Adhesion to fibronectin via α5ß1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis.

Excessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5ß1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5ß1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Absence of endothelial α5ß1 integrin triggers early onset of experimental autoimmune encephalomyelitis due to reduced vascular remodeling and compromised vascular integrity.

Early in the development of multiple sclerosis (MS) and its mouse model experimental autoimmune encephalomyelitis (EAE), vascular integrity is compromised. This is accompanied by a marked vascular remodeling response, though it is currently unclear whether this is an adaptive vascular repair mechanism or is part of the pathogenic process. In light of the well-described angiogenic role for the α5ß1 integrin, the goal of this study was to evaluate how genetic deletion of endothelial α5 integrin (α5-EC-KO mice) impacts vascular remodeling and repair following vascular disruption during EAE pathogenesis, and how this subsequently influences clinical progression and inflammatory demyelination. Immunofluorescence staining revealed that fibronectin and α5 integrin expression were strongly upregulated on spinal cord blood vessels during the pre-symptomatic phase of EAE. Interestingly, α5-EC-KO mice showed much earlier onset and faster progression of EAE, though peak disease severity and chronic disease activity were no different from wild-type mice. At the histological level, earlier disease onset in α5-EC-KO mice correlated with accelerated vascular disruption and increased leukocyte infiltration into the spinal cord. Significantly, spinal cord blood vessels in α5-EC-KO mice showed attenuated endothelial proliferation during the pre-symptomatic phase of EAE which resulted in reduced vascular density at later time-points. Under pro-inflammatory conditions, primary cultures of α5KO brain endothelial cells showed reduced proliferation potential. These findings suggest that α5ß1 integrin-mediated angiogenic remodeling represents an important repair mechanism that counteracts vascular disruption during the early stages of EAE development.

α5ß1 integrin mediates pulmonary epithelial cyst formation.

BACKGROUND: Formation of the epithelial cyst involves the establishment of apical-basolateral polarity through a series of cellular interactions that are in part mediated by the extracellular matrix (ECM). We report that in a three-dimensional multi-cellular self-assembly model of lung development, α5 integrin regulates epithelial cyst formation through organization of soluble fibronectin matrix into insoluble fibrils through a process called fibrillogenesis. RESULTS: Dissociated murine embryonic lung cells self-assemble into three-dimensional pulmonary bodies that are dependent on α5ß1 integrin mediated fibrillogenesis for cell-cell mediated self-assembly: compaction and epithelial cyst formation. Knockdown of α5 integrin resulted in a significant increase in another mediator of fibrillogenesis, αV integrin. Compensatory increased expression of another mediator of fibrillogenesis, αV integrin, was not sufficient to normalize epithelial cyst formation. Loss of α5 integrin-mediated fibrillogenesis perturbed the ability of clustered epithelial cells to establish clear polarity, loss of epithelial cell pyramidal shape, and disrupted apical F-actin-rich deposition. Lack of rich central epithelial localization of F-actin cytoskeleton and Podocalyxin suggests that loss of α5 integrin-mediated fibrillogenesis interferes with the normal cytoskeleton organization that facilitates epithelial cysts polarization. CONCLUSIONS: We conclude that lung epithelial cyst formation in development is mediated in part by α5ß1 integrin dependent fibrillogenesis. Developmental Dynamics 246:475-484, 2016. © 2017 Wiley Periodicals, Inc.

Binding of ZO-1 to α5ß1 integrins regulates the mechanical properties of α5ß1-fibronectin links.

Fundamental processes in cell adhesion, motility, and rigidity adaptation are regulated by integrin-mediated adhesion to the extracellular matrix (ECM). The link between the ECM component fibronectin (fn) and integrin α5ß1 forms a complex with ZO-1 in cells at the edge of migrating monolayers, regulating cell migration. However, how this complex affects the α5ß1-fn link is unknown. Here we show that the α5ß1/ZO-1 complex decreases the resistance to force of α5ß1-fn adhesions located at the edge of migrating cell monolayers while also increasing α5ß1 recruitment. Consistently with a molecular clutch model of adhesion, this effect of ZO-1 leads to a decrease in the density and intensity of adhesions in cells at the edge of migrating monolayers. Taken together, our results unveil a new mode of integrin regulation through modification of the mechanical properties of integrin-ECM links, which may be harnessed by cells to control adhesion and migration.

Clustering of integrin α5 at the lateral membrane restores epithelial polarity in invasive colorectal cancer cells.

Apicobasolateral polarity is a fundamental property of epithelial cells, and its loss is a hallmark of cancer. Integrin-mediated contact with the extracellular matrix defines the basal surface, setting in motion E-cadherin-mediated cell-cell contact, which establishes apicobasolateral polarity. Role(s) for lateral integrins in this polarization process and the consequences of their disruption are incompletely understood. We show that addition of an integrin ß1-activating monoclonal antibody, P4G11, to invasive colorectal cancer cells in three-dimensional type 1 collagen reverts the invasive phenotype and restores apicobasolateral polarity. P4G11 induces clustering of integrin α5ß1 at lateral, intercellular surfaces. This leads to deposition and polymerization of fibronectin and recruitment of paxillin to sites of lateral integrin α5ß1 clustering and is followed by tight junction formation, as determined by ZO-1 localization. Inducible elimination of integrin α5 abrogates the epithelial-organizing effects of P4G11. In addition, polymerization of fibronectin is required for the effects of P4G11, and addition of polymerized superfibronectin is sufficient to induce tight junction formation and apicobasolateral polarization. In the normal human colon, we show that integrin α5 localizes to the lateral membrane of terminally differentiated colonocytes and that integrin α5 staining may be reduced in colorectal cancer. Thus we propose a novel role for integrin α5ß1 in regulating epithelial morphogenesis.

A model for cyclic mechanical reinforcement.

Mechanical force regulates a broad range of molecular interactions in biology. Three types of counterintuitive mechanical regulation of receptor-ligand dissociation have been described. Catch bonds are strengthened by constant forces, as opposed to slip bonds that are weakened by constant forces. The phenomenon that bonds become stronger with prior application of cyclic forces is termed cyclic mechanical reinforcement (CMR). Slip and catch bonds have respectively been explained by two-state models. However, they assume fast equilibration between internal states and hence are inadequate for CMR. Here we propose a three-state model for CMR where both loading and unloading regulate the transition of bonds among the short-lived, intermediate, and long-lived state. Cyclic forces favor bonds in the long-lived state, hence greatly prolonging their lifetimes. The three-state model explains the force history effect and agrees with the experimental CMR effect of integrin α5ß1-fibronectin interaction. This model helps decipher the distinctive ways by which molecular bonds are mechanically strengthened: catch bonds by constant forces and CMR by cyclic forces. The different types of mechanical regulation may enable the cell to fine tune its mechanotransduction via membrane receptors.

Single Molecule Force Measurements in Living Cells Reveal a Minimally Tensioned Integrin State.

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms, yet fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. Here we use Förster resonance energy transfer-based molecular tension sensors to directly measure the distribution of loads experienced by individual integrins in living cells. We find that a large fraction of integrins bear modest loads of 1-3 pN, while subpopulations bearing higher loads are enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site specifically for α5ß1 integrin, leads to increased levels of α5ß1 integrin recruitment to adhesions but not to an increase in overall cellular traction generation. The presence of the synergy site does, however, increase cells' resistance to detachment by externally applied loads. We suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with mechanical integrity in the presence of widely varying mechanical loads.

Insulin-Like Growth Factor Binding Protein-2 Promotes Adhesion of Endothelial Progenitor Cells to Endothelial Cells via Integrin α5ß1.

The contribution of endothelial progenitor cells (EPCs) to new vessel formation has been studied in different physiological and pathological conditions for decades. As previously suggested, insulin-like growth factor binding protein-2 (IGFBP-2) may interact with integrins and promote cell migration. However, the role of IGFBP-2 in regulation of EPC functions remains largely unknown. In this present study, we found that overexpression of IGFBP-2 in human umbilical vein endothelial cells (HUVECs) promoted EPC-endothelial adhesion. Conversely, siRNA-mediated depletion of IGFBP-2 inhibited oxygen-glucose deprivation (OGD)-induced EPC-endothelial adhesion. Further, we demonstrated that the arginine-glycine-aspartic acid (RGD) motif in its C-domain is required for interaction with integrin α5ß1. In addition, treatment with IGFBP-2 significantly enhanced incorporation of EPCs into tubule networks formed by HUVECs. Thus, our findings suggest that exogenous administration of IGFBP-2 may facilitate neovascularization and improve treatment of ischemic conditions.

Mechanism of mast cell adhesion to human tenocytes in vitro.

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5ß1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5ß1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5ß1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin.

Integrin-mediated first signal for inflammasome activation in intestinal epithelial cells.

How intestinal epithelial cells (IECs) recognize pathogens and activate inflammasomes at intestinal surfaces is poorly understood. We hypothesized that IECs use integrin receptors to recognize pathogens and initiate inflammation within the intestinal tract. We find that IECs infected with Yersinia enterocolitica, an enteric pathogen, use ß1 integrins as pathogen recognition receptors detecting the bacterial adhesin invasin (Inv). The Inv-integrin interaction provides the first signal for NLRP3 inflammasome activation with the type three secretion system translocon providing the second signal for inflammasome activation, resulting in release of IL-18. During infection, Yersinia employs two virulence factors, YopE and YopH, to counteract Inv-mediated integrin-dependent inflammasome activation. Furthermore, NLRP3 inflammasome activation in epithelial cells requires components of the focal adhesion complex signaling pathway, focal adhesion kinase, and rac1. The binding of Inv to ß1 integrins rapidly induces IL-18 mRNA expression, suggesting integrins provide a first signal for NLRP3 inflammasome activation. These data suggest integrins function as pathogen recognition receptors on IECs to rapidly induce inflammasome-derived IL-18-mediated responses.

Cyclic mechanical reinforcement of integrin-ligand interactions.

Cells regulate adhesion in response to internally generated and externally applied forces. Integrins connect the extracellular matrix to the cytoskeleton and provide cells with mechanical anchorages and signaling platforms. Here we show that cyclic forces applied to a fibronectin-integrin α5ß1 bond switch the bond from a short-lived state with 1 s lifetime to a long-lived state with 100 s lifetime. We term this phenomenon "cyclic mechanical reinforcement," as the bond strength remembers the history of force application and accumulates over repeated cycles, but does not require force to be sustained. Cyclic mechanical reinforcement strengthens the fibronectin-integrin α5ß1 bond through the RGD binding site of the ligand with the synergy binding site greatly facilitating the process. A flexible integrin hybrid domain is also important for cyclic mechanical reinforcement. Our results reveal a mechanical regulation of receptor-ligand interactions and identify a molecular mechanism for cell adhesion strengthening by cyclic forces.

TßRIII/ß-arrestin2 regulates integrin α5ß1 trafficking, function, and localization in epithelial cells.

The type III TGF-ß receptor (TßRIII) is a ubiquitous co-receptor for TGF-ß superfamily ligands with roles in suppressing cancer progression, in part through suppressing cell motility. Here we demonstrate that TßRIII promotes epithelial cell adhesion to fibronectin in a ß-arrestin2 dependent and TGF-ß/BMP independent manner by complexing with active integrin α5ß1, and mediating ß-arrestin2-dependent α5ß1 internalization and trafficking to nascent focal adhesions. TßRIII-mediated integrin α5ß1 trafficking regulates cell adhesion and fibronectin fibrillogenesis in epithelial cells, as well as α5 localization in breast cancer patients. We further demonstrate that increased TßRIII expression correlates with increased α5 localization at sites of cell-cell adhesion in breast cancer patients, while higher TßRIII expression is a strong predictor of overall survival in breast cancer patients. These data support a novel, clinically relevant role for TßRIII in regulating integrin α5 localization, reveal a novel crosstalk mechanism between the integrin and TGF-ß superfamily signaling pathways and identify ß-arrestin2 as a regulator of α5ß1 trafficking.

Cell adhesion: a FERM grasp of the tail sorts out integrins.

As well as modulating integrin activation, a conserved NPxY motif in integrin cytoplasmic tails that binds the FERM-domain-containing proteins kindlin and sorting nexin 17 plays pivotal roles in integrin recycling and degradation.

Distinct roles of talin and kindlin in regulating integrin α5ß1 function and trafficking.

BACKGROUND: Integrins are heterodimeric αß transmembrane receptors that play key roles in cellular physiology and pathology. Accumulating data indicate that the two NPxY motifs in the cytoplasmic domain of the ß1 subunit synergistically promote integrin activation through the binding of talin and kindlin. However, it is unclear how the individual motifs regulate integrin function and trafficking. RESULTS: To investigate how the two NPxY motifs individually control integrin α5ß1 function and trafficking, we introduced Y > A mutations in either motif. Disruption of the membrane-proximal NPxY completely prevented α5ß1-induced morphological changes, cell scattering and migration, and fibronectin fibrillogenesis. In addition, it reduced α5ß1 internalization but not its recycling. In contrast, disruption of the membrane-distal NPxY promoted degradation of α5ß1 in late endosomes/lysosomes but did not prevent α5ß1-dependent cell scattering, migration, or fibronectin fibrillogenesis. Whereas depletion of either talin-1 or kindlin-2 reduced α5ß1 binding to fibronectin and cell adhesion, talin-1 depletion recapitulated the loss-of-function phenotype of the membrane-proximal NPxY mutation, whereas kindlin-2 depletion induced α5ß1 accumulation in lysosomes and degradation. CONCLUSIONS: The two NPxY motifs of ß1 play distinct and separable roles in controlling the function and trafficking of α5ß1. Whereas talin binding to the membrane-proximal NPxY is crucial for connecting α5ß1 to the actin cytoskeleton and thus permit the tension required for fibronectin fibrillogenesis and cell migration, kindlin binding to the membrane-distal NPxY is dispensable for these events but regulates α5ß1 surface expression and degradation.

Integrin α5ß1 plays a critical role in resistance to temozolomide by interfering with the p53 pathway in high-grade glioma.

Integrins play a role in the resistance of advanced cancers to radiotherapy and chemotherapy. In this study, we show that high expression of the α5 integrin subunit compromises temozolomide-induced tumor suppressor p53 activity in human glioblastoma cells. We found that depletion of the α5 integrin subunit increased p53 activity and temozolomide sensitivity. However, when cells were treated with the p53 activator nutlin-3a, the protective effect of α5 integrin on p53 activation and cell survival was lost. In a functional p53 background, nutlin-3a downregulated the α5 integrin subunit, thereby increasing the cytotoxic effect of temozolomide. Clinically, α5ß1 integrin expression was associated with a more aggressive phenotype in brain tumors, and high α5 integrin gene expression was associated with decreased survival of patients with high-grade glioma. Taken together, our findings indicate that negative cross-talk between α5ß1 integrin and p53 supports glioma resistance to temozolomide, providing preclinical proof-of-concept that α5ß1 integrin represents a therapeutic target for high-grade brain tumors. Direct activation of p53 may remain a therapeutic option in the subset of patients with high-grade gliomas that express both functional p53 and a high level of α5ß1 integrin.

Titanium alloy surface oxide modulates the conformation of adsorbed fibronectin to enhance its binding to α(5) ß(1) integrins in osteoblasts.

Our laboratory has previously demonstrated that heat (600°C) or radiofrequency plasma glow discharge (RFGD) pretreatment of a titanium alloy (Ti6Al4V) increased the net negative charge of the alloy's surface oxide and the attachment of osteoblastic cells to adsorbed fibronectin. The purpose of the current study was to investigate the biological mechanism by which these surface pretreatments enhance the capacity of fibronectin to stimulate osteoblastic cell attachment. Each pretreatment was found to increase the binding (measured by ELISA) of a monoclonal anti-fibronectin Ig to the central integrin-binding domain of adsorbed fibronectin, and to increase the antibody's inhibition of osteogenic cell attachment (measured by hexosaminidase assay). Pretreatments also increased the binding (measured by ELISA) of anti-integrin IgG's to the α(5) and ß(1) integrin subunits that became attached to fibronectin during cell incubation. These findings suggest that negatively charged surface oxides of Ti6Al4V cause conformational changes in fibronectin that increase the availability of its integrin-binding domain to α(5) ß(1) integrins.

Mechanical stress-activated integrin α5ß1 induces opening of connexin 43 hemichannels.

The connexin 43 (Cx43) hemichannel (HC) in the mechanosensory osteocytes is a major portal for the release of factors responsible for the anabolic effects of mechanical loading on bone formation and remodeling. However, little is known about how the Cx43 molecule responds to mechanical stimulation leading to the opening of the HC. Here, we demonstrate that integrin α5ß1 interacts directly with Cx43 and that this interaction is required for mechanical stimulation-induced opening of the Cx43 HC. Direct mechanical perturbation via magnetic beads or conformational activation of integrin α5ß1 leads to the opening of the Cx43 HC, and this role of the integrin is independent of its association with an extracellular fibronectin substrate. PI3K signaling is responsible for the shear stress-induced conformational activation of integrin α5ß1 leading to the opening of the HC. These results identify an unconventional function of integrin that acts as a mechanical tether to induce opening of the HC and provide a mechanism connecting the effect of mechanical forces directly to anabolic function of the bone.

Rv2468c, a novel Mycobacterium tuberculosis protein that costimulates human CD4+ T cells through VLA-5.

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5ß1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.

Fibronectin-integrin (alpha5beta1) modulates migration and invasion of murine melanoma cell line B16F10 by involving MMP-9.

Cell adhesion to extracellular matrix (ECM) initiates signaling cascade regulated by cell surface integrin receptors, which affects the proliferation and invasion of cells. Cells cultured in the presence of ECM ligand fibronectin (FN) stimulate secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion and metastasis. Among all the members of the MMP family, MMP-9 is of crucial importance in tumor invasion and metastasis. The present study aims at studying the effects of integrin receptor alpha5beta1 and its ligand FN on expression of MMP-9 in murine melanoma cell line B16F10 and understanding the molecular mechanism(s) involved. The main experimental methods performed in the study were gelatin zymography, immunoblot, real-time RT-PCR, immunocytochemistry, enzyme linked immunosorbent assay (ELISA), transwell chamber assay, and in vivo metastasis assay in syngenic (C57BL6J) mice. The study reports that FN induces the activity, mRNA, and protein expression of MMP-9 and initiates its proteolytic activation in B16F10 cells. Blockage of the alpha5 receptor abrogated the FN-mediated stimulatory response on MMP-9 in B16F10 cells. Inhibitor studies and immunoblot analysis strongly suggest the involvement of focal adhesion kinase (FAK), extracellular regulated kinase (ERK), and phosphatidylinositol-3-kinase (PI-3K) in the FN-mediated responses. Immunocytochemical analysis showed the nuclear localization of nuclear factor-kappaB (NF-kappaB) might lead to activation of MMP-9 gene upon FN treatment. This study demonstrates that integrin receptor alpha5beta1 and FN interaction induces the invasive potential of B16F10 cells and MMP-9 induction is the downstream effectors in the process. This system serves as a novel model system to understand the molecular mechanism of melanoma growth and invasion.

Macrophage motility requires distinct α5ß1/FAK and α4ß1/paxillin signaling events.

Macrophages function as key inflammatory mediators at sites of infection and tissue damage. Integrin and growth factor receptors facilitate recruitment of monocytes/macrophages to sites of inflammation in response to numerous extracellular stimuli. We have shown recently that FAK plays a role in regulating macrophage chemotaxis and invasion. As FAK is an established downstream mediator of integrin signaling, we sought to define the molecular circuitry involving FAK and the predominant ß1 integrin heterodimers expressed in these cells-α4ß1 and α5ß1. We show that α4ß1 and α5ß1 integrins are required for efficient haptotactic and chemotactic invasion and that stimulation of these integrin receptors leads to the adoption of distinct morphologies associated with motility. FAK is required downstream of α5ß1 for haptotaxis toward FN and chemotaxis toward M-CSF-1 and downstream of α4ß1 for the adoption of a polarized phenotype. The scaffolding molecule paxillin functions independently of FAK to promote chemotaxis downstream of α4ß1. These studies expand our understanding of ß1 integrin signaling networks that regulate motility and invasion in macrophages and thus, provide important new insights into mechanisms by which macrophages perform their diverse functions.