Bcl-2 Regulates the Onset of Shiga Toxin 1-Induced Apoptosis in THP-1 Cells

Abstract

Shiga toxins (Stxs), which are proteins expressed by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli, are potent protein synthesis inhibitors. Stx-producing organisms cause bloody diarrhea with the potential to progress to acute renal failure and central nervous system complications. Studies using animal models of these diseases have shown that Stxs are major virulence factors, and purified toxins have been shown to be capable of killing many types of cells in vitro. We showed that Stx type 1 (Stx1) rapidly induced apoptosis in undifferentiated, monocytic THP-1 cells through a mechanism involving the endoplasmic reticulum (ER) stress response. Rapid apoptosis correlated with increased expression of C/EBP homologous protein (CHOP), TRAIL, and DR5, while expression of the antiapoptotic factor Bcl-2 was downregulated. Stx1 treatment of differentiated, macrophage-like THP-1 cells was associated with cytokine production and delayed apoptosis. The mechanisms contributing to cell maturation-dependent differences in responses to Stx1 are unknown. We show here that in macrophage-like cells, Stx1 activated the proximal ER stress sensors RNA-dependent protein kinase-like ER kinase and inositol-requiring ER signal kinase 1␣ but did not activate activating transcription factor 6. Proapoptotic signaling pathways mediated by CHOP and by Bax and Bak were activated by Stx1. However, the toxin also activated prosurvival signaling through increased expression, mitochondrial translocation, and alternative phosphorylation of Bcl-2.Shiga toxins (Stxs), also called Shiga-like toxins or verotoxins, are potent protein synthesis inhibitors expressed by the enteric pathogens Shigella dysenteriae (serotype 1) and Escherichia coli (select serotypes). Stxs act as virulence factors by increasing the severity of bloody diarrhea and increasing the likelihood of development of life-threatening postdiarrheal sequelae, such as acute renal failure (hemolytic-uremic syndrome) and central nervous system complications (40). S. dysenteriae serotype 1 expresses the prototypic Stx, while closely related toxins expressed by E. coli can be classified into Stx type 1 (Stx1) and Stx2 (30). X-ray crystallographic analyses revealed that all Stxs contain six protein subunits in an arrangement described as an AB 5 structure. Stx A-subunits possess N-glycosidase activity that depurinates a specific unpaired adenine residue from eukaryotic rRNA, leading to protein synthesis inhibition in the intoxicated cell (11,35). The pentameric Bsubunits are responsible for toxin binding to the cell surface and interact with the neutral globo series glycolipid globotriaosylceramide (for a review, see reference 26). Toxin-mediated receptor cross-linking through interaction with the pentameric

Plant ribosome-inactivating proteins type II induce the unfolded protein response in human cancer cells

“…The question whether depurination is necessary for RIP-induced cell death and not just a side effect is being controversially discussed [24][25][26][27][28]. Moreover, it is still not clear how depurination leads to cell death or why an inflammatory response is activated upon RIP treatment in vivo.…”

Signaling through C/EBP Homologous Protein and Death Receptor 5 and Calpain Activation Differentially Regulate THP-1 Cell Maturation-Dependent Apoptosis Induced by Shiga Toxin Type 1

“…We have previously shown that Stx1 activates NF-B signaling in macrophage-like THP-1 cells, and TRAIL and DR5 gene expression is regulated by NF-B (20,48,54,64). DR5 expression is also regulated by CHOP (68), and Stx1 treatment of monocytic and macrophage-like THP-1 cells increases CHOP expression (33,36). Furthermore, we show here that RNAi knockdown of CHOP and DR5 expression partially protects macrophage-like cells from the cytotoxic action of Stx1.…”

“…Extracts were collected and cleared by centrifugation at 15,000 ϫ g for 10 min. Cell lysates from monocytic THP-1 cells were prepared as previously described (33). Protein concentrations in each extract were determined using the Micro bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL).…”

“…Undifferentiated, nonadherent monocytic THP-1 cells underwent rapid apoptosis when treated with Stx1, while differentiation to the adherent, mac-rophage-like state was associated with increased resistance to the cytotoxic action of the toxins, with only approximately 30% of cells undergoing delayed apoptosis (22). The induction of apoptosis by Stx1 involved the activation of the ER stress response in both monocytic and macrophage-like THP-1 cells (33,36). Stx1 induced the expression of the ER stress effectors C/EBP homologous protein (CHOP), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), and death receptor 5 (DR5) in monocytic THP-1 cells.…”

Differential Response of the Human Renal Proximal Tubular Epithelial Cell Line HK-2 to Shiga Toxin Types 1 and 2

“…Our data suggest that Stx2 cleaves procaspase 8 in HK-2 cells while Stx1 fails to do so. Stx-induced activation of caspase 8 may lead to the activation of the Bcl-2 protein family member BID, which in turn translocates to the mitochondria to facilitate mitochondrialmembrane depolarization and the release of mitochondrial intermembrane constituents into the cytoplasm (29). These events trigger the formation of the apoptosome and the activation of procaspase 9, and caspase 9 then activates caspase 3.…”