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Anthrax infection inhibits the AKT signaling involved in the E-cadherin-mediated adhesion of lung epithelial cells.

Popova T, Espina V, Bailey C, Liotta L, Petricoin E, Popov S - FEMS Immunol. Med. Microbiol. (2009)

Bottom Line: The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade (ERK, p38, and P90RSK), the PI3K cascade (AKT, GSK-3alpha/beta), and downstream in the case of the proapoptotic BAD and the transcription factor STAT3.In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals.We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

ABSTRACT
The effect of anthrax infection on phosphoprotein signaling was studied in human small airway lung epithelial cells exposed to B. anthracis spores of the plasmidless dSterne strain in comparison with the Sterne strain containing the toxigenic plasmid (pXO1). The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade (ERK, p38, and P90RSK), the PI3K cascade (AKT, GSK-3alpha/beta), and downstream in the case of the proapoptotic BAD and the transcription factor STAT3. Both strains stimulate phosphorylation of CREB and inhibit phosphorylation of 4E-BP1 required for activation of cap-dependent translation. Downregulation of the survival AKT phosphorylation by the Sterne strain inhibits the process of Ca(2+)-dependent homophilic interaction of E-cadherin (EC) upon formation or repair of cell-cell contacts. Both lethal and edema toxins produced by the Sterne strain inhibit the AKT phosphorylation induced during the EC-mediated signaling. Activity of ERK1/2 and p38 inhibitors indicates that inhibition of AKT phosphorylation takes place through the ERK1/2-PI3K crosstalk. In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals. We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.

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Fragments of MAPKK and PI3K prosurvival pathways in HSAECs exposed to Bacillus anthracis. Asterisks indicate differential phosphorylation detected by the microarray.
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fig02: Fragments of MAPKK and PI3K prosurvival pathways in HSAECs exposed to Bacillus anthracis. Asterisks indicate differential phosphorylation detected by the microarray.

Mentions: To study the dynamics of host cell signaling phosphorylation during B. anthracis infection, we used a model of HSAECs challenged with B. anthracis spores, which germinated and grew as vegetative bacteria atop of the host cells. We suggested that this model would be instructive regarding the initial interaction of lung epithelium with inhaled spores, as well as the late septicemic stage of infection characterized by dissemination of vegetative bacteria and their secreted products to the lungs and other epithelium-reach organs. Cell lysates were prepared at different times postchallenge to encompass the period of infection from 10 min to 8 h at MOI 1 and 10. For initial identification of signaling proteins the lysates were used for fabrication of RPMA on nitrocellulose membrane slides. Each slide was then probed with one of 40 different antibodies specific against phosphorylated forms of signaling proteins (see Materials and methods) selected to monitor the molecular networks involved in host responses most likely affected by bacterial exposure, namely survival, apoptosis, inflammation, growth, differentiation, and immune response. This microarray technique was extensively validated in our previous studies (Wulfkuhle et al., 2006) with regard to specificity of antibodies, sensitivity, and accuracy of phosphoprotein detection in cell lysates. Of the 40 different signaling proteins, the relative levels of 20 phosphorylated proteins showed statistically significant (P<0.05) difference from those in the control untreated cells at one or several time points upon exposure to either toxigenic or nontoxigenic B. anthracis strain (Table S1). The responses detected at both MOIs were similar but stronger at MOI 10 (not shown). To determine whether the detected phosphorylation events reflect a biologically cohesive set of signaling processes, the data were evaluated using the software package pathwayarchitect 2.0 and the associated database of biological interactions (Stratagene, CA). All proteins whose phosphorylations were significantly changed during the infection in HSAECs were found to belong to a single direct-interaction network (P<10−5) where all signaling nodes are interconnected, and no irrelevant nodes without direct connections are present. The high functional importance of these network proteins is reflected in the connectivity index measuring the number of interactions with other proteins in the database (Table S1). Eleven of the above proteins were chosen for further Western blot analyses as time course curves for MOI 10 (Fig. 1). A part of the signaling network relevant to these proteins is illustrated in Fig. 2.


Anthrax infection inhibits the AKT signaling involved in the E-cadherin-mediated adhesion of lung epithelial cells.

Popova T, Espina V, Bailey C, Liotta L, Petricoin E, Popov S - FEMS Immunol. Med. Microbiol. (2009)

Fragments of MAPKK and PI3K prosurvival pathways in HSAECs exposed to Bacillus anthracis. Asterisks indicate differential phosphorylation detected by the microarray.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2734923&req=5

fig02: Fragments of MAPKK and PI3K prosurvival pathways in HSAECs exposed to Bacillus anthracis. Asterisks indicate differential phosphorylation detected by the microarray.
Mentions: To study the dynamics of host cell signaling phosphorylation during B. anthracis infection, we used a model of HSAECs challenged with B. anthracis spores, which germinated and grew as vegetative bacteria atop of the host cells. We suggested that this model would be instructive regarding the initial interaction of lung epithelium with inhaled spores, as well as the late septicemic stage of infection characterized by dissemination of vegetative bacteria and their secreted products to the lungs and other epithelium-reach organs. Cell lysates were prepared at different times postchallenge to encompass the period of infection from 10 min to 8 h at MOI 1 and 10. For initial identification of signaling proteins the lysates were used for fabrication of RPMA on nitrocellulose membrane slides. Each slide was then probed with one of 40 different antibodies specific against phosphorylated forms of signaling proteins (see Materials and methods) selected to monitor the molecular networks involved in host responses most likely affected by bacterial exposure, namely survival, apoptosis, inflammation, growth, differentiation, and immune response. This microarray technique was extensively validated in our previous studies (Wulfkuhle et al., 2006) with regard to specificity of antibodies, sensitivity, and accuracy of phosphoprotein detection in cell lysates. Of the 40 different signaling proteins, the relative levels of 20 phosphorylated proteins showed statistically significant (P<0.05) difference from those in the control untreated cells at one or several time points upon exposure to either toxigenic or nontoxigenic B. anthracis strain (Table S1). The responses detected at both MOIs were similar but stronger at MOI 10 (not shown). To determine whether the detected phosphorylation events reflect a biologically cohesive set of signaling processes, the data were evaluated using the software package pathwayarchitect 2.0 and the associated database of biological interactions (Stratagene, CA). All proteins whose phosphorylations were significantly changed during the infection in HSAECs were found to belong to a single direct-interaction network (P<10−5) where all signaling nodes are interconnected, and no irrelevant nodes without direct connections are present. The high functional importance of these network proteins is reflected in the connectivity index measuring the number of interactions with other proteins in the database (Table S1). Eleven of the above proteins were chosen for further Western blot analyses as time course curves for MOI 10 (Fig. 1). A part of the signaling network relevant to these proteins is illustrated in Fig. 2.

Bottom Line: The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade (ERK, p38, and P90RSK), the PI3K cascade (AKT, GSK-3alpha/beta), and downstream in the case of the proapoptotic BAD and the transcription factor STAT3.In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals.We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, VA 20110, USA.

ABSTRACT
The effect of anthrax infection on phosphoprotein signaling was studied in human small airway lung epithelial cells exposed to B. anthracis spores of the plasmidless dSterne strain in comparison with the Sterne strain containing the toxigenic plasmid (pXO1). The differential regulation of phosphorylation was found in the mitogen-activated protein kinase cascade (ERK, p38, and P90RSK), the PI3K cascade (AKT, GSK-3alpha/beta), and downstream in the case of the proapoptotic BAD and the transcription factor STAT3. Both strains stimulate phosphorylation of CREB and inhibit phosphorylation of 4E-BP1 required for activation of cap-dependent translation. Downregulation of the survival AKT phosphorylation by the Sterne strain inhibits the process of Ca(2+)-dependent homophilic interaction of E-cadherin (EC) upon formation or repair of cell-cell contacts. Both lethal and edema toxins produced by the Sterne strain inhibit the AKT phosphorylation induced during the EC-mediated signaling. Activity of ERK1/2 and p38 inhibitors indicates that inhibition of AKT phosphorylation takes place through the ERK1/2-PI3K crosstalk. In Sterne spore-challenged mice, a specific inhibitor of PI3K/AKT, wortmannin, accelerates the lethal outcome, and reduction of AKT phosphorylation in the circulating blood cells coincides with the death of animals. We conclude that the PI3K/AKT pathway controlling the integrity of epithelium plays an important survival role in anthrax infection.

Show MeSH
Related in: MedlinePlus