Integrin Complex Research Articles

Envelope Glycoprotein Binding to the Integrin α 4 β 7 Is Not a General Property of Most HIV-1 Strains

The HIV-1 surface glycoprotein gp120 has been reported to bind and signal through α4β7 by means of a tripeptide motif in the V2 loop that mimics structures present in the natural ligands for α4β7, suggesting that α4β7 may facilitate HIV-1 infection of CD4(+) T cells in the gut. Furthermore, immune correlates in the RV144 vaccine efficacy trial generated the hypothesis that V1V2 antibodies to an epitope near the putative α4β7 binding motif may play a role in protection against HIV-1 infection. In the interest of developing an assay to detect antibodies that block gp120 binding to α4β7, we used retinoic acid (RA)-activated human peripheral blood mononuclear cells (PBMCs) and transfected HEK293T (293T) cells expressing the integrin complex to study the α4β7 binding properties of 16 HIV-1 envelope glycoproteins. The natural ligand for α4β7, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), bound efficiently to RA-activated PBMCs and transfected 293T cells, and this binding was blocked by antibodies to α4. gp120 from multiple HIV-1 subtypes bound to RA-activated PBMCs from three donors in a CD4-dependent manner, but little or no α4β7 binding was detected. Similarly, little or no binding to α4β7 on transfected 293T cells was detected with multiple gp120s and gp140s, including gp120s from transmitted/founder strains, or when gp120 was produced in CHO, 293T, and 293S/GnT1(-/-) cells. Finally, we found no evidence that infectious HIV-1 virions produced in either PBMCs or 293T cells could bind α4β7 on transfected 293T cells. Infectious HIV-1 virions and most gp120s/gp140s appear to be poor ligands for the α4β7 integrin complex under the conditions tested here. Certain HIV-1 gp120 envelope glycoproteins have been shown to bind the gut-homing receptor α4β7, and it has been suggested that this binding facilitates mucosal transmission and virus replication in the gut mucosa. Additional evidence has generated the hypothesis that antibodies that bind near the putative α4β7 binding motif in the V2 loop of gp120, possibly disrupting gp120-α4β7 binding, may be important for HIV-1 vaccines. Our evidence indicates that infectious HIV-1 virions and many gp120s lack detectable α4β7 binding activity, suggesting that this homing receptor may play a limited role in direct HIV-1 infection of cells.

A C-MET-ss1 INTEGRIN COMPLEX DRIVES INVASIVE RESISTANCE TO ANTI-ANGIOGENIC THERAPY

BACKGROUND: Anti-angiogenic therapies like bevacizumab are promising for cancer, but acquired resistance, which often includes increased invasiveness, can limit their utility. We investigated mediators of this invasive resistance using a large archive of bevacizumab-resistant glioblastoma (BRG) tissue and by developing novel xenograft models of anti-angiogenic therapy resistance. METHODS: Microarray analysis, immunostaining, immunoprecipitation, and proximity ligation assays (PLAs) were used to define mediators of invasive anti-angiogenic therapy resistance. RESULTS: Microarray analysis and immunostaining revealed increased hypoxia and reactive-oxygen species (ROS), along with upregulated s1 integrin and receptor tyrosine kinase c-Met, in BRGs compared to pre-treatment (P < 0.005). An siRNA screen and western blot revealed upregulation of AP-1 (P < 0.01), an ROS-activated transcription factor, in BRGs and BRG-derived xenografts with siRNA targeting AP-1 components reducing s1 integrin and c-Met expression in BRG-derived cells. Immunoprecipitation and PLA revealed formation of a c-Met-s1 integrin complex involving s1 heterodimer partners alpha5 and alphaV integrins, with the formation of this complex increased in culture by hypoxia, a feature of anti-angiogenic therapy resistance, and prolonged bevacizumab treatment in vivo. Cross-activation occurred in this complex, with c-Met activation by HGF increasing cell adhesion to fibronectin and fibronectin leading to c-Met phosphorylation. We also found that increasing VEGF concentration suppressed formation of the c-Met-s1 integrin complex (P < 0.05). We developed U87-BevR, a novel xenograft model of acquired bevacizumab resistance through serial multi-generational treatment of U87MG xenografts. U87-BevR exhibited upregulated c-Met and s1 integrin upon development of resistance. Treating U87-BevR with s1 or c-Met inhibitors transiently suppressed invasive growth and reduced c-Met-s1 complex formation. Use of 3- and 10-fold less bevacizumab to treat xenografts led to less initial efficacy but in a more durable fashion associated with less c-Met-s1 integrin complex formation (P < 0.05). CONCLUSIONS: Invasive resistance limits the efficacy of anti-angiogenic therapy. We demonstrated increased activity of s1 integrin and c-Met in BRGs and the formation of a c-Met-s1 complex. The combination of chemotactic c-Met and haptotactic s1 integrin endows BRG cells with their tremendous invasive capacity. The formation of this complex is promoted by two parallel mechanisms–increased transcription of each individual factor driven by hypoxia-induced ROS leading to AP-1 activation and relief of VEGF suppression of the formation of the complex. One option to prevent the formation of a c-Met-s1 complex and thereby counteract the evolution of therapeutic resistance is dose reduction, which may be associated with lesser, but more durable responses, suggesting that, with anti-angiogenic therapy, “less is more.” SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

A monoclonal antibody targeting neuropilin-1 inhibits adhesion of MCF7 breast cancer cells to fibronectin by suppressing the FAK/p130cas signaling pathway

Neuropilin-1 (NRP-1) is a nontyrosine kinase coreceptor for semaphorin 3A and the vascular endothelial growth factor involved in tumor angiogenesis, growth, and metastasis and is regarded as a promising target for cancer therapy. In the present study, we investigated the effects of an anti-NRP-1 monoclonal antibody (mAb) that we generated for MCF7 breast cancer cellular adhesion studies. MTT, colony formation, and adhesion assays showed that our anti-NRP-1 mAb dose-dependently inhibited MCF7 proliferation and fibronectin adhesion, leading to a rounded cellular morphology. Further, rhodamine phalloidin stain revealed that fibronectin-dependent formation of actin stress fibers was inhibited by anti-NRP-1 mAb. Immunoprecipitation and western blot showed that anti-NRP-1 mAb treatment inhibited the formation of NRP-1-α5β1 integrin complexes and suppressed the phosphorylation of focal adhesion kinase and p130cas in MCF7 cells. These findings contribute to further understanding the NRP-1 function in cell adhesion and tumor metastasis. Moreover, our anti-NRP-1 mAb is a prospective drug candidate for tumor treatment.

A multiscale approach for modeling atherosclerosis progression.

Progression of atherosclerotic process constitutes a serious and quite common condition due to accumulation of fatty materials in the arterial wall, consequently posing serious cardiovascular complications. In this paper, we assemble and analyze a multitude of heterogeneous data in order to model the progression of atherosclerosis (ATS) in coronary vessels. The patient's medical record, biochemical analytes, monocyte information, adhesion molecules, and therapy-related data comprise the input for the subsequent analysis. As indicator of coronary lesion progression, two consecutive coronary computed tomography angiographies have been evaluated in the same patient. To this end, a set of 39 patients is studied using a twofold approach, namely, baseline analysis and temporal analysis. The former approach employs baseline information in order to predict the future state of the patient (in terms of progression of ATS). The latter is based on an approach encompassing dynamic Bayesian networks whereby snapshots of the patient's status over the follow-up are analyzed in order to model the evolvement of ATS, taking into account the temporal dimension of the disease. The quantitative assessment of our work has resulted in 93.3% accuracy for the case of baseline analysis, and 83% overall accuracy for the temporal analysis, in terms of modeling and predicting the evolvement of ATS. It should be noted that the application of the SMOTE algorithm for handling class imbalance and the subsequent evaluation procedure might have introduced an overestimation of the performance metrics, due to the employment of synthesized instances. The most prominent features found to play a substantial role in the progression of the disease are: diabetes, cholesterol and cholesterol/HDL. Among novel markers, the CD11b marker of leukocyte integrin complex is associated with coronary plaque progression.

Mechanical force transmission at single integrin complexes in living cells (479.1)

Mechanical interactions between cells and the extracellular matrix (ECM) play central roles in directing cell migration, proliferation, and stem‐cell differentiation. However, fundamental aspects of how cells detect and generate mechanical forces at the cell‐ECM interface remain poorly understood. Here we describe the use of molecular tension sensors to visualize the forces experienced by single cellular adhesion molecules with nanometer, piconewton, and second resolutions. The molecular tension sensors consist of a Förster resonant energy transfer (FRET) donor and acceptor pair separated by a protein domain that acts as an extensible spring. Sensor molecules are immobilized to an avidin‐coated glass coverslip at one end and present an integrin‐binding site at the other. Cellular integrins transmit force to the FRET pairs, resulting in decreased FRET with increasing load. We find that human foreskin fibroblasts (HFFs) adhere to sensor‐functionalized surfaces and develop focal adhesions as evidenced by paxillin localization and actin stress fiber formation. We observe a bimodal distribution of FRET efficiency values for sensor molecules beneath HFFs, with one peak corresponding to zero load and the other indicating a distribution of forces between 1 and 4 pN. The relatively narrow range of forces that we observe suggests that mechanical tension at individual adhesion complexes is subject to active feedback and control. Ongoing work makes use of molecular‐scale force measurements to elucidate the mechanically activated signaling events that underlie cell migration and adhesion.

Cation‐mediated electrostatic interaction in collagen–integrin complex

The paper investigates the electrostatic interactions between organic scaffolds, made of collagen, and a specific family of integrin proteins. We focus the attention on a triple‐helical collagen peptide able to specifically recognize the transmembrane protein integrin α2β1, usually involved in cell adhesion processes. The study of molecular adhesion is performed via a boundary element code designed to solve the linearized Poisson–Boltzmann equation. The binding affinity of the two molecular structures is analyzed making reference to the electrostatic complementarity principle. The mediating role of cation Co2+ is also discussed. Copyright © 2014 John Wiley &amp; Sons, Ltd.

Junctional adhesion molecule-A suppresses platelet integrin αIIbβ3 signaling by recruiting Csk to the integrin-c–Src complex

AbstractFibrinogen binding to activated integrin induces outside-in signaling that results in stable platelet aggregates and clot retraction. How integrin αIIbβ3 is discouraged from spontaneous activation is not known. We have recently shown that junctional adhesion molecule-A (JAM-A) renders protection from thrombosis by suppressing integrin outside-in signaling. In this study, we show that JAM-A associates with integrin αIIbβ3 in resting platelets and dissociates upon platelet activation by agonists. We also show that integrin-associated JAM-A is tyrosine phosphorylated and is rapidly dephosphorylated upon platelet activation. C-terminal Src kinase (Csk) binds to tyrosine phosphorylated JAM-A through its Src homology 2 domain. Thus, JAM-A recruits Csk to the integrin-c–Src complex in resting platelets. Csk, in turn, keeps integrin-associated c-Src in an inactive state by phosphorylating Y529 in its regulatory domain. Absence of JAM-A results in impaired c-SrcY529 phosphorylation and augmentation of outside-in signaling-dependent c-Src activation. Our results strongly suggest that tyrosine-phosphorylated JAM-A is a Csk-binding protein and functions as an endogenous inhibitor of integrin signaling. JAM-A recruits Csk to the integrin-c–Src complex, where Csk negatively regulates c-Src activation, thereby suppressing the initiation of outside-in signaling. Upon agonist stimulation, JAM-A is dephosphorylated on the tyrosine, allowing the dissociation of Csk from the integrin complex, and thus facilitating outside-in signaling.

CD9 Negatively Regulates CD26 Expression and Inhibits CD26-Mediated Enhancement of Invasive Potential of Malignant Mesothelioma Cells

CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5β1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5β1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of β1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5β1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5β1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.

Abstract A07: MTI-101 induces synergistic cell kill in combination with Erlotinib in EGFR-driven lung cancer models.

Abstract The epidermal growth factor receptor (EGFR) is a well-characterized oncogene that is mutated in approximately 10% of U.S. non-small cell lung cancer (NSCLC) patients. EGFR has understandably emerged as an important target for the treatment of this molecular subset of NSCLC patients. The use of tyrosine kinase inhibitors (TKIs) such as Erlotinib has been shown to prolong survival in metastatic NSCLC. However, clinical evidence indicates that the emergence of resistance can limit the overall success of EGFR inhibitors. Gatekeeper mutations account for approximately 50% of cases in which patients relapse with resistant disease. In the remaining 50% of resistant patients, other mechanisms of resistance include overexpression of cMET and transition to an EMT phenotype. These clinical statistics highlight the immediate need for the development of novel combination strategies for the treatment of EGFR driven lung cancer that would further improve patient outcomes. Our laboratory has been developing a novel cyclic peptide that we previously showed binds to a CD44/VLA-4 integrin complex and induces necrotic cell death in myeloma models. Consistent with what we previously reported in myeloma, in this report we show that MTI-101 induces caspase independent cell death in EGFR driven NSCLC tumor cells lines, PC9 and HCC4006. Moreover, we show that MTI-101 treatment results in ATP depletion, increased ROS levels, and depolarization of the mitochondrial membrane potential, all hallmarks of necrotic cell death. Additionally, MTI-101 was not cross-resistant in PC9 cells selected for resistance to Gefitinib (PC9/GR harboring a gatekeeper mutation) nor HCC4006 cells resistant to Erlotinib (HCC4006/ER demonstrating EMT phenotype). To delineate the cell surface targets responsible for MTI-101's actions in EGFR driven NSCLC, we utilized biotinylated MTI-101 coupled to LC-MS/MS, and discovered EGFR, CD44, β1, β4, α3 and α6 integrins were among the key molecules found in the binding complex. We are currently working to analyze the causative role of these proteins in relationship to MTI-101's mechanism of cell death in EGFR driven NSCLC. Due to the fact that EGFR was contained in the complex, we reasoned that MTI-101 in combination with Erlotinib may result in increased cell kill. In this report we show that the combination is synergistic. Furthermore, pre-treatment with Erlotinib enhances MTI-101 induced depletion of ATP levels, suggesting that the mechanism underlying synergy occurs in the necrotic cell death pathway. Interestingly, as a single agent, we discovered that MTI-101 leads to decreased activity of EGFR and PI3K. In light of this promising in vitro activity, we tested MTI-101 as a single agent and in combination with Erlotinib using an in vivo model. In this report we show increased activity in the combination treatment as determined by (i) decreased bone tumor burden contained in the tibia, (ii) decreased lung tumor nodules and, (iii) increased survival data. Taken together, our data demonstrates that rationally combining MTI-101 with an EGFR inhibitor has the potential to be an effective treatment option in EGFR driven lung cancer. Citation Format: Lisa E. Blumencranz, Rajesh R. Nair, Mark L. McLaughlin, Eric B. Haura, Lori A. Hazlehurst. MTI-101 induces synergistic cell kill in combination with Erlotinib in EGFR-driven lung cancer models. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A07.

Mechanosensing through focal adhesion‐anchored intermediate filaments

Integrin-based mechanotransduction involves a complex focal adhesion (FA)-associated machinery that is able to detect and respond to forces exerted either through components of the extracellular matrix or the intracellular contractile actomyosin network. Here, we show a hitherto unrecognized regulatory role of vimentin intermediate filaments (IFs) in this process. By studying fibroblasts in which vimentin IFs were decoupled from FAs, either because of vimentin deficiency (V0) or loss of vimentin network anchorage due to deficiency in the cytolinker protein plectin (P0), we demonstrate attenuated activation of the major mechanosensor molecule FAK and its downstream targets Src, ERK1/2, and p38, as well as an up-regulation of the compensatory feedback loop acting on RhoA and myosin light chain. In line with these findings, we show strongly reduced FA turnover rates in P0 fibroblasts combined with impaired directional migration, formation of protrusions, and up-regulation of "stretched" high-affinity integrin complexes. By exploiting tension-independent conditions, we were able to mechanistically link these defects to diminished cytoskeletal tension in both P0 and V0 cells. Our data provide important new insights into molecular mechanisms underlying cytoskeleton-regulated mechanosensing, a feature that is fundamental for controlled cell movement and tumor progression.

Identification of nucleolin as a lipid-raft-dependent β1-integrin-interacting protein in A375 cell migration.

Lipid rafts are related to cell surface receptor function. Integrin is a major surface receptor protein in cell adhesion and migration on the extracellular matrix (ECM). Here, we showed that lipid rafts played a critical role in human melanoma A375 cell spreading and migration on fibronectin; an important component of the ECM that interacts with β1 integrin. We found that the disruption of lipid rafts did not markedly inhibit the expression and activation of β1 integrin. By coimmunoprecipitation and mass spectrometry, we investigated the influence of lipid rafts on the β1 integrin complex and identified nucleolin as a potential lipid-raft-dependent β1-integrin-interacting protein. Upon confirmation of the interaction between β1 integrin and nucleolin, further studies revealed that nucleolin colocalized with β1 integrin in lipid rafts and raft disruption interrupted their association. In addition, knockdown of nucleolin markedly attenuated A375 cell spreading and migration on fibronectin. Taken together, we demonstrated that nucleolin is a critical lipid-raft-dependent β1-integrin-interacting protein in A375 cell spreading and migration on fibronectin.

Anosmin-1 contributes to brain tumor malignancy through integrin signal pathways

Anosmin-1, encoded by the KAL1 gene, is an extracellular matrix (ECM)-associated protein which plays essential roles in the establishment of olfactory and GNRH neurons during early brain development. Loss-of-function mutations of KAL1 results in Kallmann syndrome with delayed puberty and anosmia. There is, however, little comprehension of its role in the developed brain. As reactivation of developmental signal pathways often takes part in tumorigenesis, we investigated if anosmin-1-mediated cellular mechanisms associated with brain tumors. Our meta-analysis of gene expression profiles of patients' samples and public microarray datasets indicated that KAL1 mRNA was significantly upregulated in high-grade primary brain tumors compared with the normal brain and low-grade tumors. The tumor-promoting capacity of anosmin-1 was demonstrated in the glioblastoma cell lines, where anosmin-1 enhanced cell motility and proliferation. Notably, anosmin-1 formed a part of active β1 integrin complex, inducing downstream signaling pathways. ShRNA-mediated knockdown of anosmin-1 attenuated motility and growth of tumor cells and induced apoptosis. Anosmin-1 may also enhance the invasion of tumor cells within the ECM by modulating cell adhesion and activating extracellular proteases. In a mouse xenograft model, anosmin-1-expressing tumors grew faster, indicating the role of anosmin-1 in tumor microenvironment in vivo. Combined, these data suggest that anosmin-1 can facilitate tumor cell proliferation, migration, invasion, and survival. Therefore, although the normal function of anosmin-1 is required in the proper development of GNRH neurons, overexpression of anosmin-1 in the developed brain may be an underlying mechanism for some brain tumors.

Postprandial VLDL lipolysis products increase monocyte adhesion and lipid droplet formation via activation of ERK2 and NFκB

Postprandial lipemia is characterized by a transient increase in circulating triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL) and has been shown to activate monocytes in vivo. Lipolysis of VLDL releases remnant particles, phospholipids, monoglycerides, diglycerides, and fatty acids in close proximity to endothelial cells and monocytes. We hypothesized that postprandial VLDL lipolysis products could activate and recruit monocytes by increasing monocyte expression of proinflammatory cytokines and adhesion molecules, and that such activation is related to the development of lipid droplets. Freshly isolated human monocytes were treated with VLDL lipolysis products (2.28 mmol/l triglycerides + 2 U/ml lipoprotein lipase), and monocyte adhesion to a primed endothelial monolayer was observed using a parallel plate flow chamber coupled with a CCD camera. Treated monocytes showed more rolling and adhesion than controls, and an increase in transmigration between endothelial cells. The increased adhesive events were related to elevated expression of key integrin complexes including Mac-1 [α(m)-integrin (CD11b)/β2-integrin (CD18)], CR4 [α(x)-integrin (CD11c)/CD18] and VLA-4 [α4-integrin (CD49d)/β1-integrin (CD29)] on treated monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes with VLDL lipolysis products increased expression of TNFα, IL-1β, and IL-8 over controls, with concurrent activation of NFkB and AP-1. NFκB and AP-1-induced cytokine and integrin expression was dependent on ERK and Akt phosphorylation. Additionally, fatty acids from VLDL lipolysis products induced ERK2-dependent lipid droplet formation in monocytes, suggesting a link to inflammatory signaling pathways. These results provide novel mechanisms for postprandial monocyte activation by VLDL lipolysis products, suggesting new pathways and biomarkers for chronic, intermittent vascular injury.

Invasion patterns in brain metastases of solid cancers

Brain metastases are generally considered to be well demarcated from the surrounding brain parenchyma, although infiltrative growth patterns have been observed. We systemically investigated infiltration patterns and expression of adhesion molecules in a large and well-defined series of autopsy cases of brain metastases. Ninety-seven autopsy specimens from 57 brain metastasis patients (primary tumor: 27 lung cancer, 6 breast cancer, 8 melanoma, 2 colorectal cancer, 1 kidney cancer, and 13 other) were evaluated for patterns of invasion into surrounding brain parenchyma. Expression of integrins αv; cytoplasmic β3, αvβ3, αvβ5, αvβ6, and αvβ8; and of E and N cadherin were evaluated using immunohistochemistry. Three main invasion patterns were seen: well-demarcated growth (29/57, 51%), vascular co-option (10/57, 18%), and diffuse infiltration (18/57, 32%). There was no statistically significant association of invasion pattern with primary tumor type, although vascular co-option was most common in melanoma brain metastases (4/10). Invasion patterns of different brain metastases of the same patient were highly concordant (P < .001, chi-square test). Distance of infiltration from the main tumor mass ranged from 12.5 µm to 450 µm (median 56.2 µm) and was not significantly different between the vascular co-option and the diffuse infiltration groups. Levels of αvβ6 were significantly higher in the well-demarcated group than in the vascular co-option and the diffuse infiltration groups (P = .033, Kruskal-Wallis test). Expression of αvβ5 in tumor cells was higher in brain metastasis lesions previously treated with stereotactic radiosurgery (P = .034, chi-square test). Distinct invasion patterns of brain metastases into the brain parenchyma are not specific for primary tumor types, seem to be influenced by expression of αv integrin complexes, and may help to guide clinical decision-making.

Identification of Novel Integrin Binding Partners for Calcium and Integrin Binding Protein 1 (CIB1): Structural and Thermodynamic Basis of CIB1 Promiscuity

The short cytoplasmic tails of the α- and β-chains of integrin adhesion receptors regulate integrin activation and cell signaling. Significantly less is known about proteins that bind to α-integrin cytoplasmic tails (CTs) as opposed to β-CTs to regulate integrins. Calcium and integrin binding protein 1 (CIB1) was previously identified as an αIIb binding partner that inhibits agonist-induced activation of the platelet-specific integrin, αIIbβ3. A sequence alignment of all α-integrin CTs revealed that key residues in the CIB1 binding site of αIIb are well-conserved, and was used to delineate a consensus binding site (I/L-x-x-x-L/M-W/Y-K-x-G-F-F). Because the CIB1 binding site of αIIb is conserved in all α-integrins and CIB1 expression is ubiquitous, we asked if CIB1 could interact with other α-integrin CTs. We predicted that multiple α-integrin CTs were capable of binding to the same hydrophobic binding pocket on CIB1 with docking models generated by all-atom replica exchange discrete molecular dynamics. After demonstrating novel in vivo interactions between CIB1 and other whole integrin complexes with co-immunoprecipitations, we validated the modeled predictions with solid-phase competitive binding assays, which showed that other α-integrin CTs compete with the αIIb CT for binding to CIB1 in vitro. Isothermal titration calorimetry measurements indicated that this binding is driven by hydrophobic interactions and depends on residues in the CIB1 consensus binding site. These new mechanistic details of CIB1-integrin binding imply that CIB1 could bind to all integrin complexes and act as a broad regulator of integrin function.

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.

AB0109 The soluble form of CD18 is associated with clinical findings, CRP and MRI activity in spondyloarthritis

BackgroundLack of measures of disease activity and prognosis is a key challenge in spondyloarthritis (SpA). Several soluble biomarkers have been proposed, but very few have been found to relate to...

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation, Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.

Identification and Analysis of Multi-Protein Complexes in Placenta

Placental malfunction induces pregnancy disorders which contribute to life-threatening complications for both the mother and the fetus. Identification and characterization of placental multi-protein complexes is an important step to integratedly understand the protein-protein interaction networks in placenta which determine placental function. In this study, blue native/sodium dodecyl sulfate polyacrylamide gel electrophoresis (BN/SDS-PAGE) and Liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to screen the multi-protein complexes in placenta. 733 unique proteins and 34 known and novel heterooligomeric multi-protein complexes including mitochondrial respiratory chain complexes, integrin complexes, proteasome complexes, histone complex, and heat shock protein complexes were identified. A novel protein complex, which involves clathrin and small conductance calcium-activated potassium (SK) channel protein 2, was identified and validated by antibody based gel shift assay, co-immunoprecipitation and immunofluorescence staining. These results suggest that BN/SDS-PAGE, when integrated with LC-MS/MS, is a very powerful and versatile tool for the investigation of placental protein complexes. This work paves the way for deeper functional characterization of the placental protein complexes associated with pregnancy disorders.

Abstract 4043: The role of CD63/Timp1/β1 integrin complex in cell survival.

Abstract Although malignant melanoma is the less frequently diagnosed skin cancer, it shows a poor prognosis due its chemoresistance and metastasis development. One of the acquired abilities of transformed cells is anoikis resistance and this property is closely related to metastasis formation. In our laboratory, we developed a model that allows us to study different steps of melanocyte malignant transformation. Melan-a melanocytes surviving after 1, 2, 3 and 4 deadhesion cycles showed modified morphology and independent PMA growth and have been named 1C, 2C, 3C and 4C cells, respectively. Different melanoma cell lines were established after submitting 4C spheroids to limiting dilution. Previous data from our group demonstrated increased expression of Timp1 and resistance to anoikis during the malignant transformation of melanocytes. Melan-a melanocytes overexpressing Timp1 acquire anoikis-resistant phenotype. In our laboratory, we observed interaction between CD63, Timp1 and β1-integrin in the 4C, 4C11- and 4C11+ cell lines established after cycles of anchorage impediment. This seems to be the first study describing the interaction between Timp1, CD63 and β1-integrin in tumor cells. However, it is unclear how Timp1 modulates resistance to anoikis. Therefore, our aim is to analyze the role of Timp1 in the context of Timp1/CD63/β1-integrin complex and the signaling pathways involved in the acquisition of anoikis resistance. Supported by CAPES and FAPESP. Citation Format: Mariana T. Pinto, Fabiana Henriques Machado de Melo, Miriam Galvonas Jasiulionis. The role of CD63/Timp1/β1 integrin complex in cell survival. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4043. doi:10.1158/1538-7445.AM2013-4043