The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Components

TLR2 is a cell surface receptor which elicits an immediate response to a wide repertoire of bacteria and viruses. Its response is usually thought to be proinflammatory rather than an antiviral. In monocytic cells TLR2 cooperates with coreceptors, e.g. CD14, CD36 and αMβ2-integrin. In an earlier work we showed that αvβ3-integrin acts in concert with TLR2 to elicit an innate response to HSV, and to lipopolysaccharide. This response is characterized by production of IFN-α and -β, a specific set of cytokines, and NF-κB activation. We investigated the basis of the cooperation between αvβ3-integrin and TLR2. We report that β3-integrin participates by signaling through Y residues located in the C-tail, known to be involved in signaling activity. αvβ3-integrin boosts the MYD88-dependent TLR2 signaling and IRAK4 phosphorylation in 293T and in epithelial, keratinocytic and neuronal cell lines. The replication of ICP0minus HSV is greatly enhanced by DN versions of MYD88, of Akt – a hub of this pathway, or by β3integrin-silencing. αvβ3-integrin enables the recruitment of TLR2, MAL, MYD88 at lipid rafts, the platforms from where the signaling starts. The PAMP of the HSV-induced innate response is the gH/gL virion glycoprotein, which interacts with αvβ3-integrin and TLR2 independently one of the other, and cross-links the two receptors. Given the preferential distribution of αvβ3-integrin to epithelial cells, we propose that αvβ3-integrin serves as coreceptor of TLR2 in these cells. The results open the possibility that TLR2 makes use of coreceptors in a variety of cells to broaden its spectrum of activity and tissue specificity.     

Growth Factor–dependent Activation of αvβ3 Integrin in Normal Epithelial Cells: Implications for Tumor Invasion

Integrin activation is a multifaceted phenomenon leading to increased affinity and avidity for matrix ligands. To investigate whether cytokines produced during stromal infiltration of carcinoma cells activate nonfunctional epithelial integrins, a cellular system of human thyroid clones derived from normal glands (HTU-5) and papillary carcinomas (HTU-34) was employed. In HTU-5 cells, αvβ3 integrin was diffused all over the membrane, disconnected from the cytoskeleton, and unable to mediate adhesion. Conversely, in HTU-34 cells, αvβ3 was clustered at focal contacts (FCs) and mediated firm attachment and spreading. αvβ3 recruitment at FCs and ligand-binding activity, essentially identical to those of HTU-34, occurred in HTU-5 cells upon treatment with hepatocyte growth factor/scatter factor (HGF/SF). The HTU-34 clone secreted HGF/SF and its receptor was constitutively tyrosine phosphorylated suggesting an autocrine loop responsible for αvβ3 activated state. Antibody-mediated inhibition of HGF/SF function in HTU-34 cells disrupted αvβ3 enrichment at FCs and impaired adhesion. Accordingly, activation of αvβ3 in normal cells was produced by HTU-34 conditioned medium on the basis of its content of HGF/SF. These results provide the first example of a growth factor–driven integrin activation mechanism in normal epithelial cells and uncover the importance of cytokine-based autocrine loops for the physiological control of integrin activation.     

Integrin αIIbβ3 Transmembrane Domain Separation Mediates Bi-Directional Signaling across the Plasma Membrane

Integrins play an essential role in hemostasis, thrombosis, and cell migration, and they transmit bidirectional signals. Transmembrane/cytoplasmic domains are hypothesized to associate in the resting integrins; whereas, ligand binding and intracellular activating signals induce transmembrane domain separation. However, how this conformational change affects integrin outside-in signaling and whether the α subunit cytoplasmic domain is important for this signaling remain elusive. Using Chinese Hamster Ovary (CHO) cells that stably expressed different integrin α_IIbβ₃ constructs, we discovered that an α_IIb cytoplasmic domain truncation led to integrin activation but not defective outside-in signaling. In contrast, preventing transmembrane domain separation abolished both inside-out and outside-in signaling regardless of removing the α_IIb cytoplasmic tail. Truncation of the α_IIb cytoplasmic tail did not obviously affect adhesion-induced outside-in signaling. Our research revealed that transmembrane domain separation is a downstream conformational change after the cytoplasmic domain dissociation in inside-out activation and indispensable for ligand-induced outside-in signaling. The result implicates that the β TM helix rearrangement after dissociation is essential for integrin transmembrane signaling. Furthermore, we discovered that the PI3K/Akt pathway is not essential for cell spreading but spreading-induced Erk1/2 activation is PI3K dependent implicating requirement of the kinase for cell survival in outside-in signaling.     

Type I Interferon and NF-κB Activation Elicited by Herpes Simplex Virus gH/gL via αvβ3 Integrin in Epithelial and Neuronal Cell Lines

αvβ3 integrin represents a novel sensing system which detects herpes simplex virus (HSV) and bacterial constituents. In cooperation with Toll-like receptor 2 (TLR2), it elicits an innate response that leads to activation of type I interferon (IFN), NF-κB, and a specific set of cytokines. We report that this defensive branch is functional in cells which represent experimental models of epithelial, including keratinocytic, and neuronal cells. These are the major targets of HSV in vivo. HSV entered the three cell lines via distinct routes. Hence, the defensive response was independent of the route of virus entry. Soluble gH/gL sufficed to elicit type I IFN and NF-κB activation and represents the viral pathogen-associated molecular pattern (PAMP) of this defense system.     

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy.     

MSH Receptors and the Response of Human A375 Melanoma Cells to Interleukin-1β

Abstract

α-Melanocyte-stimulating hormone (α-MSH, α-melanotropin) has been shown to be an inhibitory factor in many immunologic and inflammatory processes involving the cytokine interleukin-1 (IL-1). As the mechanism of the interaction between IL-1 and α-MSH at the receptor level is unknown, we have studied the role of MC1 melanocortin receptors in two variants of the human melanoma cell line A375 differing in their sensitivity to the cytostatic effects of IL-1β. Both IL-1 sensitive (A375r-) and resistant cells (A375r+) carry specific high affinity receptors for IL-1, albeit their concentration is 10-fold higher in A375r+ cells. In A375r- cells, MC1 receptors are absent or below the level for reliable detection in the binding assay. Conversion of A375r- to A375r+ cells by prolonged culture in medium not depleted of endotoxin led to the appearance of MC1 receptors (K_D 0.4 ± 0.123 nmol/l; 608 ± 134 receptors/cell). Stable transfection of A375r- cells with the human MC1 receptor did not, however, render them resistant to the cytostatic effect of IL-1β on concomitant treatment with α-MSH or result in the production of IL-6 on treatment with IL-1β Therefore, the presence of MC1 receptors on the surface of A375 cells or their binding to α-MSH does not seem to be a factor in cytokine resistance or IL-6 secretion. No interaction between IL-1β and α-MSH could be demonstrated at the cellular level in this melanoma cell line.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

A Role for the αvβ3 Integrin in the Transmigration of Monocytes

The β2 integrins and intercellular adhesion molecule-1 (ICAM-1) are important for monocyte migration through inflammatory endothelium. Here we demonstrate that the integrin αvβ3 is also a key player in this process. In an in vitro transendothelial migration assay, monocytes lacking β3 integrins revealed weak migratory ability, whereas monocytes expressing β3 integrins engaged in stronger migration. This migration could be partially blocked by antibodies against the integrin chains αL, β2, αv, or IAP, a protein functionally associated with αvβ3 integrin. Transfection of β3 integrin chain cDNA into monocytes lacking β3 integrins resulted in expression of the αvβ3 integrin and conferred on these cells an enhanced ability to transmigrate through cell monolayers expressing ICAM-1. These monocytes also engaged in αLβ2-dependent locomotion on recombinant ICAM-1 which was enhanced by αvβ3 integrin occupancy. Antibodies against IAP were able to revert this αvβ3 integrin-dependent cell locomotion to control levels. Finally, adhesion assays revealed that occupancy of αvβ3 integrin could decrease monocyte binding to ICAM-1.In conclusion, we show that αvβ3 integrin modulates αLβ2 integrin-dependent monocyte adhesion to and migration on ICAM-1. This could represent a novel mechanism to promote monocyte motility on vascular ICAM-1 and initiate subsequent transendothelial migration.     

HIV gp120 Induces Mucus Formation in Human Bronchial Epithelial Cells through CXCR4/α7-Nicotinic Acetylcholine Receptors

Lung diseases such as chronic obstructive pulmonary disease (COPD), asthma, and lung infections are major causes of morbidity and mortality among HIV-infected patients even in the era of antiretroviral therapy (ART). Many of these diseases are strongly associated with smoking and smoking is more common among HIV-infected than uninfected people; however, HIV is an independent risk factor for chronic bronchitis, COPD, and asthma. The mechanism by which HIV promotes these diseases is unclear. Excessive airway mucus formation is a characteristic of these diseases and contributes to airway obstruction and lung infections. HIV gp120 plays a critical role in several HIV-related pathologies and we investigated whether HIV gp120 promoted airway mucus formation in normal human bronchial epithelial (NHBE) cells. We found that NHBE cells expressed the HIV-coreceptor CXCR4 but not CCR5 and produced mucus in response to CXCR4-tropic gp120. The gp120-induced mucus formation was blocked by the inhibitors of CXCR4, α7-nicotinic acetylcholine receptor (α7-nAChR), and γ-aminobutyric acid (GABA)_AR but not the antagonists of CCR5 and epithelial growth factor receptor (EGFR). These results identify two distinct pathways (α7-nAChR-GABA_AR and EGFR) for airway mucus formation and demonstrate for the first time that HIV-gp120 induces and regulates mucus formation in the airway epithelial cells through the CXCR4-α7-nAChR-GABA_AR pathway. Interestingly, lung sections from HIV ± ART and simian immunodeficiency virus (SIV) ± ART have significantly more mucus and gp120-immunoreactivity than control lung sections from humans and macaques, respectively. Thus, even after ART, lungs from HIV-infected patients contain significant amounts of gp120 and mucus that may contribute to the higher incidence of obstructive pulmonary diseases in this population.     

BMP-7 Enhances Cell Migration and αvβ3 Integrin Expression via a c-Src-Dependent Pathway in Human Chondrosarcoma Cells

Bone morphogenic protein (BMP)-7 is a member of the transforming growth factor (TGF)-beta superfamily, which is originally identified based on its ability to induce cartilage and bone formation. In recent years, BMP-7 is also defined as a potent promoter of cell motility, invasion, and metastasis. However, there is little knowledge of the role of BMP-7 and its cellular function in chondrosarcoma cells. In the present study, we investigated the biological impact of BMP-7 on cell motility using transwell assay. In addition, the intracellular signaling pathways were also investigated by pharmacological and genetic approaches. Our results demonstrated that treatment with exogenous BMP-7 markedly increased cell migration by activating c-Src/PI3K/Akt/IKK/NF-κB signaling pathway, resulting in the transactivation of αvβ3 integrin expression. Indeed, abrogation of signaling activation, by chemical inhibition or expression of a kinase dead form of the protein attenuated BMP-7-induced expression of integrin αvβ3 and cell migration. These findings may provide a useful tool for diagnostic/prognostic purposes and even therapeutically in late-stage chondrosarcoma as an anti-metastatic agent.     

N-Ethylmaleimide-sensitive Factor Attachment Protein α (αSNAP) Regulates Matrix Adhesion and Integrin Processing in Human Epithelial Cells

Integrin-based adhesion to the extracellular matrix (ECM) plays critical roles in controlling differentiation, survival, and motility of epithelial cells. Cells attach to the ECM via dynamic structures called focal adhesions (FA). FA undergo constant remodeling mediated by vesicle trafficking and fusion. A soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is an essential mediator of membrane fusion; however, its roles in regulating ECM adhesion and cell motility remain unexplored. In this study, we found that siRNA-mediated knockdown of αSNAP induced detachment of intestinal epithelial cells, whereas overexpression of αSNAP increased ECM adhesion and inhibited cell invasion. Loss of αSNAP impaired Golgi-dependent glycosylation and trafficking of β1 integrin and decreased phosphorylation of focal adhesion kinase (FAK) and paxillin resulting in FA disassembly. These effects of αSNAP depletion on ECM adhesion were independent of apoptosis and NSF. In agreement with our previous reports that Golgi fragmentation mediates cellular effects of αSNAP knockdown, we found that either pharmacologic or genetic disruption of the Golgi recapitulated all the effects of αSNAP depletion on ECM adhesion. Furthermore, our data implicates β1 integrin, FAK, and paxillin in mediating the observed pro-adhesive effects of αSNAP. These results reveal novel roles for αSNAP in regulating ECM adhesion and motility of epithelial cells.     

αvβ6- and αvβ8-Integrins Serve As Interchangeable Receptors for HSV gH/gL to Promote Endocytosis and Activation of Membrane Fusion

Herpes simplex virus (HSV) - and herpesviruses in general - encode for a multipartite entry/fusion apparatus. In HSV it consists of the HSV-specific glycoprotein D (gD), and three additional glycoproteins, gH/gL and gB, conserved across the Herpesviridae family and responsible for the execution of fusion. According to the current model, upon receptor binding, gD propagates the activation to gH/gL and to gB in a cascade fashion. Questions remain about how the cascade of activation is controlled and how it is synchronized with virion endocytosis, to avoid premature activation and exhaustion of the glycoproteins. We considered the possibility that such control might be carried out by as yet unknown receptors. Indeed, receptors for HSV gB, but not for gH/gL, have been described. In other members of the Herpesviridae family, such as Epstein-Barr virus, integrin receptors bind gH/gL and trigger conformational changes in the glycoproteins. We report that αvβ6- and αvβ8-integrins serve as receptors for HSV entry into experimental models of keratinocytes and other epithelial and neuronal cells. Evidence rests on loss of function experiments, in which integrins were blocked by antibodies or silenced, and gain of function experiments in which αvβ6-integrin was expressed in integrin-negative cells. αvβ6- and αvβ8-integrins acted independently and are thus interchangeable. Both bind gH/gL with high affinity. The interaction profoundly affects the route of HSV entry and directs the virus to acidic endosomes. In the case of αvβ8, but not αvβ6-integrin, the portal of entry is located at lipid microdomains and requires dynamin 2. Thus, a major role of αvβ6- or αvβ8-integrin in HSV infection appears to be to function as gH/gL receptors and to promote virus endocytosis. We propose that placing the gH/gL activation under the integrin trigger point enables HSV to synchronize virion endocytosis with the cascade of glycoprotein activation that culminates in execution of fusion.     

Prostate cancer sheds the αvβ3 integrin in vivo through exosomes

The αvβ3 integrin has been shown to promote aggressive phenotypes in many types of cancers, including prostate cancer. We show that GFP-labeled αvβ3 derived from cancer cells circulates in the blood and is detected in distant lesions in NOD scid gamma (NSG) mice. We, therefore, hypothesized that αvβ3 travels through exosomes and tested its levels in pools of vesicles, which we designate extracellular vesicles highly enriched in exosomes (ExVs), and in exosomes isolated from the plasma of prostate cancer patients. Here, we show that the αvβ3 integrin is found in patient blood exosomes purified by sucrose or iodixanol density gradients. In addition, we provide evidence that the αvβ3 integrin is transferred through ExVs isolated from prostate cancer patient plasma to β3-negative recipient cells. We also demonstrate the intracellular localization of β3-GFP transferred via cancer cell-derived ExVs. We show that the ExVs present in plasma from prostate cancer patients contain higher levels of αvβ3 and CD9 as compared to plasma ExVs from age-matched subjects who are not affected by cancer. Furthermore, using PSMA antibody-bead mediated immunocapture, we show that the αvβ3 integrin is expressed in a subset of exosomes characterized by PSMA, CD9, CD63, and an epithelial-specific marker, Trop-2. Finally, we present evidence that the levels of αvβ3, CD63, and CD9 remain unaltered in ExVs isolated from the blood of prostate cancer patients treated with enzalutamide. Our results suggest that detecting exosomal αvβ3 integrin in prostate cancer patients could be a clinically useful and non-invasive biomarker to follow prostate cancer progression. Moreover, the ability of αvβ3 integrin to be transferred from ExVs to recipient cells provides a strong rationale for further investigating the role of αvβ3 integrin in the pathogenesis of prostate cancer and as a potential therapeutic target.     

PKD Controls αvβ3 Integrin Recycling and Tumor Cell Invasive Migration through Its Substrate Rabaptin-5

Integrin recycling is critical for cell migration. Protein kinase D (PKD) mediates signals from the platelet-derived growth factor receptor (PDGF-R) to control αvβ3 integrin recycling. We now show that Rabaptin-5, a Rab5 effector in endosomal membrane fusion, is a PKD substrate. PKD phosphorylates Rabaptin-5 at Ser407, and this is both necessary and sufficient for PDGF-dependent short-loop recycling of αvβ3, which in turn inhibits α5β1 integrin recycling. Rab4, but not Rab5, interacts with phosphorylated Rabaptin-5 toward the front of migrating cells to promote delivery of αvβ3 to the leading edge, thereby driving persistent cell motility and invasion that is dependent on this integrin. Consistently, disruption of Rabaptin-5 Ser407 phosphorylation reduces persistent cell migration in 2D and αvβ3-dependent invasion. Conversely, invasive migration that is dependent on α5β1 integrin is promoted by disrupting Rabaptin phosphorylation. These findings demonstrate that the PKD pathway couples receptor tyrosine kinase signaling to an integrin switch via Rabaptin-5 phosphorylation.     

PAR-3 Knockdown Enhances Adhesion Rate of PANC-1 Cells via Increased Expression of Integrinαv and E-Cadherin

The balance between the adhesion of cancer cells to extracellular matrix and their migratory potential, as well as their proteolytic activity, are important parameters that determine cancer cells invasiveness and metastasis. Since thrombin has been implicated in cancer progression, we studied the role(s) of thrombin-activated receptors in the adhesion process. We stably knocked down proteinase-activated receptors (PARs) -1, or -3 in human pancreatic adenocarcinoma PANC-1 cells. PANC-1 cells exhibit rapid adhesion to cell culture treated plastic and much faster kinetics of adhesion to Matrigel coated surface. Knockdown of PAR-1 had no effect on cells'' adhesiveness, while PAR-3 knockdowns (KDs) exhibited much faster adhesion kinetics. PAR-3 KDs also exhibited slower in vitro wound closure than vector-control and PAR-1 KD cells. To study the molecular mechanism(s) of PAR-3 KD cells'' enhanced rate of adhesion, we assayed the expression of the molecules that mediate cell-surface and cell-cell adhesion. ITGαv, as well as ITGα6 and ITGα10 mRNAs, were greatly enriched (>40-fold) in a rapidly-adhering sub-population of PAR-3 KD cells. The whole population of both PAR-1 and -3 KDs exhibited enhanced expression of a number of integrins (ITGs) mRNAs. However, ITGαv mRNA and protein expression was increased in PAR-3 KD and markedly decreased in PAR-1 KD. PAR-3 KD cells also expressed more E-cadherin mRNA and protein. The enhanced adhesion kinetics of PAR-3 KDs was almost fully inhibited by calcium chelation, or by a HAV-motive decapeptide that affects E-cadherin intermolecular interactions. We propose that the enhanced rate of adhesion of PAR-3 KDs results from enhanced expression of E-cadherin, leading to a greater adhesion of free-floating cells to cells rapidly bound to the surface via their integrins, and particularly ITGαv.