Gα13 Switch Region 2 Binds to the Talin Head Domain and Activates αIIbβ3 Integrin in Human Platelets

Even though GPCR signaling in human platelets is directly involved in hemostasis and thrombus formation, the sequence of events by which G protein activation leads to αIIbβ3 integrin activation (inside-out signaling) is not clearly defined. We previously demonstrated that a conformationally sensitive domain of one G protein, i.e. Gα₁₃ switch region 1 (Gα₁₃SR1), can directly participate in the platelet inside-out signaling process. Interestingly however, the dependence on Gα₁₃SR1 signaling was limited to PAR1 receptors, and did not involve signaling through other important platelet GPCRs. Based on the limited scope of this involvement, and the known importance of G₁₃ in hemostasis and thrombosis, the present study examined whether signaling through another switch region of G₁₃, i.e. Gα₁₃ switch region 2 (Gα₁₃SR2) may represent a more global mechanism of platelet activation. Using multiple experimental approaches, our results demonstrate that Gα₁₃SR2 forms a bi-molecular complex with the head domain of talin and thereby promotes β3 integrin activation. Moreover, additional studies provided evidence that Gα₁₃SR2 is not constitutively associated with talin in unactivated platelets, but becomes bound to talin in response to elevated intraplatelet calcium levels. Collectively, these findings provide evidence for a novel paradigm of inside-out signaling in platelets, whereby β3 integrin activation involves the direct binding of the talin head domain to the switch region 2 sequence of the Gα₁₃ subunit.     

The Novel Recognition Site in the C-Terminal Heparin-Binding Domain of Fibronectin by Integrin α4β1 Receptor on HL-60 Cells

The hematopoietic cell recognition sites of human fibronectin (FN) are the Arg–Gly–Asp–Ser (RGDS) sequence recognized by widely distributed integrin receptor α5β1 and the type III connecting segment (III CS) containing two cell-binding sites, designated CS1 and CS5, that are recognized by the α4β1 receptor. The C-terminal heparin-binding domain of FN (Hep II) has recently been demonstrated to support adhesion of α4β1-dependent melanoma cells [A. P. Mould and M. J. Humphries (1991)EMBO J.10, 4089–4095]. Previously we demonstrated that this region of FN mediated binding of FN to HL-60 cells (acute promyelocytic leukemia cell line) by direct interaction independently of RGD and CS1 [H. Fujitaet al.,(1995)Exp. Cell Res.217, 484–488]. In this study we have characterized a novel site in the Hep II region for binding to HL-60 cells. α4β1 and α5β1 were expressed on HL-60 cells, while α2β1 and α3β1 were not present, as shown by flow cytometry using monoclonal antibodies specific for the different integrins. Anti-α4β1 (P4C2) and anti-β1 (JB1a) antibodies inhibited binding of a 29-kDa dispase-digestive fragment of FN to HL-60 cells. This fragment contains the C-terminal heparin-binding domain of FN but lacks CS1 and CS5. Only the peptide representing the sequence from Val¹⁸⁶⁶to Arg¹⁸⁸⁰, designated E1, inhibited the binding of the 29-kDa fragment to HL-60 cells. The active region of this peptide was a sequence of Thr–Asp–Ile–Asp–Ala–Pro–Ser (TAI- DAPS), which is homologous to Leu–Asp–Val–Pro–Ser (LDVPS) derived from the active site of CS1. Furthermore, labeled E1 peptide directly bound to HL-60 cells. The anti-α4β1 antibody (P4C2) inhibited this interaction. These results indicate that the site of binding to hematopoietic cells is present in the Hep II region of FN and the definition of the chemical structure of FN clarifies a fundamental mechanism of cell invasion of the extracellular matrix.     

Release of cAMP Gating by the α6β4 Integrin Stimulates Lamellae Formation and the Chemotactic Migration of Invasive Carcinoma Cells

The α6β4 integrin promotes carcinoma in-vasion by its activation of a phosphoinositide 3-OH (PI3-K) signaling pathway (Shaw, L.M., I. Rabinovitz, H.H.-F. Wang, A. Toker, and A.M. Mercurio. Cell. 91: 949–960). We demonstrate here using MDA-MB-435 breast carcinoma cells that α6β4 stimulates chemotactic migration, a key component of invasion, but that it has no influence on haptotaxis. Stimulation of chemotaxis by α6β4 expression was observed in response to either lysophosphatidic acid (LPA) or fibroblast conditioned medium. Moreover, the LPA-dependent formation of lamellae in these cells is dependent upon α6β4 expression. Both lamellae formation and chemotactic migration are inhibited or “gated” by cAMP and our results reveal that a critical function of α6β4 is to suppress the intracellular cAMP concentration by increasing the activity of a rolipram-sensitive, cAMP-specific phosphodiesterase (PDE). This PDE activity is essential for lamellae formation, chemotactic migration and invasion based on data obtained with PDE inhibitors. Although PI3-K and cAMP-specific PDE activities are both required to promote lamellae formation and chemotactic migration, our data indicate that they are components of distinct signaling pathways. The essence of our findings is that α6β4 stimulates the chemotactic migration of carcinoma cells through its ability to influence key signaling events that underlie this critical component of carcinoma invasion.     

Loss of the TGFβ-Activating Integrin αvβ8 on Dendritic Cells Protects Mice from Chronic Intestinal Parasitic Infection via Control of Type 2 Immunity

Chronic intestinal parasite infection is a major global health problem, but mechanisms that promote chronicity are poorly understood. Here we describe a novel cellular and molecular pathway involved in the development of chronic intestinal parasite infection. We show that, early during development of chronic infection with the murine intestinal parasite Trichuris muris, TGFβ signalling in CD4+ T-cells is induced and that antibody-mediated inhibition of TGFβ function results in protection from infection. Mechanistically, we find that enhanced TGFβ signalling in CD4+ T-cells during infection involves expression of the TGFβ-activating integrin αvβ8 by dendritic cells (DCs), which we have previously shown is highly expressed by a subset of DCs in the intestine. Importantly, mice lacking integrin αvβ8 on DCs were completely resistant to chronic infection with T. muris, indicating an important functional role for integrin αvβ8-mediated TGFβ activation in promoting chronic infection. Protection from infection was dependent on CD4+ T-cells, but appeared independent of Foxp3+ Tregs. Instead, mice lacking integrin αvβ8 expression on DCs displayed an early increase in production of the protective type 2 cytokine IL-13 by CD4+ T-cells, and inhibition of this increase by crossing mice to IL-4 knockout mice restored parasite infection. Our results therefore provide novel insights into how type 2 immunity is controlled in the intestine, and may help contribute to development of new therapies aimed at promoting expulsion of gut helminths.     

Adhesion of Lymphoma Cells to Fibronectin: Differential Use of α4β1and α5β1Integrins and Stimulation by the 9EG7 mAb against the Murine β1Integrin Subunit

Murine ESb and MDAY-D2 lymphoma cells are highly metastatic, in particular to the liver, and are highly invasive in hepatocyte cultures. This may involve adhesion to hepatocyte surface-associated fibronectin (Kemperman et al., 1994, Cell Adh. and Communic. 2:45). Both ESb and MDAY-D2 cells express the fibronectin receptor α₄β₁, and MDAY-D2 cells in addition also α₅β₁. Yet, adhesion of ESb cells to fibronectin was low, and MDAY-D2 cells did not adhere at all, but adhesion of both cells was stimulated by phorbol myristate acetate (PMA) and Mn²⁺. In ESb cells, this adhesion was mediated by α₄β₁. In MDAY-D2 cells, however, only α₅β₁was involved, despite β₄β₁levels similar to ESb cells. The α₄β₁integrin was functional since it mediated adhesion of MDAY-D2 cells to VCAM-1. An α₅β₁-negative variant of MDAY-D2 adhered to fibronectin and this was mediated by α₅β₁. These results indicate that α₄β₁function in these cells is suppressed in the presence of α₅β₁. Adhesion of ESb cells to hepatocytes was inhibited by anti-α₄antibody, but only by 30%, and fibronectin adhesion was found to have no role in the interaction of MDAY-D2 cells with hepatocytes. This suggests that α₄β₁and α₅β₁function is not activated during this interaction.

The 9EG7 antibody against mouse β₁integrin was described to inhibit β₁integrins (Lenter et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 9051). In contrast, we observed that β₁stimulated Printegrin function: Adhesion of ESb and MDAY-D2 cells not only to fibronectin, but also to laminin was induced or enhanced.     

α-Mangostin Suppresses Phorbol 12-myristate 13-acetate-Induced MMP-2/MMP-9 Expressions via αvβ3 Integrin/FAK/ERK and NF-κB Signaling Pathway in Human Lung Adenocarcinoma A549 Cells

The purpose of this study is to investigate the anti-metastatic effect of α-mangostin on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expressions in A549 human lung adenocarcinoma cells. Firstly, α-mangostin could inhibit PMA-induced abilities of the adhesion, invasion, and migration. Data also showed α-mangostin could inhibit the activation of αvβ3 integrin, focal adhesion kinase (FAK), and extracellular signal-regulated kinase1/2 (ERK1/2) involved in the downregulation the enzyme activities, protein and messenger RNA levels of MMP-2 and MMP-9 induced by PMA. Next, α-mangostin also strongly inhibited PMA-induced degradation of inhibitor of kappaBα (IκBα) and the nuclear levels of nuclear factor kappa B (NF-κB). Also, a dose-dependent inhibition on the binding abilities of NF-κB by α-mangostin treatment was further observed. Furthermore, reduction of FAK or ERK1/2 phosphorylation by FAK small interfering RNA (FAK siRNA) potentiated the effect of α-mangostin. Finally, the transient transfection of ERK siRNA significantly down-regulated the expressions of MMP-2 and MMP-9 concomitantly with a marked inhibition on cell invasion and migration. Presented results indicated α-mangostin is a novel, effect, anti-metastatic agent that functions by downregulating MMP-2 and MMP-9 gene expressions.     

Transmitted/Founder and Chronic Subtype C HIV-1 Use CD4 and CCR5 Receptors with Equal Efficiency and Are Not Inhibited by Blocking the Integrin α4β7

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.     

Hydroxysafflor Yellow A Inhibits Aβ1–42-Induced Neuroinflammation by Modulating the Phenotypic Transformation of Microglia via TREM2/TLR4/NF-κB Pathway in BV-2 Cells

Neurochemical Research - Hydroxysafflor yellow A (HSYA) is an extract from Carthamus tinctorius L. dry flowers (Compositae). HSYA has been shown to have neuroprotective effects on several...     

A Novel Cryptic Binding Motif,LRSKSRSFQVSDEQY, in the C-Terminal Fragment of MMP-3/7-Cleaved Osteopontin as a Novel Ligand for α9β1 Integrin Is Involved in the Anti-Type II Collagen Antibody-Induced Arthritis

Osteopontin (OPN) is a multifunctional protein that has been linked to various intractable inflammatory diseases. One way by which OPN induces inflammation is the production of various functional fragments by enzyme cleavage. It has been well appreciated that OPN is cleaved by thrombin, and/or matrix metalloproteinase-3 and -7 (MMP-3/7). Although the function of thrombin-cleaved OPN is well characterized, little is known about the function of MMP-3/7-cleaved OPN. In this study, we found a novel motif, LRSKSRSFQVSDEQY, in the C-terminal fragment of MMP-3/7-cleaved mouse OPN binds to α9β1 integrin. Importantly, this novel motif is involved in the development of anti-type II collagen antibody-induced arthritis (CAIA). This study provides the first in vitro and in vivo evidence that OPN cleavage by MMP-3/7 is an important regulatory mechanism for CAIA.     

The α4β1 Fibronectin Ligands CS-19 HEP II,and RGD Induce Different Intracellular Events in B Lymphoid Cells. Comparison with the Effects of the Endothelial Ligand V CAM-1

The lymphocyte integrin α4β1 is the receptor for the Hep II domain and CS-1 site in fibronectin (Fn) as well as for VCAM-1. We recently showed that upon activation with anti-β1 mAb TS2/16, α4β1 also recognizes the RGD Fn sequence. To determine the physiological role of these multiple interactions, we have now studied some intracellular events induced by “resting” and activated α4β1 binding to its different ligands. Analyses of actin and tubulin reorganization upon adhesion of B lymphoid cells to Fn fragments or VCAM-1 showed that VCAM-1, a 38 kDa fragment (Hep II+CS-1), and the CS-1 synthetic peptide induced formation of transient cytoplasmic projections; however, cells attached to a 58 kDa (Hep II) or 80 kDa (RGD) fragments remained rounded. Using transfilter assays, we showed that VCAM-1, 38 kDa and CS-1 also induced dose-dependent B cell migration mediated by α4β1. Furthermore, these three ligands, but not the 80 kDa fragment or a synthetic peptide (H1) containing a sequence from Hep II shown to bind α4β1, induced tyrosine phosphorylation of a 110 kDa protein. Activation of α4β1 with TS2/16 inhibited the cytoplasmic protrusions and cell migration but did not affect the pattern of phosphorylation. Our results indicate that the various α4β1 ligands induce different cellular responses. Most importantly they show that α4β1 interaction with CS-1 is sufficient to trigger intracellular events in B cells. Furthermore, they suggest a regulation by the activation form of the receptor as well as by the ligand in events involving lymphocyte adhesion and migration.     

Enhancement of the Immunostimulatory Functions of Ex Vivo–Generated Dendritic Cells from Early-Stage Colon Cancer Patients by Consecutive Exposure to Low Doses of Sequential-Kinetic-Activated IL-4 and IL-12. A Preliminary Study

Dendritic cells (DCs), specialized antigen-presenting cells bridging innate and adaptive immunity, play a crucial role in determining specific immune response to tumors. Because of their potent immunoregulatory capacities, DCs have been exploited in anticancer vaccination, with limited success thus far. This pilot study compared low-dose interleukin (IL)-4 and IL-12 prepared by sequential kinetic activation (SKA) with standard doses of the same recombinant human cytokines on functional activity of ex vivo–generated monocyte-derived (Mo) DCs from colon carcinoma patients and normal subjects. MoDCs were exposed to medium alone, SKA-IL-4 (0.5 fg/ml), or SKA-IL-12 (2 fg/ml), alone or consecutively combined, in parallel with rhIL-4 (50 ng/ml) and rhIL-12 (1 ng/ml). Primary allogeneic one-way mixed lymphocyte reaction (MLR) was the end point to assess in vitro T-lymphocyte proliferation in response to MoDCs, and secreted IL-12p70 and interferon-γ in MLR supernatants measured by ELISA to assay for T-helper 1–promoting MoDC phenotype. No single agent enhanced the compromised allostimulatory activity of MoDCs from colon cancer patients, unlike healthy donors. However, MoDCs from nonmetastatic colon cancer patients, after sequential exposure to SKA-IL-4 (48 hours) and SKA-IL-12 (24 hours), displayed increased T-cell stimulatory capacity by MLR and acquired driving T-helper 1 polarization activity, although less markedly than the effects induced by recombinant human cytokines or found in normal subjects. These results point to an immunomodulatory capacity of low-dose SKA-IL-4 and SKA-IL-12 and encourage further investigation to provide clues for the rational development of new and more effective immunotherapeutic strategies against cancer.     

1H and13C-NMR analysis of four pentasaccharides from urine of blood-group A and B,Leb adults. Confirmation of the structure of two new oligosaccharides: Fuc(α1–2)[GalNAc(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc and Fuc(α1–2)[Gal(α1–3)]Gal(β1–3)[Fuc(α1–4)]Glc

The major pentasaccharides Fuc(1-2)[GalNAc(1-3)]Gal(1-4)[Fuc(1-3)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-4)[Fuc(1-3)]Glc, which are normally present in the urine of bloodgroup A Le^b and B Le^b healthy subjects, were each found to be contaminated by a minor component when analysed by¹H-NMR. The determination of these structures, Fuc(1-2) [GalNAc(1-3)]Gal(1-3)[Fuc(1-4)]Glc and Fuc(1-2) [Gal(1-3)]Gal(1-3)[Fuc(1-4)]Glc, was based on the results of methylation analysis and¹H/¹³C-NMR spectroscopy.Abbreviations HPLC high performance liquid chromatography - GLC gas liquid chromatography - NMR nuclear magnetic resonance - COSY correlation spectroscopy - Gal d-galactopyranose - GalNAc 2-acetamido-2-deoxy-d-galactopyranose - Glc d-glucopyranose - Fuc l-fucopyranose - LNDFH I lacto-N-difucohexaose I (Le^b determinant