110.177519

FIGURE 4.

FIGURE 4.

The human RCAN1 gene promoter contains a GRE. A, dexamethasone increased RCAN1 promoter activity in a time-dependent fashion. pRCANluc-A plasmid was cotransfected with GR expression plasmid and pCH11O into HEK293 cells. Cells were treated with 100 nm dexamethasone for 0, 12, 24, 48, and 72 h prior to the luciferase assay. The luciferase activity was measured by a luminometer and expressed in RLU. The x axis represents the time, and the y axis represents the percentage increase relative to the non-treatment control. Values represent mean ± S.E. (error bars) (n = 3–6). *, p < 0.05 by analysis of variance with post hoc Newmann-Keuls test. B, mapping of GRE in the RCAN1 promoter. The various deletion plasmid constructs were cotransfected with the GR expression plasmid and pCH11O in SH-SY5Y cells. Cells were treated with 100 nm dexamethasone for 48 h before harvest. Values represent mean ± S.E. (n = 4). *, p < 0.01 by Student's t test. C, gel shift assay. The gel shift assay was performed as described under “Experimental Procedures” using a 32P-labeled double-stranded oligonucleotide probe, RCAN1−272−237bp. Lane 1, labeled probe alone without nuclear extract. Incubation of 32P-labeled RCAN1−272−237bp with HeLa nuclear extracts retarded the migration rate of the labeled probe, which formed a new shifted DNA-protein complex band (lane 2). Competition assays were performed by further adding different concentrations of molar excess of unlabeled GRE competition consensus oligonucleotide (lanes 3–5). D, dexamethasone increased the RCAN1-1 mRNA level. RT-PCR was used to amplify RCAN1-1 from SH-SY5Y cells transfected with GR expression plasmid treated with or without 100 nm dexamethasone for 48 h. GAPDH was amplified as the internal control. E, quantification of RT-PCR shows that RCAN1-1 mRNA was elevated ∼5-fold after dexamethasone treatment. Values represent mean ± S.E. (n = 3). *, p = 0.0003 by Student's t test. F, dexamethasone increased RCAN1 protein expression. Western blot of RCAN1 in SH-SY5Y cells, which were transfected with GR expression plasmid and treated with or without 100 nm dexamethasone for 48 h. 150 μg of cell lysates were separated in a 12% Tris-glycine polyacrylamide gel. RCAN1 was detected with anti-RCAN1 antibody DCT3. β-Actin was detected by anti-β-actin and served as the internal control. G, quantification of E shows that RCAN1 protein was increased ∼2-fold following dexamethasone treatment. The values represent mean ± S.E. (n = 3). *, p = 0.0006 by Student's t test. H, differential activation of RCAN1 alternative promoters in SH-SY5Y cells by dexamethasone. The pRCANluc-A and pDE4Luc plasmids were cotransfected with GR expression plasmid and pCH11O into SH-SY5Y cells. pGL3-Basic was used as a negative control. Cells were treated with or without 100 nm dexamethasone for 48 h before the luciferase assay. The isoform 1 promoter plasmid pRCANluc-A has a higher activity in the neuronal cell line than the isoform 4 promoter plasmid pDE4Luc. *, p = 0.0002 by Student's t test. Dexamethasone increases the promoter activity of isoform 1 promoter pRCANluc-A (*, p = 0.0120 by Student's t test), whereas the drug has no effect on RCAN1 isoform 4 promoter (p > 0.05 by Student's t test). Values represent mean ± S.E., n = 3. Dex, dexamethasone.

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