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 A1, the fusion blocker of autophagosomes and lysosomes. Likewise, bafilomycin A1 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.

The 5q31-linked corneal dystrophies are heterogeneous autosomal-dominant eye disorders pathologically characterized by the progressive accumulation of aggregated proteinaceous deposits in the cornea, which manifests clinically as severe vision impairment. The 5q31-linked corneal dystrophies are commonly caused by mutations in the TGFBI (transforming growth factor-beta-induced) gene. However, despite the identification of the culprit gene, the cellular roles of TGFBI and the molecular mechanisms underlying the pathogenesis of corneal dystrophy remain poorly understood. Here we report the identification of periostin, a molecule that is highly related to TGFBI, as a specific TGFBI-binding partner. The association of TGFBI and periostin is mediated by the amino-terminal cysteine-rich EMI domains of TGFBI and periostin. Our results indicate that the endogenous TGFBI and periostin colocalize within the trans-Golgi network and associate prior to secretion. The corneal dystrophy-associated R124H mutation in TGFBI severely impairs interaction with periostin in vivo. In addition, the R124H mutation causes aberrant redistribution of the mutant TGFBI into lysosomes. We also find that the periostin-TGFBI interaction is disrupted in corneal fibroblasts cultured from granular corneal dystrophy type II patients and that periostin accumulates in TGFBI-positive corneal deposits in granular corneal dystrophy type II (also known as Avellino corneal dystrophy). Together, our findings suggest that TGFBI and periostin may play cooperative cellular roles and that periostin may be involved in the pathogenesis of 5q31-linked corneal dystrophies.