Syk tyrosine kinase participates in beta1-integrin signaling and inflammatory responses in airway epithelial cells

The protein tyrosine kinase Syk is critically involved in immunoreceptor signaling in hematopoietic cells. Recent studies demonstrate Syk expression in nonhematopoietic cells, including fibroblasts, endothelial cells, hepatocytes, and breast epithelium. However, the role of Syk in these cells is uncertain. We hypothesized that Syk is expressed in respiratory epithelial cells (EC) and that it functions as a signaling molecule involved in inflammatory responses in the epithelium. With the use of immunohistochemistry, Western blot, PCR, and laser scanning confocal microscopy, Syk was detected in human, rat, and mouse bronchial epithelium in situ and in cultured human bronchial EC in primary cells and the cell lines HS-24 and BEAS-2B. Syk-dependent signaling pathways in EC were initiated by engagement of beta1-integrin receptors. Stimulation of beta1-integrin receptors by fibronectin or antibody cross-linking caused redistribution of Syk from a cytoplasmic to plasma membrane localization. In stimulated cells, Syk and beta1-integrin colocalized. In addition, following beta1-integrin receptor engagement, tyrosine phosphorylation of Syk was observed. Expression of the intercellular adhesion molecule-1 (ICAM-1) and production of IL-6, both important molecules in lung inflammation, was downregulated in EC treated with Syk small interfering RNA or Syk inhibitor piceatannol. We propose that Syk is involved in signaling pathways induced by integrin engagement in airway EC. Syk-mediated signaling regulates IL-6 and ICAM-1 expression and may be important in the pathophysiology of lung inflammation.     

Induced focal adhesion kinase expression suppresses apoptosis by activating NF-kappaB signaling in intestinal epithelial cells

Focal adhesion kinase (FAK) integrates various extracellular and intracellular signals and is implicated in a variety of biological functions, but its exact role and downstream targeting signals in the regulation of apoptosis in intestinal epithelial cells (IECs) remains unclear. The current study tested the hypothesis that FAK has an antiapoptotic role in the IEC-6 cell line by altering NF-B signaling. Induced FAK expression by stable transfection with the wild-type (WT)-FAK gene increased FAK phosphorylation, which was associated with an increase in NF-B activity. These stable WT-FAK-transfected IECs also exhibited increased resistance to apoptosis when they were exposed to TNF- plus cycloheximide (TNF-/CHX). Specific inhibition of NF-B by the recombinant adenoviral vector containing the IB superrepressor prevented increased resistance to apoptosis in WT-FAK-transfected cells. In contrast, inactivation of FAK by ectopic expression of dominant-negative mutant of FAK (DNM-FAK) inhibited NF-B activity and increased the sensitivity to TNF-/CHX-induced apoptosis. Furthermore, induced expression of endogenous FAK by depletion of cellular polyamines increased NF-B activity and resulted in increased resistance to TNF-/CHX-induced apoptosis, both of which were prevented by overexpression of DNM-FAK. These results indicate that increased expression of FAK suppresses TNF-/CHX-induced apoptosis, at least partially, through the activation of NF-B signaling in IECs. polyamines; -difluoromethylornithine; X-linked inhibitor of apoptosis protein; IB     

Enterophilin-1 interacts with focal adhesion kinase and decreases beta1 integrins in intestinal Caco-2 cells

Intestinal cell growth and differentiation are tightly regulated by growth factors and extracellular matrix components along the crypt-villus axis. We previously described enterophilin-1 (Ent-1) as a new intestinal protein associated with growth arrest and enterocyte differentiation. Ent-1 interacted with sorting nexin 1 and decreased cell surface epidermal growth factor receptor. Because beta(1) integrins are mostly found in vivo in the proliferative crypt cells, we investigated the role of Ent-1 in the fate of beta(1) integrin subunits. In undifferentiated intestinal Caco-2 cells, overexpression of Ent-1 induces a marked decrease of alpha(5)beta(1) integrin pools, whereas alpha(2)beta(1) integrin is weakly affected. Conversely, overexpression of sorting nexin 1 has no effect on integrin levels despite its ability to interact with Ent-1. Interestingly, we identified focal adhesion kinase as a new Ent-1 partner using yeast two-hybrid screening and co-precipitation experiments. Furthermore by confocal microscopy, we observed that Ent-1 and beta(1) integrins partly co-localize on vesicular structures, suggesting a role for Ent-1 in integrin trafficking. Because focal adhesion kinase is able to bind both Ent-1 and beta(1) integrins, the kinase might act as a molecular bridge between the two proteins. Altogether, these results support a role of Ent-1 in regulating beta(1) integrin expression that could favor intestinal differentiation.     

Lipid rafts mediate internalization of beta1-integrin in migrating intestinal epithelial cells

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of beta(1)-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, beta(1)-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent beta(1)-integrin internalization. However, beta(1)-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized beta(1)-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased beta(1)-integrin endocytosis. Our data suggest that, in migrating IEC, beta(1)-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.     

Induction of osteopontin gene expression during mammary gland involution and effects of glucocorticoid on its expression in mammary epithelial cells

To understand the molecular mechanisms of mammary gland involution, an involution-induced clone was identified from a cDNA library of mouse mammary gland by differential screening. Characterization of a clone by sequencing and northern analysis showed that expression of the osteopontin gene was induced during involution of mouse mammary gland. But induction of the osteopontin gene was not observed in apoptotic HC11 mammary epithelial cells under serum starvation. In HC11 cells, dexamethasone treatment from the seeding stage showed five-fold induction of osteopontin gene expression, but the expression was not changed when dexamethasone was added to confluent cells.     

hsp72 inhibits focal adhesion kinase degradation in ATP-depleted renal epithelial cells

Prior heat stress (HS) or the selective overexpression of hsp72 prevents apoptosis caused by exposure to metabolic inhibitors by protecting the mitochondrial membrane and partially reducing caspase-3 activation. Focal adhesion kinase (FAK), a tyrosine kinase, exhibits anti-apoptotic properties and is a potential target for degradation by caspase-3. This study tested the hypothesis that hsp72 interacts with FAK, preventing caspase-3-mediated degradation during ATP depletion. ATP depletion (5 mm NaCN and 5 mm 2-deoxy-d-glucose in the absence of medium glucose) caused FAK degradation within 15 min. FAK degradation was completely prevented by a caspase-3-specific inhibitor. HS induced the accumulation of hsp72, increased the interaction between hsp72 and FAK, and significantly inhibited FAK degradation during ATP depletion. Selective overexpression of wild-type hsp72 (but not hsp72DeltaEEVD) reproduced the protective effects of HS on FAK cleavage. Purified hsp72 prevented the degradation of FAK by caspase-3 in vitro in a dose-dependent manner without affecting caspase-3 activity. Interaction between hsp72 and FAK is critical because both exogenous ATP and deletion of the substrate-binding site decreased protection of FAK by hsp72. These data indicate that FAK is an early target of injury in cells exposed to metabolic inhibitors and demonstrate that hsp72 reduces caspase-3-mediated proteolysis of FAK, an anti-apoptotic protein.     

Regulation of trophoblast beta1-integrin expression by contact with endothelial cells

Background  

In human and non-human primates, migratory trophoblasts penetrate the uterine epithelium, invade uterine matrix, and enter the uterine vasculature. Invasive trophoblasts show increased expression of β1 integrin. Since trophoblast migration within the uterine vasculature involves trophoblast attachment to endothelial cells lining the vessel walls, this raises the possibility that cell-cell contact and/or factors released by endothelial cells could regulate trophoblast integrin expression. To test this, we used an in vitro system consisting of early gestation macaque trophoblasts co-cultured on top of uterine microvascular endothelial cells.     

Oxazolone-induced over-expression of focal adhesion kinase in colonic epithelial cells of colitis mouse model

We examined the change of protein tyrosine kinases (PTKs) expression levels in colonic epithelial cells isolated from mice in which colitis was induced by oxazolone administration, using the monoclonal antibody YK34, which cross-reacts with a wide variety of PTKs. We identified focal adhesion kinase (FAK) and found the expression level increased due to the induction of colitis. Furthermore, we found that there was a positive correlation between FAK expression and the severity of colitis. Also, FAK expression localized in the colonic epithelium but not in the lamina propria, implying FAK functions in epithelial cells during colitis formation and/or wound repairing.     

Involvement of alpha5beta1-integrin and TNF-alpha in Staphylococcus aureus alpha-toxin-induced death of epithelial cells

Staphylococcus aureus causes suppurative infections which are often associated with tissue destruction and cell death. In the present study, we investigated the molecular and cellular basis of S. aureus-induced apoptosis and death in a human lung epithelial cell line (A549). We found that staphylococcal alpha-toxin is an important mediator of cytotoxicity in these epithelial cells. Specifically, we found that downregulating alpha-toxin production eliminated the cytotoxicity of S. aureus, whereas the addition of alpha-toxin to the cell culture medium significantly increased cell death in a dose-dependent manner. Importantly, we found that alpha-toxin-mediated cell death may partially function through alpha5beta1-integrin, because both the beta1-integrin antibody and the ligand fibronectin inhibited the cytotoxicity of alpha-toxin. Furthermore, we found that the overexpression of the inflammatory cytokine interferon (TNF)-alpha is associated with alpha-toxin-induced cell death, because both the TNF-alpha release inhibitor and antibody effectively inhibited the cytotoxicity of alpha-toxin. In contrast, the cytotoxicity of alpha-toxin was enhanced by the inhibition of the MAPK p38 and NF-kappaB pathways. Taken together, our results suggest that the activation of the MAPK p38 and NF-kappaB pathways are stress responses for survival, rather than direct contributes to alpha-toxin-induced cell death, and that the interaction of alpha-toxin with alpha5beta1-integrin and overproduction of TNF-alpha may contribute to destruction of epithelial cells during S. aureus infection.     

The cytoplasmic tyrosines of integrin subunit beta1 are involved in focal adhesion kinase activation

We have previously shown that mutation of the two tyrosines in the cytoplasmic domain of integrin subunit beta1 (Y783 and Y795) to phenylalanines markedly reduces the capability of beta1A integrins to mediate directed cell migration. In this study, beta1-dependent cell spreading was found to be delayed in GD25 cells expressing beta1A(Y783/795F) compared to that in wild-type GD25-beta1A. Focal adhesion kinase (FAK) tyrosine phosphorylation and activation were severely impaired in response to beta1-dependent adhesion in GD25-beta1A(Y783/795F) cells compared to that in wild-type GD25-beta1A or mutants in which only a single tyrosine was altered (beta1A(Y783F) or beta1A(Y795F)). Phosphorylation site-specific antibodies selective for FAK phosphotyrosine 397 indicated that the defect in FAK phosphorylation via beta1A(Y783/795F) lies at the level of the initial autophosphorylation step. Indeed, beta1A-dependent tyrosine phosphorylation of tensin and paxillin was lost in the beta1A(Y783/795F) cells, consistent with the impairment in FAK activation. In contrast, p130(CAS) overall tyrosine phosphorylation was unaffected by the beta1 mutations. Despite the defect in beta1-mediated FAK activation, FAK was still localized to focal adhesions. Taken together, the phenotype of the GD25-beta1A(Y783/795F) cells resembles, but is distinct from, the phenotype observed in FAK-null cells. These observations argue that tyrosines 783 and 795 within the cytoplasmic tail of integrin subunit beta1A are critical mediators of FAK activation and cell spreading in GD25 cells.