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Monosodium urate activates Src/Pyk2/PI3 kinase and cathepsin dependent unconventional protein secretion from human
Elina Välimäki1, Juho J Miettinen, Niina Lietzén
1Institute of Biotechnology, University of Helsinki, University of Helsinki, Finland.
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-1β 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.
Insights
Monosodium urate (MSU) crystals activate macrophages to secrete inflammatory proteins via unconventional pathways. This study reveals cathepsins and kinases like Src are key regulators of this MSU-induced immune response.
Area of Science:
- Immunology
- Cell Biology
- Proteomics
Background:
- Monosodium urate (MSU) crystals act as danger signals activating inflammatory responses in macrophages.
- The precise molecular mechanisms underlying MSU-induced macrophage activation and protein secretion remain largely uncharacterized.
- Understanding macrophage secretome changes is crucial for deciphering early innate immune defense mechanisms.
Purpose of the Study:
- To comprehensively analyze the secretome of human primary macrophages stimulated with MSU.
- To elucidate the molecular mechanisms and pathways involved in MSU-induced protein secretion.
- To identify novel regulators of unconventional protein secretion in macrophages.
Main Methods:
- Quantitative proteomics using two-dimensional gel electrophoresis.
- High-throughput qualitative GeLC-MS/MS analysis of macrophage secretomes.
- Bioinformatic analysis of secreted proteins and functional studies involving kinase inhibitors.
Main Results:
- MSU stimulation robustly induced protein secretion from lipopolysaccharide-primed human macrophages.
- MSU strongly activated unconventional, vesicle-mediated protein secretion, including pro-inflammatory cytokines (IL-1β, IL-18) and danger signals.
- Active cathepsins were secreted and essential for MSU-induced unconventional protein secretion; Src, Pyk2, and PI3 kinases act upstream of cathepsins.
Conclusions:
- This study provides the first detailed characterization of protein secretion pathways activated by MSU in human macrophages.
- A novel role for cathepsins in mediating MSU-induced unconventional protein secretion is revealed.
- Src, Pyk2, and PI3 kinases are identified as upstream activators of cathepsin-dependent unconventional protein secretion.
