Novel Regulators of Nuclear Hormone Action: Anthrax Lethal Factor Represses Glucocorticoid and Progesterone Receptor Activity

J. I. Webster¹, L. H. Tonelli¹, M. Moayeri², S. Stoney Simons³, S. H. Leppla², E. M. Sternberg¹

¹ Section on Neuroendocrine Immunology and Behavior, National Institute of Mental Health,² National Institute of Allergy and Infectious Diseases³ National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, U.S.A.

Death by anthrax is reported to result from systemic shock resembling lipopolysaccharide-induced shock, although the precise mechanisms are not understood. Anthrax lethal toxin (LeTx) is composed of two proteins, protective antigen (PA) and lethal factor (LF). PA binds to a cell surface receptor and allows entry of LF into the cell. This mechanism of entry into the cell is fairly well understood. However, the actions of LF once inside the cell are not so clear. LF has been shown to be a metalloprotease that cleaves members of the MAP kinase family. Much research has focused on this cleavage and inactivation of MAPKKs, however, this MAPKK cleavage has been shown to be similar in LeTx sensitive and resistant cells.

Fischer (F344/N) rats have long been known to be extremely sensitive to LeTx with death occurring within 40 minutes after exposure to a lethal dose. F334/N rats are also known to exhibit a hyper-HPA axis and are resistant to autoimmune/inflammatory diseases. Intact glucocorticoid responses have been shown to be critical for survival from several proinflammatory or infectious insults, such as streptococcal cells walls (SCW), MCMV virus or Shiga toxin. Intervention of the HPA axis at any point results in increased mortality rates that can be subsequently reversed by glucocorticoid administration. We, therefore, hypothesized that the lethal toxin might act as a glucocorticoid repressor, thereby removing the protective anti-inflammatory properties of an intact glucocorticoid response.

We found that the fully active anthrax lethal toxin (LeTx), LF + PA, represses dexamethasone-induced glucocorticoid receptor (GR) transactivation in a transient transfection system at very low concentrations (Fig. [1]). An LF mutant that is catalytically inactive was unable to repress GR, suggesting that the protease activity of LF may be required for GR repression. The activity of the endogenous GR-regulated gene, the liver enzyme tyrosine aminotransferase, was similarly repressed by LeTx in both a cellular system and in vivo in BALB/cJ mice. This repression is non-competitive 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.

Fig. 1 Repression of dexamethsone-induced GR transactions by LF (□), LF + PA (), E867C (○) or E867C + PA (•).

Fig. 2

This LeTx - nuclear hormone receptor repression is also selective, repressing GR, progesterone receptor B (PR-B) and estrogen receptor α (ERα), but not the mineralocorticoid receptor (MR) or ERβ. This is the first report of a bacterial product directly repressing the activity of GR. As known targets of LeTx are members of the MAPK family including MEK1/2 and MKK3/6 we tested the ability of inhibitors of the MAPK pathways to repress GR. Selected MEK/ERK inhibitors and JNK inhibitors had no effect on GR mediated transactivation. However, selected p38 MAP kinase inhibitors did repress GR in this transient transfection system, suggesting that the LeTx action may result in part from its known inactivation of MAP kinases.

A 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 anti-inflammatory effects of these hormones, similar to the increased mortality seen in animals exposed to both GR antagonists and infectious agents or bacterial products. Precise definition of mechanisms by which LeTx represses GR and PR may lead to potential new avenues of therapy for anthrax.