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

The CB1 receptor antagonist AM251 impairs reconsolidation of pavlovian fear memory in the rat basolateral amygdala

We have investigated the requirement for signaling at CB1 receptors in the reconsolidation of a previously consolidated auditory fear memory, by infusing the CB1 receptor antagonist AM251, or the FAAH inhibitor URB597, directly into the basolateral amygdala (BLA) in conjunction with memory reactivation. AM251 disrupted memory restabilization, but only when administered after reactivation. URB597 produced a small, transient enhancement of memory restabilization when administered after reactivation. The amnestic effect of AM251 was rescued by coadministration of the GABAA receptor antagonist bicuculline at reactivation, indicating that the disruption of reconsolidation was mediated by altered GABAergic transmission in the BLA. These data show that the endocannabinoid system in the BLA is an important modulator of fear memory reconsolidation and that its effects on memory are mediated by an interaction with the GABAergic system. Thus, targeting the endocannabinoid system may have therapeutic potential to reduce the impact of maladaptive memories in neuropsychiatric disorders such as posttraumatic stress disorder.This work was conducted within the Behavioural and Clinical Neuroscience Institute, a joint initiative funded by the Wellcome Trust and the UK Medical Research Council, in the Department of Psychology at the University of Cambridge. This work was funded by a UK Medical Research Council programme grant (no. G1002231) awarded to BJE and ALM. PR was supported by a Department of Physiology and Pharmacology Fellowship at the Sapienza University of Rome, and an Italian Society of Pharmacology Fellowship. ALM is the Ferreras-Willetts Fellow in Neuroscience at Downing College, Cambridge. The manuscript was partly prepared while ALM was an Erskine Visiting Cambridge Fellow at the University of Canterbury, Christchurch, New Zealand

Helicobacter pylori Induces Interleukin-8 Secretion by Toll-Like Receptor 2- and Toll-Like Receptor 5-Dependent and -Independent Pathways

Helicobacter pylori is an important human pathogen that causes gastritis and is strongly associated with gastric ulcers, gastric adenocarcinomas, and mucosa-associated lymphoid tissue lymphomas. In response to H. pylori, interleukin-8 (IL-8) is secreted from host cells to attract components of the innate and adaptive immune systems to the site of infection. Toll-like receptor 2 (TLR2) and TLR5 have been shown to recognize H. pylori and to initiate signaling pathways that result in enhanced activation of NF-κB. Here, we evaluated the contribution of mitogen-activated protein kinase signaling pathways to TLR2-dependent and TLR5-dependent secretion of IL-8. Secretion of IL-8 from H. pylori-infected HEK293 cells was augmented by the expression of TLR2 or TLR5. While H. pylori infection resulted in the activation of ERK, JNK, and p38, the enhanced IL-8 secretion from TLR2- and TLR5-expressing cells coincided with increased p38 activation and phosphorylation of the transcription factor ATF2. When p38 activity was inhibited in TLR2- or TLR5-expressing cells, H. pylori-dependent IL-8 secretion returned to the level observed in infected parental HEK293 cells that did not express TLR2 or TLR5; inhibition of p38 had no effect on IL-8 secretion from infected parental HEK cells. In contrast, inhibition of JNK and/or ERK resulted in substantially less IL-8 secretion from infected cells, independent of TLR2 or TLR5 expression. Based on these data, we propose that H. pylori induces IL-8 secretion through a dual mechanism that includes a TLR2/5-independent component involving the activities of JNK and ERK and a TLR2/5-dependent component that requires p38 activity

Breast cancer antiestrogen resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.

Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process

Activation of Focal Adhesion Kinase by <i>Salmonella</i> Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages

<div><p>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 <i>Salmonella</i> 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 <i>Salmonella</i> SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the <i>Salmonella</i>-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). <i>In vivo</i>, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with <i>S. typhimurium</i>. 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 <i>S. typhimurium</i> as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.</p></div

FAK is selectively recruited to LAMP-1-positive SCVs.

<p>(A) FAK immunoblotting was performed on macrophages isolated from the peritoneum of WT and FAK^Δmyeloid mice. (B) WT and FAK^−/− PEMs were incubated with <i>S. typhimurium</i> strain <i>ΔinvG</i> for 0–5 hours before immunoblotting with the indicated antibodies. (C, E and G) WT PEMs expressing GFP-FAK were incubated with either <i>ΔinvG Salmonella</i> (C), LPS-coated beads (E) or <i>E. coli</i> (G) for a total of 5 hours before analysis by immunofluorescence. Cells were co-stained with antibodies recognizing LAMP1 (red). Dapi was used to visualize nuclei and bacteria (blue). Bars represent 10 µm. Arrowheads indicate bacteria or beads in enlarged panel where bars represent 2 µm. (D, F and H) The percent of LAMP1-positive (gray bars) and LAMP1+GFP+ (black bars) <i>ΔinvG Salmonella</i> (D), LPS-coated beads (F) and <i>E. coli</i> (H) was quantified. At least 100 bacteria were counted per condition; N = 3.</p