Subject(s)
Atherosclerosis/genetics , NADPH Oxidase 5/genetics , Acetylcholine/pharmacology , Animals , Aorta, Thoracic/drug effects , Clustered Regularly Interspaced Short Palindromic Repeats , Diet, Atherogenic , Epinephrine/pharmacology , Male , NADPH Oxidase 5/deficiency , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/pathology , Potassium/pharmacology , RabbitsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cytoskeleton/genetics , Extracellular Matrix/genetics , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Animals , Cell Movement/genetics , Endothelial Cells/metabolism , Gene Silencing , Human Umbilical Vein Endothelial Cells , Humans , Mice , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/pathology , Neovascularization, Physiologic/genetics , Podosomes/genetics , Pseudopodia/genetics , Signal Transduction/geneticsABSTRACT
Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Cell Movement , Pseudopodia/metabolism , Sequence Homology, Amino Acid , src Homology Domains , Actins/metabolism , Cortactin/metabolism , ErbB Receptors/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , HeLa Cells , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Macrophages/cytology , Macrophages/metabolism , Phosphatidylinositols/metabolism , Protein Binding , Protein TransportABSTRACT
Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about H(v)1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of H(v)1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of H(v)1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that H(v)1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that H(v)1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of H(v)1 and Nox2 expression was observed in PLB-985 cells. The upregulation of H(v)1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between H(v)1 expression and I(Hv) density in PLB-985 cells. It is also demonstrated that a massive reduction in H(v)1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native H(v)1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of H(v)1 in granulocytes is optimized to support intense NADPH oxidase activity.
Subject(s)
Granulocytes/metabolism , Ion Channels/metabolism , Membrane Glycoproteins/metabolism , NADPH Oxidases/metabolism , Animals , Blotting, Western , COS Cells , Cell Differentiation , Cell Line, Tumor , Cells, Cultured , Chlorocebus aethiops , Eosinophils/cytology , Eosinophils/metabolism , Gene Expression , Granulocytes/cytology , Humans , Intracellular Membranes/metabolism , Ion Channels/chemistry , Ion Channels/genetics , Jurkat Cells , Membrane Glycoproteins/genetics , Microscopy, Confocal , NADPH Oxidase 2 , NADPH Oxidases/genetics , Neutrophils/cytology , Neutrophils/metabolism , Phagosomes/metabolism , Protein Multimerization , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Superoxides/metabolismABSTRACT
Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-alpha, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-kappaB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.
