α-2-Macroglobulin induces the shedding of microvesicles from cutaneous wound myofibroblasts

Microvesicles (MVs) are recognized as an important class of cell-to-cell messengers. Although the properties of MVs are increasingly documented, the mechanisms regulating MV biogenesis remain debated. Myofibroblasts are a key cellular component of wound healing and have been shown to produce MVs upon stimulation with serum. However, the mediator(s) responsible for the observed effect of serum on MV release have yet to be identified. To isolate the molecule(s) of interest, serum proteins were sequentially separated using chromatography, selective precipitation, and electrophoresis. MV production was assessed throughout the purification and after stimulation of myofibroblasts with two potent purified molecules. α-2-Macroglobulin (A2M) was thereby found to dose-dependently stimulate MV release. We confirmed the presence of the A2M receptor, low-density lipoprotein receptor-related protein-1 (LRP1), on myofibroblasts. Inhibition of LRP1 resulted in a significant decrease in MV production. Together, our results suggest that A2M positively regulates MV shedding through the activation of LRP1 on myofibroblasts.     

Microglia convert aggregated amyloid-β into neurotoxic forms through the shedding of microvesicles

Alzheimer''s disease (AD) is characterized by extracellular amyloid-β (Aβ) deposition, which activates microglia, induces neuroinflammation and drives neurodegeneration. Recent evidence indicates that soluble pre-fibrillar Aβ species, rather than insoluble fibrils, are the most toxic forms of Aβ. Preventing soluble Aβ formation represents, therefore, a major goal in AD. We investigated whether microvesicles (MVs) released extracellularly by reactive microglia may contribute to AD degeneration. We found that production of myeloid MVs, likely of microglial origin, is strikingly high in AD patients and in subjects with mild cognitive impairment and that AD MVs are toxic for cultured neurons. The mechanism responsible for MV neurotoxicity was defined in vitro using MVs produced by primary microglia. We demonstrated that neurotoxicity of MVs results from (i) the capability of MV lipids to promote formation of soluble Aβ species from extracellular insoluble aggregates and (ii) from the presence of neurotoxic Aβ forms trafficked to MVs after Aβ internalization into microglia. MV neurotoxicity was neutralized by the Aβ-interacting protein PrP and anti-Aβ antibodies, which prevented binding to neurons of neurotoxic soluble Aβ species. This study identifies microglia-derived MVs as a novel mechanism by which microglia participate in AD degeneration, and suggest new therapeutic strategies for the treatment of the disease.     

Short-term TNFα shedding is independent of cytoplasmic phosphorylation or furin cleavage of ADAM17

Proteolysis of transmembrane molecules is an irreversible post-translational modification enabling autocrine, paracrine and endocrine signaling of many cytokines. The pro-inflammatory activities of membrane bound TNFα (pro-TNFα) strongly depend on ectodomain shedding mediated by the A Disintegrin And Metalloprotease family member ADAM17. Despite the well-documented role of ADAM17 in pro-TNFα cleavage during inflammation, little is known about its regulation. Mitogen-activated protein kinase-induced phosphorylation of the ADAM17 cytoplasmic tail has been described to be required for proper activation. To address, if pro-TNFα shedding depends on cytosolic phosphorylation we analyzed ADAM17 mutants lacking the cytoplasmic domain. ADAM17 mediated shedding of pro-TNFα was induced by PMA, Anisomycin and the phosphatase inhibitors Cantharidin and Calyculin A. Deletion of the entire cytoplasmic portion of ADAM17 abolished furin-dependent proteolytic maturation and pro-TNFα cleavage. Interestingly, we could exclude that resistance to proconvertase processing is the reason for the enzymatic inactivity of ADAM17 lacking the cytoplasmic portion as furin-resistant ADAM17 mutants rescued genetic ADAM17 deficiency after mitogen-activated protein kinase activation. Adding only 6 cytoplasmic amino acids completely restored ADAM17 maturation and shedding of pro-TNFα as well as of both TNF-receptors Finally, we showed that a pro-TNFα mutant lacking the cytoplasmic portion was also shed from the cell surface. We conclude that pro-TNFα cleavage by its major sheddase ADAM17 does not depend on cytosolic phosphorylation and/or interaction. These results have general implications on understanding the activation mechanism controlling the activity of ADAM17.     

TGFβ induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

Background and aims: Transforming growth factor-beta (TGFβ) is known to potently inhibit cell growth. Loss of responsiveness to TGFβ inhibition on cell growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGFβ and HB-EGF signal transduction via ADAM activation.Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGFβ. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGFβ was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGFβ was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown.Result: TGFβ-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGFβ induced shedding of proHB-EGF allowing HB-EGF-CTF to translocate to the nucleus. ADAM inhibitors blocked this nuclear translocation. TGFβ enhanced gastric cancer cell growth and ADAM inhibitors suppressed this effect. EGFR phosphorylation, HB-EGF-CTF nuclear translocation, and cell growth were suppressed in ADAM17 knockdown cells.Conclusion: HB-EGF-CTF nuclear translocation and EGFR transactivation from proHB-EGF shedding mediated by ADAM17 activated by TGFβ might be an important pathway of gastric cancer cell proliferation by TGFβ.     

Interleukin-1α promotes extracellular shedding of syndecan-2 via induction of matrix metalloproteinase-7 expression

The cell surface heparan sulfate proteoglycan, syndecan-2, is known to play an important role in the tumorigenic activity of colon cancer cells. In addition, the extracellular domain of syndecan-2 is cleaved by matrix metalloproteinase-7 (MMP-7) in various colon cancer cells, but factors involved in regulating this process remain unknown. Here, we demonstrate a role for interleukin-1α (IL-1α) in syndecan-2 shedding in colon cancer cells. Treatment of low metastatic (HT-29) and highly metastatic (HCT-116) colon cancer cells with various soluble growth factors and cytokines revealed that IL-1α specifically increased extracellular shedding of syndecan-2 in a concentration- and time-dependent manner. IL-1α did not affect the expression of syndecan-2, but did significantly reduce its cell surface levels. Notably, IL-1α increased the mRNA expression and subsequent secreted levels of MMP-7 protein and enhanced the phosphorylation of p38 and ERK mitogen-activated protein kinases. Furthermore, increased syndecan-2 shedding was dependent on the mitogen-activated protein kinase-mediated MMP-7 expression. Taken together, these data suggest that IL-1α regulates extracellular domain shedding of syndecan-2 through regulation of the MAP kinase-mediated MMP-7 expression in colon cancer cells.     

3′-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

In this report, we establish that 3-azido-3-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these results may have important implications regarding the potential use of AZT or related glycosylation inhibitors as cancer chemotherapeutics.Abbreviations AZT 3-azido-3-deoxythymidine - Cer ceramide - Crbr cerebroside - Gal galactose - GalNAc N-acetylgalactosamine - Glc glucose - GlcNAc N-acetylglucosamine - GD3 disialyl lactosylceramide - GM3 sialyl lactosylceramide - HPTLC high-performance thin layer chromatography - LacCer lactosylceramide - MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide - NeuAc N-acetylneuraminic acid - PA phosphatidic acid - PBS phosphate-buffered saline - PC phosphatidylcholine - PDMP 1-phenyl-2-decanoylamino-3-morpholino-1-propanol - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - SM sphingomyelin     

PKCα and PKCδ regulate ADAM17-mediated ectodomain shedding of heparin binding-EGF through separate pathways

Epidermal growth factor receptor (EGFR) signalling is initiated by the release of EGFR-ligands from membrane-anchored precursors, a process termed ectodomain shedding. This proteolytic event, mainly executed by A Disintegrin And Metalloproteases (ADAMs), is regulated by a number of signal transduction pathways, most notably those involving protein kinase C (PKC). However, the molecular mechanisms of PKC-dependent ectodomain shedding of EGFR-ligands, including the involvement of specific PKC isoforms and possible functional redundancy, are poorly understood. To address this issue, we employed a cell-based system of PMA-induced PKC activation coupled with shedding of heparin binding (HB)-EGF. In agreement with previous studies, we demonstrated that PMA triggers a rapid ADAM17-mediated release of HB-EGF. However, PMA-treatment also results in a protease-independent loss of cell surface HB-EGF. We identified PKCα as the key participant in the activation of ADAM17 and suggest that it acts in parallel with a pathway linking PKCδ and ERK activity. While PKCα specifically regulated PMA-induced shedding, PKCδ and ERK influenced both constitutive and inducible shedding by apparently affecting the level of HB-EGF on the cell surface. Together, these findings indicate the existence of multiple modes of regulation controlling EGFR-ligand availability and subsequent EGFR signal transduction.     

‘Disk shedding’ in the cone outer segments of the teleost,Poecilia reticulata P.

Summary Electron microscopical observations show that the cones in the retina of the diurnal Poecilia reticulata shed their membranous outer segment disks. This occurs at the side of the disk which is open to the extracellular space. Shedding is observed in single and twin cones and occurs at any level of the outer segment. The disks are not discarded in packages or as single disks, but are shed in small vesicular portions. This mode of disk shedding may explain why in cone outer segments radioactively labelled replacement protein is diffusely distributed.The authors wish to thank Dr. C. Wise of this Department for helpful discussions     

Do captive conditions favor shedding of parasites in the reared Atlantic bluefin tuna (Thunnus thynnus)?

Tuna (Thunnus spp.) has been characterized by long distance migrations, highly predatory behavior and longevity, all of which in turn, enable infections with a wide spectrum of different parasitic groups, reflecting in a remarkable diversity of tuna parasite communities. Since 2003, we have been monitoring parasite communities of Atlantic bluefin tuna (Thunnus thynnus) that are caught from the wild and transferred into cages during spring-summer months, as well as assemblages in fish that exit rearing cycle during the winter harvest period after 1.5 years. Interestingly in reared tuna, parasitic populations exhibit a significant decreasing trend at the end of the rearing cycle, rarely observed in other intensive productions that represent a suitable environment for the emergence, establishment and transmission of pathogens. In order to assess epizootiological behavior of tuna parasites assemblages at the beginning (B group) and at the end (A group) of 1.5 year rearing cycle, we examined data on parasite prevalence and abundance over 4 years. The aim was to evaluate parasite diversity indices and emerging differences between newly caught and harvested fish, as well as community compositions and their nestedness in respect to the event in the rearing cycle (capture or harvest time). In order to be able to predict classification of tuna in two categories (newly caught or heavily infected and harvested or less infected fish), based on empirical didymozoids abundances and year of sampling, we built a decision tree model. Results suggest that specificities of parasite assemblages and their dynamics in tuna before and after farming have no similar precedents in aquaculture. A trend of parasitic pauperization repeating in each rearing cycle over four-years time, in once diverse and species rich parasite communities is observed, however, structures of both B and A group rearing assemblages remain nested, with the same species being core parasites (Didymosulcus katsuwonicola and Koellikerioides intestinalis). The B group exhibited significantly higher total parasite richness and mean parasites abundance, as well as the heteroxenous species richness and abundance in comparison to A group, where monoxenous species were not recorded at all. Eleven parasite species out of 26 taxa were selected as important in discriminating between B and A groups' parasites assemblages, while significantly the most abundant in B group were D. katsuwonicola, Platocystis alalongae, K. intestinalis, Koellikerioides internogastricus, Didymocystis abdominalis and Anisakis sp. It is hard to postulate the combination of factors affecting these parasite populations, but environmental, anthropogenic or host intrinsic influence has to be taken into account for further investigation.     

Use of RapidChek® SELECT™ Salmonella to detect shedding of live attenuated Salmonella enterica serovar Typhi vaccine strains

Identification of individuals shedding Salmonella enterica serovar Typhi in stool is imperative during clinical trial safety evaluations. Recovery of live attenuated S. Typhi vaccine strains can be difficult because the mutations necessary for safety in humans often compromise survival in stringent selective enrichment media. RapidChek? SELECT? Salmonella is a highly sensitive detection method for S. enterica species which utilizes a bacteriophage cocktail designed to reduce the growth of competitor microbes in mildly selective enrichment medium. Detection of Salmonella is enhanced by means of a Salmonella-specific antibody strip targeted to lipopolysaccharide. The RapidChek? SELECT? Salmonella method was compared to conventional enrichment and plating methods to determine the most sensitive method for detecting attenuated S. Typhi strains in human stool samples. Although traditional enrichment strategies were more sensitive to the presence of wild-type S. Typhi, RapidChek? SELECT? Salmonella was superior at detecting attenuated strains of S. Typhi. Strains containing a wide variety of attenuating mutations were detected with equal sensitivity as the wild type by RapidChek? SELECT? Salmonella. The presence of Vi capsule or mutations which affected O-antigen synthesis (Δpmi, ΔgalE) did not decrease the sensitivity of the RapidChek? SELECT? Salmonella assay.     

An six-amino acid motif in the α3 domain of MICA is the cancer therapeutic target to inhibit shedding

Expression of the MHC class I chain related molecules A and B (MICA/B) on tumor cell surface can signal the immune receptor NKG2D for tumor immune destruction. However, MIC was found to be shed by tumors in cancer patients, which negatively regulates host immunity and promotes tumor immune evasion and progression. The mechanisms by which tumors shed MIC are not well understood although diverse groups of enzymes are suggested to be involved. The functional complexity of these enzymes makes them unfeasible therapeutic targets for inhibiting MIC shedding. Here we identified an six-amino acid (6-aa) motif in the α3 domain of MIC that is critical for the interaction of MIC with ERp5 to enable shedding. Mutations in this motif prevented MIC shedding but did not interfere with NKG2D-mediated recognition of MIC. Our study suggests that the 6-aa motif is a feasible target to inhibit MIC shedding for cancer therapy.     

Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein

Background  

The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.     

Is the prevalence and shedding concentrations of E. coli O157 in beef cattle in Scotland seasonal?

The prevalence of Escherichia coli O157 in Scottish beef cattle at abattoir was found to be greater during the cooler months [11.2% (95% CI, 8.4-13.9%)] compared to the warmer months [7.5% (95% CI, 5.4-9.6%)]; the reverse of seasonality of human infections. However, high shedding beef cattle (excreting 10(-4) g(-1)) appear to shed greater concentrations of E. coli O157 in the warmer months which may partly explain increased human infection seasonality at this time.     

TGF-β1 influence on TNF-α production and sTNF-Rs shedding in a coculture of colon carcinoma cell spheroids with normal cells

In this study, the levels of TNF-α and its soluble receptors sTNF-Rp55 and sTNF-Rp75 were analyzed in cocultures of human colon carcinoma cell spheroids prepared from different grades of tumors with normal human colon epithelium, myofibroblast, and endothelial cell monolayers. Additionally, the influence of exogenously added rhTGF-β1 (2 ng/ml) on the TNF-α and sTNF-Rs levels was tested. Direct interactions of colon carcinoma spheroids with normal cells caused decreases in TNF-α levels and normal cell-dependent changes in sTNF-Rs amounts as compared to normal cells cultured alone. The addition of rhTGF-β1 to the cocultures caused a significant increase in TNF-α levels with a simultaneous decrease in the amounts of both sTNF-Rs. During direct interactions of colon carcinoma cells with normal tissue, paracrine effects are very important. We showed that TGF-β1 acts synergistically with TNF-α and significantly limits sTNF-Rs shedding. Therefore, TNF-Rs bound to cellular membranes, but not their soluble forms, play an important role in tumor/normal cell interactions. TGF-β1 and sTNF-Rs, in turn, may be valuable factors in colon cancer development and metastasis.     

Characterization of degradable polyelectrolyte multilayers fabricated using DNA and a fluorescently-labeled poly(β-amino ester): shedding light on the role of the cationic polymer in promoting surface-mediated gene delivery

Polyelectrolyte multilayers (PEMs) fabricated from cationic polymers and DNA have been investigated broadly as materials for surface-mediated DNA delivery. One attractive aspect of this "multilayered" approach is the potential to exploit the presence of cationic polymer "layers" in these films to deliver DNA to cells more effectively. Past studies demonstrate that these films can promote transgene expression in vitro and in vivo, but significant questions remain regarding roles that the cationic polymers could play in promoting the internalization and processing of DNA. Here, we report physicochemical and in vitro cell-based characterization of DNA-containing PEMs fabricated using fluorescently end-labeled derivatives of a degradable polycation (polymer 1) used in past studies of surface-mediated transfection. This approach permitted simultaneous characterization of polymer and DNA in solution and in cells using fluorescence-based techniques, and provided information about the locations and behaviors of polymer 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells and that they were colocalized to a significant extent inside cells (e.g., ~58% of DNA was colocalized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1, which is incorporated into these materials as "layers" rather than as part of optimized, preformed "polyplexes", can act to promote or enhance surface-mediated DNA delivery. More broadly, our results suggest opportunities to improve the delivery properties of DNA-containing PEMs by incorporation of additional "layers" of other conventional cationic polymers designed to address specific intracellular barriers to transfection, such as endosomal escape, more effectively.