title: Complement modulates the function of the ubiquitin-proteasome system and endoplasmic reticulum-associated degradation in glomerular epithelial cells.
abstract: In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.
authors.affiliation: Department of Medicine, McGill University Health Centre, McGill University, Montreal, Quebec, Canada.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Kitzler
authors.forename: Joan
authors.initials: J
authors.lastname: Papillon
authors.forename: Julie
authors.initials: J
authors.lastname: Guillemette
authors.forename: Simon S
authors.initials: SS
authors.lastname: Wing
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Cybulsky
mesh_terms: D000818:Animals; D015938:Complement Membrane Attack Complex / Q000378:metabolism; D003165:Complement System Proteins / Q000378:metabolism*; D019925:Cyclin A / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress; D060746:Endoplasmic Reticulum-Associated Degradation; D004847:Epithelial Cells / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D015433:Glomerulonephritis, Membranous / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007678:Kidney Glomerulus / Q000473:pathology*; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D059748:Proteolysis; D051381:Rats; D025801:Ubiquitin / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D015938:Complement Membrane Attack Complex; D019925:Cyclin A; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D003165:Complement System Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D046988:Proteasome Endopeptidase Complex