title: Internalized PCSK9 dissociates from recycling LDL receptors in PCSK9-resistant SV-589 fibroblasts.
abstract: Secreted PCSK9 binds to cell surface LDL receptor (LDLR) and directs the receptor for lysosomal degradation. PCSK9 is potent at inducing LDLR degradation in cultured liver-derived cells, but it is considerably less active in immortalized fibroblasts. We examined PCSK9 trafficking in SV-589 human skin fibroblasts incubated with purified recombinant wild-type PCSK9 or gain-of-function mutant PCSK9-D374Y with increased LDLR binding affinity. Despite LDLR-dependent PCSK9 uptake, cell surface LDLR levels in SV-589 fibroblasts were only modestly reduced by wild-type PCSK9, even at high nonphysiological concentrations (20 µg/ml). Internalized (125)I-labeled wild-type PCSK9 underwent lysosomal degradation at high levels, indicating its dissociation from recycling LDLRs. PCSK9-D374Y (2 µg/ml) reduced cell surface LDLRs by approximately 50%, but this effect was still blunted compared with HepG2 hepatoma cells. Radioiodinated PCSK9-D374Y was degraded less efficiently in SV-589 fibroblasts, and Alexa488-labeled PCSK9-D374Y trafficked to both lysosomes and endocytic recycling compartments. Endocytic recycling assays showed that more than 50% of internalized PCSK9-D374Y recycled to the cell surface compared with less than 10% for wild-type PCSK9. These data support that wild-type PCSK9 readily dissociates from the LDLR within early endosomes of SV-589 fibroblasts, contributing to PCSK9-resistance. Although a large proportion of gain-of-function PCSK9-D374Y remains bound to LDLR in these cells, degradative activity is still diminished.
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Ottawa Heart Institute, Ottawa, Ontario, Canada K1Y 4W7.
authors.forename: My-Anh
authors.initials: MA
authors.lastname: Nguyen
authors.forename: Tanja
authors.initials: T
authors.lastname: Kosenko
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Lagace
mesh_terms: D002460:Cell Line; D004705:Endocytosis; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D009154:Mutation; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D059748:Proteolysis; D011973:Receptors, LDL / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012867:Skin / Q000166:cytology; D013261:Sterols / Q000378:metabolism
chemical_list: D011973:Receptors, LDL; D013261:Sterols; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases
keywords: degradation; endosome; ligand dissociation