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Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

ABSTRACT: Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel the details of early phases of defense responses. Here, we have analyzed the secretome of human primary macrophages stimulated with MSU using quantitative two-dimensional gel electrophoresis based proteomics as well as high-throughput qualitative GeLC-MS/MS approach combining protein separation by SDS-PAGE and protein identification by liquid chromatography-MS/MS. Both methods showed that MSU stimulation induced robust protein secretion from lipopolysaccharide-primed human macrophages. Bioinformatic analysis of the secretome data showed that MSU stimulation strongly activates unconventional, vesicle mediated protein secretion. The unconventionally secreted proteins included pro-inflammatory cytokines like IL-1? and IL-18, interferon-induced proteins, and danger signal proteins. Also active forms of lysosomal proteases cathepsins were secreted on MSU stimulation, and cathepsin activity was essential for MSU-induced unconventional protein secretion. Additionally, proteins associated to phosphorylation events including Src family tyrosine kinases were increased in the secretome of MSU-stimulated cells. Our functional studies demonstrated that Src, Pyk2, and PI3 kinases act upstream of cathepsins to activate the overall protein secretion from macrophages. In conclusion, we provide the first comprehensive characterization of protein secretion pathways activated by MSU in human macrophages, and reveal a novel role for cathepsins and Src, Pyk2, PI3 kinases in the activation of unconventional protein secretion.

SUBMITTER: Valimaki E

PROVIDER: S-EPMC3591666 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

Välimäki Elina E Miettinen Juho J JJ Lietzén Niina N Matikainen Sampsa S Nyman Tuula A TA

Molecular & cellular proteomics : MCP 20130104 3

Monosodium urate (MSU) is an endogenous danger signal that is crystallized from uric acid released from injured cells. MSU is known to activate inflammatory response in macrophages but the molecular mechanisms involved have remained uncharacterized. Activated macrophages start to secrete proteins to activate immune response and to recruit other immune cells to the site of infection and/or tissue damage. Secretome characterization after activation of innate immune system is essential to unravel t ...[more]

PMID: 23292187

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Project description:Before stimulation, the cells were washed and the culture media was changed to RPMI-1640. After stimulation, cell culture media were collected, concentrated, and the proteins were precipitated with 2-D Clean-Up Kit (GE Healthcare) followed by protein alkylation, trypsin digestion and iTRAQ labelling of the resulting peptides according to manufacturer´s instructions (Applied Biosystems). Control sample was labeled with 114, LPS-stimulated with 115, Curdlan-stimulated with 116, and GBY-stimulated with 117 isobaric tag. After labelling the samples were pooled and dried, and the peptides were fractionated by strong cation exchange chromatography using an Ettan HPLC system (Amersham Biosciences) connected to a PolySULFOETHYL A column. Each SCX-fraction containing labelled peptides was analyzed twice with nano-liquid chromatography-tandem mass spectrometry using Ultimate 3000 nano-liquid chromatograph (Dionex) and QSTAR Elite hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems / MDS Sciex) with nano-ESI ionization as previously described (Lietzén et al., 2011). MS data were acquired automatically using Analyst QS 2.0 software. Protein identification and relative quantitation were performed with ParagonTM search algorithm (Shilov et al., 2007) using ProteinPilot 2.0 interface (AB Sciex). Data files from both technical replicates of an iTRAQ sample set were processed together. Database searching was done against UniProt human database (version 2008-01-28 with 20330 human sequences) and ´decoy` database (the reverse amino acid sequence for false discovery rate estimation). The search criteria were: cysteine alkylation with MMTS, trypsin digestion, biological modifications allowed, thorough search and detected protein threshold of 95% confidence (Unused ProtScore > 1.3). Importantly, automatic bias correction was not used in quantitation. The false discovery rates were calculated as previously described (Elias and Gygi, 2007) and were 1.4% and 2% for the two biological replicates.

Project description:Background:Marantodes pumilum is traditionally used for dysentery, gonorrhea, and sickness in the bones. Previous studies revealed its antibacterial and xanthine oxidase inhibitory activities. Objective:To evaluate the inhibitory effects of three M. pumilum varieties on the secretion of lipopolysaccharide (LPS)- and monosodium urate crystal (MSU)-induced cytokines and plasma prostaglandin E2 (PGE2) in vitro. Materials and Methods:The leaves and roots of M. pumilum var. alata (MPA), M. pumilum var. pumila (MPP), and M. pumilum var. lanceolata (MPL) were successively extracted with dichloromethane (DCM), methanol, and water. Human peripheral blood mononuclear cells and ELISA technique were used for the cytokine assay, whereas human plasma and radioimmunoassay technique were used in the PGE2 assay. Flavonoids content was determined using a reversed-phase high-performance liquid chromatography. Results:DCM extract of MPL roots showed the highest inhibition of LPS-stimulated cytokine secretion with IC50 values of 29.87, 7.62, 5.84, 25.33, and 5.40 ?g/mL for interleukin (IL)-1?, IL-1?, IL-6, IL-8, and tumor necrosis factor (TNF)-?, respectively; while that of plasma PGE2 secretion was given by DCM extract of MPP roots (IC50 31.10 ?g/mL). Similarly, the DCM extract of MPL roots demonstrated the highest inhibition against MSU-stimulated IL-1?, IL-1?, IL-6, IL-8, TNF-?, and PGE2 secretion with IC50 values of 11.2, 8.92, 12.29, 49.51, 9.60, and 31.58 ?g/mL, respectively. Apigenin in DCM extracts of MPL (0.051 mg/g) and MPP (0.064 mg/g) roots could be responsible for the strong inhibitory activity against IL-1?, IL-6, TNF-?, and PGE2. Conclusion:The results suggested that DCM extracts of MPL and MPP roots are potential anti-inflammatory agents by inhibiting the secretion of LPS- and MSU-stimulated pro-inflammatory cytokines and PGE2. SUMMARY:Amongst 18 tested extracts, DCM extracts of MPL and MPP roots remarkably inhibited LPS- and MSU-stimulated pro-inflammatory cytokines and PGE2 secretionPhytochemical analysis was performed for the active extracts using RP-HPLC systemThe presence of flavonoids particularly apigenin could be responsible for the anti-inflammatory activity. Abbreviations used: BSA: Bovine serum albumin, COX-2: Cyclooxygenase-2, CPM: Count per minute, DAMP: Danger-associated molecular pattern, DCM: Dichloromethane, DMSO: Dimethyl sulfoxide, ELISA: Enzyme-linked immunosorbent assay, FBS: Fetal bovine serum, H2O: Water, HEPES: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, HMC-1: Human mast cell-1, HMGB1: High-mobility group box 1, ICAM: Intercellular adhesion molecule, IFN: Interferon, IgG: Immunoglobulin G, IKK: IkB kinase, IL: Interleukin, iNOS: Inducible nitric oxide synthase, LPS: Lipopolysaccharide, MeOH: Methanol, MPA: Marantodes pumilum var. alata, MPL: Marantodes pumilum var. lanceolata, MPP: Marantodes pumilum var. pumila, MSU: Monosodium urate, MTT: Methylthiazole tetrazolium, NF-?B: Nuclear factor-kappa B, NLR: NOD-like receptor, NLRP3: NLR family pyrin domain containing protein 3, NO: Nitric oxide, NOD: Nucleotide-binding oligomerization domain, NSAID: Nonsteroidal anti-inflammatory drug, PAMP: Pathogen-associated molecular pattern, PBMC: Peripheral blood mononuclear cell, PBS: Phosphate buffered saline, PGE2: Prostaglandin E2, PMACI: Phorbol-12-myristate 13-acetate and calcium ionosphere A23187, PRR: Pathogen recognition receptor, PTFE: Polytetrafluoroethylene, RIA: Radioimmunoassay, RIG: Retinoic acid-inducible gene I, RLR: RIG I-like receptor, RP-HPLC: Reversed-phase high-performance liquid chromatography, RPMI-1640: Roswell Park Memorial Institute-1640, TLR: Toll-like receptor, TNF: Tumor necrosis factor, VCAM: Vascular cell adhesion molecule.

Dataset Information

Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

Publications

Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human primary macrophages.

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