MAB2253 Sigma-AldrichAnti-Integrin β1 Antibody, clone 6S6

Pathogenic bacteria such as Listeria and Yersinia gain initial entry by binding to host target cells and stimulating their internalization. Bacterial uptake entails successive, increasingly strong associations between receptors on the surface of bacteria and hosts. Even with genetically identical cells grown in the same environment, there are vast differences in the number of bacteria entering any given cell. To gain insight into this variability, we examined uptake dynamics of Escherichia coli engineered to express the invasin surface receptor from Yersinia, which enables uptake via mammalian host β1-integrins. Surprisingly, we found that the uptake probability of a single bacterium follows a simple power-law dependence on the concentration of integrins. Furthermore, the value of a power-law parameter depends on the particular host-bacterium pair but not on bacterial concentration. This power-law captures the complex, variable processes underlying bacterial invasion while also enabling differentiation of cell lines.

Intercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems. ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remains poorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synaptic contacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies against ICAM-5 and β1 integrins altered spine maturation. Furthermore, we found that β1 integrins serve as binding partners for ICAM-5. β1 integrins were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. β1 integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, β1 integrins covered the mushroom spines. Loss of β1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage decreased or increased when the interaction between ICAM-5 and β1 integrins was potentiated or weakened, respectively, using antibodies. These results suggest that the interaction between ICAM-5 and β1 integrins is important in formation of functional synapses.

As an obligatory pathogen, influenza virus co-opts host cell machinery to harbor infection and to produce progeny viruses. In order to characterize the virus-host cell interactions, several genome-wide siRNA screens and proteomic analyses have been performed recently to identify host factors involved in influenza virus infection. CD81 has emerged as one of the top candidates in two siRNA screens and one proteomic study. The exact role played by CD81 in influenza infection, however, has not been elucidated thus far. In this work, we examined the effect of CD81 depletion on the major steps of the influenza infection. We found that CD81 primarily affected virus infection at two stages: viral uncoating during entry and virus budding. CD81 marked a specific endosomal population and about half of the fused influenza virus particles underwent fusion within the CD81-positive endosomes. Depletion of CD81 resulted in a substantial defect in viral fusion and infection. During virus assembly, CD81 was recruited to virus budding site on the plasma membrane, and in particular, to specific sub-viral locations. For spherical and slightly elongated influenza virus, CD81 was localized at both the growing tip and the budding neck of the progeny viruses. CD81 knockdown led to a budding defect and resulted in elongated budding virions with a higher propensity to remain attached to the plasma membrane. Progeny virus production was markedly reduced in CD81-knockdown cells even when the uncoating defect was compensated. In filamentous virus, CD81 was distributed at multiple sites along the viral filament. Taken together, these results demonstrate important roles of CD81 in both entry and budding stages of the influenza infection cycle.

Cyclin D1 plays significant roles in cell cycle entry and migration. We have documented that both integrin α3β1 expressions and the number of podocytes were reduced in focal segmental glomerulosclerosis. We wondered whether integrin-extracellular matrix (ECM) interaction was involved in the regulation of cyclin D1 expression, and the possible signaling pathways in mitogen-stimulating podocytes. Cultured podocytes were divided into serum (mitogens/growth factors)-starved and serum-stimulated groups. Reverse transcription polymerase chain reaction was used to detect cyclin D1 mRNA, and Western blot analysis was used to measure protein concentrations of cyclin D1 and extracellular signal-regulated kinase (ERK) activation (p-ERK/ERK). The integrin-ECM interaction was blocked by anti-β1-integrin monoclonal antibody or RGDS (Arg-Gly-Asp-Ser). The MEK inhibitor, U0126, was used to inhibit ERK activation. The results showed that there was little cyclin D1 protein in serum-starved groups, but it was abundant in serum-stimulated groups. Both cyclin D1 mRNA and protein levels were reduced in serum-stimulated podocytes after blocking integrin-ECM interaction. ERK activation in serum-stimulated podocytes was significantly decreased after blocking integrin-ECM interaction. Cyclin D1 mRNA and protein concentrations in serum-stimulated podocytes were reduced after blocking ERK activation by U0126. We demonstrate that integrin-ECM interaction collaborates with mitogens to activate ERK/mitogen-activated protein kinase pathways which are essential for cyclin D1 expression in podocytes.

Epithelial ovarian carcinoma (EOC) is a deadly disease, and little is known about the mechanisms underlying its metastatic progression. Using human specimens and established cell lines, we determined that the G-protein-coupled seven-transmembrane fractalkine receptor (CX(3)CR1) is expressed in primary and metastatic ovarian carcinoma cells. Ovarian carcinoma cells robustly migrated toward CX(3)CL1, a specific ligand of CX(3)CR1, in a CX(3)CR1-dependent manner. Silencing of CX(3)CR1 reduced migration toward human ovarian carcinoma ascites fluid by approximately 70%. Importantly, adhesion of ovarian carcinoma cells to human peritoneal mesothelial cells was dependent on CX(3)CL1/CX(3)CR1 signaling. In addition, CX(3)CL1 was able to induce cellular proliferation. Together, our data suggest that the fractalkine network may function as a major contributor to the progression of EOC, and further attention to its role in the metastasis of this deadly malignancy is warranted.

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

The effect of reactive oxygen species (ROS) and blocking integrin-extracellular matrix (ECM) interaction on apoptosis in podocytes, and the related signal transduction pathways remain unclear. Primary cultured rat podocytes were exposed to ROS. Integrin-ECM interaction was inhibited with anti-β1-integrin monoclonal antibody (mAb) or RGDS (Arg-Gly-Asp-Ser). Extracellular signal-regulated kinase (ERK) activation was evaluated with Western blotting. U0126 was used to inhibit ERK activation. Terminal deoxynucleotidyl transferase-mediated dUTP-peroxidase nick end-labeling of DNA (TUNEL) was used to evaluate apoptosis. We found that ROS-treated podocytes exhibited increased apoptosis, and both anti-β1-integrin mAb and RGDS induce apoptosis. Addition of ROS to either anti-β1-integrin mAb or RGDS enhanced apoptosis in both conditions. ERK activation was increased by either ROS or blocking integrin-ECM interaction. Preincubation with U0126 decreased apoptosis induced by ROS, anti-β1-integrin mAb or RGDS, respectively. Our study demonstrated that ROS and blocking integrin-ECM interaction induce podocyte apoptosis, which is mediated by ERK activation.

Integrins are used as prognostic indicators in breast cancer. Following engagement with extracellular matrix proteins, their signaling influences numerous cellular processes including migration, proliferation, and death. Integrin signaling varies between cell types through differential expression of integrin subunits, and changes within a given cell upon exposure to a cell agonist or through changes in its surroundings. These variations in signaling can profoundly affect the phenotypic, tumorogenecity and metastatic properties of cancer cells. In the present study, we investigated if there were differences in the expression of integrins, integrin structures, and integrin co-receptors within three breast cancer cells and if these differences effected integrin signaling.Expression of integrins, urokinase receptor and vascular endothelial cell growth factor receptor (VEGFR) in metastatic MDA-MB-435 and MDA-MB-231, non-metastatic MCF7 and non-breast cancer Hek-293 cells was measured by flow cytometry. Cell adhesion was assessed using collagen, fibrinogen, fibronectin and vitronectin coated plates. Changes in kinase levels following PMA stimulation, and cell adhesion-induced activation of kinases were determined by western blot analysis. Distribution of actin stress fibers and focal adhesions was assessed by immunocytochemistry.All cells expressed αv integrins, while high β5 and αvβ5 expression was restricted to the cancer cells and high β3 and αvβ3 expression was restricted to MDA-MB-435 cells. The two metastatic cells were the least adhesive, but all cells adhered well to most proteins in the absence of PMA. All proliferating cells expressed activated pSrc, but only proliferating metastatic cells expressed high pMEK levels. PMA treatment resulted in time-dependent changes in activated kinase levels, and only MDA-MB-231 cells constitutively expressed high levels of activated pMEK. MDA-MB-435 cells formed more stress fibers and focal adhesions and only exhibited adhesion-induced activation of pMEK and pFAK. All cells expressed the urokinase receptor, but MCF7 cells had markedly higher VEGFR expression. Adhesion induced differential expression of pFAK, pMEK and pERK.This study demonstrates that breast cancers vary in their expression of integrins, their capacity to form focal adhesion and to signal through integrins. These differences likely contribute to phenotypic variations between cancer lines and account for some of the heterogeneity of breast cancer.

The best available treatment of peripheral nerve lesions involves transplantation of an autologous nerve. This approach, however, entails sensory deficits at the donor site and requires additional surgery. Such limitations have motivated the search for a bioengineering solution to design artificial implants. For this purpose we are producing orientated biodegradable microfibers of poly(ε-caprolactone) (PCL) with electrospinning. The present study describes the functionalization of these electrospun fibers with biologically active peptides to produce guidance structures for Schwann cell migration and axonal regeneration. For the chemical modification PCL was blended with star-shaped NCO-poly(ethylene glycol)-stat-poly(propylene glycol) (PCL/sPEG) as a covalent linker for the peptide GRGDS, derived from extracellular matrix proteins. To test biological functions of electrospun fibers, Schwann cell migration and axonal growth from dorsal root ganglia explants were investigated with time lapse video microscopy. Migrating Schwann cells as well as growing sensory axons closely followed the electrospun fibers with occasional leaps between adjacent fibers. Cell migration was characterized by frequent changes in velocity and direction reversals. Comparison of substrates showed that functionalized fibers caused more Schwann cells to move out of the explants, supported faster cell migration and axonal growth than the nonfunctional fibers. Using inhibitors of intracellular signaling kinases, we found that these biological effects required activation of the phosphatidyl inositol-3-kinase pathway. Since sPEG-containing fibers also showed low levels of nonspecific protein adsorption, which is desirable in the context of artificial implant design, the peptide modification of fibers appears to provide good substrates for nerve repair.

Fibronectin (FN) is an endogenous ligand of integrins, which plays a critical role in cell adhesion and growth. Here, we converted globular FN (G-FN) into a fibrillar form (F-FN) and found that, even though both G-FN and F-FN interacted with integrin alpha5beta1, G-FN induced cellular proliferation, whereas F-FN resulted in apoptosis that was associated with deactivation of Akt/GSK-3beta and phosphorylation of SHP-2. SHP-2 inhibitor and anti-sense oligodeoxynucleotide decreased SHP-2 level and reversed the F-FN mediated apoptosis. F-FN also induced stress fiber formation associated with activation of RhoA, Rho kinase (ROCK), and filamin. Inhibition of ROCK by ROCK inhibitor or dominant negative plasmid treatment modulated F-FN mediated apoptosis. Pharmacological studies revealed that F-FN was effective in inhibiting the survival of SKOV-3 and MCF-7 cancer cells. These findings thus demonstrate that unlike G-FN, F-FN exhibits fibrillar structure to induce cell apoptosis that is associated with phosphorylation of SHP-2, activation of RhoA/ROCK and formation of stress fibers as well as deactivation of Akt/GSK-3beta.