Rama P. Cherla

Shiga toxin 1 induces apoptosis through the endoplasmic reticulum stress response in human monocytic cells

Shiga toxins (Stxs) expressed by the enteric pathogens Shigella dysenteriae 1 and enterohaemorrhagic Escherichia coli are potent protein synthesis inhibitors. Shiga toxins have also been shown to induce apoptosis in epithelial, endothelial and monocytic cells. The precise relationship between protein synthesis inhibition and induction of apoptosis is not known. We show that stimulation of the myelogenous leukaemia cell line THP‐1 with purified Stx1 induced the endoplasmic reticulum (ER) stress response. Stx1 treatment increased activation of the ER stress sensors IRE1, PERK and ATF6. Toxin treatment increased expression of the transcriptional regulator CHOP and the death domain‐containing receptor DR5 at mRNA and protein levels. Following Stx1 intoxication, levels of the survival factor Bcl‐2 decreased, while secretion of the death‐inducing ligand TRAIL increased. Stx1 enzymatic activity was required for optimal activation of PERK and ATF6, but not IRE1. ER stress elicited by Stx1 increased the release of Ca2+ from ER stores and the activation of the protease calpain. Inhibition of calpain activity led to reductions in Stx1‐induced cleavage of procaspase‐8 and apoptosis. Collectively, these data suggest that Shiga toxins trigger monocytic cell apoptosis through the ER stress response, the increased expression of DR5 and TRAIL, and activation of caspase‐8 via a calpain‐dependent mechanism.

Shiga toxins and apoptosis

The enteric pathogens Shigella dysenteriae serotype 1 and Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrheal diseases that may progress to life-threatening extraintestinal complications. Although the S. dysenteriae and STEC differ in the expression of a number of virulence determinants, they share the capacity to produce one or more potent cytotoxins, called Shiga toxins (Stxs). Following the ingestion of the organisms, the expression of Stxs is critical for the development of vascular lesions in the colon, kidneys and central nervous system. It has been known for some time that following the intracellular routing of Stxs to the endoplasmic reticulum and nuclear membrane, the toxins translocate into the cytoplasm and target ribosomes for damage. However, numerous recent studies have shown that Stxs trigger programmed cell death signaling cascades in intoxicated cells. The mechanisms of apoptosis induction by these toxins are newly emerging, and the data published to date suggest that the toxins may signal apoptosis in different cells types via different mechanisms. Here we review the Stxs and the known mechanistic aspects of Stx-induced apoptosis, and present a model of apoptosis induction.

Shiga toxin 1-induced cytokine production is mediated by MAP kinase pathways and translation initiation factor eIF4E in the macrophage-like THP-1 cell line

Upon binding to the glycolipid receptor globotriaosylceramide, Shiga toxins (Stxs) undergo retrograde transport to reach ribosomes, cleave 28S rRNA, and inhibit protein synthesis. Stxs induce the ribotoxic stress response and cytokine and chemokine expression in some cell types. Signaling mechanisms necessary for cytokine expression in the face of toxin‐mediated protein synthesis inhibition are not well characterized. Stxs may regulate cytokine expression via multiple mechanisms involving increased gene transcription, mRNA transcript stabilization, and/or increased translation initiation efficiency. We show that treatment of differentiated THP‐1 cells with purified Stx1 resulted in prolonged activation of c‐Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK) cascades, and lipopolysaccharides (LPS) rapidly triggered transient activation of JNK and p38 and prolonged activation of extracellular signal‐regulated kinase cascades. Simultaneous treatment with Stx1 + LPS mediated prolonged p38 MAPK activation. Stx1 increased eukaryotic translation initiation factor 4E (eIF4E) activation by 4.3‐fold within 4–6 h, and LPS or Stx1 + LPS treatment increased eIF4E activation by 7.8‐ and 11‐fold, respectively, within 1 h. eIF4E activation required Stx1 enzymatic activity and was mediated by anisomycin, another ribotoxic stress inducer. A combination of MAPK inhibitors or a MAPK‐interacting kinase 1 (Mnk1)‐specific inhibitor blocked eIF4E activation by all stimulants. Mnk1 inhibition blocked the transient increase in total protein synthesis detected in Stx1‐treated cells but failed to block long‐term protein synthesis inhibition. The MAPK inhibitors or Mnk1 inhibitor blocked soluble interleukin (IL)‐1β and IL‐8 production or release by 73–96%. These data suggest that Stxs may regulate cytokine expression in part through activation of MAPK cascades, activation of Mnk1, and phosphorylation of eIF4E.

Shiga Toxin 1 Induces Apoptosis in the Human Myelogenous Leukemia Cell Line THP-1 by a Caspase-8-Dependent, Tumor Necrosis Factor Receptor-Independent Mechanism

ABSTRACT Shiga toxins (Stxs) induce apoptosis in a variety of cell types. Here, we show that Stx1 induces apoptosis in the undifferentiated myelogenous leukemia cell line THP-1 in the absence of tumor necrosis factor alpha (TNF-α) or death receptor (TNF receptor or Fas) expression. Caspase-8 and -3 inhibitors blocked, and caspase-6 and -9 inhibitors partially blocked, Stx1-induced apoptosis. Stx1 induced the mitochondrial pathway of apoptosis, as activation of caspase-8 triggered the (i) cleavage of Bid, (ii) disruption of mitochondrial membrane potential, and (iii) release of cytochrome c into the cytoplasm. Caspase-8, -9, and -3 cleavage and functional activities began 4 h after toxin exposure and peaked after 8 h of treatment. Caspase-6 may also contribute to Stx1-induced apoptosis by directly acting on caspase-8. It appears that functional Stx1 holotoxins must be transported to the endoplasmic reticulum to initiate apoptotic signaling through the ribotoxic stress response. These data suggest that Stxs may activate monocyte apoptosis via a novel caspase-8-dependent, death receptor-independent mechanism.

Simultaneous Induction of Apoptotic and Survival Signaling Pathways in Macrophage-Like THP-1 Cells by Shiga Toxin 1

ABSTRACT Shiga toxins have been shown to induce apoptosis in many cell types. However, Shiga toxin 1 (Stx1) induced only limited apoptosis of macrophage-like THP-1 cells in vitro. The mechanisms regulating macrophage death or survival following toxin challenge are unknown. Differentiated THP-1 cells expressed tumor necrosis factor receptors and membrane-associated tumor necrosis factor alpha (TNF-α) and produced soluble TNF-α after exposure to Stx1. However, the cells were refractory to apoptosis induced by TNF-α, although the cytokine modestly increased apoptosis in the presence of Stx1. Despite the partial resistance of macrophage-like THP-1 cells to Stx1-mediated killing, treatment of these cells with Stx1 activated a broad array of caspases, disrupted the mitochondrial membrane potential (ΔΨm), and released cytochrome c into the cytoplasm. The ΔΨm values were greatest in cells that had detached from plastic surfaces. Specific caspase inhibitors revealed that caspase-3, caspase-6, caspase-8, and caspase-9 were primarily involved in apoptosis induction. The antiapoptotic factors involved in macrophage survival following toxin challenge include inhibitors of apoptosis proteins and X-linked inhibitor of apoptosis protein. NF-κB and JNK mitogen-activated protein kinases (MAPKs) appeared to activate survival pathways, while p38 MAPK was involved in proapoptotic signaling. The JNK and p38 MAPKs were shown to be upstream signaling pathways which may regulate caspase activation. Finally, the protein synthesis inhibitors Stx1 and anisomycin triggered limited apoptosis and prolonged JNK and p38 MAPK activation, while macrophage-like cells treated with cycloheximide remained viable and showed transient activation of MAPKs. Collectively, these data suggest that Stx1 activates both apoptotic and cell survival signaling pathways in macrophage-like THP-1 cells.

Shiga Toxin 1-Induced Proinflammatory Cytokine Production Is Regulated by the Phosphatidylinositol 3-Kinase/Akt/Mammalian Target of Rapamycin Signaling Pathway

ABSTRACT Shiga toxin 1 (Stx1) transiently increases the expression of proinflammatory cytokines by macrophage-like THP-1 cells in vitro. Increased cytokine production is partly due to activation of the translation initiation factor eIF4E through a mitogen-activated protein kinase (MAPK)- and Mnk1-dependent pathway. eIF4E availability for translation initiation is regulated by association with eIF4E binding proteins (4E-BP). In this study, we showed that Stx1 transiently induced 4E-BP hyperphosphorylation, which may release eIF4E for translation initiation. Phosphorylation of 4E-BP at priming sites T37 and T46 was not altered by Stx1 but was transiently increased at S65, concomitant with increased cytokine expression. Using kinase inhibitors, we showed that 4E-BP phosphorylation was dependent on phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) activation but did not require MAPKs. Stx1 treatment resulted in increased levels of cytosolic Ca2+. PI3K and Akt activation led to the phosphorylation and inactivation of the positive cytokine regulator glycogen synthase kinase 3α/β (GSK-3α/β). PI3K, Akt, and mTOR inhibitors and small interfering RNA knockdown of Akt expression all increased, whereas a GSK-3α/β inhibitor decreased, Stx1-induced soluble tumor necrosis factor alpha and interleukin-1β production. Overall, these findings suggest that despite transient activation of 4E-BP, the PI3K/Akt/mTOR pathway negatively influences cytokine induction by inactivating the positive regulator GSK-3α/β.

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 1α 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: Intracellular Trafficking to the ER Leading to Activation of Host Cell Stress Responses

Despite efforts to improve hygenic conditions and regulate food and drinking water safety, the enteric pathogens, Shiga toxin-producing Escherichia coli (STEC) and Shigella dysenteriae serotype 1 remain major public health concerns due to widespread outbreaks and the severity of extra-intestinal diseases they cause, including acute renal failure and central nervous system complications. Shiga toxins are the key virulence factors expressed by these pathogens mediating extra-intestinal disease. Delivery of the toxins to the endoplasmic reticulum (ER) results in host cell protein synthesis inhibition, activation of the ribotoxic stress response, the ER stress response, and in some cases, the induction of apoptosis. Intrinsic and/or extrinsic apoptosis inducing pathways are involved in executing cell death following intoxication. In this review we provide an overview of the current understanding Shiga toxin intracellular trafficking, host cellular responses to the toxin and ER stress-induced apoptosis with an emphasis on recent findings.

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

ABSTRACT Shiga toxins (Stxs) induce apoptosis via activation of the intrinsic and extrinsic pathways in many cell types. Toxin-mediated activation of the endoplasmic reticulum (ER) stress response was shown to be instrumental in initiating apoptosis in THP-1 myeloid leukemia cells. THP-1 cells responded to Shiga toxin type 1 (Stx1) in a cell maturation-dependent manner, undergoing rapid apoptosis in the undifferentiated state but reduced and delayed apoptosis in differentiated cells. The onset of apoptosis was associated with calpain activation and changes in expression of C/EBP homologous protein (CHOP), Bcl-2 family members, and death receptor 5 (DR5). Ligation of DR5 by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) activates the extrinsic pathway of apoptosis. We show here that expression of TRAIL and DR5 is increased by Stx1 treatment. Addition of exogenous TRAIL enhances, and anti-TRAIL antibodies inhibit, Stx1-induced apoptosis of THP-1 cells. Silencing of CHOP or DR5 expression selectively prevented caspase activation, loss of mitochondrial membrane potential, and Stx1-induced apoptosis of macrophage-like THP-1 cells. In contrast, the rapid kinetics of apoptosis induction in monocytic THP-1 cells correlated with rates of calpain cleavage. The results suggest that CHOP-DR5 signaling and calpain activation differentially contribute to cell maturation-dependent Stx1-induced apoptosis. Inhibition of these signaling pathways may protect cells from Stx cytotoxicity.

Global Transcriptional Response of Macrophage-Like THP-1 Cells to Shiga Toxin Type 1

ABSTRACT Shiga toxins (Stxs) are bacterial cytotoxins produced by the enteric pathogens Shigella dysenteriae serotype 1 and some serotypes of Escherichia coli that cause bacillary dysentery and hemorrhagic colitis, respectively. To date, approaches to studying the capacity of Stxs to alter gene expression in intoxicated cells have been limited to individual genes. However, it is known that many of the signaling pathways activated by Stxs regulate the expression of multiple genes in mammalian cells. To expand the scope of analysis of gene expression and to better understand the underlying mechanisms for the various effects of Stxs on host cell functions, we carried out comparative microarray analyses to characterize the global transcriptional response of human macrophage-like THP-1 cells to Shiga toxin type 1 (Stx1) and lipopolysaccharides. The data were analyzed by using a rigorous combinatorial approach with three separate statistical algorithms. A total of 36 genes met the criteria of upregulated expression in response to Stx1 treatment, with 14 genes uniquely upregulated by Stx1. Microarray data were validated by real-time reverse transcriptase PCR for genes encoding early growth response 1 (Egr-1) (transcriptional regulator), cyclooxygenase 2 (COX-2; inflammation), and dual specificity phosphatase 1 (DUSP1), DUSP5, and DUSP10 (regulation of mitogen-activated protein kinase signaling). Stx1-mediated signaling through extracellular signal-regulated kinase 1/2 and Egr-1 appears to be involved in the increased expression and production of the proinflammatory mediator tumor necrosis factor alpha. Activation of COX-2 is associated with the increased production of proinflammatory and vasoactive eicosanoids. However, the capacity of Stx1 to increase the expression of genes encoding phosphatases suggests that mechanisms to dampen the macrophage proinflammatory response may be built into host response to the toxins.