Β1 Integrin Activation Research Articles

High-Affinity Bent β2-Integrin Molecules in Arresting Neutrophils Face Each Other through Binding to ICAMs In cis

SUMMARYLeukocyte adhesion requires β2-integrin activation. Resting integrins exist in a bent-closed conformation—i.e., not extended (E−) and not high affinity (H−)—unable to bind ligand. Fully activated E+H+ integrin binds intercellular adhesion molecules (ICAMs) expressed on the opposing cell in trans. E−H− transitions to E+H+ through E+H− or through EH+, which binds to ICAMs on the same cell in cis. Spatial patterning of activated integrins is thought to be required for effective arrest, but no high-resolution cell surface localization maps of activated integrins exist. Here, we developed Super-STORM by combining super-resolution microscopy with molecular modeling to precisely localize activated integrin molecules and identify the molecular patterns of activated integrins on primary human neutrophils. At the time of neutrophil arrest, E−H+ integrins face each other to form oriented (non-random) nanoclusters. To address the mechanism causing this pattern, we blocked integrin binding to ICAMs in cis, which significantly relieved the face-to-face orientation.

Type VI collagen promotes lung epithelial cell spreading and wound-closure

Basement membrane (BM) is an essential part of the extracellular matrix (ECM) that plays a crucial role in mechanical support and signaling to epithelial cells during lung development, homeostasis and repair. Abnormal composition and remodeling of the lung ECM have been associated with developmental abnormalities observed in multiple pediatric and adult respiratory diseases. Collagen VI (COL6) is a well-studied muscle BM component, but its role in the lung and its effect on pulmonary epithelium is largely undetermined. We report the presence of COLVI immediately subjacent to human airway and alveolar epithelium in the pediatric lung, in a location where it is likely to interact with epithelial cells. In vitro, both primary human lung epithelial cells and human lung epithelial cell lines displayed an increased rate of “wound healing” in response to a scratch injury when plated on COL6 as compared to other matrices. For the 16HBE cell line, wounds remained >5-fold larger for cells on COL1 (p<0.001) and >6-fold larger on matrigel (p<0.001), a prototypical basement membrane, when compared to COL6 (>96% closure at 10 hr). The effect of COL6 upon lung epithelial cell phenotype was associated with an increase in cell spreading. Three hours after initial plating, 16HBE cells showed >7-fold less spreading on matrigel (p<0.01), and >4-fold less spreading on COL1 (p<0.01) when compared to COL6. Importantly, the addition of COL6 to other matrices also enhanced cell spreading. Similar responses were observed for primary cells. Inhibitor studies indicated both integrin β1 activity and activation of multiple signaling pathways was required for enhanced spreading on all matrices, with the PI3K/AKT pathway (PI3K, CDC42, RAC1) showing both significant and specific effects for spreading on COL6. Genetic gain-of-function experiments demonstrated enhanced PI3K/AKT pathway activity was sufficient to confer equivalent cell spreading on other matrices as compared to COL6. We conclude that COL6 has significant and specific effects upon human lung epithelial cell-autonomous functions.

Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity

AbstractMegakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished β1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.

Peri/epicellular protein disulfide isomerase-A1 acts as an upstream organizer of cytoskeletal mechanoadaptation in vascular smooth muscle cells.

Although redox processes closely interplay with mechanoresponses to control vascular remodeling, redox pathways coupling mechanostimulation to cellular cytoskeletal organization remain unclear. The peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) supports postinjury vessel remodeling. Using distinct models, we investigated whether pecPDIA1 could work as a redox-dependent organizer of cytoskeletal mechanoresponses. In vascular smooth muscle cells (VSMCs), pecPDIA1 immunoneutralization impaired stress fiber assembly in response to equibiaxial stretch and, under uniaxial stretch, significantly perturbed cell repositioning perpendicularly to stretch orientation. During cyclic stretch, pecPDIA1 supported thiol oxidation of the known mechanosensor β1-integrin and promoted polarized compartmentalization of sulfenylated proteins. Using traction force microscopy, we showed that pecPDIA1 organizes intracellular force distribution. The net contractile moment ratio of platelet-derived growth factor-exposed to basal VSMCs decreased from 0.90 ± 0.09 (IgG-exposed controls) to 0.70 ± 0.08 after pecPDI neutralization ( P < 0.05), together with an enhanced coefficient of variation for distribution of force modules, suggesting increased noise. Moreover, in a single cell model, pecPDIA1 neutralization impaired migration persistence without affecting total distance or velocity, whereas siRNA-mediated total PDIA1 silencing disabled all such variables of VSMC migration. Neither expression nor total activity of the master mechanotransmitter/regulator RhoA was affected by pecPDIA1 neutralization. However, cyclic stretch-induced focal distribution of membrane-bound RhoA was disrupted by pecPDI inhibition, which promoted a nonpolarized pattern of RhoA/caveolin-3 cluster colocalization. Accordingly, FRET biosensors showed that pecPDIA1 supports localized RhoA activity at cell protrusions versus perinuclear regions. Thus, pecPDI acts as a thiol redox-dependent organizer and noise reducer mechanism of cytoskeletal repositioning, oxidant generation, and localized RhoA activation during a variety of VSMC mechanoresponses. NEW & NOTEWORTHY Effects of a peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) during mechanoregulation in vascular smooth muscle cells (VSMCs) were highlighted using approaches such as equibiaxial and uniaxial stretch, random single cell migration, and traction force microscopy. pecPDIA1 regulates organization of the cytoskeleton and minimizes the noise of cell alignment, migration directionality, and persistence. pecPDIA1 mechanisms involve redox control of β1-integrin and localized RhoA activation. pecPDIA1 acts as a novel organizer of mechanoadaptation responses in VSMCs.

The membrane tethered matrix metalloproteinase MT1-MMP triggers an outside-in DNA damage response that impacts chemo- and radiotherapy responses of breast cancer

Breast cancer is the second leading cause of death among women in the US. Targeted therapies exist, however resistance is common and patients resort to chemotherapy. Chemotherapy is also a main treatment for triple negative breast cancer (TNBC) patients; while radiation is delivered to patients with advanced disease to counteract metastasis. Yet, resistance to both chemo- and radiotherapy is still frequent, highlighting a need to provide novel sensitizers. We discovered that MT1-MMP modulates DNA damage responses (DDR) in breast cancer. MT1-MMP expression inversely correlates to chemotherapy response of breast cancer patients. Inhibition of MT1-MMP sensitizes TNBC cells to IR and doxorubicin in vitro, and in vivo in an orthotopic breast cancer model. Specifically, depletion of MT1-MMP causes stalling of replication forks and Double Strand Breaks (DBSs), leading to increased sensitivity to additional genotoxic stresses. These effects are mediated by integrinβ1, as a constitutive active integrinβ1 reverts replication defects and protects cells depleted of MT1-MMP from IR and chemotherapy. These data highlight a novel DNA damage response triggered by MT1-MMP-integrinβ1 and provide a new point of therapeutic targeting that may improve breast cancer patient outcomes.

Podoplanin suppresses the cell adhesion of epidermal keratinocytes via functional regulation of β1-integrin.

Epidermal stem cells adhere more efficiently to the extracellular matrix (ECM) than the less adhesive differentiating cells due to their high expression of cell adhesion molecules including β1-integrin. Podoplanin is majorly expressed in the markedly proliferative and differentiating basal cells of the wounded and psoriatic epidermis. This study was designed to reveal podoplanin's function in human epidermal keratinocytes (HEK) focusing on its interaction with β1-integrin. We analyzed the adhesion and differentiation of HEK in both podoplanin-overexpressing and -knock-down cells, considering their β1-integrin levels. The basal layer of IL-22-treated hyperproliferative reconstituted epidermis cells (simulating basal hyperproliferative psoriatic epidermal basal cells) expressed higher podoplanin levels than the untreated control cells. The adhesiveness of HaCaT cells, which do not express podoplanin, was reduced after the overexpression of podoplanin. HEK with podoplanin overexpression suppressed the cell adhesion to type I collagen (while downregulating β1-integrin functions) and podoplanin silencing augmented it (by increasing active ECM-bound β1-integrin). The increased cell adhesion to type I collagen induced by podoplanin silencing could be reversed by addition of P5D2, a neutralizing antibody against β1-integrin. In the psoriatic epidermis, podoplanin expression was especially upregulated on the rete ridges of the basal cell layer. This expression pattern was inversely correlated with the total/ECM-bound active β1-integrin-expression, which was stronger at the basal cell layer covering the dermal papillae. Our results indicate that podoplanin inhibits the cell ECM attachment by suppressing β1-integrin and initiating HEK differentiation. Podoplanin is presumably involved in the pathogenesis of psoriasis.

IL-1β promotes transendothelial migration of PBMCs by upregulation of the FN/α5β1 signalling pathway in immortalised human brain microvascular endothelial cells

Neuroinflammation is often associated with pathological changes in the function of the blood-brain barrier (BBB) caused by disassembly of tight and adherens junctions that under physiological conditions are important for the maintenance of the BBB integrity. Consequently, in inflammation the BBB becomes dysfunctional, facilitating leukocyte traversal of the barrier and accumulation of immune cells within the brain.The extracellular matrix (ECM) also contributes to BBB integrity but the significance of the main ECM receptors, the β1 integrins also expressed on endothelial cells, is less well understood. To evaluate whether β1 integrin function is affected during inflammation and impacts barrier function, we used a transformed human brain microvascular endothelial cell (THBMEC)-based Interleukin 1β (IL-1β)-induced inflammatory in vitro BBB model. We demonstrate that IL-1β increases cell-matrix adhesion and induces a redistribution of active β1 integrins to the basal surface. In particular, binding of α5β1 integrin to its ligand fibronectin is enhanced and α5β1 integrin-dependent signalling is upregulated. Additionally, localisation of the tight junction protein claudin-5 is altered. Blockade of the α5β1 integrin reduces the IL-1β-induced transendothelial migration of peripheral blood mononuclear cells (PBMCs).These data imply that IL-1β-induced inflammation not only destabilizes tight junctions but also increases α5β1 integrin-dependent cell-matrix adhesion to fibronectin.

Abstract A18: Netrin-1/Neogenin-1 promotes neuroblastoma cell migration via activation of Integrin β1

Abstract Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It is derived from the sympatho-adrenal lineage of the neural crest, during development of the sympathetic nervous system, and usually arises in the adrenal gland or sympathetic ganglia. Since &amp;gt;50% of diagnosed cases are metastatic, it is important to know the mechanisms underlying its dissemination. During metastasis, cells migrate, invade, and colonize local and distant organs. Focal Adhesion Kinase (FAK) and Integrin β1 are key regulators of focal adhesion formation and cell migration. FAK induces Integrin β1 activation, focal adhesion turnover, and consequently cell migration, and it has been shown to be upregulated in tumors. FAK binds several upstream receptors including Neogenin-1 (NEO1), which is involved in axonal guidance, angiogenesis, and neuronal cell migration. On the other hand, NEO1 binds to the Netrin family of ligands. Analysis of public databases of NB tumors reveals that high expression of NEO1 correlates with poor patient survival. The aim of this work is to evaluate the role of migrationrelated signaling pathways downstream of NEO1/Netrin-1 (main ligand of the Netrin family). To this end, we focused on FAK and Integrin β1 interaction/activation. Western blot in SK-N-SH (MYCN WT) and LAN-1 (MYCN high) NB cell lines indicated that NEO1 and Netrin-4 are expressed while Netrin-1 is only expressed in LAN-1. Also, NEO1 associated with Netrin-1, as shown by co-immunoprecipitation assays in both cell lines. To evaluate the contribution of NEO1 in cell migration, integrin β1 activation, and metastasis, stable NEO1 knockdown cells (shNEO1) were generated using shRNAs via lentiviral infection. Cell migration was decreased in shNEO1 cells, as shown in transwell assays using Netrin-1 as chemoattractant, which could be due to decreased sensibility to the chemotactic stimulus of the ligand. We also assessed FAK dependency by pharmacologic inhibition with the PF562,271 compound in a spheroid-based cell migration assay. To further evaluate the association between NEO1 with Integrin β1 and FAK, we performed co-immunoprecipitation assays. Our data show that NEO1 associates in a complex with Integrin β1 and FAK in SK-N-SH cells. Immunoprecipitation of activated Integrin β1 with a specific conformational antibody shows that activation of Integrin β1 is less in shNEO1 cells compared to control cells. In an in vivo analysis of metastasis, SK-N-SK shNEO1 and control cells were injected into flank of immunodeficient mice (NSG strain). Metastasis was evaluated via luciferase activity in several organs, revealing that NEO1 is important to NB metastases. Our work suggests that NEO1 is a tumor progression-promoting protein, with an active role in metastasis. We propose a possible mechanism whereby NEO1, via its interaction with Netrin-1, is involved in cell migration and metastasis of NB cells. NEO1 likely plays its role by associating with FAK, allowing its phosphorylation and complex formation with Integrin β1, inducing its activation. Our results provide further information on the potential use of NEO1 as a therapeutic target to reduce metastasis in neuroblastoma. Citation Format: Andrea A. Villanueva, Ernesto Muñoz-Palma, Carlos Cubo, Vicente A. Torres, Pilar Sanchez-Gomez, Veronica Palma. Netrin-1/Neogenin-1 promotes neuroblastoma cell migration via activation of Integrin β1 [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A18.

Integrin β1 activity is required for cardiovascular formation in zebrafish.

Integrins are transmembrane molecules that facilitate cell-to-cell and cell-to-extracellular matrix (ECM) interactions. Integrin molecules are heterodimers that consist of α- and β-subunits. The integrin β1 gene is widely expressed in vivo and is the major β molecule in many tissues; however, tissue-specific roles of integrin β1 are still elusive. In this study, we investigated integrin β1 function in endothelial cells of zebrafish. An integrin β1b mutant zebrafish exhibited morphological abnormalities in blood vessel formation, cephalic hemorrhage and a decreased responsiveness to tactile stimulation during development. To determine the role of integrin β1b in vascular formation, we developed a Gal4/UAS-mediated conditional inactivation of integrin β1 by expressing the cytoplasmic region of integrin β1 that acts as a dominant-negative (DN) isoform. Expression of integrin β1 DN in endothelial cells induced blood vessel abnormalities as in integrin β1b mutants. These results show that endothelial cells require integrin activity for the formation and/or maintenance of blood vessels in zebrafish. Furthermore, our time-lapse recording visualized the breakpoint of cephalic vessels and the hemorrhage onset. Taken together, our tissue-specific inactivation of integrin β1 in zebrafish is powerful tools for functional analysis of integrin β1 in developing tissues.

Cochaperone Mzb1 is a key effector of Blimp1 in plasma cell differentiation and β1-integrin function

Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1-/-Prdm1+/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1-/- plasmablasts show a reduced activation of β1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.

Mechanosensing by β1 integrin induces angiocrine signals for liver growth and survival.

Angiocrine signals derived from endothelial cells are an important component of intercellular communication and have a key role in organ growth, regeneration and disease1-4. These signals have been identified and studied in multiple organs, including the liver, pancreas, lung, heart, bone, bone marrow, central nervous system, retina and some cancers1-4. Here we use the developing liver as a model organ to study angiocrine signals5,6, and show that the growth rate of the liver correlates both spatially and temporally with blood perfusion to this organ. By manipulating blood flow through the liver vasculature, we demonstrate that vessel perfusion activates β1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3). Notably, both β1 integrin and VEGFR3 are strictly required for normal production of hepatocyte growth factor, survival of hepatocytes and liver growth. Ex vivo perfusion of adult mouse liver and in vitro mechanical stretching of human hepatic endothelial cells illustrate that mechanotransduction alone is sufficient to turn on angiocrine signals. When the endothelial cells are mechanically stretched, angiocrine signals trigger in vitro proliferation and survival of primary human hepatocytes. Our findings uncover a signalling pathway in vascular endothelial cells that translates blood perfusion and mechanotransduction into organ growth and maintenance.

Matrix stiffness mediates stemness characteristics via activating the Yes-associated protein in colorectal cancer cells.

Matrix stiffness is an essential physical microenvironment in solid cancer. However, its influence on cancer stemness still remains elusive. Colorectal cancer (CRC) cell line HCT-116 was cultured in the matrix with various stiffness. The siYAP was applied to detect the changes of stemness markers. The cancer stemness markers, Yes-associated protein (YAP), Lamin A/C and downstream protein molecules, and their activation were measured after the treatment with anti-β1-integrin and FAK inhibitors. In CRC tissue samples, collagen deposition and the expression of α-SMA and CD133 were detected. The study found that the expression level of stemness markers and Lamin A/C increased as the matrix stiffness raised and was regulated by YAP activation in CRC stem cells. Inhibition of β1-integrin and FAK activation in a high stiffness cell culture medium significantly decreased the activation of YAP, PI3K, and AKT. Collagen was highly deposited in the CRC invasive tumor front (ITF), and the expression of CD133 was higher in ITF compared with normal tissue and the tumor cells. Moreover, the expression level of α-SMA was positively correlated with CD133 expression level. Together, our results suggest that activation of YAP in CRC plays an important role in the promotion of cancer stem cell properties by extracellular matrix stiffness in CRC.

Endothelial Dab1 signaling orchestrates neuro-glia-vessel communication in the central nervous system.

The architecture of the neurovascular unit (NVU) is controlled by the communication of neurons, glia, and vascular cells. We found that the neuronal guidance cue reelin possesses proangiogenic activities that ensure the communication of endothelial cells (ECs) with the glia to control neuronal migration and the establishment of the blood-brain barrier in the mouse brain. Apolipoprotein E receptor 2 (ApoER2) and Disabled1 (Dab1) expressed in ECs are required for vascularization of the retina and the cerebral cortex. Deletion of Dab1 in ECs leads to a reduced secretion of laminin-α4 and decreased activation of integrin-β1 in glial cells, which in turn control neuronal migration and barrier properties of the NVU. Thus, reelin signaling in the endothelium is an instructive and integrative cue essential for neuro-glia-vascular communication.

ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism.

Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. NA.

ADAP deficiency impairs megakaryocyte polarization with ectopic proplatelet release and causes microthrombocytopenia

AbstractBone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in thrombopoiesis can lead to thrombocytopenia associated with increased bleeding tendency. Recently, the platelet disorder congenital autosomal-recessive small-platelet thrombocytopenia (CARST) was described; it is caused by mutations in the adhesion and degranulation-promoting adaptor protein (ADAP; synonym: FYB, SLAP130/120) gene, and characterized by microthrombocytopenia and bleeding symptoms. In this study, we used constitutive ADAP-deficient mice (Adap−/−) as a model to investigate mechanisms underlying the microthrombocytopenia in CARST. We show that Adap−/− mice display several characteristics of human CARST, with moderate thrombocytopenia and smaller-sized platelets. Adap−/− platelets had a shorter life span than control platelets, and macrophage depletion, but not splenectomy, increased platelet counts in mutant mice to control levels. Whole-sternum 3-dimensional confocal imaging and intravital 2-photon microscopy revealed altered morphology of ADAP-deficient MKs with signs of fragmentation and ectopic release of (pro)platelet-like particles into the BM compartment. In addition, cultured BM-derived MKs lacking ADAP showed reduced spreading on extracellular matrix proteins as well as activation of β1 integrins, impaired podosome formation, and displayed defective polarization of the demarcation membrane system in vitro. MK-/platelet-specific ADAP-deficient mice (PF4-cre) also produced fewer and smaller-sized platelets and released platelets ectopically. These data demonstrate that the abnormal platelet production in the mutant mice is an MK-intrinsic defect. Taken together, these results point to an as-yet-unidentified role of ADAP in the process of MK polarization and platelet biogenesis.

Proteomics reveals Rictor as a noncanonical TGF-β signaling target during aneurysm progression in Marfan mice.

The objective of the present study was to 1) analyze the ascending aortic proteome within a mouse model of Marfan syndrome (MFS; Fbn1C1041G/+) at early and late stages of aneurysm and 2) subsequently test a novel hypothesis formulated on the basis of this unbiased proteomic screen that links changes in integrin composition to transforming growth factor (TGF)-β-dependent activation of the rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling pathway. Ingenuity Pathway Analysis of over 1,000 proteins quantified from the in vivo MFS mouse aorta by data-independent acquisition mass spectrometry revealed a predicted upstream regulator, Rictor, that was selectively activated in aged MFS mice. We validated this pattern of Rictor activation in vivo by Western blot analysis for phosphorylation on Thr1135 in a separate cohort of mice and showed in vitro that TGF-β activates Rictor in an integrin-linked kinase-dependent manner in cultured aortic vascular smooth muscle cells. Expression of β3-integrin was upregulated in the aged MFS aorta relative to young MFS mice and wild-type mice. We showed that β3-integrin expression and activation modulated TGF-β-induced Rictor phosphorylation in vitro, and this signaling effect was associated with an altered vascular smooth muscle cell proliferative-migratory and metabolic in vitro phenotype that parallels the in vivo aneurysm phenotype in MFS. These results reveal that Rictor is a novel, context-dependent, noncanonical TGF-β signaling effector with potential pathogenic implications in aortic aneurysm. NEW & NOTEWORTHY We present the most comprehensive quantitative analysis of the ascending aortic aneurysm proteome in Marfan syndrome to date resulting in novel and potentially wide-reaching findings that expression and signaling by β3-integrin constitute a modulator of transforming growth factor-β-induced rapamycin-independent component of mammalian target of rapamycin (Rictor) signaling and physiology in aortic vascular smooth muscle cells.

Abstract 5224: hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis

Abstract The splicing of the actin regulator hMENA generates different isoforms and we have demonstrated that the two alternatively expressed isoforms, hMENA11a and hMENAΔv6, have opposite functions in cell invasiveness. This general mechanism is of great clinical relevance in early NSCLC patients, where the pattern of hMENA isoform expression is a powerful prognostic factor. However the mechanism of action of the two isoforms have remained unclear. Herein, we evaluated whether hMENA and its isoforms influence β1 integrin expression and signaling considering the role of this integrin in cancer cell invasiveness and tumor progression. We performed hMENA silencing by siRNA and shRNA, to evaluate by QRT-PCR and biochemical approaches the expression of β1 integrin; by immunofluorescence the MRTF1 localization, by in vivo assay G-Actin/F-Actin ratio and by luciferase reporter assay the SRF activity. β1 integrin activation and signaling was evaluated by flow cytometry using an antibody specific for the β1 active conformation and by biochemical analysis of the phosphorylation of FAK, SRC and Paxillin. The secretoma of hMENA11a transfected cancer cell lines was analyzed by LC-MS/MS. Immunohistochemical analysis was performed using pan-hMENA, hMENA11a, and fibronectin antibodies in primary cancer tissues from node negative NSCLC patients. The Chi-Square or Fisher Exact tests were used to estimate associations among categorical variables and disease-free survival was calculated by the Kaplan-Meier product limit method. We show that the depletion of all hMENA isoforms inhibits the Serum Response Factor (SRF) activity, and the expression of its target gene β1 Integrin, by affecting G-Actin/F-Actin ratio, critical for the nuclear localization of the SRF co-factor myocardin related transcription factor 1 (MRTF1). Furthermore, we provide new insights into the mechanisms involved in the opposite functions of hMENA11a and hMENAΔv6 in cell invasiveness and we identify a new role of these isoforms in the β1 integrin-ECM signalling axis. Indeed, hMENAΔv6-drives cancer cell invasion by increasing β1 integrin activation and signalling, which is reduced by the anti-invasive hMENA11a isoform. Moreover, exogenous expression of hMENA11a in hMENAΔv6 positive cancer cells dramatically reduces secretion of extracellular matrix (ECM) components, including β1 integrin ligands and metalloproteinases. On the other hand overexpression of the pro-invasive hMENAΔv6 increases fibronectin production. In primary tumors high hMENA11a correlates with low stromal fibronectin and favorable clinical outcome of early node-negative non-small cell lung cancer patients. This newly discovered signature, which pays attention to the alternative splicing of hMENA and ECM components such as fibronectin in the stroma, might help fill in the gap in the still controversial clinical management of early node-negative NSCLC patients. Citation Format: Francesca Di Modugno, Sheila Spada, Belinda Palermo, Paolo Visca, Pierluigi Iapicca, Anna Di Carlo, Barbara Antoniani, Isabella Sperduti, Anna Di Benedetto, Irene Terrenato, Marcella Mottolese, Francesco Gandolfi, Francesco Facciolo, Emily Chen, Martin A. Schwartz, Angela Santoni, Mina J. Bissell, Paola Nisticò. hMENA isoforms impact NSCLC patient outcome through fibronectin/β1 integrin axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5224.

Abstract 3149: Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis

Abstract Increased collagen expression and deposition is a risk factor for breast cancer development. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse model show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. However, function of collagen XIII in cancer development and progression largely remains to be determined. We found that collagen XIII expression is significantly induced during breast cancer development, especially in triple negative breast cancer. Increased collagen XIII mRNA level is associated with poor prognosis. Importantly, silencing collagen XIII expression in MDA MB 231 cells slow down xenograft growth and significantly reduced cancer metastasis in mice. Using gain- and loss-of function approaches, we showed that collagen XIII enhanced cell migration, invasion, and mammosphere/tumorsphere formation. We also found that collagen XIII induced integrin-β1 activation, and subsequently enhanced TGF-β signaling. Blocking integrin-β1 activation with AIIB2 antibody significantly reduced collagen XIII-induced cell migration and mammosphere formation. These findings demonstrate a novel function of collagen XIII in promoting cancer metastasis through enhancing β1 integrin-dependent cell migration and invasion Citation Format: Hui Zhang, Tricia Fredericks, Ren Xu. Collagen XIII induces β1-integrin activation and triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3149.

ADAM17 promotes cell migration and invasion through the integrin β1 pathway in hepatocellular carcinoma

ADAM17 is believed to promote tumor development by facilitating both cell proliferation and migration. In this study, we investigated the involvement of ADAM17 and the activation of the integrin pathway in the regulation of the malignant properties of hepatocellular carcinoma cells and tissues. ADAM17 was positively correlated with active integrin β1, which was determined using a human tissue microarray and an N-nitrosodiethylamine-induced HCC mouse model. We found elevated ADAM17 and active integrin β1 levels in HCC tissues compared with adjacent liver tissues, and the active integrin β1 levels were associated with tumor size and TNM grade. High ADAM17 and active integrin β1 levels in tumor tissues were significantly associated with poor survival of HCC patients. RNAi-mediated ADAM17 knockdown and integrin β1 blockade significantly attenuated the migration and invasion of HCC cells, and overexpression of ADAM17 showed the reverse effects. ADAM17 interference attenuated the intrahepatic growth and metastasis of HCC cells in an orthotopic xenograft model. ADAM17-knockdown cells showed diminished levels of active integrin β1, p-FAK, p-AKT, MMP-2 and MMP-9. ADAM17 knockdown significantly attenuated the translocation of the Notch1 intracellular domain into the nucleus, whereas overexpression of the Notch1 intracellular domain rescued the translocation and enhanced the activation of integrin β1. Our data provide evidence for ADAM17 as an important determinant of malignant properties via regulation of integrin β1 activation and Notch1 signaling. Inhibition of ADAM17 may provide viable therapeutic potential for preventing HCC metastasis.

Intracellular β2 integrin (CD11/CD18) interacting partners in neutrophil trafficking.

Neutrophil recruitment during acute inflammation critically depends on the spatial and temporal regulation of β2 integrins (CD11/CD18). This regulation occurs by inside-out and outside-in signalling via interaction of cytoplasmic proteins with the intracellular domains of the integrin α- and β-subunits. The underlying molecular mechanisms regulating β2 integrins in neutrophils are still incompletely understood. This review provides a comprehensive overview of our current knowledge on proteins interacting with the cytoplasmic tail of CD18, the conserved β-subunit of β2 integrins, their regulation and their functional importance for neutrophil trafficking during acute inflammation. A total of 22 proteins including Talin, Kindlin 3 and Coronin 1A have been reported to interact with the CD18 cytoplasmic tail. Here, proteins binding to the cytoplasmic domain of CD18 in experiments using purified, recombinant proteins or peptides in, for example, pull-down assays, are defined as direct interactors. Proteins that have been shown to interact with the cytoplasmic domain of CD18 using whole cell lysates in, for example, pull-down experiments are claimed as interacting proteins without evidence for direct interaction. In summary, β2 integrin activation and signalling depend on a specific subset of proteins interacting with CD18 and their precise regulation. If disturbed, profound defects of neutrophil recruitment and activation become evident compromising the innate immune response. The knowledge of proteins interacting with β2 integrins and their regulation during neutrophil trafficking does not only improve our basic understanding of innate immunity but may pave the way to novel therapeutic strategies in the treatment of inflammatory diseases.