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. Author manuscript; available in PMC: 2021 Jun 30.
Published in final edited form as: Cell Rep. 2021 Jun 1;35(9):109185. doi: 10.1016/j.celrep.2021.109185

Figure 4. MR drives FKBP5 expression under baseline conditions, which fine-tunes GR stress responsiveness in mouse primary hippocampal neurons.

Figure 4.

The effects of altered MR levels on FKBP5 expression as well as on receptor binding to GREs within Fkbp5’s promoter region were examined under baseline conditions, in medium supplemented with charcoal-stripped serum (CSS, steroid-free; resulting in no receptor activation) and following Dex stimulation using biotinylated oligoIP in primary hippocampal neurons.

(A) Transfection of a MR-expressing plasmid concentration dependently increased MR protein expression (F3,8 = 18.30, p < 0.001).

(B–D) Under baseline conditions, OE of MR significantly increased FKBP5 expression (F3,8 = 7.578, p < 0.01) as well as MR binding to the Fkbp5-GRE oligonucleotide (F3,8 = 12.98, p < 0.01), while GR binding was decreased (F3,8 = 6.461, p < 0.05).

(E) Example blots of (A)–(D). Ctrl 1: magnetic beads lacking conjugated streptavidin. Ctrl 2: cells transfected with empty Ctrl vector.

(F–I) Only under normal media (NM) conditions, OE of MR (main treatment effect F1,8 = 57.60, p < 0.0001) significantly increased FKBP5 expression (treatment-by-condition interaction F1,8 = 12.71, p < 0.01) as well as MR binding to the Fkbp5-GRE oligonucleotide (t4 = 8.241, p < 0.01), while GR binding was decreased (t4 = 2.351, p = 0.07). These effects were abolished in neurons cultured in medium supplemented with CSS.

(J) Example blots of (F)–(I). Ctrl 1: magnetic beads lacking conjugated streptavidin. Ctrl 2: cells transfected with empty Ctrl vector.

(K) Knockdown (KD) of MR led to significantly reduced MR expression under vehicle (Veh) and Dex conditions. In addition, Dex treatment increased MR expression under control conditions (treatment-by-condition interaction F1,8 = 11.71, p < 0.01).

(L) MR KD significantly reduced FKBP5 expression under vehicle conditions. In contrast, Dex treatment significantly increased FKBP5 expression, which was even more pronounced under MR KD conditions (treatment-by-condition interaction F1,8 = 5.168, p < 0.05).

(M) Dex treatment significantly increased GR binding to the Fkbp5-GRE oligonucleotide independent of MR expression (main treatment effect F1,8 = 83.18, p < 0.0001).

(N) KD of MR significantly decreased MR binding to the Fkbp5-GRE oligonucleotide. MR binding was significantly decreased following Dex treatment under control conditions (treatment-by-condition interaction F1,8 = 14.34, p < 0.01).

(O) Example blots of (K)–(N). Ctrl 1: magnetic beads lacking conjugated streptavidin. Ctrl 2: vehicle treated cells transfected with scrambled small interfering RNA (scr-siRNA) Ctrl vector.

One-way ANOVA + Bonferroni post hoc test, two-way ANOVA + Bonferroni post hoc test, and unpaired, two-tailed Student’s t test for simple comparisons: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ####p < 0.0001 (two-way ANOVA main treatment effect). Data are presented as mean + SEM; n = mean of three independent experiments.