title: Autophagy modulates articular cartilage vesicle formation in primary articular chondrocytes.
abstract: 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.
authors: From the Division of Rheumatology, Department of Medicine, Zablocki VA Medical Center, Milwaukee, Wisconsin 53295, ann.rosenthal@va.gov.
authors: Ann K
authors: AK
authors: Rosenthal
authors: From the Division of Rheumatology, Department of Medicine, Zablocki VA Medical Center, Milwaukee, Wisconsin 53295.
authors: Claudia M
authors: CM
authors: Gohr
authors: From the Division of Rheumatology, Department of Medicine, Zablocki VA Medical Center, Milwaukee, Wisconsin 53295.
authors: Elizabeth
authors: E
authors: Mitton-Fitzgerald
authors: From the Division of Rheumatology, Department of Medicine, Zablocki VA Medical Center, Milwaukee, Wisconsin 53295.
authors: Rupinder
authors: R
authors: Grewal
authors: Departments of Orthopedic Surgery and.
authors: James
authors: J
authors: Ninomiya
authors: Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15219.
authors: Carolyn B
authors: CB
authors: Coyne
authors: Microbiology and Molecular Genetics and Center for Infectious Disease Research, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and.
authors: William T
authors: WT
authors: Jackson
mesh_terms: D000328:Adult; D000818:Animals; D017209:Apoptosis; D001343:Autophagy; D001692:Biological Transport; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology* / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008875:Middle Aged; D015388:Organelles / Q000378:metabolism*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology*; D010588:Phagosomes / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020123:Sirolimus / Q000494:pharmacology; D013552:Swine; D058570:TOR Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; D012333:RNA, Messenger; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D053148:Caspase 3; D020123:Sirolimus
keywords: articular cartilage; articular cartilage vesicles; autophagy; caspase 3; chondrocyte; exosome; extracellular vesicles