Polymodal Transient Receptor Potential Vanilloid (TRPV) Ion Channels in Chondrogenic Cells

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.     

Bacterial Outer Membrane Protein OmpX Regulates β1 Integrin and Epidermal Growth Factor Receptor (EGFR) Involved in Invasion of M-HeLa Cells by Serratia proteamaculans

Opportunistic pathogen Serratia proteamaculans are able to penetrate the eukaryotic cells. The penetration rate can be regulated by bacterial surface protein OmpX. OmpX family proteins are able to bind to host cell surface to the epidermal growth factor receptor (EGFR) and the extracellular matrix protein fibronectin, whose receptors are in return the α5 β1 integrins. Here we elucidated the involvement of these host cell proteins in S. proteamaculans invasion. We have shown that, despite the absence of fibronectin contribution to S. proteamaculans invasion, β1 integrin was directly involved in invasion of M-HeLa cells. Herewith β1 integrin was not the only receptor that determines sensitivity of host cells to bacterial invasion. Signal transfer from EGFR was also involved in the penetration of these bacteria into M-HeLa cells. However, M-HeLa cells have not been characterized by large number of these receptors. It turned out that S. proteamaculans attachment to the host cell surface resulted in an increment of EGFR and β1 integrin genes expression. Such gene expression increment also caused Escherichia coli attachment, transformed with a plasmid encoding OmpX from S. proteamaculans. Thus, an OmpX binding to the host cell surface caused an increase in the EGFR and β1 integrin expression involved in S. proteamaculans invasion.     

Update on Novel Targeted Therapy for Pleural Organization and Fibrosis

Pleural injury and subsequent loculation is characterized by acute injury, sustained inflammation and, when severe, pathologic tissue reorganization. While fibrin deposition is a normal part of the injury response, disordered fibrin turnover can promote pleural loculation and, when unresolved, fibrosis of the affected area. Within this review, we present a brief discussion of the current IPFT therapies, including scuPA, for the treatment of pathologic fibrin deposition and empyema. We also discuss endogenously expressed PAI-1 and how it may affect the efficacy of IPFT therapies. We further delineate the role of pleural mesothelial cells in the progression of pleural injury and subsequent pleural remodeling resulting from matrix deposition. We also describe how pleural mesothelial cells promote pleural fibrosis as myofibroblasts via mesomesenchymal transition. Finally, we discuss novel therapeutic targets which focus on blocking and/or reversing the myofibroblast differentiation of pleural mesothelial cells for the treatment of pleural fibrosis.     

Cytokine Receptors—Regulators of Antimycobacterial Immune Response

Cytokine receptors are critical regulators of the antimycobacterial immune response, playing a key role in initiating and coordinating the recruitment and activation of immune cells during infection. They recognize and bind specific cytokines and are involved in inducing intracellular signal transduction pathways that regulate a diverse range of biological functions, including proliferation, differentiation, metabolism and cell growth. Due to mutations in cytokine receptor genes, defective signaling may contribute to increased susceptibility to mycobacteria, allowing the pathogens to avoid killing and immune surveillance. This paper provides an overview of cytokine receptors important for the innate and adaptive immune responses against mycobacteria and discusses the implications of receptor gene defects for the course of mycobacterial infection.     

Complex target SELEX

Aptamers are non-naturally occurring structured oligonucleotides that may bind to small molecules, peptides, and proteins. Typically, aptamers are generated by an in vitro selection process referred to as SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind with high affinity and specificity to proteins that reside on the cell surface have potential utility as therapeutic antagonists, agonists, and diagnostic agents. When the target protein requires the presence of the cell membrane (e.g., G-protein-coupled receptors, ion channels) or a co-receptor to fold properly, it is difficult or impossible to program the SELEX experiment with purified, soluble protein target. Recent advances in which the useful range of SELEX has been extended from comparatively simple purified forms of soluble proteins to complex mixtures of proteins in membrane preparations or in situ on the surfaces of living cells offer the potential to discover aptamers against previously intractable targets. Additionally, in cases in which a cell-type specific diagnostic is sought, the most desirable target on the cell surface may not be known. Successful application of aptamer selection techniques to complex protein mixtures can be performed even in the absence of detailed target knowledge and characterization. This Account presents a review of recent work in which membrane preparations or whole cells have been utilized to generate aptamers to cell surface targets. SELEX experiments utilizing a range of target "scaffolds" are described, including cell fragments, parasites and bacteria, viruses, and a variety of human cell types including adult mesenchymal stem cells and tumor lines. Complex target SELEX can enable isolation of potent and selective aptamers directed against a variety of cell-surface proteins, including receptors and markers of cellular differentiation, as well as determinants of disease in pathogenic organisms, and as such should have wide therapeutic and diagnostic utility.     

Beta-1,3 Oligoglucans Specifically Bind to Immune Receptor CD28 and May Enhance T Cell Activation

Beta glucans are known to have immunomodulatory effects that mediated by a variety of mechanisms. In this article, we describe experiments and simulations suggesting that beta-1,3 glucans may promote activation of T cells by a previously unknown mechanism. First, we find that treatment of a T lymphoblast cell line with beta-1,3 oligoglucan significantly increases mRNA levels of T cell activation-associated cytokines, especially in the presence of the agonistic anti-CD3 antibody. This immunostimulatory activity was observed in the absence of dectin-1, a known receptor for beta-1,3 glucans. To clarify the molecular mechanism underlying this activity, we performed a series of molecular dynamics simulations and free-energy calculations to explore the interaction of beta-1,3 oligoglucans with potential immune receptors. While the simulations reveal little association between beta-1,3 oligoglucan and the immune receptor CD3, we find that beta-1,3 oligoglucans bind to CD28 near the region identified as the binding site for its natural ligands CD80 and CD86. Using a rigorous absolute binding free-energy technique, we calculate a dissociation constant in the low millimolar range for binding of 8-mer beta-1,3 oligoglucan to this site on CD28. The simulations show this binding to be specific, as no such association is computed for alpha-1,4 oligoglucan. This study suggests that beta-1,3 glucans bind to CD28 and may stimulate T cell activation collaboratively with T cell receptor activation, thereby stimulating immune function.     

Plasminogen Receptors and Fibrinolysis

The ability of cells to promote plasminogen activation on their surfaces is now well recognized, and several distinct cell surface proteins have been demonstrated to function as plasminogen receptors. Here, we review studies demonstrating that plasminogen bound to cells, in addition to plasminogen directly bound to fibrin, plays a major role in regulating fibrin surveillance. We focus on the ability of specific plasminogen receptors on eukaryotic cells to promote fibrinolysis in the in vivo setting by reviewing data obtained predominantly in murine models. Roles for distinct plasminogen receptors in fibrin surveillance in intravascular fibrinolysis, immune cell recruitment in the inflammatory response, wound healing, and lactational development are discussed.     

Oestrogen Activates the MAP3K1 Cascade and β-Catenin to Promote Granulosa-like Cell Fate in a Human Testis-Derived Cell Line

Sex determination triggers the differentiation of the bi-potential gonad into either an ovary or testis. In non-mammalian vertebrates, the presence or absence of oestrogen dictates gonad differentiation, while in mammals, this mechanism has been supplanted by the testis-determining gene SRY. Exogenous oestrogen can override this genetic trigger to shift somatic cell fate in the gonad towards ovarian developmental pathways by limiting the bioavailability of the key testis factor SOX9 within somatic cells. Our previous work has implicated the MAPK pathway in mediating the rapid cellular response to oestrogen. We performed proteomic and phosphoproteomic analyses to investigate the precise mechanism through which oestrogen impacts these pathways to activate β-catenin—a factor essential for ovarian development. We show that oestrogen can activate β-catenin within 30 min, concomitant with the cytoplasmic retention of SOX9. This occurs through changes to the MAP3K1 cascade, suggesting this pathway is a mechanism through which oestrogen influences gonad somatic cell fate. We demonstrate that oestrogen can promote the shift from SOX9 pro-testis activity to β-catenin pro-ovary activity through activation of MAP3K1. Our findings define a previously unknown mechanism through which oestrogen can promote a switch in gonad somatic cell fate and provided novel insights into the impacts of exogenous oestrogen exposure on the testis.     

Biomedical Application of Low Molecular Weight Heparin/Protamine Nano/Micro Particles as Cell- and Growth Factor-Carriers and Coating Matrix

Low molecular weight heparin (LMWH)/protamine (P) nano/micro particles (N/MPs) (LMWH/P N/MPs) were applied as carriers for heparin-binding growth factors (GFs) and for adhesive cells including adipose-derived stromal cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs). A mixture of LMWH and P yields a dispersion of N/MPs (100 nm–3 μm in diameter). LMWH/P N/MPs can be immobilized onto cell surfaces or extracellular matrix, control the release, activate GFs and protect various GFs. Furthermore, LMWH/P N/MPs can also bind to adhesive cell surfaces, inducing cells and LMWH/P N/MPs-aggregate formation. Those aggregates substantially promoted cellular viability, and induced vascularization and fibrous tissue formation in vivo. The LMWH/P N/MPs, in combination with ADSCs or BMSCs, are effective cell-carriers and are potential promising novel therapeutic agents for inducing vascularization and fibrous tissue formation in ischemic disease by transplantation of the ADSCs and LMWH/P N/MPs-aggregates. LMWH/P N/MPs can also bind to tissue culture plates and adsorb exogenous GFs or GFs from those cells. The LMWH/P N/MPs-coated matrix in the presence of GFs may provide novel biomaterials that can control cellular activity such as growth and differentiation. Furthermore, three-dimensional (3D) cultures of cells including ADSCs and BMSCs using plasma-medium gel with LMWH/P N/MPs exhibited efficient cell proliferation. Thus, LMWH/P N/MPs are an adequate carrier both for GFs and for stromal cells such as ADSCs and BMSCs, and are a functional coating matrix for their cultures.     

Increased Histone Deacetylase Activity Involved in the Suppressed Invasion of Cancer Cells Survived from ALA-Mediated Photodynamic Treatment

Previously, we have found that cancer cells survived from 5-Aminolevulinic acid-mediated photodynamic therapy (ALA-PDT) have abnormal mitochondrial function and suppressed cellular invasiveness. Here we report that both the mRNA expression level and enzymatic activity of histone deacetylase (HDAC) were elevated in the PDT-derived variants with dysfunctional mitochondria. The activated HDAC deacetylated histone H3 and further resulted in the reduced migration and invasion, which correlated with the reduced expression of the invasion-related genes, matrix metalloproteinase 9 (MMP9), paternally expressed gene 1 (PEG1), and miR-355, the intronic miRNA. Using chromatin immunoprecipitation, we further demonstrate the reduced amount of acetylated histone H3 on the promoter regions of MMP9 and PEG1, supporting the down-regulation of these two genes in PDT-derived variants. These results indicate that HDAC activation induced by mitochondrial dysfunction could modulate the cellular invasiveness and its related gene expression. This argument was further verified in the 51-10 cybrid cells with the 4977 bp mtDNA deletion and A375 ρ⁰ cells with depleted mitochondria. These results indicate that mitochondrial dysfunction might suppress tumor invasion through modulating histone acetylation.     

Conserved and Distinct Elements of Phagocytosis in Human and C. elegans

Endocytosis provides the cellular nutrition and homeostasis of organisms, but pathogens often take advantage of this entry point to infect host cells. This is counteracted by phagocytosis that plays a key role in the protection against invading microbes both during the initial engulfment of pathogens and in the clearance of infected cells. Phagocytic cells balance two vital functions: preventing the accumulation of cell corpses to avoid pathological inflammation and autoimmunity, whilst maintaining host defence. In this review, we compare elements of phagocytosis in mammals and the nematode Caenorhabditis elegans. Initial recognition of infection requires different mechanisms. In mammals, pattern recognition receptors bind pathogens directly, whereas activation of the innate immune response in the nematode rather relies on the detection of cellular damage. In contrast, molecules involved in efferocytosis—the engulfment and elimination of dying cells and cell debris—are highly conserved between the two species. Therefore, C. elegans is a powerful model to research mechanisms of the phagocytic machinery. Finally, we show that both mammalian and worm studies help to understand how the two phagocytic functions are interconnected: emerging data suggest the activation of innate immunity as a consequence of defective apoptotic cell clearance.     

A Nanobody Activation Immunotherapeutic that Selectively Destroys HER2‐Positive Breast Cancer Cells

We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti‐dinitrophenyl (anti‐DNP) antibodies to the surface of HER2‐positive breast cancer cells, resulting in their targeted destruction by antibody‐dependent cellular cytotoxicity. As nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease‐relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.     

Hetero‐bivalent GLP‐1/Glibenclamide for Targeting Pancreatic β‐Cells

G protein‐coupled receptor (GPCR) cell signalling cascades are initiated upon binding of a specific agonist ligand to its cell surface receptor. Linking multiple heterologous ligands that simultaneously bind and potentially link different receptors on the cell surface is a unique approach to modulate cell responses. Moreover, if the target receptors are selected based on analysis of cell‐specific expression of a receptor combination, then the linked binding elements might provide enhanced specificity of targeting the cell type of interest, that is, only to cells that express the complementary receptors. Two receptors whose expression is relatively specific (in combination) to insulin‐secreting pancreatic β‐cells are the sulfonylurea‐1 (SUR1) and the glucagon‐like peptide‐1 (GLP‐1) receptors. A heterobivalent ligand was assembled from the active fragment of GLP‐1 (7–36 GLP‐1) and glibenclamide, a small organic ligand for SUR1. The synthetic construct was labelled with Cy5 or europium chelated in DTPA to evaluate binding to β‐cells, by using fluorescence microscopy or time‐resolved saturation and competition binding assays, respectively. Once the ligand binds to β‐cells, it is rapidly capped and presumably removed from the cell surface by endocytosis. The bivalent ligand had an affinity approximately fivefold higher than monomeric europium‐labelled GLP‐1, likely a result of cooperative binding to the complementary receptors on the βTC3 cells. The high‐affinity binding was lost in the presence of either unlabelled monomer, thus demonstrating that interaction with both receptors is required for the enhanced binding at low concentrations. Importantly, bivalent enhancement was accomplished in a cell system with physiological levels of expression of the complementary receptors, thus indicating that this approach might be applicable for β‐cell targeting in vivo.     

The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.     

Single-molecule imaging of cell surfaces using near-field nanoscopy

Living cells use surface molecules such as receptors and sensors to acquire information about and to respond to their environments. The cell surface machinery regulates many essential cellular processes, including cell adhesion, tissue development, cellular communication, inflammation, tumor metastasis, and microbial infection. These events often involve multimolecular interactions occurring on a nanometer scale and at very high molecular concentrations. Therefore, understanding how single-molecules localize, assemble, and interact on the surface of living cells is an important challenge and a difficult one to address because of the lack of high-resolution single-molecule imaging techniques. In this Account, we show that atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) provide unprecedented possibilities for mapping the distribution of single molecules on the surfaces of cells with nanometer spatial resolution, thereby shedding new light on their highly sophisticated functions. For single-molecule recognition imaging by AFM, researchers label the tip with specific antibodies or ligands and detect molecular recognition signals on the cell surface using either adhesion force or dynamic recognition force mapping. In single-molecule NSOM, the tip is replaced by an optical fiber with a nanoscale aperture. As a result, topographic and optical images are simultaneously generated, revealing the spatial distribution of fluorescently labeled molecules. Recently, researchers have made remarkable progress in the application of near-field nanoscopy to image the distribution of cell surface molecules. Those results have led to key breakthroughs: deciphering the nanoscale architecture of bacterial cell walls; understanding how cells assemble surface receptors into nanodomains and modulate their functional state; and understanding how different components of the cell membrane (lipids, proteins) assemble and communicate to confer efficient functional responses upon cell activation. We anticipate that the next steps in the evolution of single-molecule near-field nanoscopy will involve combining single-molecule imaging with single-molecule force spectroscopy to simultaneously measure the localization, elasticity, and interactions of cell surface molecules. In addition, progress in high-speed AFM should allow researchers to image single cell surface molecules at unprecedented temporal resolution. In parallel, exciting advances in the fields of photonic antennas and plasmonics may soon find applications in cell biology, enabling true nanoimaging and nanospectroscopy of individual molecules in living cells.     

The Tyrosine Phosphatase hPTPRβ Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X₇ receptor (P2X₇R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X₇R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRβ and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson’s Disease.     

GCN5 Potentiates Glioma Proliferation and Invasion via STAT3 and AKT Signaling Pathways

The general control of nucleotide synthesis 5 (GCN5), which is one kind of lysine acetyltransferases, regulates a number of cellular processes, such as cell proliferation, differentiation, cell cycle and DNA damage repair. However, its biological role in human glioma development remains elusive. In the present study, we firstly reported that GCN5 was frequently overexpressed in human glioma tissues and GCN5 was positively correlated with proliferation of cell nuclear antigen PCNA and matrix metallopeptidase MMP9. Meanwhile, down-regulation of GCN5 by siRNA interfering inhibited glioma cell proliferation and invasion. In addition, GCN5 knockdown reduced expression of p-STAT3, p-AKT, PCNA and MMP9 and increased the expression of p21 in glioma cells. In conclusion, GCN5 exhibited critical roles in glioma development by regulating cell proliferation and invasion, which suggested that GCN5 might be a potential molecular target for glioma treatment.