title: Impaired autophagy and delayed autophagic clearance of transforming growth factor β-induced protein (TGFBI) in granular corneal dystrophy type 2.
abstract: Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease characterized by a progressive age-dependent extracellular accumulation of transforming growth factor β-induced protein (TGFBI). Corneal fibroblasts from GCD2 patients also have progressive degenerative features, but the mechanism underlying this degeneration remains unknown. Here we observed that TGFBI was degraded by autophagy, but not by the ubiquitin/proteasome-dependent pathway. We also found that GCD2 homozygous corneal fibroblasts displayed a greater number of fragmented mitochondria. Most notably, mutant TGFBI (mut-TGFBI) extensively colocalized with microtubule-associated protein 1 light chain 3β (MAP1LC3B, hereafter referred to as LC3)-enriched cytosolic vesicles and CTSD in primary cultured GCD2 corneal fibroblasts. Levels of LC3-II, a marker of autophagy activation, were significantly increased in GCD2 corneal fibroblasts. Nevertheless, levels of SQSTM1/p62 and of polyubiquitinated protein were also significantly increased in GCD2 corneal fibroblasts compared with wild-type (WT) cells. However, LC3-II levels did not differ significantly between WT and GCD2 cells, as assessed by the presence of bafilomycin A 1, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A 1 caused a similar change in levels of SQSTM1. Thus, the increase in autophagosomes containing mut-TGFBI may be due to inefficient fusion between autophagosomes and lysosomes. Rapamycin, an autophagy activator, decreased mut-TGFBI, whereas inhibition of autophagy increased active caspase-3, poly (ADP-ribose) polymerase 1 (PARP1) and reduced the viability of GCD2 corneal fibroblasts compared with WT controls. These data suggest that defective autophagy may play a critical role in the pathogenesis of GCD2.
authors.affiliation: Corneal Dystrophy Research Institute and Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Seung-Il
authors.initials: SI
authors.lastname: Choi
authors.forename: Bong-Yoon
authors.initials: BY
authors.lastname: Kim
authors.forename: Shorafidinkhuja
authors.initials: S
authors.lastname: Dadakhujaev
authors.forename: Jun-Young
authors.initials: JY
authors.lastname: Oh
authors.forename: Tae-Im
authors.initials: TI
authors.lastname: Kim
authors.forename: Joo Young
authors.initials: JY
authors.lastname: Kim
authors.forename: Eung Kweon
authors.initials: EK
authors.lastname: Kim
mesh_terms: D000328:Adult; D001343:Autophagy / Q000187:drug effects; D015415:Biomarkers / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D003315:Cornea / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D003317:Corneal Dystrophies, Hereditary / Q000378:metabolism* / Q000473:pathology*; D004789:Enzyme Activation / Q000187:drug effects; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008561:Membrane Fusion / Q000187:drug effects; D008875:Middle Aged; D050505:Mutant Proteins / Q000378:metabolism; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D059748:Proteolysis / Q000187:drug effects; D020123:Sirolimus / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D055815:Young Adult; D000099222:betaIG-H3 Protein
chemical_list: D015415:Biomarkers; D053148:Caspase 3; D016326:Extracellular Matrix Proteins; D050505:Mutant Proteins; D020123:Sirolimus; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein