Anthrax Lethal Toxin Repression of GR

We have recently shown that LeTx does inhibit dexamethasone-induced GR transactivation in two cellular systems. In a transient transfection system, the ability of GR to activate a reporter gene was repressed 50% by LeTx, and in cells endogenously expressing GR the ability of dexametha-sone to induce the tyrosine aminotransferase gene was also repressed 50% by LeTx. In addition, we also showed that the ability of dexamethasone to induce the liver tyrosine aminotransferase gene in vivo in BALB/c mice was also repressed 50% by LeTx (Figure 4.1) (Webster et al., 2003).This LeTx-mediated repression of GR function is specific to gene activation as it has no effect on GR repression of NF-kB (Webster and Sternberg, 2005). LeTx does not function as a true GR antagonist, such as RU486, to repress GR gene activation as it does not prevent ligand binding to the receptor (Webster et al., 2003).

LeTx not only represses GR but also represses the progesterone receptor B (PR-B) and estrogen receptor a (ER-a) to different extents. However, not all nuclear hormone receptors are repressed by LeTx, for example ER-P is not repressed by LeTx (Webster et al., 2003). Upon further analysis, we have shown that LeTx shows receptor specificity but also some promoter specificity. For example, on a simple promoter, (GRE)2 tk-luc, which contains 2 GRE sequences before the minimal tk promoter, LeTx represses GR and PR-B but has no effect on MR. LeTx also has no effect on the androgen receptor (AR) on its simple promoter, (ARE)4 luc. However, on the complex promoter, mouse mammary tumor virus (MMTV), which contains 1000 base pairs of the MMTV promoter, LeTx is able to repress GR, PR-B, AR, and MR (Webster and Sternberg, 2005). Therefore, LeTx shows some receptor and some promoter specificity in the repression of nuclear hormone receptors.

The features that determine LeTx repression are a matter of current research. LeTx, unlike a true antagonist such as RU486, does not fully repress these receptors. This led to the suggestion that LeTx may be removing/inactivating one or more of the many cofactors involved in the interaction of nuclear hormone receptors and the transcriptional machinery (Webster et al., 2003). It is feasible that removal of one or more of these multiple pathways may result in partial repression as some activity could still be afforded through the remaining intact pathways.

We had previously shown that LeTx does not interfere with the GR-DNA interaction, at least on a simple GRE sequence in an in vitro elec-trophoretic mobility shift assay (EMSA) experiment (Webster et al., 2003). However, further analysis using chromatin immunoprecipitation (ChIP) assays showed that LeTx did, in fact, prevent GR binding to DNA in the context of native chromatin (Webster and Sternberg, 2005). The observation of this difference in assays employing an oligonucleotide versus native chromatin eludes toward the molecular mechanism of action of LeTx. As stated above, we had previously suggested that the site of action of LeTx is one of the many cofactors involved in nuclear hormone receptor gene activation (Webster et al., 2003), but this could now be extended to included the accessory proteins that interact with GR at the level of DNA binding.

Interestingly, arsenic has been shown to inhibit GR in a similar manner. Low levels of arsenic inhibit GR-mediated gene activation but not gene repression, and this appears to be also dependent on the DNA binding region of the receptor (Bodwell et al., 2004). This suggests that it is possible that other toxins (bacterial or environmental) may be able to interfere with GR signaling through a similar mechanism. Other bacterial proteins have previously been shown to affect the HPA axis and glucocorticoid signaling [reviewed by us (Webster and Sternberg, 2004)], but this mechanism of repression of nuclear hormone receptors by LeTx is novel. Recently, activation of some nuclear hormone receptors, namely RXR and LXR, has been shown to prevent cell death and apoptosis of macrophage cell lines infected by Bacillus anthracis (Valledor et al., 2004). These data suggest that nuclear hormone receptors may play a role in bacteria-mediated cell killing.

One unanswered question has been the role of HPA axis/glucocorticoid responses during exposure to anthrax lethal toxin. As we have previously described, an intact HPA axis response is essential for survival from a range of proinflammatory, bacterial, and viral insults.We have recently shown that adrenalectomy also enhances sensitivity to LeTx in all mouse strains tested including DBA/2J mice, which are completely resistant to LeTx when the adrenals are intact (Moayeri et al., 2005). However, unlike the studies described earlier, dexamethasone treatment does not prevent this increased mortality rate but rather sensitizes mice to LeTx (Moayeri et al., 2005). Although this data is contrary to what one might initially expect, there is data to demonstrate that the HPA axis needs to be carefully balanced for optimal host responses in bacterial exposures. Perturbations in either direction are detrimental to health. Indeed, prolonged treatment of septic shock with high doses of glucocorticoids has been shown to be detrimental to health, and increased cortisol levels have been shown in critically ill patients (Vermes and Beishuizen, 2001; Thompson, 2003; Hamrahian et al., 2004). These data suggest that a carefully balanced HPA axis is required for survival from LeTx. In agreement with this is the observation that blood cortisol levels increase in BALB/cJ mice, which are normally sensitive to LeTx, after LeTx administration, whereas there is no change in DBA/2J blood cortisol levels (Moayeri et al., 2005).

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