Epigenetic upregulation of FKBP5 by aging and stress contributes to NF-κB-driven inflammation and cardiovascular risk

Abstract

Aging and psychosocial stress are associated with increased inflammation and disease risk, but the underlying molecular mechanisms are unclear. Because both aging and stress are also associated with lasting epigenetic changes, a plausible hypothesis is that stress along the lifespan could confer disease risk through epigenetic effects on molecules involved in inflammatory processes. Here, by combining large-scale analyses in human cohorts with experiments in cells, we report that FKBP5, a protein implicated in stress physiology, contributes to these relations. Across independent human cohorts (total n > 3,000), aging synergized with stress-related phenotypes, measured with childhood trauma and major depression questionnaires, to epigenetically up-regulate FKBP5 expression. These age/stress-related epigenetic effects were recapitulated in a cellular model of replicative senescence, whereby we exposed replicating human fibroblasts to stress (glucocorticoid) hormones. Unbiased genome-wide analyses in human blood linked higher FKBP5 mRNA with a proinflammatory profile and altered NF-κB-related gene networks. Accordingly, experiments in immune cells showed that higher FKBP5 promotes inflammation by strengthening the interactions of NF-κB regulatory kinases, whereas opposing FKBP5 either by genetic deletion (CRISPR/Cas9-mediated) or selective pharmacological inhibition prevented the effects on NF-κB. Further, the age/stress-related epigenetic signature enhanced FKBP5 response to NF-κB through a positive feedback loop and was present in individuals with a history of acute myocardial infarction, a disease state linked to peripheral inflammation. These findings suggest that aging/stress-driven FKBP5-NF-κB signaling mediates inflammation, potentially contributing to cardiovascular risk, and may thus point to novel biomarker and treatment possibilities.

Keywords: FKBP5; aging; epigenetics; inflammation; psychosocial stress.

Fig. 1.

Aging and stress are together associated with decreased DNA methylation at selected FKBP5 CpGs. (A) Methylation decreases at selected FKBP5 CpGs along the human lifespan (GTP: β_age = −0.0045, SE = 0.0008, P = 8 × 10⁻⁸; KORA: β_age = −0.0055, SE = 0.0005, P < 2 × 10⁻¹⁶; MPIP: β_age = −0.0064, SE = 0.0012, P = 7 × 10⁻⁸; total n = 2,523). (B) Depressive phenotypes are associated with accelerated age-related decrease in FKBP5 methylation (total n = 2,249, meta-analysis interaction P = 2.6 × 10⁻², heterogeneity P = 2.7 × 10⁻¹). Statistics per cohort: GTP: interaction P = 1.9 × 10⁻², β_age for moderate/severe depression = −0.0075 (SE = 0.0014) vs. β_age for no/mild depression = −0.0032 (SE = 0.0011); KORA: interaction P = 6.3 × 10⁻¹, β_age for higher levels of depression = −0.0063 (SE = 0.0011) vs. β_age for lower levels of depression = −0.0047 (SE = 0.0007); MPIP: interaction P = 1.9 × 10⁻¹, β_age for depressed = −0.0077 (SE = 0.0015) vs. β_age for nondepressed = −0.0044 (SE = 0.0019). (C) Early life separation is associated with lower methylation of the age-related FKBP5 CpGs in the HBCS (β_separation = −0.0932, SE = 0.0343, P = 7.4 × 10⁻³, mean DNA methylation difference = 1.4%). The y axis in A, B, and C depicts the residuals of the average DNA methylation levels (M-values) of the two age-related FKBP5 CpGs (cg20813374 and cg00130530) and reported statistics are after adjustment for all covariates for each cohort (SI Appendix, Supplementary Methods); for a more intuitive visualization, selected panels are also depicted as percent DNA methylation (Beta-values) in SI Appendix, Fig. S1. (D) In vitro aging and exposure to the stress hormone (glucocorticoid) receptor agonist DEX additively decrease methylation at the age-related FKBP5 CpGs in the IMR-90 fibroblast model of replicative senescence (F_1,6 = 6.3, interaction P = 4.6 × 10⁻², n = 4 replicates per group). Statistical comparisons were performed with two-way mixed-design ANOVA (per experimental design), using replicative age as the between-subject and DEX treatment as the within-subject factor. Statistically significant effects were followed with Bonferroni-corrected pairwise comparisons, shown as follows: *P < 5 × 10⁻², statistically significant pairwise comparisons for young vs. old replicative age; ^#P < 5 × 10⁻², statistically significant pairwise comparison for vehicle vs. DEX-treated old cells. Error bars depict the SE around the group mean. The y axis in D depicts the average percent DNA methylation of the two FKBP5 CpGs.

Fig. 2.

Aging and stress-related phenotypes are associated with epigenetic up-regulation of FKBP5 in peripheral blood in the GTP (n = 355). (A) FKBP5 mRNA levels are negatively associated with average methylation of the age/stress-related CpGs (β = −0.3835, SE = 0.1585, P = 1.6 × 10⁻²). (B and C) The cortisol–FKBP5 relationship is stronger at lower methylation levels of the age-related FKBP5 CpGs: interaction P = 1.4 × 10⁻³, β_cortisol for lower methylation = 0.0299 (SE = 0.0044) vs. β_cortisol for higher methylation = 0.0069 (SE = 0.0039). The cortisol–FKBP5 relationship is stronger in older ages: interaction P = 2.4 × 10⁻⁵, β_cortisol for older subjects = 0.0376 (SE = 0.0050) vs. β_cortisol for younger subjects = 0.0075 (SE = 0.0035). (D) Higher levels of depressive symptoms are associated with stronger cortisol–FKBP5 relationship in subjects with higher levels of childhood trauma (cortisol–depression interaction P = 7.3 × 10⁻⁵) but not in subjects with lower levels of childhood trauma (cortisol–depression interaction P = 1.4 × 10⁻¹). In all panels, FKBP5 mRNA residuals are after controlling for all covariates (SI Appendix, Supplementary Methods).

Fig. 3.

FKBP5 up-regulation promotes NF-κB–driven peripheral inflammation. (A) FKBP5-related genes in peripheral blood show enrichment for inflammation-related genes and NF-κB gene targets. The number of genes for each analysis is shown in parentheses. Statistical details are provided in Datasets S5–S9. (B) Western blotting confirming FKBP5 overexpression in Jurkat T cells transfected with FKBP51-FLAG vs. cells transfected with the control vector. (C) FKBP5 overexpression nearly doubles IL-8 secretion by Jurkat T cells stimulated overnight with 25 ng/mL of phorbol-12-myristate-13-acetate and 375 ng/mL of ionomycin (PMA/I). The bar graph depicts IL-8 secretion in stimulated cell supernatants measured with ELISA from two independent experiments (t = 8.8, P = 4.4 × 10⁻⁷, n = 8 per condition). For each experiment, fold ratios of IL-8 secretion were calculated relative to stimulated cells expressing the control vector. IL-8 was not detectable in nonstimulated cells. (D) FKBP5 overexpression increases NF-κB activity in stimulated Jurkat T cells. The bar graph depicts NF-κB reporter activity in stimulated cells measured with dual-luciferase reporter assays from three independent experiments (t = 3.2, P = 5.5 × 10⁻³, n = 9 per condition). For each experiment, fold ratios of NF-κB activity were calculated relative to nonstimulated cells expressing the control vector. (E) FKBP5 expression changes are associated with extensive alterations in the NF-κB coexpression network in peripheral blood. The circles depict genes encoding molecular partners of the NF-κB pathway. Continuous lines (edges) represent positive and dotted lines negative pairwise correlations corrected for expression levels of all other genes in the pathway (details in SI Appendix, Supplementary Methods). Edge widths are proportional to the absolute value of the respective correlation coefficient. The gene pair with the most robust difference in correlation between the two groups (CHUK-MAP3K14) is highlighted in orange. Statistical details for all gene pairs are provided in Dataset S10. Error bars depict the SE around the group mean. **P < 10⁻²; ***P < 10⁻³.

Fig. 4.

FKBP5 up-regulation promotes NF-κB signaling by strengthening the binding of key regulatory kinases, and these effects are prevented by selective FKBP5 antagonism. (A and B) Immunoprecipitation (IP) for either FKBP5 or NIK followed by Western blotting and binding quantification in lysates from Jurkat cells or PBMC treated with the stress hormone (glucocorticoid) receptor agonist DEX, which robustly induces FKBP5 expression, and/or the selective FKBP5 antagonist SAFit1. IgG: control IP without primary antibody. Jurkat: FKBP5 to NIK binding, DEX × SAFit1 F_1,8 = 9.3, interaction P = 1.6 × 10⁻²; IKKα to FKBP5 binding, DEX × SAFit1 F_1,8 = 4.7, interaction P = 6.2 × 10⁻²; IKKα to NIK binding, DEX × SAFit1 F_1,8 = 5.8, interaction P = 4.3 × 10⁻². PBMC: FKBP5 to NIK binding, DEX × SAFit1 F_1,8 = 5.7, interaction P = 4.4 × 10⁻²; IKKα to FKBP5 binding, DEX × SAFit1 F_1,8 = 11.2, interaction P = 1 × 10⁻²; IKKα to NIK binding, DEX × SAFit1 F_1,8 = 3.9, interaction P = 8.4 × 10⁻². n = 3 biological replicates per condition. (C) Western blotting of Jurkat cell and PBMC lysates (n = 3 replicates per condition) showing increase in the functional phosphorylation of IKKα at serine 176 (pIKKα) by DEX that is prevented by SAFit1 (Jurkat: DEX × SAFit1 F_1,8 = 12.9, interaction P = 7 × 10⁻³; PBMC: DEX × SAFit1 F_1,8 = 0.6, interaction P = 4.6 × 10⁻¹). (D) Similar effects are observed when Jurkat cells are transfected with an expression construct encoding FKBP5 (ect. FKBP5) and treated with SAFit1 (ect. FKBP5 × SAFit1 F_1,12 = 6.6, interaction P = 2.5 × 10⁻², n = 4 replicates per condition). (E) FKBP5 overexpression increases NF-κB activity in Jurkat cells stimulated overnight with 25 ng/mL of phorbol-12-myristate-13-acetate and 375 ng/mL of ionomycin, and this increase is prevented by concomitant treatment with SAFit1 (ect. FKBP5 × SAFit1 F_1,32 = 4.5, interaction P = 4.2 × 10⁻², n = 9 replicates per condition). NF-κB reporter activity was measured with dual-luciferase reporter assays in three independent experiments. (F) Scheme summarizing the results from protein–protein binding and reporter gene experiments. All treatments with DEX and SAFit1 were performed for 24 h at 100 nM concentration. All data are shown as fold changes compared with the control-vector vehicle-treated cells. All statistical comparisons were performed with two-way ANOVA, using either DEX treatment or FKBP5 overexpression as the first factor and SAFit1 treatment as the second factor. Statistically significant effects were followed with Bonferroni-corrected pairwise comparisons, shown as follows: *P < 5 × 10⁻², **P < 10⁻², ***P < 10⁻³, statistically significant pairwise comparisons for control vs. DEX or ect. FKBP5; ^#P < 5 × 10⁻², ^##P < 10⁻², ^###P < 10⁻³, pairwise comparisons for vehicle vs. SAFit1 treatment (shown only for significant interaction terms of the respective two-way ANOVAs). Error bars depict the SE around the group mean.

Fig. 5.

NF-κB signaling drives FKBP5 expression via a response element gated by the age/stress-related CpGs. (A) Data from dual-luciferase reporter gene assays using a CpG-free luciferase reporter construct, which includes the FKBP5 sequence surrounding the NF-κB response element and the age/stress related CpGs (insert sequence shown in SI Appendix, Fig. S9) but completely lacks other CpGs. This reporter construct was in vitro-methylated and transfected into monocyte-derived human cell lines (THP-1). Cells were then stimulated overnight with 25 ng/mL phorbol-12-myristate-13-acetate and 375 ng/mL ionomycin (PMA/I), a combination that robustly induces NF-κB signaling. Data are derived from two independent experiments (n = 12 replicates per condition). Comparison was performed using two-way ANOVA with methylation and treatment as factors (F_1,44 = 59.5, interaction P < 10⁻³), and statistically significant effects were followed with Bonferroni-corrected pairwise comparisons. (B–D) The effect of in vitro DNA methylation on PMA/I-induced NF-κB binding to the NF-κB response element was examined using biotinylated oligonucleotide-mediated ChIP in THP-1 cells (oligonucleotide sequence shown in SI Appendix, Fig. S9). Schematic summary of the experimental setup is shown in B. After ChIP, NF-κB/p65 binding was quantified by Western blotting using antibodies specific for NF-κB (C: example blots; D: quantifications). CTRL (control) 1: magnetic beads lacking conjugated streptavidin; CTRL 2: cells transfected with nonbiotinylated oligonucleotide. Bar graph shows data derived from four independent experiments (t = 2.5, P = 4.4 × 10⁻², n = 4 per condition). Statistical t test compared cells carrying the unmethylated probe treated overnight with vehicle or PMA/I. Binding was not quantifiable for cells carrying the methylated probe. Data are always shown as fold changes compared with the vehicle-unmethylated cells. Error bars depict the SE around the group mean. P values for pairwise comparison are shown as follows: ***P < 10⁻³, statistically significant pairwise comparisons for methylated vs. unmethylated. ^#P < 5 × 10⁻²; ^###P < 10⁻³, statistically significant pairwise comparisons for vehicle vs. drug treatment.

Fig. 6.

Association of age/stress-related FKBP5 decrease in DNA methylation with a history of MI and overall scheme summarizing study findings. (A) Age/stress-related decrease in FKBP5 methylation is associated with a history of MI in two independent cohorts: KORA, n = 1,648 subjects without vs. 62 with history of MI, β_MI = −0.0535, SE = 0.0201, P = 7.9 × 10⁻³, mean DNA methylation difference = 1.8% and MPIP, n = 310 subjects without vs. 8 with history of MI, β_MI = −0.1992, SE = 0.0611, P = 1.2 × 10⁻³, mean DNA methylation difference = 5.3%. The y axis depicts average DNA methylation levels of the two age/stress-related FKBP5 CpGs (cg20813374 and cg00130530), after adjusting for all covariates (SI Appendix, Supplementary Methods). Error bars depict the SE around the group mean. (B) Schematic summary of study’s findings showing how aging, childhood trauma, and depressive symptoms interact to decrease FKBP5 methylation at selected CpGs (cg00130530 and cg20813374) located proximally upstream of the TSS. These epigenetic changes enhance FKBP5 responses in immune cells, an effect that in turn promotes NF-κB signaling, whereas this is prevented when cells are concomitantly treated with selective FKBP5 antagonists. Notably, NF-κB signaling is not only activated by FKBP5 but it can also trigger FKBP5 transcription through an NF-κB response element that is flanked and moderated by the age/stress-related CpGs. This forms a positive feedback loop of FKBP5–NF-κB signaling that may be enhanced in individuals with lower methylation at this site. Enhanced FKBP5 responses and NF-κB activity may in turn promote chemotaxis of proinflammatory cells and peripheral inflammation, potentially contributing to cardiovascular risk.