Anthrax lethal factor represses glucocorticoid and progesterone receptor activity

Abstract

We report here that a bacterial toxin, anthrax lethal toxin (LeTx), at very low concentrations represses glucocorticoid receptor (GR) transactivation in a transient transfection system and the activity of an endogenous GR-regulated gene in both a cellular system and an animal model. This repression is noncompetitive and does not affect ligand binding or DNA binding, suggesting that anthrax lethal toxin (LeTx) probably exerts its effects through a cofactor(s) involved in the interaction between GR and the basal transcription machinery. LeTx-nuclear receptor repression is selective, repressing GR, progesterone receptor B (PR-B), and estrogen receptor alpha (ERalpha), but not the mineralocorticoid receptor (MR) or ERbeta. GR repression was also caused by selected p38 mitogen-activated protein (MAP) kinase inhibitors, suggesting that the LeTx action may result in part from its known inactivation of MAP kinases. Simultaneous loss of GR and other nuclear receptor activities could render an animal more susceptible to lethal or toxic effects of anthrax infection by removing the normally protective antiinflammatory effects of these hormones, similar to the increased mortality seen in animals exposed to both GR antagonists and infectious agents or bacterial products. These finding have implications for development of new treatments and prevention of the toxic effects of anthrax.

Figure 1

LeTx repression of Dex-induced GR transactivation in Cos7 cells. GR-transfected Cos7 cells were treated with 100 nM Dex either with increasing concentrations of LF, alone (■) or in the presence of 500 ng/ml PA (□); or with increasing concentrations of E687C, either alone (●) or in the presence of 500 ng/ml PA (○). Means and standard deviations of eight (WT LF) or three (E687C LF) experiments are shown, and data are analyzed by a one-way ANOVA followed by a Dunnett post hoc test (*, P = 0.01–0.05; **, P = 0.001–0.01; ***, P ≤ 0.001).

Figure 2

Comparison of the effects of RU486 and LeTx on the dose–response curve of Dex in GR-transfected Cos7 cells. GR-transfected Cos7 cells were treated with increasing concentrations of Dex, either alone (■) or in the presence of 0.2 μM RU486 (□), 500 ng/ml PA + 10 ngml LF (●) or 50 ng/ml LF + 5 ng/ml PA (□). (A) Means and standard deviations of three experiments are shown. (B) Data normalized as percentage of maximal for each treatment.

Figure 3

LeTx repression of Dex-induced TAT in systems endogenously expressing GR. (A) HTC cells were treated with Dex either alone (■) or together with 2 ng/ml LF with 500 ng/ml PA (□) for 18 h, and TAT activity was assayed. Mean and standard deviations are shown, and a one-way ANOVA followed by a Bonferroni post hoc test was performed. (B) BALB/cJ mice were injected with LeTx, and 30 min later with Dex. After 6 and 12 h, liver TAT activity was assayed. Means and standard deviations of 6–10 animals are shown, and a two-way ANOVA followed by a Scheffé post hoc test was performed.

Figure 4

Comparison of the effects of LeTx and inhibitors of MEK1 and JNK pathways on the response of a Dex-induced GRE luciferase and a constitutive luciferase. Cos7 cells were transfected with GR and GRE-TK luc (■) or with GR and the constitutive luciferase vector pGL3 control (Promega) (□) and treated with 100 nM Dex, and increasing concentrations of LF with 500 ng/ml PA (A), or increasing concentrations of the MEK1 inhibitors PD98059 (B) and U0126 (C) or the JNK inhibitor SP600126 (D). Means and standard deviations are shown, and data analyzed by using a two-way ANOVA followed by a Scheffé post hoc test.

Figure 5

Effect of p38 MAP kinase inhibitors on the response of a Dex-induced GRE luciferase and a constitutive luciferase and on inhibition of p38. Cos7 cells were transfected with GR and GRE-TK luc (■) or with GR and the constitutive luciferase vector pGL3 control (□) and treated with 100 nM Dex, and increasing concentrations of the p38 MAP kinase inhibitors SB203580 (A), SB220025 (C), and p38 MAP kinase inhibitor (E). Means and standard deviations are shown, and data analyzed by using a two-way ANOVA followed by a Scheffé post hoc test. Cos7 cells were pretreated for 30 min with various concentrations of SB203580 (B), SB220025 (D), or p38 MAP kinase inhibitor (F) and then further incubated with 10 μg/ml anisomycin for 30 min. Proteins were then subjected to SDS/PAGE and Western blotting using an anti-phospho-p38 antibody.

Figure 6

Effect of the LeTx on hormone-induced activity of other nuclear hormone receptors in Cos7 cells. Cos7 cells were transfected with the expression plasmids for MR (A), ERα (B), ERβ (C), or PR-B (D) and their respective reporters and treated with 100 nM aldosterone (A), 1 nM 17β-estradiol (B), 100 nM 17β-estradiol (C), or 100 nM progesterone (D), together with increasing concentrations of LF either alone (■) or in the presence of 500 ng/ml PA (□). Means and standard deviations are shown, and data analyzed by using a one-way ANOVA followed by a Dunnett post hoc test.