Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion.

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 Å resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca2+ increases are transient and oscillatory, defining the so-called Ca2+ code that links cell responses to specific Ca2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca2+ signals. Here, we show that by hydrolyzing phosphatidylinositol-(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol-(1,4,5)trisphosphate (InsP3) levels. By modifying InsP3 dynamics and diffusion, IpgD favors the elicitation of long-lasting local Ca2+ signals at Shigella invasion sites and converts Shigella-induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP3-dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca2+-dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.

Cytoskeletal mechanics during Shigella invasion and dissemination in epithelial cells.

The actin cytoskeleton is key to the barrier function of epithelial cells, by permitting the establishment and maintenance of cell-cell junctions and cell adhesion to the basal matrix. Actin exists under monomeric and polymerized filamentous form and its polymerization following activation of nucleation promoting factors generates pushing forces, required to propel intracellular microorganisms in the host cell cytosol or for the formation of cell extensions that engulf bacteria. Actin filaments can associate with adhesion receptors at the plasma membrane via cytoskeletal linkers. Membrane anchored to actin filaments are then subjected to the retrograde flow that may pull membrane-bound bacteria inside the cell. To induce its internalization by normally non-phagocytic cells, bacteria need to establish adhesive contacts and trick the cell into apply pulling forces, and/or to generate protrusive forces that deform the membrane surrounding its contact site. In this review, we will focus on recent findings on actin cytoskeleton reorganization within epithelial cells during invasion and cell-to-cell spreading by the enteroinvasive pathogen Shigella, the causative agent of bacillary dysentery.

The inside story of Shigella invasion of intestinal epithelial cells.

As opposed to other invasive pathogens that reside into host cells in a parasitic mode, Shigella, the causative agent of bacillary dysentery, invades the colonic mucosa but does not penetrate further to survive into deeper tissues. Instead, Shigella invades, replicates, and disseminates within the colonic mucosa. Bacterial invasion and spreading in intestinal epithelium lead to the elicitation of inflammatory responses responsible for the tissue destruction and shedding in the environment for further infection of other hosts. In this article, we highlight specific features of the Shigella arsenal of virulence determinants injected by a type III secretion apparatus (T3SA) that point to the targeting of intestinal epithelial cells as a discrete route of invasion during the initial event of the infectious process.

Tips and tricks about Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery, invades the colonic epithelium where it elicits an intense inflammation leading to tissular destruction. Key to bacterial virulence, type III effectors injected into host cells reorganize the actin cytoskeleton and regulate inflammatory responses. Much progress has been made recently in the characterization of these type III effectors. These findings have reshaped our view of Shigella invasion, suggesting a strategy to invade epithelial cells 'discretely' as an initial route of invasion, contrasting with the devastating inflammatory response associated with the disease's acute phase. The diverse roles of Shigella type III effectors highlight the complexity of an infection process where as little as a thousand bacteria are estimated sufficient to cause the disease in humans.

ATP-mediated Erk1/2 activation stimulates bacterial capture by filopodia, which precedes Shigella invasion of epithelial cells.

Shigella, the causative agent of bacillary dysentery in humans, invades epithelial cells, using a type III secretory system (T3SS) to inject bacterial effectors into host cells and remodel the actin cytoskeleton. ATP released through connexin hemichanels on the epithelial membrane stimulates Shigella invasion and dissemination in epithelial cells. Here, we show that prior to contact with the cell body, Shigella is captured by nanometer-thin micropodial extensions (NMEs) at a distance from the cell surface, in a process involving the T3SS tip complex proteins and stimulated by ATP- and connexin-mediated signaling. Upon bacterial contact, NMEs retract, bringing bacteria in contact with the cell body, where invasion occurs. ATP stimulates Erk1/2 activation, which controls actin retrograde flow in NMEs and their retraction. These findings reveal previously unappreciated facets of interaction of an invasive bacterium with host cells and a prominent role for Erk1/2 in the control of filopodial dynamics.

Role of interferon {alpha} (IFN{alpha})-inducible Schlafen-5 in regulation of anchorage-independent growth and invasion of malignant melanoma cells.

IFNα exerts potent inhibitory activities against malignant melanoma cells in vitro and in vivo, but the mechanisms by which it generates its antitumor effects remain unknown. We examined the effects of interferon α (IFNα) on the expression of human members of the Schlafen (SLFN) family of genes, a group of cell cycle regulators that mediate growth-inhibitory responses. Using quantitative RT-real time PCR, we found detectable basal expression of all the different human SLFN genes examined (SLFN5, SLFN11, SLFN12, SLFN13, and SLFN14), in malignant melanoma cells and primary normal human melanocytes, but SLFN5 basal expression was suppressed in all analyzed melanoma cell lines. Treatment of melanoma cells with IFNα resulted in induction of expression of SLFN5 in malignant cells, suggesting a potential involvement of this gene in the antitumor effects of IFNα. Importantly, stable knockdown of SLFN5 in malignant melanoma cells resulted in increased anchorage-independent growth, as evidenced by enhanced colony formation in soft agar assays. Moreover, SLFN5 knockdown also resulted in increased invasion in three-dimensional collagen, suggesting a dual role for SLFN5 in the regulation of invasion and anchorage-independent growth of melanoma cells. Altogether, our findings suggest an important role for the SLFN family of proteins in the generation of the anti-melanoma effects of IFNα and for the first time directly implicate a member of the human SLFN family in the regulation of cell invasion.

Critical roles for mTORC2- and rapamycin-insensitive mTORC1-complexes in growth and survival of BCR-ABL-expressing leukemic cells.

mTOR-generated signals play critical roles in growth of leukemic cells by controlling mRNA translation of genes that promote mitogenic responses. Despite extensive work on the functional relevance of rapamycin-sensitive mTORC1 complexes, much less is known on the roles of rapamycin-insensitive (RI) complexes, including mTORC2 and RI-mTORC1, in BCR-ABL-leukemogenesis. We provide evidence for the presence of mTORC2 complexes in BCR-ABL-transformed cells and identify phosphorylation of 4E-BP1 on Thr37/46 and Ser65 as RI-mTORC1 signals in primary chronic myelogenous leukemia (CML) cells. Our studies establish that a unique dual mTORC2/mTORC1 inhibitor, OSI-027, induces potent suppressive effects on primitive leukemic progenitors from CML patients and generates antileukemic responses in cells expressing the T315I-BCR-ABL mutation, which is refractory to all BCR-ABL kinase inhibitors currently in clinical use. Induction of apoptosis by OSI-027 appears to negatively correlate with induction of autophagy in some types of BCR-ABL transformed cells, as shown by the induction of autophagy during OSI-027-treatment and the potentiation of apoptosis by concomitant inhibition of such autophagy. Altogether, our studies establish critical roles for mTORC2 and RI-mTORC1 complexes in survival and growth of BCR-ABL cells and suggest that dual therapeutic targeting of such complexes may provide an approach to overcome leukemic cell resistance in CML and Ph+ ALL.

Activation of the p38 Map kinase pathway is essential for the antileukemic effects of dasatinib.

Dasatinib, a dual Src/Abl tyrosine kinase inhibitor, has significant antileukemic effects against various imatinib mesylate-resistant BCR/ABL mutants. Despite well-documented inhibitory effects of dasatinib on BCR/ABL kinase, the exact downstream cellular events leading to generation of its potent antileukemic effects remain to be defined. We provide evidence that p38 Map kinase (MAPK) pathway is activated leading to increased upregulation of mixed lineage kinase 3, MKK3/6, MSK1, and Mapkapk2, upon treatment of BCR/ABL expressing cells with dasatinib, including cells expressing various imatinib-resistant mutants, except for T315I. Our data demonstrate that such dasatinib-dependent activation of p38 MAPK and its effectors plays a critical role in the generation of antileukemic responses, since pharmacological inhibition of p38 or siRNA-mediated knockdown of its expression reverse dasatinib-mediated apoptosis, cell cycle arrest, and anti-proliferative effects. p38 MAPK inhibition also reversed dasatinib-induced suppression of CML patient-derived leukemic colony-forming units progenitor growth in vitro, as well as BCR/ABL expressing KT-1 cell-derived leukemic progenitor growth. Altogether, our findings suggest a critical role for p38 MAPK pathway in the generation of antileukemic effects of dasatinib, and raise the possibility that development of novel means to enhance p38 MAPK activation in BCR/ABL expressing cells may be an approach to promote antileukemic responses and, possibly, reverse T315I mutation-mediated resistance.

Role of Schlafen 2 (SLFN2) in the generation of interferon alpha-induced growth inhibitory responses.

The precise STAT-regulated gene targets that inhibit cell growth and generate the antitumor effects of Type I interferons (IFNs) remain unknown. We provide evidence that Type I IFNs regulate expression of Schlafens (SLFNs), a group of genes involved in the control of cell cycle progression and growth inhibitory responses. Using cells with targeted disruption of different STAT proteins and/or the p38 MAP kinase, we demonstrate that the IFN-dependent expression of distinct Schlafen genes is differentially regulated by STAT complexes and the p38 MAP kinase pathway. We also provide evidence for a key functional role of a member of the SLFN family, SLFN2, in the induction of the growth-suppressive effects of IFNs. This is shown in studies demonstrating that knockdown of SLFN2 enhances hematopoietic progenitor colony formation and reverses the growth-suppressive effects of IFNalpha on normal hematopoietic progenitors. Importantly, NIH3T3 or L929 cells with stable knockdown of SLFN2 form more colonies in soft agar, implicating this protein in the regulation of anchorage-independent growth. Altogether, our data implicate SLFN2 as a negative regulator of the metastatic and growth potential of malignant cells and strongly suggest a role for the SLFN family of proteins in the generation of the antiproliferative effects of Type I IFNs.

Regulation of arsenic trioxide-induced cellular responses by Mnk1 and Mnk2.

Arsenic trioxide (As(2)O(3)) is a potent inducer of apoptosis of malignant cells in vitro and in vivo, but the precise mechanisms by which it mediates such effects are not well defined. We provide evidence that As(2)O(3) induces phosphorylation/activation of the MAPK signal-integrating kinases (Mnks) 1 and 2 in leukemia cell lines. Such activation is defective in cells with targeted disruption of the p38alpha MAPK gene, indicating that it requires upstream engagement of the p38 MAPK pathway. Studies using Mnk1(-/-) or Mnk2(-/-), or double Mnk1(-/-)Mnk2(-/-) knock-out cells, establish that activation of Mnk1 and Mnk2 by arsenic trioxide regulates downstream phosphorylation of the eukaryotic initiation factor 4E at Ser-209. Importantly, arsenic-induced apoptosis is enhanced in cells with targeted disruption of the Mnk1 and/or Mnk2 genes, suggesting that these kinases are activated in a negative-feedback regulatory manner, to control generation of arsenic trioxide responses. Consistent with this, pharmacological inhibition of Mnk activity enhances the suppressive effects of arsenic trioxide on primary leukemic progenitors from patients with acute leukemias. Taken together, these findings indicate an important role for Mnk kinases, acting as negative regulators for signals that control generation of arsenic trioxide-dependent apoptosis and antileukemic responses.

Suppression of interferon (IFN)-inducible genes and IFN-mediated functional responses in BCR-ABL-expressing cells.

The interferons (IFNs) are cytokines that play key roles in host defense against viral infections and immune surveillance against cancer. We report that BCR-ABL transformation of hematopoietic cells results in suppression of IFN-dependent responses, including transcription of IFN-inducible genes and generation of IFN-mediated antiviral effects. BCR-ABL transformation suppresses expression of several IFN-regulated genes containing IFN-sensitive response element (ISRE) or GAS elements in their promoters, including Isg15, Irf1, Irf9, and Ifit2 (interferon-induced protein with tetratricopeptide repeats 2). Suppression of transcription of ISRE-containing genes is also seen in cells expressing various BCR-ABL kinase domain mutants, including T315I, H396P, Y253F, and E255K, but not kinase-defective BCR-ABL. Such effects are associated with impaired IFN-dependent phosphorylation of Stat1 on Tyr(701) and Stat3 on Tyr(705) and defective binding of Stat complexes to ISRE or GAS elements. Beyond suppression of Stat activities, BCR-ABL inhibits IFN-inducible phosphorylation/activation of the p38 MAPK, suggesting a dual mechanism by which this abnormal fusion protein blocks IFN transcriptional responses. The inhibitory activities of BCR-ABL ultimately result in impaired IFNalpha-mediated protection against encephalomyocarditis virus infection and reversal of IFN-dependent growth suppression. Altogether, our data provide evidence for a novel mechanism by which BCR-ABL impairs host defenses and promotes malignant transformation, involving dual suppression of IFN-activated signaling pathways.

Suppression of programmed cell death 4 (PDCD4) protein expression by BCR-ABL-regulated engagement of the mTOR/p70 S6 kinase pathway.

There is accumulating evidence that mammalian target of rapamycin (mTOR)-activated pathways play important roles in cell growth and survival of BCR-ABL-transformed cells. We have previously shown that the mTOR/p70 S6 kinase (p70 S6K) pathway is constitutively activated in BCR-ABL transformed cells and that inhibition of BCR-ABL kinase activity by imatinib mesylate abrogates such activation. We now provide evidence for the existence of a novel regulatory mechanism by which BCR-ABL promotes cell proliferation, involving p70 S6K-mediated suppression of expression of programmed cell death 4 (PDCD4), a tumor suppressor protein that acts as an inhibitor of cap-dependent translation by blocking the translation initiation factor eIF4A. Our data also establish that second generation BCR-ABL kinase inhibitors block activation of p70 S6K and downstream engagement of the S6 ribosomal protein in BCR-ABL transformed cells. Moreover, PDCD4 protein expression is up-regulated by inhibition of the BCR-ABL kinase in K562 cells and BaF3/BCR-ABL transfectants, suggesting a mechanism for the generation of the proapoptotic effects of such inhibitors. Knockdown of PDCD4 expression results in reversal of the suppressive effects of nilotinib and imatinib mesylate on leukemic progenitor colony formation, suggesting an important role for this protein in the generation of antileukemic responses. Altogether, our studies identify a novel mechanism by which BCR-ABL may promote leukemic cell growth, involving sequential engagement of the mTOR/p70 S6K pathway and downstream suppression of PDCD4 expression.

A dominant function of IKK/NF-kappaB signaling in global lipopolysaccharide-induced gene expression.

Porphyromonas gingivalis is an etiologic pathogen of periodontitis that is one of the most common inflammatory diseases. Recently, we found that P. gingivalis LPS activated the transcription factor nuclear factor-kappaB (NF-kappaB) through the IkappaB kinase complex (IKK). NF-kappaB is a transcription factor that controls inflammation and host responses. In this study, we examined the role of IKK/NF-kappaBin P. gingivalis LPS-induced gene expression on a genome-wide basis using a combination of microarray and biochemical approaches. A total of 88 early response genes were found to be induced by P. gingivalis LPS in a human THP.1 monocytic cell lines. Interestingly, the induction of most of these genes was abolished or attenuated under the inactivation of IKK/NF-kappaB. Among those IKK/NF-kappaB-dependent genes, 20 genes were NF-kappaB-inducible genes reported previously, and 59 genes represented putative novel NF-kappaB target genes. Using transcription factor binding analysis, we found that most of these putative NF-kappaB target genes contained one or multiple NF-kappaB-binding sites. Also, some transcription factor-binding motifs were overrepresented in the promoter of both known and putative NF-kappaB-dependent genes, indicating that these genes may be regulated in a similar fashion. Furthermore, we found that several transcription factors associated with metabolic and inflammatory responses, including nuclear receptors, activator of protein-1, and early growth responses, were induced by P. gingivalis LPS through IKK/NF-kappaB, indicating that IKK/NF-kappaB may utilize these transcription factors to mediate secondary responses. Taken together, our results demonstrate that IKK/NF-kappaB signaling plays a dominant role in P. gingivalis LPS-induced early response gene expression, suggesting that IKK/NF-kappaB is a therapeutic target for periodontitis.

IKKalpha stabilizes cytosolic beta-catenin by inhibiting both canonical and non-canonical degradation pathways.

Beta-catenin is a bi-functional protein. It is not only a major component of the cellular adhesion machinery, but is also a transcription co-activator of the Wnt signaling pathway. The cytosolic levels of the beta-catenin protein, as well as its subcellular localization, are tightly regulated due to its oncogenic potentials. Two independent pathways are found to regulate beta-catenin. The canonical pathway is induced by the Axin/adenomatous polyposis coli (APC)/glycogen synthase kinase-3beta (GSK-3beta) complex which is dependent on GSK-3beta phosphorylation. The non-canonical pathway is mediated by p53-induced Siah-1 which is GSK-3beta phosphorylation-independent. Recently, several studies reported that IkappaB kinase alpha (IKKalpha) could stabilize beta-catenin and stimulate beta-catenin/T cell factor (Tcf)-dependent transcription. Here we report that IKKalpha could inhibit beta-catenin degradation mediated not only by the Axin/APC/GSK-3beta complex, but also by the Siah-1 pathway. Consistently, we found that IKKalpha abolished the inhibition of beta-catenin/Tcf-dependent transcription by Siah-1. Furthermore, we found that IKKalpha interacted with beta-catenin and inhibited beta-catenin ubiquitination. Taken together, our results provide a new insight into IKKalpha-mediated beta-catenin stabilization.

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells.

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.