Figure 3.

Depolarization-induced upregulation of mRNA levels and promoter gene expressions of NMDAR and COX subunits in neurons and the effects of NRF-1 silencing or binding site mutation. A, KCl depolarization induced an upregulation of NRF-1, Grin 1, Grin2b, COX2, and COX6c in primary neurons analyzed with real-time quantitative PCR. *p < 0.05, **p < 0.01, ***p < 0.001 compared with controls. B, Comparable results as in A were obtained in N2a cells. NRF-1 silencing with shRNA prevented the upregulation of these transcripts by KCl. Grin2a and Grin3a were also upregulated by KCl, indicating a general stimulatory effect of KCl on neurons. However, NRF-1 silencing did not prohibit these transcripts from being induced by KCl, as their levels are significantly different from controls, but are not different from those of KCl alone. *p < 0.05 compared with controls. All ^#p values (< 0.05) were compared with 20 mm KCl alone. Values in A and B each represent mean ± SEM of combined data from three independent experiments. C, Site-directed mutations (mut) of NRF-1 binding sites on Grin1 and Grin2b promoters resulted in a significant reduction in luciferase activity compared with their wild-type (wt) controls. KCl depolarization increased promoter activity in the wild type but not in mutated Grin1 and Grin2b. (n = 6 for each construct). *p < 0.05, **p < 0.01 compared with Grin1 and Grin2b wild type. X = p < 0.01 compared with Grin1 and Grin2b wt with KCl depolarization.