Autophagy Modulates Articular Cartilage Vesicle Formation in Primary Articular Chondrocytes*
Autor: | James T. Ninomiya, Carolyn B. Coyne, Ann K. Rosenthal, Elizabeth Mitton-Fitzgerald, Claudia M. Gohr, Rupinder Grewal, William T. Jackson |
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Jazyk: | angličtina |
Rok vydání: | 2015 |
Předmět: |
musculoskeletal diseases
Adult Cartilage Articular Small interfering RNA Swine viruses ATG5 Blotting Western Glycobiology and Extracellular Matrices Apoptosis Biology Real-Time Polymerase Chain Reaction Biochemistry Exosome Chondrocyte Chondrocytes Phagosomes Osteoarthritis Extracellular medicine Autophagy Animals Humans RNA Messenger skin and connective tissue diseases Molecular Biology Cells Cultured Cell Proliferation Organelles Sirolimus Caspase 3 Reverse Transcriptase Polymerase Chain Reaction Cartilage TOR Serine-Threonine Kinases virus diseases Biological Transport Cell Biology Middle Aged musculoskeletal system Flow Cytometry Cell biology medicine.anatomical_structure Intracellular Immunosuppressive Agents |
Popis: | Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair. |
Databáze: | OpenAIRE |
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