Katherine A. Owen

Regulation of lamellipodial persistence, adhesion turnover, and motility in macrophages by focal adhesion kinase.

Macrophages are a key component of the innate immune system. In this study, we investigate how focal adhesion kinase (FAK) and the related kinase Pyk2 integrate adhesion signaling and growth factor receptor signaling to regulate diverse macrophage functions. Primary bone marrow macrophages isolated from mice in which FAK is conditionally deleted from cells of the myeloid lineage exhibited elevated protrusive activity, altered adhesion dynamics, impaired chemotaxis, elevated basal Rac1 activity, and a marked inability to form stable lamellipodia necessary for directional locomotion. The contribution of FAK to macrophage function in vitro was substantiated in vivo by the finding that recruitment of monocytes to sites of inflammation was impaired in the absence of FAK. Decreased Pyk2 expression in primary macrophages also resulted in a diminution of invasive capacity. However, the combined loss of FAK and Pyk2 had no greater effect than the loss of either molecule alone, indicating that both kinases function within the same pathway to promote invasion.

Physical and functional interactions between Cas and c-Src induce tamoxifen resistance of breast cancer cells through pathways involving epidermal growth factor receptor and signal transducer and activator of transcription 5b.

High expression of the adaptor molecule Cas has been linked to resistance to the antiestrogen tamoxifen, both in tissue culture and in human tumors. The aim of this study was to elucidate the mechanism(s) by which overexpression of Cas confers resistance to tamoxifen. Cas overexpression in MCF-7 breast cancer cells was shown to alleviate both tamoxifen-mediated growth inhibition and induction of apoptosis. This enhancement of cell proliferation/survival occurred in the absence of detectable effects on estrogen receptor (ER) transcriptional activity under conditions where tamoxifen was present, indicating that Cas-dependent tamoxifen resistance is not the result of a switch to an ER-negative phenotype or enhanced responses to the partial agonist activity of tamoxifen. Instead, we present evidence, suggesting that Cas promotes tamoxifen resistance by deregulation of alternative cell proliferation pathways, particularly those mediated through enhanced c-Src protein tyrosine kinase activity arising from Cas/c-Src interactions. Overexpression of Cas was found to drive endogenous c-Src into complex with Cas, a process that has been shown previously to cause up-regulation of c-Src tyrosine kinase activity. MCF-7 cells overexpressing Cas exhibited increased phosphorylation of two c-Src substrates, Tyr845 in the kinase domain of the epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) 5b. Importantly, Cas-dependent protection from the antiproliferative effects of tamoxifen was reversed by the expression of dominant inhibitory variants of these substrates (Y845F EGFR and COOH-terminally truncated STAT5b). Based on these findings, we suggest that the Cas/c-Src/EGFR/STAT5 signaling axis is a major regulator of tamoxifen-resistant breast cancer cell growth and survival.

Brain angiogenesis inhibitor 1 (BAI1) is a pattern recognition receptor that mediates macrophage binding and engulfment of Gram-negative bacteria.

Bacterial recognition by host cells is essential for initiation of infection and the host response. Bacteria interact with host cells via multiple pattern recognition receptors that recognize microbial products or pathogen-associated molecular patterns. In response to this interaction, host cell signaling cascades are activated that lead to inflammatory responses and/or phagocytic clearance of attached bacteria. Brain angiogenesis inhibitor 1 (BAI1) is a receptor that recognizes apoptotic cells through its conserved type I thrombospondin repeats and triggers their engulfment through an ELMO1/Dock/Rac1 signaling module. Because thrombospondin repeats in other proteins have been shown to bind bacterial surface components, we hypothesized that BAI1 may also mediate the recognition and clearance of pathogenic bacteria. We found that preincubation of bacteria with recombinant soluble BAI1 ectodomain or knockdown of endogenous BAI1 in primary macrophages significantly reduced binding and internalization of the Gram-negative pathogen Salmonella typhimurium. Conversely, overexpression of BAI1 enhanced attachment and engulfment of Salmonella in macrophages and in heterologous nonphagocytic cells. Bacterial uptake is triggered by the BAI1-mediated activation of Rac through an ELMO/Dock-dependent mechanism, and inhibition of the BAI1/ELMO1 interaction prevents both Rac activation and bacterial uptake. Moreover, inhibition of ELMO1 or Rac function significantly impairs the proinflammatory response to infection. Finally, we show that BAI1 interacts with a variety of Gram-negative, but not Gram-positive, bacteria through recognition of their surface lipopolysaccharide. Together these findings identify BAI1 as a pattern recognition receptor that mediates nonopsonic phagocytosis of Gram-negative bacteria by macrophages and directly affects the host response to infection.

Activation of focal adhesion kinase by Salmonella suppresses autophagy via an Akt/mTOR signaling pathway and promotes bacterial survival in macrophages.

Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK−/− macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing.

Background Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo. Methodology and Principal Findings To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression. Conclusions In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.

The differential expression of Yersinia pseudotuberculosis adhesins determines the requirement for FAK and/or Pyk2 during bacterial phagocytosis by macrophages

Phagocytosis of Yersinia pseudotuberculosis by macrophages is initiated by interactions between host cell integrin receptors and the bacterial adhesins, invasin and YadA. Two non‐receptor protein tyrosine kinases, FAK and Pyk2, have been implicated in this process. In this study, we investigated the mechanisms of activation and functional requirements for these kinases during phagocytosis. A panel of Yersinia strains that differentially express invasin and YadA were used to infect cells in which FAK and/or Pyk2 expression was reduced by RNA interference. Bacterial strains that simultaneously express invasin and YadA activated FAK and Pyk2 signalling pathways that perform non‐redundant functions required for Yersinia internalization. In contrast, FAK activation was found to be sufficient for phagocytosis of bacteria expressing invasin alone, and Pyk2 activation was sufficient when YadA was expressed in the absence of invasin. Based on these data, we suggest that the activation states of FAK and Pyk2, as well as the subsequent signalling events that lead to phagocytosis, are differentially regulated through the unique mechanisms of integrin engagement utilized by invasin and YadA. These findings lend insight into the molecular events that control bacterial phagocytosis as well as other integrin‐based processes such as cell adhesion and migration.

Macrophage motility requires distinct α5β1/FAK and α4β1/paxillin signaling events

Macrophages function as key inflammatory mediators at sites of infection and tissue damage. Integrin and growth factor receptors facilitate recruitment of monocytes/macrophages to sites of inflammation in response to numerous extracellular stimuli. We have shown recently that FAK plays a role in regulating macrophage chemotaxis and invasion. As FAK is an established downstream mediator of integrin signaling, we sought to define the molecular circuitry involving FAK and the predominant β1 integrin heterodimers expressed in these cells—α4β1 and α5β1. We show that α4β1 and α5β1 integrins are required for efficient haptotactic and chemotactic invasion and that stimulation of these integrin receptors leads to the adoption of distinct morphologies associated with motility. FAK is required downstream of α5β1 for haptotaxis toward FN and chemotaxis toward M‐CSF‐1 and downstream of α4β1 for the adoption of a polarized phenotype. The scaffolding molecule paxillin functions independently of FAK to promote chemotaxis downstream of α4β1. These studies expand our understanding of β1 integrin signaling networks that regulate motility and invasion in macrophages and thus, provide important new insights into mechanisms by which macrophages perform their diverse functions.

The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria

A macrophage adhesion receptor that mediates internalization of Gram-negative bacteria also stimulates the machinery that kills them. Bacteria come to a sticky end Pattern recognition receptors (PRRs) detect microbial products and stimulate the innate immune response to infections. BAI1 is a G protein–coupled receptor of the adhesion GPCR family and is also a PRR that binds to lipopolysaccharide on the surface of Gram-negative bacteria to facilitate their internalization by macrophages. Billings et al. found that BAI1 triggered the killing of the internalized bacteria by stimulating the production of reactive oxygen species. When engaged by bacteria, BAI1 activated Rac1 to stimulate the activity of the NADPH oxidase complex Nox2 in macrophages. Mice deficient in BAI1 were inefficient at clearing Gram-negative bacteria and were likely to die from the infection. Together, these data suggest that BAI1 connects bacterial internalization with their killing. The detection of microbes and initiation of an innate immune response occur through pattern recognition receptors (PRRs), which are critical for the production of inflammatory cytokines and activation of the cellular microbicidal machinery. In particular, the production of reactive oxygen species (ROS) by the NADPH oxidase complex is a critical component of the macrophage bactericidal machinery. We previously characterized brain-specific angiogenesis inhibitor 1 (BAI1), a member of the adhesion family of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors (GPCRs), as a PRR that mediates the selective phagocytic uptake of Gram-negative bacteria by macrophages. We showed that BAI1 promoted phagosomal ROS production through activation of the Rho family guanosine triphosphatase (GTPase) Rac1, thereby stimulating NADPH oxidase activity. Primary BAI1-deficient macrophages exhibited attenuated Rac GTPase activity and reduced ROS production in response to several Gram-negative bacteria, resulting in impaired microbicidal activity. Furthermore, in a peritoneal infection model, BAI1-deficient mice exhibited increased susceptibility to death by bacterial challenge because of impaired bacterial clearance. Together, these findings suggest that BAI1 mediates the clearance of Gram-negative bacteria by stimulating both phagocytosis and NADPH oxidase activation, thereby coupling bacterial detection to the cellular microbicidal machinery.

Salmonella Suppresses the TRIF-Dependent Type I Interferon Response in Macrophages

ABSTRACT Salmonella enterica is an intracellular pathogen that causes diseases ranging from gastroenteritis to typhoid fever. Salmonella bacteria trigger an autophagic response in host cells upon infection but have evolved mechanisms for suppressing this response, thereby enhancing intracellular survival. We recently reported that S. enterica serovar Typhimurium actively recruits the host tyrosine kinase focal adhesion kinase (FAK) to the surface of the Salmonella-containing vacuole (SCV) (K. A. Owen et al., PLoS Pathog 10:e1004159, 2014). FAK then suppresses autophagy through activation of the Akt/mTORC1 signaling pathway. In FAK−/− macrophages, bacteria are captured in autophagosomes and intracellular survival is attenuated. Here we show that the cell-autonomous bacterial suppression of autophagy also suppresses the broader innate immune response by inhibiting production of beta interferon (IFN-β). Induction of bacterial autophagy (xenophagy), but not autophagy alone, triggers IFN-β production through a pathway involving the adapter TRIF and endosomal Toll-like receptor 3 (TLR3) and TLR4. Selective FAK knockout in macrophages resulted in rapid bacterial clearance from mucosal tissues after oral infection. Clearance correlated with increased IFN-β production by intestinal macrophages and with IFN-β-dependent induction of IFN-γ by intestinal NK cells. Blockade of either IFN-β or IFN-γ increased host susceptibility to infection, whereas experimental induction of IFN-β was protective. Thus, bacterial suppression of autophagy not only enhances cell-autonomous survival but also suppresses more-systemic innate immune responses by limiting type I and type II interferons. IMPORTANCE Salmonella enterica serovar Typhimurium represents one of the most commonly identified bacterial causes of foodborne illness worldwide. S. Typhimurium has developed numerous strategies to evade detection by the host immune system. Autophagy is a cellular process that involves the recognition and degradation of defective proteins and organelles. More recently, autophagy has been described as an important means by which host cells recognize and eliminate invading intracellular pathogens and plays a key role in the production of cytokines. Previously, we determined that Salmonella bacteria are able to suppress their own autophagic capture and elimination by macrophages. Building on that study, we show here that the inhibition of autophagy by Salmonella also prevents the induction of a protective cytokine response mediated by beta interferon (IFN-β) and IFN-γ. Together, these findings identify a novel virulence strategy whereby Salmonella bacteria prevent cell autonomous elimination via autophagy and suppress the activation of innate immune responses. Salmonella enterica serovar Typhimurium represents one of the most commonly identified bacterial causes of foodborne illness worldwide. S. Typhimurium has developed numerous strategies to evade detection by the host immune system. Autophagy is a cellular process that involves the recognition and degradation of defective proteins and organelles. More recently, autophagy has been described as an important means by which host cells recognize and eliminate invading intracellular pathogens and plays a key role in the production of cytokines. Previously, we determined that Salmonella bacteria are able to suppress their own autophagic capture and elimination by macrophages. Building on that study, we show here that the inhibition of autophagy by Salmonella also prevents the induction of a protective cytokine response mediated by beta interferon (IFN-β) and IFN-γ. Together, these findings identify a novel virulence strategy whereby Salmonella bacteria prevent cell autonomous elimination via autophagy and suppress the activation of innate immune responses.

Registered report: BET bromodomain inhibition as a therapeutic strategy to target c-Myc

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by replicating selected results from a substantial number of high-profile papers in the field of cancer biology published between 2010 and 2012. This Registered report describes the proposed replication plan of key experiments from ‘BET bromodomain inhibition as a therapeutic strategy to target c-Myc’ by Delmore and colleagues, published in Cell in 2011 (Delmore et al., 2011). The key experiments that will be replicated are those reported in Figures 3B and 7C-E. Delmore and colleagues demonstrated that treatment with JQ1, a small molecular inhibitor targeting BET bromodomains, resulted in the transcriptional down-regulation of the c-Myc oncogene in vitro (Figure 3B; Delmore et al., 2011). To assess the therapeutic efficacy of JQ1 in vivo, mice bearing multiple myeloma (MM) lesions were treated with JQ1 before evaluation for tumor burden and overall survival. JQ1 treatment significantly reduced disease burden and increased survival time (Figure 7C-E; Delmore et al., 2011). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.07072.001