Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Eosinophil Adhesion and Maturation is Modulated by Laminin

Eosinophils (Eo) participate in the inflammatory response to parasites, allergins, toxins, and epitopes recognized by autoimmune antibodies. Nonetheless, little attention has heretofore been paid to the interactions of Eo with extracellular matrix (ECM) proteins during their migration through the subendothelial basement membrane and into the surrounding tissue. Therefore, we have studied the adhesion of Eo to specific ECM proteins and the effect of this adhesion on Eo viability and maturation. Control Eo (from normal donors) adhere no better to substrates coated with laminin (LM), fibronectin (FN), cytotactin (CT), or collagen types I or IV (Col IV) than they do to human serum albumin coated substrates. In contrast, Eo activated in vitro with IL-5 or in vivo in patients with eosinophilia bind well to LM, FN and Col IV. LM is by far the most avid ligand among these molecules. For example, 43% of input cells bind to a substrate bearing 200 fmol/cm² of LM; a similar level of adhesion to FN requires 30 times as much adsorbed protein. Antibody inhibition experiments suggest that the αβ1 integrin heterodimer is the predominant LM receptor on these cells. Flow cytometry showed similar levels of these subunits on control and activated Eo, suggesting that Eo adhesion to LM is not regulated simply by cell surface integrin concentration. The effects of ECM proteins on Eo behavior were also examined. A LM-coated substrate (with no added cytokine) was found to be almost as effective as IL-5 in maintaining Eo viability while an equally adhesive FN-coated substrate had much less effect. Normally, even in the presence of 10% serum, no Eo survive a 5-day incubation in vitro unless IL-3, IL-5, or GM-CSF is added to the medium. Conditions that inhibit adhesion to LM (anti-integrin antibodies in the medium or CT on the substrate) and certain anti-cy-tokine antibodies inhibited the promotion of Eo viability by LM. During incubation on LM, Eo become hypodense, as they do in the presence of IL-5, indicating that they have become activated. These observations suggest that the interactions of Eo and ECM proteins may be important both for their potential to direct Eo migration and for their ability to regulate Eo viability, cytokine production, and maturation.     

3D Coupling of Fibronectin Fibril Arrangement with Topology of Ventral Plasma Membrane

Abstract

Interaction of integrins with extracellular matrices is essential for cell adhesion to substrata. Ventral surfaces of fibroblasts adhering to flat substrata are not flat but have uneven 3D topology. However, spatial relationship between the topology of the ventral cell surface and arrangement of extracellular matrix fibrils remains unclear. Here, we report a novel and simple method based on total internal reflection fluorescence microscopy to quantify the distance between the ventral plasma membrane and the glass substratum. We observe that the distance varies from <?25 nm at focal adhesions to 40–50 nm at close contacts and >?80 nm in other regions. Furthermore, by applying this novel method, we show that fibronectin fibrils are also separated from the substratum in regions where the ventral cell surface-substratum distance is >?80 nm. Our results reveal that fibronectin fibrils are not simply adsorbed to the glass substratum but follow the ventral cell surface topology.     

Effects of a low-intensity electromagnetic field on fibroblast migration and proliferation

The aim of this study was to test if an extremely weak 1 GHz electromagnetic field (EMF), known to be in resonance with clusters of water molecules, has biological effects on human fibroblasts. We demonstrated that in an in vitro model of wound healing, this EMF can activate fibroblast migration. [³H]thymidine incorporation experiments demonstrated that the EMF could also activate fibroblast proliferation. Activation of the expression of human fibroblast growth factor 1 (HFGF1) after EMF exposure showed that molecular wound healing pathways are activated in response to this water-resonant EMF.     

Role of the extracellular matrix in epithelial morphogenesis

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

Development of tibulizumab,a tetravalent bispecific antibody targeting BAFF and IL-17A for the treatment of autoimmune disease

ABSTRACT

We describe a bispecific dual-antagonist antibody against human B cell activating factor (BAFF) and interleukin 17A (IL-17). An anti-IL-17 single-chain variable fragment (scFv) derived from ixekizumab (Taltz®) was fused via a glycine-rich linker to anti-BAFF tabalumab. The IgG-scFv bound both BAFF and IL-17 simultaneously with identical stoichiometry as the parental mAbs. Stability studies of the initial IgG-scFv revealed chemical degradation and aggregation not observed in either parental antibody. The anti-IL-17 scFv showed a high melting temperature (Tm) by differential scanning calorimetry (73.1°C), but also concentration-dependent, initially reversible, protein self-association. To engineer scFv stability, three parallel approaches were taken: labile complementary-determining region (CDR) residues were replaced by stable, affinity-neutral amino acids, CDR charge distribution was balanced, and a H44-L100 interface disulfide bond was introduced. The Tm of the disulfide-stabilized scFv was largely unperturbed, yet it remained monodispersed at high protein concentration. Fluorescent dye binding titrations indicated reduced solvent exposure of hydrophobic residues and decreased proteolytic susceptibility was observed, both indicative of enhanced conformational stability. Superimposition of the H44-L100 scFv (PDB id: 6NOU) and ixekizumab antigen-binding fragment (PDB id: 6NOV) crystal structures revealed nearly identical orientation of the frameworks and CDR loops. The stabilized bispecific molecule LY3090106 (tibulizumab) potently antagonized both BAFF and IL-17 in cell-based and in vivo mouse models. In cynomolgus monkey, it suppressed B cell development and survival and remained functionally intact in circulation, with a prolonged half-life. In summary, we engineered a potent bispecific antibody targeting two key cytokines involved in human autoimmunity amenable to clinical development.     

Characterization of a surrogate murine antibody to model anti-human CD3 therapies

Fc-modified anti-human CD3ε monoclonal antibodies (mAbs) are in clinical development for the treatment of autoimmune diseases. These next generation mAbs have completed clinical trials in patients with type-1 diabetes and inflammatory bowel disease demonstrating a narrow therapeutic window. Lowered doses are ineffective, yet higher pharmacologically-active doses cause an undesirable level of adverse events. Thus, there is a critical need for a return to bench research to explore ways of improving clinical outcomes. Indeed, we recently reported that a short course of treatment affords synergy, providing long-term disease amelioration when combining anti-mouse CD3 and anti-mouse tumor necrosis factor mAbs in experimental arthritis. Such strategies may widen the window between risk and benefit; however, to more accurately assess experimentally the biology and pharmacology, reagents that mimic the current development candidates were required. Consequently, we engineered an Fc-modified anti-mouse CD3ε mAb, 2C11-Novi. Here, we report the functional characterization of 2C11-Novi demonstrating that it does not bind FcγR in vitro and elicits little cytokine release in vivo, while maintaining classical pharmacodynamic effects (CD3-TCR downregulation and T cell killing). Furthermore, we observed that oral administration of 2C11-Novi ameliorated progression of remitting-relapsing experimental autoimmune encephalitis in mice, significantly reducing the primary acute and subsequent relapse phase of the disease. With innovative approaches validated in two experimental models of human disease, 2C11-Novi represents a meaningful tool to conduct further mechanistic studies aiming at exploiting the immunoregulatory properties of Fc-modified anti-CD3 therapies via combination therapy using parenteral or oral routes of administration.     

A novel pattern recognition protein of the Chinese oak silkmoth,Antheraea pernyi,is involved in the pro-PO activating system

In this paper, we firstly reported a C-type lectin cDNA clone of 1029 bps from the larvae of A. Pernyi (Ap-CTL) using PCR and RACE techniques. The full-length cDNA contains an open reading frame encoding 308 amino acid residues which has two different carbohydrate-recognition domains (CRDs) arranged in tandem. To investigate the biological activities in the innate immunity, recombinant Ap-CTL was expressed in E. coli with a 6-histidine at the amino-terminus (Ap-rCTL). Besides acted as a broad-spectrum recognition protein binding to a wide range of PAMPs and microorganisms, Ap-rCTL also had the ability to recognize and trigger the agglutination of bacteria and fungi. In the proPO activation assay, Ap-rCTL specifically restored the PO activity of hemolymph blocked by anti-Ap-rCTL antibody in the presence of different PAMPs or microorganisms. In summary, Ap-rCTL plays an important role in insect innate immunity as an pattern recognition protein. [BMB Reports 2013; 46(7): 358-363]     

No social distancing between ORAI channels

ORAI1 is established as an essential component of Ca²⁺ release-activated Ca²⁺ (CRAC) channel which mediates store-operated Ca²⁺ entry (SOCE). However, the contributions of ORAI2 and ORAI3 to SOCE are not understood. We highlight a recent study which shows that ORAI proteins form heteromeric channels which tune SOCE over a range of stimulus intensities.