Complement receptor 3 mediates Aspergillus fumigatus internalization into alveolar epithelial cells with the increase of intracellular phosphatidic acid by activating FAK.

Complement receptor 3 (CD11b/CD18) is an important receptor that mediates adhesion, phagocytosis and chemotaxis in various immunocytes. The conidia of the medically-important pathogenic fungus, Aspergillus fumigatus can be internalized into alveolar epithelial cells to disseminate its infection in immunocompromised host; however, the role of CR3 in this process is poorly understood. In the present study, we investigated the potential role of CR3 on A. fumigatus internalization into type II alveolar epithelial cells and its effect on host intracellular PA content induced by A. fumigatus. We found that CR3 is expressed in alveolar epithelial cells and that human serum and bronchoalveolar lavage fluid (BALF) could improve A. fumigatus conidial internalization into A549 type II alveolar epithelial cell line and mouse primary alveolar epithelial cells, which were significantly inhibited by the complement C3 quencher and CD11b-blocking antibody. Serum-opsonization of swollen conidia, but not resting conidia led to the increase of cellular phosphatidic acid (PA) in A549 cells during infection. Moreover, both conidial internalization and induced PA production were interfered by CD11b-blocking antibody and dependent on FAK activity, but not Syk in alveolar epithelial cells. Overall, our results revealed that CR3 is a critical modulator of Aspergillus fumigatus internalization into alveolar epithelial cells.

FAK inhibitor PF-431396 suppresses IgE-mediated mast cell activation and allergic inflammation in mice.

Mast cells (MCs) initiate and maintain allergic inflammation. Upon being stimulated with immunoglobulin (Ig)E and antigen (Ag), MCs exhibit FcεRI (high-affinity IgE) receptor-mediated degranulation, cytokine secretion, and increased focal adhesion kinase (FAK) activity. The aims of this study were to examine mechanisms of FAK regulation in IgE-mediated MC activation and the effects of FAK inhibition on MC-mediated allergic responses. FAK activity was manipulated with short hairpin RNA (shRNA) knockdown, FAK overexpression, and the FAK inhibitor PF-431396 (PF). Gene expression and kinase activation were analyzed with quantitative molecular biology assays. PF effects were tested in the passive cutaneous anaphylaxis (PCA), active systemic anaphylaxis (ASA), and allergic conjunctivitis (AC) mouse models. Our results showed that FAK overexpression increased IgE-mediated degranulation and reduced the dexamethasone inhibitory effect on MCs activation. The FAK inhibitor PF diminished MC release of ß-hexosaminidase (ß-hex), histamine, and inflammatory cytokines, via a mechanism that involves MAPK and NF-κB signaling pathways. CaMKII was identified as a robust FAK-associating protein. Inhibition of CaMKII activation by KN-93 suppressed FAK activity and its downstream pathway. PF attenuated inflammatory responses in our PCA and ASA models, and relieved signs of allergic disease in AC model mice. In conclusions, MC degranulation and production of inflammatory mediators in allergic disease may be consequent to FcεRI crosslinking inducing CaMKII-mediated activation of FAK activity. FAK inhibition may represent a new MC-suppressing treatment strategy for the treatment of allergic diseases.

Anti-GPR56 monoclonal antibody potentiates GPR56-mediated Src-Fak signaling to modulate cell adhesion.

GPR56 is a member of the adhesion G-protein-coupled receptor family shown to play important roles in cell adhesion, brain development, immune function, and tumorigenesis. GPR56 is highly upregulated in colorectal cancer and correlates with poor prognosis. Several studies have shown GPR56 couples to the Gα12/13 class of heterotrimeric G-proteins to promote RhoA activation. However, due to its structural complexity and lack of a high-affinity receptor-specific ligand, the complete GPR56 signaling mechanism remains largely unknown. To delineate the activation mechanism and intracellular signaling functions of GPR56, we generated a monoclonal antibody (mAb) that binds with high affinity and specificity to the extracellular domain (ECD). Using deletion mutants, we mapped the mAb binding site to the GAIN domain, which mediates membrane-proximal autoproteolytic cleavage of the ECD. We showed that GPR56 overexpression in 293T cells leads to increased phosphorylation of Src, Fak, and paxillin adhesion proteins and activation of the Gα12/13-RhoA-mediated serum response factor (SRF) pathway. Treatment with the mAb potentiated Src-Fak phosphorylation, RhoA-SRF signaling, and cell adhesion. Consistently, GPR56 knockdown in colorectal cancer cells decreased Src-Fak pathway phosphorylation and cell adhesion. Interestingly, GPR56-mediated activation of Src-Fak phosphorylation occurred independent of RhoA, yet mAb-induced potentiation of RhoA-SRF signaling was Src-dependent. Furthermore, we show that the C-terminal portion of the Serine-Threonine-Proline-rich (STP) region, adjacent to the GAIN domain, was required for Src-Fak activation. However, autoproteolytic cleavage of the ECD was dispensable. These data support a new ECD-dependent mechanism by which GPR56 functions to regulate adhesion through activation of Src-Fak signaling.

EPH receptor B2 stimulates human monocyte adhesion and migration independently of its EphrinB ligands.

The molecular basis of atherosclerosis is not fully understood and mice studies have shown that Ephrins and EPH receptors play a role in the atherosclerotic process. We set out to assess the role for monocytic EPHB2 and its Ephrin ligands in human atherosclerosis and show a role for EPHB2 in monocyte functions independently of its EphrinB ligands. Immunohistochemical staining of human aortic sections at different stages of atherosclerosis showed that EPHB2 and its ligand EphrinB are expressed in atherosclerotic plaques and that expression proportionally increases with plaque severity. Functionally, stimulation with EPHB2 did not affect endothelial barrier function, nor did stimulation with EphrinB1 or EphrinB2 affect monocyte-endothelial interactions. In contrast, reduced expression of EPHB2 in monocytes resulted in decreased monocyte adhesion to endothelial cells and a decrease in monocyte transmigration, mediated by an altered morphology and a decreased ability to phosphorylate FAK. Our results suggest that EPHB2 expression in monocytes results in monocyte accumulation by virtue of an increase of transendothelial migration, which can subsequently contribute to atherosclerotic plaque progression.

T-cell co-stimulation in combination with targeting FAK drives enhanced anti-tumor immunity.

Focal Adhesion Kinase (FAK) inhibitors are currently undergoing clinical testing in combination with anti-PD-1 immune checkpoint inhibitors. However, which patients are most likely to benefit from FAK inhibitors, and what the optimal FAK/immunotherapy combinations are, is currently unknown. We identify that cancer cell expression of the T-cell co-stimulatory ligand CD80 sensitizes murine tumors to a FAK inhibitor and show that CD80 is expressed by human cancer cells originating from both solid epithelial cancers and some hematological malignancies in which FAK inhibitors have not been tested clinically. In the absence of CD80, we identify that targeting alternative T-cell co-stimulatory receptors, in particular OX-40 and 4-1BB in combination with FAK, can drive enhanced anti-tumor immunity and even complete regression of murine tumors. Our findings provide rationale supporting the clinical development of FAK inhibitors in combination with patient selection based on cancer cell CD80 expression, and alternatively with therapies targeting T-cell co-stimulatory pathways.

mTOR- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration through the iNOS/Src/FAK Axis.

Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43+/- mice compared with TGEMs from Cx43+/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80+CD11b+ macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.

Disruption of outer blood-retinal barrier by Toxoplasma gondii-infected monocytes is mediated by paracrinely activated FAK signaling.

Ocular toxoplasmosis is mediated by monocytes infected with Toxoplasma gondii that are disseminated to target organs. Although infected monocytes can easily access to outer blood-retinal barrier due to leaky choroidal vasculatures, not much is known about the effect of T. gondii-infected monocytes on outer blood-retinal barrier. We prepared human monocytes, THP-1, infected with T. gondii and human retinal pigment epithelial cells, ARPE-19, grown on transwells as an in vitro model of outer blood-retinal barrier. Exposure to infected monocytes resulted in disruption of tight junction protein, ZO-1, and decrease in transepithelial electrical resistance of retinal pigment epithelium. Supernatants alone separated from infected monocytes also decreased transepithelial electrical resistance and disrupted tight junction protein. Further investigation revealed that the supernatants could activate focal adhesion kinase (FAK) signaling in retinal pigment epithelium and the disruption was attenuated by FAK inhibitor. The disrupted barrier was partly restored by blocking CXCL8, a FAK activating factor secreted by infected monocytes. In this study, we demonstrated that monocytes infected with T. gondii can disrupt outer blood-retinal barrier, which is mediated by paracrinely activated FAK signaling. FAK signaling can be a target of therapeutic approach to prevent negative influence of infected monocytes on outer blood-retinal barrier.

A novel role of bone morphogenetic protein-7 in the regulation of adhesion and migration of human monocytic cells.

BACKGROUND: Bone morphogenetic protein (BMP) 7 is abundant in atherosclerotic plaques and increases monocyte pro-coagulant activity by enhancing tissue factor (TF) expression. While several members of the BMP superfamily are able to serve as chemotactic agents for monocytes, the role of BMP-7 in regulation of monocyte motility is not known. AIMS: To assess the effect of BMP-7 on adhesive and migratory properties of human monocytes. METHODS: Chemokinesis, adhesion, and transendothelial migration of BMP-7-treated THP-1 cells and human monocytes were analysed using live-cell imaging, orbital shear, and Boyden chamber assays. Surface presentation of ß2 integrins and phosphorylation status of Akt & focal adhesion kinase (FAK) were studied by flow cytometry and Western blot. RESULTS: High levels of BMP-7 protein were detectable in intimal regions of atherosclerotic plaques; BMP-7 significantly enhanced THP-1 and monocyte chemokinetic properties in vitro (1.21+0.01 and 1.76+0.21 fold increase in crawling distance, respectively). Under orbital shear, adhesion of monocytic cells to microvascular endothelial cell (MVEC) monolayers was also significantly increased by BMP-7 (3.89+1.56 and 2.57+0.97 fold over vehicle). Moreover, BMP-7 accelerated transendothelial migration of THP-1 cells and monocytes towards MCP-1 (5.91+0.88 and 2.96±0.65 fold increase, respectively). BMP-7 enhanced cell surface presentation of ß2 integrins in the active conformation. Observed effects were determined to be Akt and FAK dependent, as shown by pharmacological inhibition. CONCLUSION: BMP-7 directly upregulates adhesion and migration of human monocytic cells via activation of ß2 integrins, Akt, and FAK. Our findings suggest that BMP-7 may serve as a novel contributor to atherogenesis.

Lipopolysaccharide Regulation of Intestinal Tight Junction Permeability Is Mediated by TLR4 Signal Transduction Pathway Activation of FAK and MyD88.

Gut-derived bacterial LPS plays an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor in necrotizing enterocolitis and inflammatory bowel disease. The defective intestinal tight junction barrier was shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, causes an increase in intestinal tight junction permeability (TJP) via a TLR4-dependent process; however, the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal tight junction barrier using in vitro and in vivo model systems. LPS caused a TLR4-dependent activation of membrane-associated adaptor protein focal adhesion kinase (FAK) in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and -independent pathways. Small interfering RNA silencing of MyD88 prevented an LPS-induced increase in TJP. LPS caused MyD88-dependent activation of IL-1R-associated kinase 4. TLR4, FAK, and MyD88 were colocalized. Small interfering silencing of TLR4 inhibited TLR4-associated FAK activation, and FAK knockdown prevented MyD88 activation. In vivo studies also confirmed that the LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented an LPS-induced increase in intestinal permeability. Additionally, high-dose LPS-induced intestinal inflammation was dependent on the TLR4/FAK/MyD88 signal transduction axis. To our knowledge, our data show for the first time that the LPS-induced increases in intestinal TJP and intestinal inflammation were regulated by TLR4-dependent activation of the FAK/MyD88/IL-1R-associated kinase 4 signaling pathway.

Focal adhesion kinase-mediated activation of glycogen synthase kinase 3ß regulates IL-33 receptor internalization and IL-33 signaling.

IL-33, a relatively new member of the IL-1 cytokine family, plays a crucial role in allergic inflammation and acute lung injury. Long form ST2 (ST2L), the receptor for IL-33, is expressed on immune effector cells and lung epithelia and plays a critical role in triggering inflammation. We have previously shown that ST2L stability is regulated by the ubiquitin-proteasome system; however, its upstream internalization has not been studied. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates ST2L internalization and IL-33 signaling. IL-33 treatment induced ST2L internalization, and an effect was attenuated by inhibition or downregulation of GSK3ß. GSK3ß was found to interact with ST2L on serine residue 446 in response to IL-33 treatment. GSK3ß binding site mutant (ST2L(S446A)) and phosphorylation site mutant (ST2L(S442A)) are resistant to IL-33-induced ST2L internalization. We also found that IL-33 activated focal adhesion kinase (FAK). Inhibition of FAK impaired IL-33-induced GSK3ß activation and ST2L internalization. Furthermore, inhibition of ST2L internalization enhanced IL-33-induced cytokine release in lung epithelial cells. These results suggest that modulation of the ST2L internalization by FAK/GSK3ß might serve as a unique strategy to lessen pulmonary inflammation.

Functions of the FAK family kinases in T cells: beyond actin cytoskeletal rearrangement.

T cells control the focus and extent of adaptive immunity in infectious and pathological diseases. The activation of T cells occurs when the T cell antigen receptor (TCR) and costimulatory and/or adhesion receptors are engaged by their ligands. This process drives signaling that promotes cytoskeletal rearrangement and transcription factor activation, both of which regulate the quality and magnitude of the T cell response. However, it is not fully understood how different receptor-induced signals combine to alter T cell activation. The related non-receptor tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are phosphorylated downstream of the TCR and several costimulatory and adhesion receptors. FAK family proteins integrate receptor-mediated signals that influence actin cytoskeletal rearrangement and effector T cell responses. In this review, we summarize the receptor-specific roles that FAK and Pyk2 control to influence T cell development and activation.

Phosphoinositide 3-kinase δ regulates migration and invasion of synoviocytes in rheumatoid arthritis.

Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3Kδ isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3Kδ in RA synoviocyte migration and invasion. We observed that PI3Kδ inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)-mediated migration and invasion of FLS. We then showed that PI3Kδ regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3Kδ inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3Kδ signaling, whereas FAK and JNK are not involved. Thus, PI3Kδ contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3Kδ regulates FLS growth and survival, suggest that PI3Kδ inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion.

FAK inhibition abrogates the malignant phenotype in aggressive pediatric renal tumors.

UNLABELLED: Despite the tremendous advances in the treatment of childhood kidney tumors, there remain subsets of pediatric renal tumors that continue to pose a therapeutic challenge, mainly malignant rhabdoid kidney tumors and nonosseous renal Ewing sarcoma. Children with advanced, metastatic, or relapsed disease have a poor disease-free survival rate. Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that is important in many facets of tumor development and progression. FAK has been found in other pediatric solid tumors and in adult renal cellular carcinoma, leading to the hypothesis that FAK contributes to pediatric kidney tumors and would affect cellular survival. In the current study, FAK was present and phosphorylated in pediatric kidney tumor specimens. Moreover, the effects of FAK inhibition upon G401 and SK-NEP-1 cell lines were examined using a number of parallel approaches to block FAK, including RNA interference and small-molecule FAK inhibitors. FAK inhibition resulted in decreased cellular survival, invasion and migration, and increased apoptosis. Furthermore, small-molecule inhibition of FAK led to decreased SK-NEP-1 xenograft growth in vivo. These data deepen the knowledge of the tumorigenic process in pediatric renal tumors, and provide desperately needed therapeutic strategies and targets for these rare, but difficult to treat, malignancies. IMPLICATIONS: This study provides a fundamental understanding of tumorigenesis in difficult to treat renal tumors and provides an impetus for new avenues of research and potential for novel, targeted therapies.

CD68(+)HLA-DR(+) M1-like macrophages promote motility of HCC cells via NF-κB/FAK pathway.

TAM is a prominent component of inflammatory microenvironment, presenting M1 and M2 polarized states in HCC. The objective of this study is to investigate the relationship between M1-polarized macrophages and metastasis in HCC. We used immunohistochemical double-staining method to inspect the infiltration of CD68(+)HLA-DR(+) M1-like macrophages in HCC tissues. The M1-polarized macrophage was derived from THP-1 cell treated by LPS and IFN-γ in vitro. Transwell migration assay was used to evaluate whether the M1-polarized macrophage enhanced motility of HCC cells in the presence or absence of NF-κB inhibitor Bay 11-7802. The activation of NF-κB and FAK signaling pathways was examined by Western blot assay. Our results showed that the density of CD68(+)HLA-DR(+) TAM in the HCC with metastasis is significantly higher than that in the HCC without metastasis. Moreover, the conditioned medium from the M1 macrophages promote the migration of HCC cells and induced the activation of NF-κB and FAK signaling. The promoted migration of HCC cells was abrogated by the Bay 11-7802, as well as the activation of NF-κB and FAK pathway. Our findings implied a pro-metastatic role of M1-like TAM in HCC.

D-pinitol inhibits prostate cancer metastasis through inhibition of αVß3 integrin by modulating FAK, c-Src and NF-κB pathways.

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. D-pinitol, a 3-methoxy analogue of d-chiro-inositol, was identified as an active principle in soy foods and legumes, and it has been proven to induce tumor apoptosis and metastasis of cancer cells. In this study, we investigated the anti-metastasis effects of D-pinitol in human prostate cancer cells. We found that D-pinitol reduced the migration and the invasion of prostate cancer cells (PC3 and DU145) at noncytotoxic concentrations. Integrins are the major adhesive molecules in mammalian cells and have been associated with the metastasis of cancer cells. Treatment of prostate cancer cells with D-pinitol reduced mRNA and cell surface expression of αvß3 integrin. In addition, D-pinitol exerted its inhibitory effects by reducing focal adhesion kinase (FAK) phosphorylation, c-Src kinase activity and NF-kB activation. Thus, D-pinitol may be a novel anti-metastasis agent for the treatment of prostate cancer metastasis.

Effect of tumor gangliosides on tyrosine phosphorylation of p125FAK in platelet adhesion to collagen.

The exact mechanisms as to how platelets influence blood-borne metastasis remain poorly understood. Gangliosides, sialic acid-containing glycosphingolipids, are associated with tumor progression and metastasis in humans. Gangliosides isolated from tumor cells promote collagen-stimulated platelet aggregation and ATP secretion and enhance platelet adhesion to immobilized collagen. Gangliosides interact with a number of cell surface receptors including integrin receptors. In this study, we examined the effects of α2ß1 integrin-mediated platelet adhesion to collagen and phosphotyrosine signaling of focal adhesion kinase, p125FAK (FAK). platelets pre-incubated with neuroblastoma tumor gangliosides (NBTGs) or their major component GD2 (disialoganglioside) were more adherent to immobilized collagen (OD570 0.43±0.12, 0.39±0.13) compared to platelets pre-incubated with MTB (0.14±0.06, p<0.001); the effect of NBTGs was blocked by F-17 anti-α2 antibody. Pre-incubation of platelets with NBTGs resulted in a marked increase in the phosphotyrosine content of p125FAK in the adherent platelets compared to the MTB-pre-incubated adherent platelets. F-17 anti-α2 antibody decreased protein tyrosine phosphorylation of NBTG-incubated platelets adherent to collagen. These results indicate that the tumor gangliosides enhance platelet adhesion to extracellular matrix collagen by upregulating integrin α2ß1-mediated tyrosine phosphorylation of p125FAK, thereby providing insight into how this interaction may be involved in neuroblastoma metastasis.

Antibody ligation of human leukocyte antigen class I molecules stimulates migration and proliferation of smooth muscle cells in a focal adhesion kinase-dependent manner.

Chronic rejection manifests as transplant vasculopathy, which is characterized by intimal thickening of the vessels of the allograft. Intimal thickening is thought to result from the migration and proliferation of vascular smooth muscle cells (SMC) in the vessel media, followed by deposition of extracellular matrix proteins. The development of post-transplantation anti-human leukocyte antigen (HLA) antibodies (Ab) is strongly correlated with the development of transplant vasculopathy and graft loss. Here we demonstrate that cross-linking of HLA class I molecules on the surface of human SMC with anti-HLA class I Ab induced cell proliferation and migration. Class I ligation also increased phosphorylation of focal adhesion kinase (FAK), Akt, and ERK1/2 in SMC. Knockdown of FAK by siRNA attenuated class I-induced phosphorylation of Akt and ERK1/2, as well as cell proliferation and migration. These results indicate that ligation of HLA class I molecules induces SMC migration and proliferation in a FAK-dependent manner, which may be important in promoting transplant vasculopathy.

Bacillus Calmette-Guerin and BCG cell wall skeleton suppressed viability of bladder cancer cells in vitro.

AIM: Bacillus Calmette-Guerin (BCG) is one of therapeutic options for urothelial carcinoma (UC). The objectives of this study were to determine the direct effect of viable or heat-killed BCG and BCG cell wall skeleton (BCG-CWS) on UC cells in vitro. MATERIALS AND METHODS: UC cell lines were co-cultured with viable or heat-killed BCG Immunobladder® (Tokyo 172 strain) and BCG-CWS. Viability of the cells, apoptosis and BrdU incorporation were estimated. RESULTS: BCG induced cell growth retardation in highly malignant UC bearing integrin α5ß1 (VLA5). VLA5-blocking antibody partially abrogated this effect. BCG treatment induced a modest increase in the sub-G(1) fraction of cells and a decrease of BrdU incorporation. Cell growth retardation effect of viable BCG was reproduced by both heat-killed BCG and BCG-CWS. CONCLUSION: The results indicate that VLA5 may be a biomarker of UC with sensitivity to BCG. Moreover, BCG-CWS is a promising substance which might replace BCG, preventing life-threatening complications of viable BCG treatment.

Talin-dependent integrin signalling in vivo.

Integrins are heterodimeric adhesion receptors essential for metazoan life. In addition to mediating cell-extracellular matrix and cell-cell interactions, integrins are bona fide signalling receptors in that they transmit information in both directions across the plasma membrane. The affinity of integrins for extracellular ligands is regulated through a process termed integrin activation or "inside-out signalling". On the other hand, ligand binding to integrins can induce the recruitment and activation of a number of enzymes and adaptors such as pp125(FAK) and Src family kinases, to initiate "outside-in signalling". Intensive investigation into the mechanisms of integrin signalling has revealed many of the key players; amongst these, one of the most important is talin. Our understanding of how many of these molecules interact is now understood at the atomic level thanks to detailed structural studies. Indeed structural information and model cell systems have provided unique opportunities to dissect the molecular mechanisms of many aspects of integrin signalling. Recent studies have begun testing the biological significance of these mechanisms using in-vivo models, particular genetically modified mice. The generation and characterisation of in-vivo models to study integrin signalling has provided valuable information into the functional significance of integrin signalling in fundamental physiological processes as well as within the context of human disease. Here, I will review recent insights that have been gained into integrin signalling through the use of genetically modified mice focusing on integrin alphaIIbbeta3 (GPIIb-IIIa) and the regulation of its function in haemostasis and thrombosis.

Association of FAK activation with lentivirus-induced disruption of blood-brain barrier tight junction-associated ZO-1 protein organization.

Expression of tight junction proteins between brain microvascular endothelial cells (BMECs) of the blood-brain barrier (BBB) is lost during development of human immunodeficiency virus (HIV) encephalitis (HIVE). Although many studies have focused on the strains of virus that induce neurological sequelae or on the macrophages/microglia that are associated with development of encephalitis, the molecular signaling pathways within the BMECs involved have yet to be resolved. We have previously shown that there is activation and disruption of an in vitro BBB model using lentivirus-infected CEMx174 cells. We and others have shown similar disruption in vivo. Therefore, it was of interest to determine if the presence of infected cells could disrupt intact cerebral microvessels immediately ex vivo, and if so, which signaling pathways were involved. The present data demonstrate that disruption of tight junctions between BMECs is mediated through activation of focal adhesion kinase (FAK) by phosphorylation at TYR-397. Inhibition of FAK activation is sufficient to prevent tight junction disruption. Thus, it may be possible to inhibit the development of HIVE by using inhibitors of FAK.