B cell mechanosensing: A mechanistic overview.

B cells are essential to the adaptive immune system for providing the humoral immunity against cohorts of pathogens. The presentation of antigen to the B cell receptor (BCR) leads to the initiation of B cell activation, which is a process sensitive to the stiffness features of the substrates presenting the antigens. Mechanosensing of the B cells, potentiated through BCR signaling and the adhesion molecules, efficiently regulates B cell activation, proliferation and subsequent antibody responses. Defects in sensing of the antigen-presenting substrates can lead to the activation of autoreactive B cells in autoimmune diseases. The use of high-resolution, high-speed live-cell imaging along with the sophisticated biophysical materials, has uncovered the mechanisms underlying the initiation of B cell activation within seconds of its engagement with the antigen presenting substrates. In this chapter, we reviewed studies that have contributed to uncover the molecular mechanisms of B cell mechanosensing during the initiation of B cell activation.

Targeting the proviral host kinase, FAK, limits influenza a virus pathogenesis and NFkB-regulated pro-inflammatory responses.

Influenza A virus (IAV) infections result in ∼500,000 global deaths annually. Host kinases link multiple signaling pathways at various stages of infection and are attractive therapeutic target. Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, regulates several cellular processes including NFkB and antiviral responses. We investigated how FAK kinase activity regulates IAV pathogenesis. Using a severe infection model, we infected IAV-susceptible DBA/2 J mice with a lethal dose of H1N1 IAV. We observed reduced viral load and pro-inflammatory cytokines, delayed mortality, and increased survival in FAK inhibitor (Y15) treated mice. In vitro IAV-induced NFkB-promoter activity was reduced by Y15 or a dominant negative kinase-dead FAK mutant (FAK-KD) independently of the viral immune modulator, NS1. Finally, we observed reduced IAV-induced nuclear localization of NFkB in FAK-KD expressing cells. Our data suggest a novel mechanism where IAV hijacks FAK to promote viral replication and limit its ability to contribute to innate immune responses.

Selective Killing of Melanoma Cells With Non-Thermal Atmospheric Pressure Plasma and p-FAK Antibody Conjugated Gold Nanoparticles.

Melanomas are fast growing high-mortality tumors, and specific treatments for melanomas are needed. Melanoma cells overexpress focal adhesion kinase (FAK) compared to normal keratinocytes, and we sought to exploit this difference to create a selectively lethal therapy. We combined gold nanoparticles (GNP) with antibodies targeting phosphorylated FAK (p-FAK). These conjugates (p-FAK-GNP) entered G361 melanoma cells and bound p-FAK. Treatment with p-FAK-GNP decreased the viability of G361 cells in a time dependent manner by inducing apoptosis. To maximize the preferential killing of G361 cells, non-thermal atmospheric pressure plasma was used to stimulate the GNP within p-FAK-GNP. Combined treatment with plasma and p-FAK-GNP showed much higher lethality against G361 cells than HaCaT keratinocyte cells. The p-FAK-GNP induced apoptosis over 48 hours in G361 cells, whereas plasma and p-FAK-GNP killed G361 cells immediately. This study demonstrates that combining plasma with p-FAK-GNP results in selective lethality against human melanoma cells.

Cell type-specific differences in ß-glucan recognition and signalling in porcine innate immune cells.

ß-glucans exert receptor-mediated immunomodulating activities, including oxidative burst activity and cytokine secretion. The role of the ß-glucan receptors dectin-1 and complement receptor 3 (CR3) in the response of immune cells towards ß-glucans is still unresolved. Dectin-1 is considered as the main ß-glucan receptor in mice, while recent studies in man show that CR3 is more important in ß-glucan-mediated responses. This incited us to elucidate which receptor contributes to the response of innate immune cells towards particulate ß-glucans in pigs as the latter might serve as a better model for man. Our results show an important role of CR3 in ß-glucan recognition, as blocking this receptor strongly reduced the phagocytosis of ß-glucans and the ß-glucan-induced ROS production by porcine neutrophils. Conversely, dectin-1 does not seem to play a major role in ß-glucan recognition in neutrophils. However, recognition of ß-glucans appeared cell type-specific as both dectin-1 and CR3 are involved in the ß-glucan-mediated responses in pig macrophages. Moreover, CR3 signalling through focal adhesion kinase (FAK) was indispensable for ß-glucan-mediated ROS production and cytokine production in neutrophils and macrophages, while the Syk-dependent pathway was only partly involved in these responses. We may conclude that CR3 plays a cardinal role in ß-glucan signalling in porcine neutrophils, while macrophages use a more diverse receptor array to detect and respond towards ß-glucans. Nonetheless, FAK acts as a master switch that regulates ß-glucan-mediated responses in neutrophils as well as macrophages.

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells.

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.

Activation of integrin ß1-focal adhesion kinase-RasGTP pathway plays a critical role in TGF beta1-induced podocyte injury.

The depletion of glomerular podocytes is the key mechanism of glomerulosclerosis and progressive renal failure. Transforming growth factor-ß (TGFß) is a central mediator of signaling networks that control a diverse set of cellular processes, such as cell proliferation, differentiation, and apoptosis. Though many key events in TGFß1 signaling have been documented at cellular and molecular level in podocytes, the complete effects of TGFß1 on podocyte integrity are still elusive. In this study, the function of adhesion protein integrin ß1, focal adhesion kinase (FAK), and a small GTPase Ras was explored in TGFß1-induced podocyte injury. In cultured mouse podocyte, caspase 3-positive cells were counted by flow cytometry to evaluate podocyte damage at different time points after TGFß1 treatment. Immunoblotting assay showed that integrin ß1, FAK, Src kinase, and an adaptor protein Grb2 were activated rapidly after TGFß1 stimulation. Active Ras Pull-Down assay revealed that the active Ras (GTP-bound Ras) level was upregulated in TGFß1-treated cell. Immunoprecipitation results displayed that TGFß1 enhanced the complex formation of integrin ß1, FAK and Src kinase, as well as FAK, Grb2 and Ras. The FAK inhibitor TAE226 and the specific knockdown of Grb2 remarkably alleviated TGFß1-induced podocyte apoptosis. The activation of p38MAPK and Erk1/2, and the nuclear translocation of NFκB(p65) were increased evidently in TGFß1-treated cell, which could be dramatically prohibited by the application of the p38MAPK inhibitor SB202190 and the Ras inhibitor FPT Inhibitor III. The Src kinase inhibitor PP2 obviously prevented the activation of FAK and Ras, as well as the translocation of NFκB(p65) from cytoplasm to nuclei. The PP2, FPT Inhibitor III, and SB202190 significantly decreased TGFß1-induced podocyte apoptosis. Taken together, these data demonstrated that the activation of integrin ß1/Src/FAK and Grb2/RasGTP should be responsible for TGFß1-induced podocyte damage through the p38MAPK and Erk1/2-mediated nuclear translocation of NFκB(p65).

Inhibition of FAK prevents blister formation in the neonatal mouse model of pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis and by autoantibodies against desmoglein 3 localized on desmosomes. In addition, caspases also seem to participate in this blistering disease. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase involved in cytoskeleton remodelling and formation and disassembly of cell adhesion structures. We have previously demonstrated that HER (human epidermal growth factor receptor related) isoforms, Src (Rous sarcoma) and mammalian target of rapamycin (mTOR), three molecules implicated in signalling processes, take part in suprabasal acantholysis and apoptosis induced by PV-IgG in a mouse model. Our aim was to investigate whether upregulation of FAK is implicated in the development of PV lesions. Herein, using a mouse model, PV-IgG administration showed an increased level of FAK phosphorylated on 397 and 925 tyrosine residues in the basal layer of epidermis. When mice were pretreated with a FAK inhibitor (FI), the acantholysis of the basal layer of epidermis was absent. More interestingly, we observed that phosphorylated FAK (Y397/925) decreased when HER isoforms, Src, mTOR and pan-caspases inhibitors were employed before PV-IgG administration. In addition, pretreatment with the FI before PV-IgG injection prevented the changes in both Bax and Bcl-2 expression and caspase-9 and caspase-3 activities induced by PV-IgG. Finally, FI reduced the expression of phosphorylated Src and mTOR in the basal cells of epidermis. In conclusion, our data reveal a novel role of phosphorylated FAK (Y397/925) in PV development involving HER isoforms, Src and mTOR kinases.

Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration.

During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins ß1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.

Expression of focal adhesion proteins in the developing rat kidney.

Focal adhesions play a critical role as centers that transduce signals by cell-matrix interactions and regulate fundamental processes such as proliferation, migration, and differentiation. Focal adhesion kinase (FAK), paxillin, integrin-linked kinase (ILK), and hydrogen peroxide-inducible clone-5 (Hic-5) are major proteins that contribute to these events. In this study, we investigated the expression of focal adhesion proteins in the developing rat kidney. Western blotting analysis revealed that the protein levels of FAK, p-FAK(397), paxillin, p-paxillin(118), and Hic-5 were high in embryonic kidneys, while ILK expression persisted from the embryonic to the mature stage. Immunohistochemistry revealed that FAK, p-FAK(397), paxillin, and p-paxillin(118) were strongly expressed in condensed mesenchymal cells and the ureteric bud. They were detected in elongating tubules and immature glomerular cells in the nephrogenic zone. Hic-5 was predominantly expressed in mesenchymal cells as well as immature glomerular endothelial and mesangial cells, suggesting that Hic-5 might be involved in mesenchymal cell development. ILK expression was similar to that of FAK in the developmental stages. Interestingly, ILK was strongly expressed in podocytes in mature glomeruli. ILK might play a role in epithelial cell differentiation as well as kidney growth and morphogenesis. In conclusion, the temporospatially regulated expression of focal adhesion proteins during kidney development might play a role in morphogenesis and cell differentiation.

RIG-like helicase innate immunity inhibits vascular endothelial growth factor tissue responses via a type I IFN-dependent mechanism.

RATIONALE: Vascular endothelial growth factor (VEGF) regulates vascular, inflammatory, remodeling, and cell death responses. It plays a critical role in normal pulmonary physiology, and VEGF excess and deficiency have been implicated in the pathogenesis of asthma and chronic obstructive pulmonary disease, respectively. Although viruses are an important cause of chronic obstructive pulmonary disease exacerbations and innate responses play an important role in these exacerbations, the effects of antiviral responses on VEGF homeostasis have not been evaluated. OBJECTIVES: We hypothesized that antiviral innate immunity regulates VEGF tissue responses. METHODS: We compared the effects of transgenic VEGF(165) in mice treated with viral pathogen-associated molecular pattern polyinosinic:polycytidylic acid [poly(I:C)], mice treated with live virus, and control mice. MEASUREMENTS AND MAIN RESULTS: Transgenic VEGF stimulated angiogenesis, edema, inflammation, and mucin accumulation. Each of these was abrogated by poly(I:C). These inhibitory effects were dose dependent, noted when poly(I:C) was administered before and after transgene activation, and mediated by a Toll-like receptor-3-independent and RIG-like helicase (RLH)- and type I IFN receptor-dependent pathway. VEGF stimulated the expression of VEGF receptor-1 and poly(I:C) inhibited this stimulation. Poly(I:C) also inhibited the ability of VEGF to activate extracellular signal-regulated kinase-1, Akt, focal adhesion kinase, and endothelial nitric oxide synthase, and aeroallergen-induced adaptive helper T-cell type 2 inflammation. Influenza and respiratory syncytial virus also inhibited VEGF-induced angiogenesis. CONCLUSIONS: These studies demonstrate that poly(I:C) and respiratory viruses inhibit VEGF-induced tissue responses and adaptive helper T-cell type 2 inflammation and highlight the importance of a RLH- and type I IFN receptor-dependent pathway(s) in these regulatory events. They define a novel link between VEGF and antiviral and RLH innate immune responses and a novel pathway that regulates pulmonary VEGF activity.

Antibody binding to cell surface amyloid precursor protein induces neuronal injury by deregulating the phosphorylation of focal adhesion signaling related proteins.

The biological function of full-length amyloid-beta protein precursor (APP), the precursor of Abeta, is not fully understood. Mounting studies reported that antibody binding to cell surface APP causes neuronal injury. However, the mechanism of cell surface APP mediating neuronal injury remains to be determined. Colocalization of APP with integrin on cell surface leads us to suppose that focal adhesion (FA) related mechanism is involved in surface APP-mediated neuronal injury. In the present study, results demonstrated that primary cultured neurons treated with antibody against APP-N-terminal not only caused neuronal injury and aberrant morphologic changes of neurite, but also induced reaction of FA proteins appearing an acute increase then decrease pattern. Moreover, the elevation of tyrosine phosphorylation of FA proteins including paxillin and focal adhesion kinase (FAK), and down-regulated expression of protein tyrosine phosphatase (PTP1B) induced by APP antibody were prevented by inhibitor of Src protein kinases 4-amino-5-(4-chlorophenyl)-7(t-butyl) pyrazol (3,4-D) pyramide (PP2) and G protein inhibitor pertussis toxin (PTX), implying that Src family kinase and G protein play roles in APP-induced FA signals. In addition, pretreatment with PTX and PP2 was able to suppress APP-antibody induced neuronal injury. Taken together, the results suggest a novel mechanism for APP mediating neuronal injury through deregulating FA signals.

Focal adhesion kinase (FAK) immunocytochemical expression in breast ductal invasive carcinoma (DIC): correlation with clinicopathological parameters and tumor proliferative capacity.

BACKGROUND: Focal adhesion kinase (FAK) is an enzyme of the tyrosine kinase group linked to signaling pathways between cells and the extracellular matrix. In tumor cells in vitro, FAK expression correlated with their ability for invasion and metastasis. Additionally, in vivo FAK has been implicated in malignant transformation and disease progression. The aim of the present study was to evaluate the clinical significance of FAK expression in breast ductal invasive carcinoma (DIC). MATERIAL/METHODS: Immunocytochemical techniques were used to assess FAK expression on cytological material obtained from 73 patients with breast DIC. FAK expression status (positivity, overexpression, and intensity of immunostaining) was compared with clinicopathological parameters and the tumor cells' proliferative capacity. RESULTS: Sixty-four of the 73 DIC cases (88%) were FAK positive and FAK protein overexpression was noted in 15 of the 73 (21%). In the DIC cases examined, FAK positivity correlated with tumor size (p=0.016) and FAK protein overexpression with tumor histological grade (p=0.034) and the tumor cells' proliferative capacity (p=0.003). The intensity of FAK protein staining did not significantly correlate with any of the examined clinicopathological parameters. CONCLUSIONS: In breast DIC it becomes evident that FAK protein positivity and overexpression correlate with important clinicopathological parameters. Further molecular and clinical studies are required to delineate the significance of FAK as a factor for better prognosis and management of breast cancer patients.

Focal adhesion kinase as an immunotherapeutic target.

BACKGROUND: Focal adhesion kinase (FAK) is a ubiquitously expressed non-receptor tyrosine kinase involved in cancer progression and metastasis that is found overexpressed in a large number of tumors such as breast, colon, prostate, melanoma, head and neck, lung and ovary. Thus, FAK could be an attractive tumor associated antigen (TAA) for developing immunotherapy against a broad type of malignancies. In this study, we determined whether predicted T cell epitopes from FAK would be able to induce anti-tumor immune cellular responses. METHODS: To validate FAK as a TAA recognized by CD4 helper T lymphocytes (HTL), we have combined the use of predictive peptide/MHC class II binding algorithms with in vitro vaccination of CD4 T lymphocytes from healthy individuals and melanoma patients. RESULTS: Two synthetic peptides, FAK(143-157) and FAK(1,000-1,014), induced HTL responses that directly recognized FAK-expressing tumor cells and autologous dendritic cells pulsed with FAK-expressing tumor cell lysates in an HLA class II-restricted manner. Moreover, since the FAK peptides were recognized by melanoma patient's CD4 T cells, this is indicative that T cell precursors reactive with FAK already exist in peripheral blood of these patients. CONCLUSIONS: Our results provide evidence that FAK functions as a TAA and describe peptide epitopes that may be used for designing T cell-based immunotherapy for FAK-expressing cancers, which could be used in combination with newly developed FAK inhibitors.

FAK-mediated activation of ERK for eosinophil migration: a novel mechanism for infection-induced allergic inflammation.

Bacterial and viral infections often induce the exacerbation of allergic diseases. In this study, we investigated the activation of human eosinophils by different microbial products via Toll-like receptors (TLRs). The underlying intracellular mechanism involving activation of extracellular signal-regulated kinase (ERK) and focal adhesion kinase (FAK), an integrin-associated focal adhesion molecule, was also examined. Seven TLR ligands were studied for their abilities in promoting survival, modulating the expression of adhesion molecules and facilitating chemotactic migration of eosinophils. While peptidoglycan (PGN) (TLR2 ligand) showed the most prominent effects, flagellin (TLR5 ligand) and imiquimod R837 (TLR7 ligand) were also effective in activating eosinophils. However, little or no effect was observed for double-stranded polyinosinic-polycytidylic acid (TLR3 ligand), ultra-purified LPS (TLR4 ligand), single-stranded RNA (ssRNA) (TLR8 ligand) and CpG-DNA (TLR9 ligand). Further investigation confirmed that PGN, flagellin and R837 commonly transmitted signals through ERK activation that required prior phosphorylation of tyrosine 925, but not tyrosine 577, on FAK. Moreover, the inhibition of ERK activation by selective inhibitor PD98059 and FAK expression by FAK-specific RNA interference could significantly abolish the stimulatory effects induced by PGN, flagellin and R837. Taken together, our findings indicate the involvement of FAK-dependent activation of ERK1 in TLR-mediated eosinophil stimulation. A potential role of eosinophils was also suggested in exacerbating allergic inflammation in response to microbial infections.

Distinct signalling pathways promote phagocytosis of bacteria, latex beads and lipopolysaccharide in medfly haemocytes.

In insects, phagocytosis is an important innate immune response against pathogens and parasites, and several signal transduction pathways regulate this process. The focal adhesion kinase (FAK)/Src and mitogen activated protein kinase (MAPK) pathways are of central importance because their activation upon pathogen challenge regulates phagocytosis via haemocyte secretion and activation of the prophenoloxidase (proPO) cascade. The goal of this study was to explore further the mechanisms underlying the process of phagocytosis. In particular, in this report, we used flow cytometry, RNA interference, enzyme-linked immunosorbent assay, Western blot and immunoprecipitation analysis to demonstrate that (1) phagocytosis of bacteria (both Gram-negative and Gram-positive) is dependent on RGD-binding receptors, FAK/Src and MAPKs, (2) latex bead phagocytosis is RGD-binding-receptor-independent and dependent on FAK/Src and MAPKs, (3) lipopolysaccharide internalization is RGD-binding-receptor-independent and FAK/Src-independent but MAPK-dependent and (4) in unchallenged haemocytes in suspension, FAK, Src and extracellular signal-regulated kinase (ERK) signalling molecules participating in phagocytosis show both a functional and a physical association. Overall, this study has furthered knowledge of FAK/Src and MAPK signalling pathways in insect haemocyte immunity and has demonstrated that distinct signalling pathways regulate the phagocytic activity of biotic and abiotic components in insect haemocytes. Evidently, the basic phagocytic signalling pathways among insects and mammals appear to have remained unchanged during evolution.

Local calcium transients contribute to disappearance of pFAK, focal complex removal and deadhesion of neuronal growth cones and fibroblasts.

Cell adhesion is crucial for migration of cells during development, and cell-substrate adhesion of motile cells is accomplished through the formation and removal of focal complexes that are sites of cell-substrate contact. Because Ca2+ signaling regulates the rate of axon outgrowth and growth cone turning, we investigated the potential role of Ca2+ in focal complex dynamics. We describe a novel class of localized, spontaneous transient elevations of cytosolic Ca2+ observed both in Xenopus neuronal growth cones and fibroblasts that are 2-6 mum in spatial extent and 2-4 s in duration. They are distributed throughout growth cone lamellipodia and at the periphery of fibroblast pseudopodia, which are regions of high motility. In both cell types, these Ca2+ transients lead to disappearance of phosphorylated focal adhesion kinase (pFAK) and deadhesion from the substrate as assessed by confocal and internal reflection microscopy, respectively. The loss of pFAK is inhibited by cyclosporin A, suggesting that these Ca2+ transients exert their effects via calcineurin. These results identify an intrinsic mechanism for local cell detachment that may be modulated by agents that regulate motility.