Tissue Inhibitor of Metalloproteinase–3 (TIMP-3) induces FAS dependent apoptosis in human vascular smooth muscle cells

Over expression of Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) in vascular smooth muscle cells (VSMCs) induces apoptosis and reduces neointima formation occurring after saphenous vein interposition grafting or coronary stenting. In studies to address the mechanism of TIMP-3-driven apoptosis in human VSMCs we find that TIMP-3 increased activation of caspase-8 and apoptosis was inhibited by expression of Cytokine response modifier A (CrmA) and dominant negative FAS-Associated protein with Death Domain (FADD). TIMP-3 induced apoptosis did not cause mitochondrial depolarisation, increase activation of caspase-9 and was not inhibited by over-expression of B-cell Lymphoma 2 (Bcl2), indicating a mitochondrial independent/type-I death receptor pathway. TIMP-3 increased levels of the First Apoptosis Signal receptor (FAS) and depletion of FAS with shRNA showed TIMP-3-induced apoptosis was FAS dependent. TIMP-3 induced formation of the Death-Inducing Signalling Complex (DISC), as detected by immunoprecipitation and by immunofluorescence. Cellular-FADD-like IL-1 converting enzyme-Like Inhibitory Protein (c-FLIP) localised with FAS at the cell periphery in the absence of TIMP-3 and this localisation was lost on TIMP-3 expression with c-FLIP adopting a perinuclear localisation. Although TIMP-3 inhibited FAS shedding, this did not increase total surface levels of FAS but instead increased FAS levels within localised regions at the cell surface. A Disintegrin And Metalloproteinase 17 (ADAM17) is inhibited by TIMP-3 and depletion of ADAM17 with shRNA significantly decreased FAS shedding. However ADAM17 depletion did not induce apoptosis or replicate the effects of TIMP-3 by increasing localised clustering of cell surface FAS. ADAM17-depleted cells could activate caspase-3 when expressing levels of TIMP-3 that were otherwise sub-apoptotic, suggesting a partial role for ADAM17 mediated ectodomain shedding in TIMP-3 mediated apoptosis. We conclude that TIMP-3 induced apoptosis in VSMCs is highly dependent on FAS and is associated with changes in FAS and c-FLIP localisation, but is not solely dependent on shedding of the FAS ectodomain

Characterization of a heat resistant beta-glucosidase as a new reporter in cells and mice.

BACKGROUND: Reporter genes are widely used in biology and only a limited number are available. We present a new reporter gene for the localization of mammalian cells and transgenic tissues based on detection of the bglA (SYNbglA) gene of Caldocellum saccharolyticum that encodes a thermophilic beta-glucosidase. RESULTS: SYNbglA was generated by introducing codon substitutions to remove CpG motifs as these are associated with gene silencing in mammalian cells. SYNbglA expression can be localized in situ or detected quantitatively in colorimetric assays and can be co-localized with E. coli beta-galactosidase. Further, we have generated a Cre-reporter mouse in which SYNbglA is expressed following recombination to demonstrate the general utility of SYNbglA for in vivo analyses. SYNbglA can be detected in tissue wholemounts and in frozen and wax embedded sections. CONCLUSIONS: SYNbglA will have general applicability to developmental and molecular studies in vitro and in vivo.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Metabolic glycan imaging by isonitrile-tetrazine click chemistry.

Seeing the sugar coating: N-Acetyl-glucosamine and mannosamine derivatives tagged with an isonitrile group are metabolically incorporated into cell-surface glycans and can be detected with a fluorescent tetrazine. This bioorthogonal isonitrile-tetrazine ligation is also orthogonal to the commonly used azide-cyclooctyne ligation, and so will allow simultaneous detection of the incorporation of two different sugars

Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer.

Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer

ADAM17 depletion in hVSMCs regulates FAS shedding and increases sensitisation to TIMP-3-dependent apoptosis.

<p>A. Western blot for ADAM17 (A17) or FAS in hVSMCs (Cont), infected with lentivirus conferring puromycin resistance alone (Puro), control non-targeting shRNA (shCont) or shRNA targeting ADAM17 (A17-3 and A17-4). ADAM17 is the middle band, flanked by non-specific bands. B. Soluble FAS levels measured by ELISA in cell medium after 72 h from cells in A. Data are means ± SEM, n = 3. *** = P < 0.001, NS = Not Significant. C. Caspase-3 activity in hVSMC lysates from cell transduced with lentivirus expressing control shRNA or ADAM17 targeting shRNA (A17-3 and A17-4) infected with 300 pfu cell^-1 RAd60 or RAdT3-expressing adenovirus. Data are means ± SEM, n = 3. * = P < 0.05, ** = P < 0.01, NS = Not Significant. D. Quantification of surface FAS by single cell image analysis, data shows the mean number of spot-like FAS surface structures per cell, data are means ± SEM, n = 3. * = P < 0.05, *** = P < 0.001.</p

TIMP-3 induced apoptosis shows independence of FASL.

<p>A. hVSMCs were transduced with control adenovirus (RAd60) or adenovirus expressing TIMP-3 (RAdT3) for 48 h before staining with 2 μg ml^-1 isotype control IgG or an anti-FASL antibody and an anti-IgG Alexfluor488 conjugate. Cell-surface alexafluor488 mean fluorescence staining intensity was measured using flowcytometry. Data is the mean ± SEM, n = 3. NS = Not Significant. B. hVSMCs were transduced with control adenovirus (RAd60) or adenovirus expressing TIMP-3 (RAdT3) for 16 h before the addition of PBS (vehicle control), 2 μg ml^-1 isotype control IgG or a function blocking anti-FASL antibody. The antibody was added every 24 h before cell lysates were harvested 72 h post adenoviral transduction and caspase-3 activity measured. Data is the mean ± SEM, n = 3. *** = P < 0.001, NS = Not Significant. C. In order to demonstrate the function blocking FASL antibody could block FASL dependent apoptosis, hVSMCs were stimulated with either PBS vehicle control or 10 ng ml^-1 IFN-gamma for 24 h before the addition of FASL in membrane vesicles (vFASL), 2 μg ml^-1 isotype control IgG or a functional blocking FASL antibody. 24 h after the addition of antibody lysates were harvested and caspase-3 activity measured. Data are the mean ± SEM, n = 3. *** = P < 0.001, NS = Not Significant.</p

TIMP-3 induces the cellular redistribution of c-FLIP.

<p>Immunocytochemistry for c-FLIP and FAS in VSMCs infected with either control (RAd60) or RAdT3 adenovirus for 48 h. c-FLIP is shown in green, FAS in red and nuclei in blue (DAPI) in the overlay images. Scale bar represents 67 μm.</p

TIMP-3 increases FAS at the surface of hVSMCs at distinct regions.

<p>A. Soluble FAS (pg ml^-1) in the culture media of hVSMCs transduced with control (RAd60) or TIMP-3 (RAdT3)-expressing adenovirus at the time points indicated. Data are the mean ± SEM, n = 3. *** = P < 0.001. B. Surface levels of FAS analysed after 48 h by flowcytometry in hVSMCs infected with RAd60 or RAdT3-expressing adenovirus. Cells were stained with an IgM isotype control antibody or with anti-FAS IgM (mAb CH-11) followed by an anti-IgM Alexfluor-488 secondary antibody. Data is the mean fluorescence intensity ± SEM, n = 3, NS = Not Significant. C. Left; example hVSMCs in μ-slides stained with CellMask^TM Deep Red dye and for surface FAS with mAb CH-11 and an anti IgM Alexafluor-488 secondary antibody acquired by the iCys system. Middle; Image analysis contours superimposed on the left panel image generated by the iCys system. Contours in green are at the detection limit for the cell tracker dye, contours in blue are an enlargement of the contour in green to enable detection of the true cell periphery. Contours in white are the focal FAS surface staining detected. Right; Confocal image of surface staining of FAS with mAb CH-11 and an anti IgM Alexafluor-488 secondary antibody and counterstained with DAPI and Phalloidin-Alexafluor633. D–G. Quantification of FAS surface staining per cell in μ-slides by image analysis with the iCys system with approximately 70–100 cells detected per well. D. Analysis of total FAS fluorescence within the cell periphery analogous to the flowcytometry experiment in B. E. Number of spot-like FAS positive structures at the cell surface. F. Analysis of the intensity of staining within the spot-like FAS positive structures. G. Analysis of the area of spot-like FAS positive structures. Data are mean ± SEM, n = 6. * = P < 0.05, ** = P < 0.01, NS = Not Significant.</p