Chronic corticosterone exposure increases expression and decreases deoxyribonucleic acid methylation of Fkbp5 in mice

Abstract

There is evidence for hypercortisolemia playing a role in the generation of psychiatric symptoms and for epigenetic variation within hypothalamic-pituitary-adrenal (HPA) axis genes mediating behavioral changes. We tested the hypothesis that expression changes would be induced in Fkbp5 and other HPA axis genes by chronic exposure to corticosterone and that these changes would occur through the epigenetic mechanism of loss or gain of DNA methylation (DNAm). We administered corticosterone (CORT) to C57BL/6J mice via their drinking water for 4 wk and tested for behavioral and physiological changes and changes in gene expression levels using RNA extracted from hippocampus, hypothalamus, and blood for the following HPA genes: Fkbp5, Nr3c1, Hsp90, Crh, and Crhr1. The CORT mice exhibited anxiety-like behavior in the elevated plus maze test. Chronic exposure to CORT also caused a significant decrease in the hippocampal and blood mRNA levels of Nr3c1 and a decrease in Hsp90 in blood and caused an increase in Fkbp5 for all tissues. Differences were seen in Fkbp5 methylation in hippocampus and hypothalamus. To isolate a single-cell type, we followed up with an HT-22 mouse hippocampal neuronal cell line exposed to CORT. After 7 d, we observed a 2.4-fold increase in Fkbp5 expression and a decrease in DNAm. In the CORT-treated mice, we also observed changes in blood DNAm in Fkbp5. Our results suggest DNAm plays a role in mediating effects of glucocorticoid exposure on Fkbp5 function, with potential consequences for behavior.

Figures

Figure 1

Organ weights and physiological measurements. A, Thymus, spleen, and adrenal glands were weighed for corticosterone-treated (black bars) and vehicle-treated (white bars) mice. The absence of data for thymic mass of corticosterone-treated mice reflects a complete atrophy of the organ after 4 wk of treatment with corticosterone. B, Weight measurements of the same organs from corticosterone and vehicle-treated mice after the mice were allowed to recover for 4 wk in the absence of corticosterone in the drinking water. C and D, Effect of corticosterone (4 wk CORT) on the blood glucose level and total adipose tissue mass, respectively. The right panel of each graph shows corresponding measurements after the recovery period (4 wk REC). Adipose tissues were further analyzed by visceral (black bars) and sc (white bars) fat (E). Asterisks indicate differences that are statistically significant (P < 0.05) between the CORT and control groups. Organ weights were not normalized to body weights because mean body weight between the CORT-treated and the control groups were virtually identical.

Figure 2

Mice were challenged with the elevated plus maze (EPM) and tail suspension test (TST) to test for anxiety-like and depressive-like symptoms, respectively. EPM: After 4 wk of treatment (A), corticosterone (black bars) and vehicle-treated (white bars) animals were each tested for a 10-min period, and the amount of time spent in each of the open arms (OPEN), closed arms (CLOSED), and the center (CENTER) is displayed as a percentage of the total time spent (10 min) on the apparatus. Same measurements were made at the conclusion of the 4-wk recovery period (B). TST: After 4 wk of treatment (C), corticosterone (black bars) and vehicle-treated (white bars) animals were tested for a 6-min period, and the amount of time spent struggling (STRUGGLING) or immobile (IMMOBILE) is displayed as a percentage of the total time spent (6 min) on the suspension apparatus. The same measurements were made at the conclusion of the 4-wk recovery period (D). Asterisks indicate differences that are statistically significant (P < 0.05).

Figure 3

Relative HPA axis gene expression levels in hippocampus (A), hypothalamus (B), and blood (C). The white bars show the gene expression levels of CORT treated mice compared with those of the vehicle solution-treated control mice at the conclusion of 4 wk of the treatment phase of the experiment (n = 5). The black bars show the gene expression levels of CORT-treated mice compared with those of the corresponding control mice at the conclusion of 4 wk of the recovery phase of the experiment, in the absence of corticosterone (n = 5). Expression levels were normalized to those of β-actin. The dotted line in each graph is used to indicate the normalized levels of gene expression for the control group. Asterisks represent statistically significant differences between the CORT group and the corresponding control group of the same time period. In blood, levels of Crh and Crhr1 were undetectable.

Figure 4

Assessment of hippocampal and hypothalamic Fkbp5 methylation in the two intronic regions. Percent methylation at two CpG positions is shown for hippocampal Fkbp5 intron 1 region (A) and four CpG positions for the intron 5 region (B). Each intronic region was assayed for both CORT (black bars) and control (white bars) groups. Solid bars on the left indicate the treatment phase, whereas striped bars on the right indicate the recovery phase. The comparable data for the hypothalamus is shown in C and D. Asterisks represent statistically significant differences between the CORT and control groups.

Figure 5

Expression and methylation levels for Fkbp5 in the mouse hippocampal neuronal cell line HT-22. Cells were treated with 1 μm corticosterone for 7 d and cultured in the absence of the hormone for an additional 7 d. Media collected from corticosterone (black triangles with dashed lines) and vehicle-treated (white circles with solid lines) samples showed average hormone levels of 264 and 19.9 ng/ml, respectively. Cells were harvested for mRNA and genomic DNA to determine the expression levels of Fkbp5 (A) and methylation differences at intron 5, CpG position 3 (B) and intron 1, and CpG position 2 (C). Asterisks indicate differences that are statistically significant (P < 0.05).

Figure 6

Gene expression and DNAm for Fkbp5 in blood for cohort 2. A, A time-course change (weeks) in blood DNAm of Fkbp5 intron 1, CpG position 1 in the second cohort, in which significant methylation differences were observed between corticosterone-treated (black triangles with dashed lines) and vehicle-treated groups (white circles with solid lines) during the 4-wk treatment period and the subsequent 4-wk recovery period. B, Expression levels of Fkbp5 in blood during the 8-wk period that includes 4 wk of treatment with corticosterone and 4 wk of recovery. Asterisks indicate differences that are statistically significant (P < 0.05).