Proteins were transferred to poly(vinylidene difluoride) and probed with antibodies against phospho-p38 MAP kinase (Thr-180/Tyr-182), phospho-p44/42 MAP kinase (Thr-202/Tyr-204), and phospho-c-Jun (Ser-63) (Cell Signaling Technology). 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 ENMD-2076 Tartrate in animals exposed to both ENMD-2076 Tartrate 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. Death from anthrax toxin is reported to result from systemic shock (1) resembling lipopolysaccharide (LPS)-induced shock (2, 3) although the role of inflammatory cytokines in this process and the precise mechanism of this shock have not been determined (4). Anthrax toxin is composed of three proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a recent review, see refs. 5 and 6). PA and EF comprise the edema toxin and PA and LF the lethal toxin (LeTx). It is this lethal toxin produced by that causes death of the infected host (7). The mechanism of entry of this toxin into the cell is now well understood. PA binds to the anthrax toxin receptor (8), is cleaved (9), oligomerizes, and then binds LF and/or EF, facilitating internalization of these proteins into the cell (10, 11). Translocation of LF and EF to the cytosol is by a pH- and voltage-dependent mechanism (12C14). The mechanism of action of LF inside the cell is less well understood. LF is a metalloprotease that cleaves the mitogen ENMD-2076 Tartrate activation protein (MAP) kinase kinases (MAPKK/MEK), including MEK1, MEK2, MKK3, MKK4, MKK6, and MKK7 but not MEK5 (15C19). However, the fact that LeTx-resistant and -sensitive cells show similar internalization of LF (20) and similar MEK cleavage in response to LF (17, 18) suggests that these factors cannot alone account for differential susceptibility or resistance to the toxin. Other factors that have been proposed to play a role in toxicity of LeTx include the proteosome (21), intracellular calcium stores (22, 23), calmodulin (23), a calyculin A-sensitive protein phosphatase (24), protein synthesis (25), and reactive oxygen intermediates (26). It is not known which of these or other unknown factors contribute to the well-described differential cell line and rodent strain sensitivities to toxic effects of LeTx. Recently, the gene has been determined to be different between resistant and sensitive strains although the implication of this finding is not understood (27). Fischer (F344/N) rats have long been known to be particularly susceptible to the LeTx (28), with death occurring within 40 min after exposure to a lethal dose (29). F344/N rats are also known to be relatively inflammatory disease resistant, due in part to their hypothalamic-pituitary-adrenal (HPA) axis hyperresponsiveness and resultant hypersecretion of glucocorticoids from the adrenal glands in response to proinflammatory and other stimuli. Similar to F344/N rats, BALB/c mice have a hyperresponsive HPA axis (30) and are also susceptible to LeTx (31). Ordinarily this hyper-HPA axis responsiveness protects against inflammatory/autoimmune disease, including shock through the antiinflammatory and immunosuppressive effects NCR2 of the glucocorticoids. However, F344/N rats and other inflammatory-resistant rodent strains become highly susceptible to inflammation and rapid death after simultaneous glucocorticoid receptor (GR) or HPA axis blockade and exposure to proinflammatory or infectious stimuli, including bacterial products such as streptococcal cell walls (SCW) or bacterial lipopolysaccharide (LPS) (32C37). Here, we report that the LF and PA proteins comprising LeTx selectively and specifically repress GR and other nuclear hormone receptors. To our knowledge there have been no previous reports showing that a bacterial product interferes with nuclear hormone receptor function. This provides a previously uncharacterized explanation for how such agents might contribute to the pathogenesis of bacterial infections. Materials and Methods Materials. The recombinant proteins LF and PA were produced as described (38, 39). All MEK inhibitors were purchased from Calbiochem except PD98059, which was purchased from Cell Signaling Technology (Beverly, MA). Cell Culture. Cos7 and HTC cells were grown at 37C and 5% CO2 in DMEM containing 10% serum, 10 g/ml penicillin-streptomycin, and 2 mM glutamine. Transient Transfections. Cos7 cells were plated in 24-well plates at a density of 5 105 cells per well in DMEM containing 10%.
Proteins were transferred to poly(vinylidene difluoride) and probed with antibodies against phospho-p38 MAP kinase (Thr-180/Tyr-182), phospho-p44/42 MAP kinase (Thr-202/Tyr-204), and phospho-c-Jun (Ser-63) (Cell Signaling Technology)