Β1-integrin Binding Research Articles

Abstract A114: Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF), the trigger of blood coagulation. Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. We recently reported that asTF is abundant in PDAC lesions and, when overexpressed in PDAC cells, it promotes tumor growth, metastatic spread, monocyte recruitment, and elevates procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we sought to determine whether i) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; ii) asTF mechanistically contributes to PDAC cell migration; and iii) antibody-based targeting of asTF slows PDAC progression and/or asTF release into systemic circulation. Methods: Cohorts of nude mice (n=5/cohort) were orthotopically transplanted with grade III human PDAC cells Pt45P1 harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi, 5x105 cells/mouse); mice began receiving Dox (2µg/mL) in sucrose at day 1 of the study (“Dox”), day 25 of the study (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo via SapC-DOPS imaging over 7 wks. PDAC cell migration toward serum was assessed using laminin-coated transwell inserts seeded with Pt45P1/asTFi cells. Plasma samples from mice bearing PDAC tumors were assayed for asTF using a custom sandwich ELISA. asTF-specific rabbit monoclonal antibody Rb1 was co-implanted with Pt45P1 cells, and tumor progression was monitored as above over 5 wks. Tumor specimens were assessed for vessel density (isolectin B4) and monocyte/macrophage infiltration (anti-F4/80 antibody). Results: “Late Dox” mice developed tumors that were comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); interestingly, tumor spread was also significantly less pronounced in “late Dox” mice compared to “Dox” mice ([7.52e+7] vs [1.22e+8]P/s/mm2, p=0.01). “Dox” mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p<0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells; anti-α6 antibody had a partial effect. Strikingly, migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with untreated cells and/or Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when co-implanted with just 100μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells co-implanted with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/LPF; PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p<0.005 Rb1 vs PBS/IgG). Mice in the Pt45P1/Rb1 cohort had ~2 fold decrease in the levels of circulating asTF compared to mice in the Pt45P1/PBS and/or isotype IgG cohorts (0.27 ng/mL vs 0.60/0.54 ng/mL; p<0.001). Conclusion: asTF can promote PDAC progression during earlier as well as later stages of the disease. asTF-β1 integrin interactions render PDAC cells more motile, likely via competing with laminin for β1 integrin binding; Rb1 fully inhibits asTF-potentiated PDAC cell migration. When co-implanted with Rb1, Pt45P1 cells grow much smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel therapeutic strategy to stem PDAC progression. Citation Format: Dusten Unruh, Xiaoyang Qi, Zhengtao Chu, Robert Sturm, Ryan Keil, Syed Ahmad, Henri H. Versteeg, Vladimir Y. Bogdanov. Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A114.

Toll-like receptor 2 (TLR2) induces migration and invasive mechanisms in rheumatoid arthritis.

IntroductionThis study investigates the role of Toll-like receptor 2 (TLR2) in the regulation of migratory and invasive mechanisms in rheumatoid arthritis (RA).MethodsInvasion, migration, matrix metalloproteinase (MMP)-1, -3 and tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) expression, β-integrin binding, cytoskeletal rearrangement and Ras-related C3 botulinum toxin substrate 1 (Rac1) activation in response to a TLR2-ligand, Pam3CSK4 (1 μg/ml), in ex vivo RA synovial tissue explants, primary RA synovial fibroblasts (RASFC) and microvascular endothelial cells (HMVEC) were assessed by Transwell Matrigel™ invasion chambers, enzyme-linked immunosorbent assay (ELISA), multiplex adhesion binding assay, reverse transcription polymerase chain reaction (RT-PCR), F-actin immunofluorescent staining, matrigel synovial outgrowths, Rac1 pull-down assays/Western blot and zymography. β1-integrin expression in RA/control synovial tissue was assessed by immunohistology. The effect of Pam3CSK4 on cell migration, invasion, MMP-3 and Rac1 activation was examined in the presence or absence of anti-β1-integrin (10 μg/ml) or anti-IgG control (10 μg/ml). The effect of an anti-TLR-2 mAb (OPN301)(1 μg/ml) or immunoglobulin G (IgG) control (1 μg/ml) on RASFC migration and RA synovial tissue MMP activity was assessed by wound assays, ELISA and zymography.ResultsPam3CSK4 significantly induced cell migration, invasion, MMP-1, MMP-3, MMP-2 and MMP-9 expression and induced the MMP-1/TIMP-3 and MMP-3/TIMP-3 ratio in RASFC and explants (p <0.05). β1-integrin expression was significantly higher in RA synovial tissue compared to controls (p <0.05). Pam3CSK4 specifically induced β1-integrin binding in RASFC (p <0.05), with no effect observed for β2-4, β6, αvβ5 or α5β1. Pam3CSK4 increased β1-integrin mRNA expression, Rac1 activation, RASFC outgrowths and altered cytoskeletal dynamic through induction of filopodia formation. Pam3CSK4-regulated cell migration and invasion processes, but not MMP-3, were inhibited in the presence of anti-β1-integrin (p <0.05), with no effect observed for anti-IgG control. Furthermore, anti-β1-integrin inhibited Pam3CSK4-induced Rac1 activation. Finally, blockade of TLR2 with OPN301 significantly decreased spontaneous release of MMP-3, MMP-2 and MMP-9 and increased TIMP-3 secretion from RA synovial explant cultures (p <0.05). Incubation of RASFC with OPN301 RA ex vivo conditioned media inhibited migration and invasion compared to IgG control.ConclusionsTLR2 activation induces migrational and invasive mechanisms, which are critically involved in the pathogenesis of RA, suggesting TLR2 as a potential therapeutic target for the treatment of RA.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0664-8) contains supplementary material, which is available to authorized users.

Identification of G α 12 and G α 13 Determinants of Binding to Cell Adhesion Proteins

The trimeric G protein α-subunits Gα12 and Gα13 have 66% amino acid homology but only partial overlap in their signaling function and downstream target proteins. Previously we reported that Gα12 and Gα13 bind the cytoplasmic domain of several cadherins, and other investigators have shown Gα13, but not Gα12, to bind the cytoplasmic tail of β3-integrin. The switch I region of Gα13 has been implicated in binding to vascular endothelial (VE) cadherin and β3-integrin, suggesting key residues arose in the G12/13 class to allow interaction with these cell-surface adhesion proteins. To more precisely define determinants in the G12/13 class mediating these interactions, we utilized epitope-tagged forms of Gα12 and Gα13 to examine their relative affinity for epithelial (E), neural (N), and VE cadherin, as well as β3-integrin. Gα12 showed more robust interaction than Gα13 with both types of cell adhesion protein, and Gα12-to-Gα13 substitutions revealed several residues contributing to their different affinity for these proteins. Surprisingly, GFP tagging in the Gα12 helical domain disrupted binding to β3-integrin but not cadherins, whereas Gα12 with a myc tag at the same location bound robustly to both proteins. These findings suggest that helical domain residues contributing to β3-integrin binding are blocked by the larger GFP adduct but not the myc tag. Cassette substitutions in the Gα12 helical domain implicated several regions in binding cadherins, and these showed partial overlap with Gα12 mutations disruptive to β3-integrin binding. These findings provide structural details of G12/13 engagement of cadherins and integrins and suggest distinct mechanisms of interaction with these adhesion proteins. Supported by the GSK Foundation and Lineberger Comprehensive Cancer Center

Yersinia enterocolitica exploits different pathways to accomplish adhesion and toxin injection into host cells.

The current paradigm suggests that Yersinia enterocolitica (Ye) adheres to host cells via the outer membrane proteins Yersinia adhesin A (YadA) or invasin (Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, shRNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with αV integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Ye and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix (ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Ye infection and differential expression of integrins by various host cell populations, the data demonstrate that Ye is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy.

Cyclic isoDGR and RGD peptidomimetics containing bifunctional diketopiperazine scaffolds are integrin antagonists.

The cyclo[DKP-isoDGR] peptidomimetics 2-5, containing bifunctional diketopiperazine (DKP) scaffolds that differ in the configuration of the two DKP stereocenters and in the substitution at the DKP nitrogen atoms, were prepared and examined in vitro in competitive binding assays with purified αv β3 and αv β5 integrin receptors. IC50 values ranged from low nanomolar (ligand 3) to submicromolar with αv β3 integrin. The biological activities of ligands cyclo[DKP3-RGD] 1 and cyclo[DKP3-isoDGR] 3, bearing the same bifunctional DKP scaffold and showing similar αV β3 integrin binding values, were compared in terms of their cellular effects in human U373 glioblastoma cells. Compounds 1 and 3 displayed overlapping inhibitory effects on the FAK/Akt integrin activated transduction pathway and on integrin-mediated cell infiltration processes, and qualify therefore, despite the different RGD and isoDGR sequences, as integrin antagonists. Both compounds induced apoptosis in glioma cells after 72 hour treatment.

β-1,4-galactosyltransferase III suppresses extravillous trophoblast invasion through modifying β1-integrin glycosylation

IntroductionGlycosylation controls diverse protein functions and regulates various cellular phenotypes. Trophoblast invasion is essential for normal placental development. However, the role of glycosylation in human placenta throughout pregnancy is still unclear. The β-1,4-galactosyltransferase III (B4GALT3) has been found to regulate cancer cell invasion. We therefore investigated the expression of B4GALT3 in placenta and its roles in trophoblast. MethodsB4GALT3 protein expression was examined by quantitative Western blotting analysis in human placentas. For identification of B4GALT3-positive cells in normal human placenta, immunohistochemistry and immunofluorescence methods were used. To investigate effects of B4GALT3 on extravillous trophoblast (EVT)-like cell and primary EVT cells, we analyzed cell growth, adhesion, migration, and invasion in mock and B4GALT3-transfected cell. ResultsB4GALT3 expression significantly increased in third trimester human placenta. Immunostaining revealed that B4GALT3 expressed in placental villous cytotrophoblast, syncytiotrophoblast, and a subpopulation of EVT cells throughout pregnancy. Interestingly, we found increases in the expression level and percentage of B4GALT3-positive cells in third trimester EVT, but not in syncytiotrophoblasts and cytotrophoblasts of placental villi. Overexpression of B4GALT3 in HTR8/SVneo cells and primary trophoblast cells significantly suppressed cell migration. In addition, B4GALT3 suppressed cell invasion, and enhanced cell adhesion to laminin in HTR8/SVneo cells. Notably, we found that B4GALT3 modified glycans on β1-integrin, suppressed focal adhesion kinase (FAK) signaling, and enhanced β1-integrin degradation. DiscussionWe propose that B4GALT3-mediated glycosylation change not only enhances β1-integrin binding to laminin, but also attenuates β1-integrin stability. Our findings suggest that B4GALT3 is a critical regulator for suppressing EVT invasion in the late stages of pregnancy.

Abstract 5287: Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells

Abstract Protein Phosphatase 2A (PP2A) is a major serine/threonine phosphatase in cells. It consists of a catalytic (C), structural (A) and regulatory/variable B-type subunits. PP2A has a critical role to play in homeostasis where it functions as a tumour suppressor. Changes in the activity of PP2A have a direct role in maintaining the transformed phenotype in cancer. However, the mechanism of action of PP2A during this process is poorly understood. Our understanding is limited because of the complex nature of PP2A holoenzyme assembly, and because it acts through a wide variety of signalling pathways. We have identified the scaffolding protein RACK1 as a PP2A interacting protein and we showed that RACK1 regulates PP2A activity to mediate crosstalk between growth factor and adhesion signalling by an IGF-I-independent mechanism(1, 2) . Our hypothesis is that RACK1 plays a critical role in determining how PP2A functions in signalling pathways. Using a series of truncation mutations and peptide array technology, we have identified and mapped the binding sites between RACK1 and the catalytic subunit of PP2A. Using a series of specific point mutations of these PP2A sites, we show that the RACK1/PP2A interaction is critical to maintain PP2A activity. Disruption of the RACK1/PP2A interaction has dramatic consequences for cell adhesion, proliferation and migration in MCF-7 cells. Our hypothesis is that RACK1 regulates the cellular distribution of PP2A and scaffolds PP2A to a specific subset of cellular targets. Using 3D in vitro breast cancer models and mass spectroscopy approaches, we have identified several proteins that interact with the RACK1/PP2A complex. One of these proteins, Metadherin has been shown to play a role in drug resistance(3). It has also been linked to the progression of many cancers(4). Using a series of novel approaches we have mapped the Metadherin interaction site on RACK1 and PP2A and confirmed that RACK1 and Metadherin colocalise at the cell membrane. Overexpression of Metadherin promotes IGF-I-mediated cell migration and invasion. Taken together, our data suggest that RACK1 scaffolds PP2A to Metadherin where it regulates Metadherin activity and cell migration in breast cancer. 1. Kiely, PA., et al. “Tyrosine 302 in RACK1 is essential for insulin-like growth factor-I-mediated competitive binding of PP2A and β1 integrin and for tumor cell proliferation and migration.” Journal of Biological Chemistry 283.34 (2008): 22952-22961. 2. Kiely, PA., et al. “Insulin-like growth factor I controls a mutually exclusive association of RACK1 with protein phosphatase 2A and β1 integrin to promote cell migration.” Molecular and cellular biology 26.11 (2006): 4041-4051. 3. Wei, Yong, Guohong Hu, and Yibin Kang. “Metadherin as a link between metastasis and chemoresistance.” Cell Cycle 8.14 (2009): 2131-2137. 4. Brown, DM., and Erkki R. “Metadherin, a cell surface protein in breast tumors that mediates lung metastasis.” Cancer cell 5.4 (2004): 365-374. Citation Format: Maeve L. Kiely, Rosemary O'Connor, Patrick A. Kiely. Identification of metadherin in a complex with RACK1/PP2A in mammary epithelial cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5287. doi:10.1158/1538-7445.AM2014-5287

Beta 1 integrin binding plays a role in the constant traction force generation in response to varying stiffness for cells grown on mature cardiac extracellular matrix

We have previously reported a unique response of traction force generation for cells grown on mature cardiac ECM, where traction force was constant over a range of stiffnesses. In this study we sought to further investigate the role of the complex mixture of ECM on this response and assess the potential mechanism behind it. Using traction force microscopy, we measured cellular traction forces and stresses for mesenchymal stem cells (MSCs) grown on polyacrylamide gels at a range of stiffnesses (9, 25, or 48kPa) containing either adult rat heart ECM, different singular ECM proteins including collagen I, fibronectin, and laminin, or ECM mimics comprised of varying amounts of collagen I, fibronectin, and laminin. We also measured the expression of integrins on these different substrates as well as probed for β1 integrin binding. There was no significant change in traction force generation for cells grown on the adult ECM, as previously reported, whereas cells grown on singular ECM protein substrates had increased traction force generation with an increase in substrate stiffness. Cells grown on ECM mimics containing collagen I, fibronectin and laminin were found to be reminiscent of the traction forces generated by cells grown on native ECM. Integrin expression generally increased with increasing stiffness except for the β1 integrin, potentially implicating it as playing a role in the response to adult cardiac ECM. We inhibited binding through the β1 integrin on cells grown on the adult ECM and found that the inhibition of β1 binding led to a return to the typical response of increasing traction force generation with increasing stiffness. Our data demonstrates that cells grown on the mature cardiac ECM are able to circumvent typical stiffness related cellular behaviors, likely through β1 integrin binding to the complex composition.

The β3-Integrin Binding Protein β3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1

Integrins are multifunctional heterodimeric adhesion receptors that mediate the attachment between a cell and the extracellular matrix or other surrounding cells. In endothelial cells, integrins can modulate cell migration and motility. In particular, β3-integrin is expressed in angiogenic vessels. Signal transduction by β3-integrins requires the recruitment of intracellular signaling molecules. β3-endonexin is a highly spliced molecule that has been identified as a β3-integrin binding protein. β3-endonexin isoforms are expressed in endothelial cells and have been suggested to act as shuttle proteins between the membrane and the nucleus. However, their functional role in angiogenesis is unclear. In this study, we investigated whether β3-endonexin isoforms are involved in endothelial angiogenic processes under hypoxia. The overexpression of β3-endonexin isoforms decreased endothelial proliferation and tube formation under hypoxia, while the depletion of β3-endonexin by RNAi promoted angiogenic responses in vitro and in vivo. In hypoxia, β3-endonexin accumulated in the nucleus, and prevention of this response by depletion of β3-endonexin increased hypoxic activation and induction of the hypoxia-inducible factor (HIF)-1 and its target genes VEGF and PAI-1. β3-endonexin diminished nuclear factor kappa B (NFκB) activation and decreased NFκB binding to the HIF-1α promoter under hypoxia, subsequently diminishing NFκB-dependent transcription of HIF-1α under hypoxia. Our results indicate for the first time that the overexpression of β3-endonexin can decrease hypoxic induction and activation of HIF-1α and can prevent hypoxic endothelial proliferation and angiogenic responses. β3-endonexin can act as a novel anti-angiogenic factor specifically in the response to hypoxia due to its negative impact on the activation of HIF-1.

THU0039 B1-Integrin is a Critical Mediator of TLR2-Induced Migrational and Invasive Mechanisms in RA Synovial Fibroblast Cells

BackgroundRheumatoid arthritis (RA) is a chronic, progressive autoimmune disease, characterized by synovial proliferation and destruction of cartilage and bone. Engagement of integrin receptors induces cell migration/invasion through attachment to the...

AB0108 BETA-1 integrin is a critical mediator of TLR2-induced cell migration and invasion in RA

BackgroundInflammatory arthritis (IA) is a chronic disease characterised by inflammation and proliferation of synovial tissue, leading to progressive degradation of articular cartilage and bone.ObjectivesThis aim of this study was to...

β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.

68Ga]NS3-RGD and [68Ga] Oxo-DO3A-RGD for imaging αvβ3 integrin expression: synthesis, evaluation, and comparison

⁶⁸Ga-labeled RGD peptides in combination with PET allow non-invasive determination of α(v)β₃ integrin expression which is highly increased during tumor-induced angiogenesis. The aim of this study was to synthesize and evaluate two RGD peptides containing alternative chelating systems, namely [⁶⁸Ga]NS₃-RGD-RGD and [⁶⁸Ga]Oxo-DO3A-RGD and to compare their in vitro and in vivo properties with [⁶⁸Ga]DOTA- and [⁶⁸Ga]NODAGA-RGD. Syntheses of both radiotracers followed standard SPPS protocols. For in vitro characterization distribution coefficients, protein binding abilities, serum stabilities, and α(v)β₃ integrin binding affinities were determined. For in vitro tests as well as for the biodistribution assay α(v)β₃ positive human melanoma M21 and α(v)β₃ negative M21-L cells were used. ⁶⁸Ga-labeling of NS₃-RGD resulted in good radiochemical purity, whereas HPLC analysis showed two peaks with a ratio of 1:6 for [⁶⁸Ga]Oxo-DO3A-RGD. Distribution coefficients were -3.4 for [⁶⁸Ga]Oxo-DO3A-RGD and -2.9 for [⁶⁸Ga]NS₃-RGD. Both radiotracers were stable in PBS solution at 37°C for 2 h but lack stability in human serum. Protein binding was approximately 40% of the total activity for [⁶⁸Ga]NS₃-RGD and 70% for [⁶⁸Ga]Oxo-DO3A-RGD, respectively, resulting in high blood pool activities. Biodistribution assays confirmed these findings and showed an additional high uptake in liver and kidneys, especially for [⁶⁸Ga]NS₃-RGD. Furthermore, [⁶⁸Ga]Oxo-DO3A-RGD showed nearly the same activity concentrations in α(v)β₃ positive and α(v)β₃ negative tumors. [⁶⁸Ga]Oxo-DO3A-RGD and [⁶⁸Ga]NS₃-RGD have inferior characteristics compared to already existing ⁶⁸Ga-labeled RGD peptides and thus, both are not suited to image α(v)β₃ integrin expression. Of all our tested RGD peptides, [⁶⁸Ga]NODAGA-RGD still possesses the most favorable imaging properties. Moreover this study shows that the use of appropriate chelators to achieve good targeting properties of ⁶⁸Ga-labeled biomolecules and careful in vitro and in vivo evaluation including comparative studies of different strategies are essential components in designing an effective imaging agent for PET.

The β3‐integrin ligand of Borrelia burgdorferi is critical for infection of mice but not ticks

P66 is a Borrelia burgdorferi surface protein with β₃ integrin binding and channel forming activities. In this study, the role of P66 in mammalian and tick infection was examined. B. burgdorferiΔp66 strains were not infectious in wild-type, TLR2⁻/⁻- or MyD88⁻/⁻-deficient mice. Strains with p66 restored to the chromosome restored near wild-type infectivity, while complementation with p66 on a shuttle vector did not restore infectivity. Δp66 mutants are cleared quickly from the site of inoculation, but analyses of cytokine expression and cellular infiltrates at the site of inoculation did not reveal a specific mechanism of clearance. The defect in these mutants cannot be attributed to nutrient limitation or an inability to adapt to the host environment in vivo as Δp66 bacteria were able to survive as well as wild type in dialysis membrane chambers in the rat peritoneum. Δp66 bacteria were able to survive in ticks through the larva to nymph moult, but were non-infectious in mice when delivered by tick bite. Independent lines of evidence do not support any increased susceptibility of the Δp66 strains to factors in mammalian blood. This study is the first to define a B. burgdorferi adhesin as essential for mammalian, but not tick infection.

Src Family Kinases collaborate with distinct TNF‐alpha‐induced signaling pathways to regulate actin dynamics at cell‐cell junctions and barrier function in endothelial cells

We previously showed that activation of Src Family Kinases (SFK) by expression of dominant negative C‐terminal Src Kinase (DN‐CSK) is not sufficient to decrease endothelial AJ integrity. However, SFK activation by expression DN‐CSK results in the formation of very dynamic lamellae that show a distinct reticular pattern of adherens junction (AJ) proteins, localization of cortactin at the leading edge of the lamellae and activation of SFK at the cell‐cell junctions. DN‐Csk also induced β3 integrin activation and phosphorylation at the AJ. TIRF live imaging showed actin polymerization preceding AJ formation at the borders of lamellae in close contact with the extracellular matrix. Interestingly, low doses of TNF‐α that do not decrease TEER or cortical actin in control cells did produce a decrease in TEER in cells expressing DN‐Csk, together with a loss in the cortical actin band and the formation of short, thick bundles of actin. These actin changes were prevented by inhibition of ROCK activity or β3 integrin binding, even though TEER was still decreased. Preliminary data show that TEER decrease can be prevented by inhibiting p38 activity. These results suggest that the loss of endothelial barrier function induced by TNF‐α and SFK activation utilizes a p38‐dependent pathway, while a β3 integrin‐ and Rho‐ROCK‐dependent pathway is required for the cytoskeletal changes. Supported by NHLB‐HL077870.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Acute serum amyloid A regulates cytoskeletal rearrangement, cell matrix interactions and promotes cell migration in rheumatoid arthritis

ObjectiveSerum amyloid A (A-SAA) is an acute-phase protein with cytokine-like properties implicated in the pathogenesis of rheumatoid arthritis (RA), atherosclerosis, diabetes and Alzheimer's disease. This study characterises the mechanism of...

CD98hc (SLC3A2) is a key regulator of keratinocyte adhesion

Adhesion of keratinocytes is crucial for maintaining the integrity of the skin, as demonstrated by the number of dermatological disorders of genetic origin that are associated with a defect of basal keratinocyte adhesion. Integrins are the main component of the molecular networks involved in this phenomenon, but there are many others. In a recent description of proteins associated to caveolae at the plasma membrane of human basal epidermal cells, we demonstrated that CD98hc is localized with β1 integrin. We investigated the CD98hc proteins interactions and the role of CD98hc in keratinocyte adhesion. CD98hc protein interaction was identified following co-immunoprecipitation and proteomic analysis using LTQ-FT mass spectrometer. Extinction of CD98hc gene expression using specific short hairpin RNA or over-expression of CD98hc lacking the β1 integrin binding site was used to evaluate the role of this protein in keratinocyte fate. We show that CD98hc forms molecular complexes with β1 and β4 integrins in primary human keratinocytes and, using immunofluorescence, that these complexes are localized at the plasma membrane, in keeping with a role in adhesion. We confirmed that this protein is a key player of keratinocyte adhesion because in absence of interaction between CD98hc and integrins, β1 integrin failed to translocate from the cytoplasm to the plasma membrane and keratinocytes expressed epidermal differentiation markers. All these data strongly suggested that CD98hc is involved in integrin trafficking and by consequence, in keratinocyte adhesion and differentiation.

Role of Altered Sialylation of the I-Like Domain of β1 Integrin in the Binding of Fibronectin to β1 Integrin: Thermodynamics and Conformational Analyses

N-glycosylation of the I-like domain of β1 integrin plays an essential role in integrin structure and function, and the altered sialylation of β1 integrin regulates β1 integrin binding to fibronectin. However, the structural basis underlying the effect of altered sialylation of the β1 I-like domain on β1 integrin binding to fibronectin remains largely unknown. In this study, we used a combination of molecular dynamics simulations and binding free energy analyses to investigate changes in binding thermodynamics and in conformation of the glycosylated β1 I-like domain-FN-III 9-10 complex caused by altered sialylation of the β1 I-like domain. Binding free energy analyses showed that desialylation of β1 I-like domain increased β1 integrin binding to fibronectin, consistent with experimental results. Interaction analyses showed that altered sialylation of the β1 I-like domain resulted in significant changes in the interaction of the N-glycans of the I-like domain with both the I-like domain and fibronectin, and these changes could directly affect the allosteric regulation of the interaction between the I-like domain and fibronectin. Altered sialylation of the β1 I-like domain caused significant conformational changes in key functional sites of both the β1 I-like domain and fibronectin. In addition, altered sialylation of the β1 I-like domain resulted in changes in the degree of correlated motions between residues in the I-like domain and residues in fibronectin, and in the degree of motion changes in fibronectin, which could affect β1 integrin binding to fibronectin. We believe results from this study provide thermodynamic and structural evidence for a role of altered sialylation of β1 integrin in regulating β1 integrin binding to fibronectin and it's induced cellular activities.

β1 integrin mediates tamoxifen resistance in breast cancer cells.

Abstract Abstract #1055 Even though it is now well established that the microenvironment plays a central role in modulating tumor progression and response to therapy, its function as a regulator of endocrine resistance in breast cancer has not yet been explored. To do so we have established a bicellular in vitro cell line, called LM05-Mix, from the estrogen dependent M05 mouse mammary tumor (BCRT, in press). The epithelial and fibroblastic cell populations of the LM05-Mix cell line were further separated to generate the LM05-E and LM05-F cell lines respectively, both of which are estrogen receptor (ER) and progesterone receptor positive. We have previously shown that treatment with 10-6M 4-OH-tamoxifen (TAM) induces apoptosis of the LM05-E cells, and that this effect is inhibited when the epithelial cells are co-cultured with the fibroblastic cell population. Immunofluorescence staining to the extracellular matrix component fibronectin (FN) was found to be especially associated to the fibroblastic cells in the co-culture. To investigate whether FN conferred TAM resistance, LM05-E cells and human MCF-7 cells were cultured on recombinant FN and treated for 48 hours with TAM. In both cases FN conferred resistance (P≤0.001). To understand what signaling pathways were involved, the experiments were repeated in the presence of specific inhibitors and both PD98059 and LY294002 reverted the protective effect, implicating the participation of both the MAPK/ERK and PI3K/AKT pathways. Next, to corroborate the activation of these pathways by FN, starved LM05-E cells were treated with FN (30 μg/ml). Phosphorylation of FAK, MAPK/ERK1/2 and AKT was detected by western blot between 3 and 15 minutes of treatment. To find out whether the activation of these pathways by FN could lead to ligand independent activation of the ER and thus account for the induction of TAM resistance, immunofluorescence staining was carried out to serine 118-phosphorylated ER. An increase in the phosphorylation of ER at this site was evident at 15 minutes of treatment both in LM05-E and MCF-7 cells. Finally, to confirm that the protective effect was mediated by the binding of β1 integrin to FN, cells were pre-incubated with either a specific β1 blocking antibody (AIIB2) or mouse IgG and seeded on FN. The protective effect of FN over TAM induced cell death was only reverted in the AIIB2 treated group (P≤0.01). Our results show that FN through its binding to β1 integrin confers TAM resistance and suggest that the mechanism involved would be through the modulation of the activity of ER. Targeting the interaction of breast cancer cells with the microenvironment could be a novel and future alternative for patients with breast cancer whose tumors progress to TAM resistance. This work is supported by a grant from the Susan G. Komen for the Cure Foundation to MS (BCTR0600341). Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1055.