Critical Role of the Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase-3β Signaling Pathway in Recovery from Anthrax Lethal Toxin-induced Cell Cycle Arrest and MEK Cleavage in Macrophages

Abstract

Anthrax lethal toxin (LeTx) is a virulence factor causing immune suppression and toxic shock of Bacillus anthracis infected host. It inhibits cytokine production and cell proliferation/differentiation in various immune cells. This study showed that a brief exposure of LeTx caused a continual MEK1 cleavage and prevented tumor necrosis factor-alpha (TNF) production in response to lipopolysaccharide (LPS) in non-proliferating cells such as human peripheral blood mononuclear cells or mouse primary peritoneal macrophages. In human monocytic cell lines U-937 and THP-1, LeTx induced cell cycle arrest in G0-G1 phase by rapid down-regulation of cyclin D1/D2 and checkpoint kinase 1 through MEK1 inhibition. However, THP-1 cells adaptively adjusted to LeTx and overrode cell cycle arrest by activating the phosphatidylinositol 3-kinase/Akt signaling pathway. Inhibitory Ser-9 phosphorylation of glycogen synthase kinase 3beta (GSK3beta) by Akt prevented proteasome-mediated cyclin D1 degradation and induced cell cycle progress in LeTx-intoxicated THP-1 cells. Recovery from cell cycle arrest was required before recovering from on-going MEK1 cleavage and suppression of TNF production. Furthermore, pretreatment with LeTx or the GSK3-specific inhibitor SB-216763, or transfection with dominant active mutant Akt or degradation-defected mutant cyclin D1 protected cells from LeTx-induced cell cycle arrest, on-going MEK1 cleavage and suppression of TNF production. These results indicate that modulation of phosphatidylinositol 3-kinase/Akt/GSK3beta signaling cascades can be beneficial for protecting or facilitating recovery from cellular LeTx intoxication in cells that depend on basal MEK1 activity for proliferation.

Inhibition of Interleukin 1β (IL-1β) Expression by Anthrax Lethal Toxin (LeTx) Is Reversed by Histone Deacetylase 8 (HDAC8) Inhibition in Murine Macrophages

“…Response to LPS in LeTx-exposed Murine Macrophages-LeTx suppresses expression of various inflammatory cytokines by inactivating MEKs (3)(4)(5). Consistent with these reports, a sublethal dose of LeTx (100 ng/ml PA and LF, each for 4 h) completely cleaved MEK1 and inhibited expression of pro-IL-1␤ induced by LPS (100 ng/ml) for more than 2 days in RAW264.7 macrophages (Fig.…”

“…The causative agent of anthrax is Bacillus anthracis, and LeTx is a main virulence factor that causes immune paralysis and mortality (1). LeTx is known to cause immune suppression by targeting the MAPK pathway that is involved in many aspects of immune responses (2)(3)(4)(5). It includes two proteins, protective antigen (PA) 2 and lethal factor (LF) (6 -10).…”

Cathepsin B Is Involved in the Trafficking of TNF-α-Containing Vesicles to the Plasma Membrane in Macrophages

“…Total cell lysate preparation and immunoblotting procedures were performed as previously described (25). Briefly, cells were lysed in ice-cold lysis buffer (20 mM MOPS, 2 mM EGTA, 5 mM EDTA, 1 mM Na 3 VO 4 , 40 mM ␤-glycerophosphate, 30 mM NaF, and 20 mM sodium pyrophosphate (pH 7.2)) containing a protease inhibitor cocktail (Roche).…”

“…Cells were grown at 37°C in the humidified atmosphere of 5% CO 2 . Human PBMC were isolated from whole blood as previously described (25).…”

Cytotoxicity of the matrix metalloproteinase–activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells

“…We determined whether the phosphatidylinositol 3-kinase/PTEN/Akt/GSK3h pathway had a role in the observed PA/LF resistance. It has recently been shown that the activation of Akt causes the inhibition of GSK3h-mediated cyclin D1 degradation and consequential G 0 -G 1 cell cycle arrest (23). This activation is critical for recovery from LeTx-mediated cell cycle arrest and resistance to subsequent toxin challenge in human macrophages (23).…”