Autophagy is induced by raptor degradation via the ubiquitin/proteasome system in granular corneal dystrophy type 2

Abstract

Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disorder that is caused by a point mutation in transforming growth factor-β-induced gene-h3 (TGFBI), which encodes transforming growth factor-β-induced protein (TGFBIp). Recently, we found that the autophagic clearance of mutant-TGFBIp is delayed in GCD2 corneal fibroblasts; however, any potential correlation between mutant-TGFBIp turnover and autophagy–lysosome pathway remains unknown. Here, we report that mutant-TGFBIp is accumulated and that autophagy, a key clearance pathway for mutant-TGFBIp, is induced in primary cultured GCD2 homozygous (HO) and wild-type (WT) corneal fibroblasts that express exogenously introduced mutant-TGFBIp. Mutant-TGFBI colocalized with LC3-enriched cytosolic vesicles and cathepsin D in primary cultured GCD2 corneal fibroblasts. We also observed reduced levels of raptor (regulatory-associated protein of the mammalian target of rapamycin [mTOR]) in GCD2 corneal fibroblasts and WT corneal fibroblasts expressing mutant-TGFBIp. Strikingly, treatment with MG132, a ubiquitin/proteasome system inhibitor, significantly increased the levels of both total and ubiquitinated raptor in GCD2 corneal fibroblasts. The levels of the autophagy marker LC3-II were also increased in WT corneal fibroblasts that were treated with shRNA against raptor. However, mutant-TGFBIp accumulated in autophagosomes or/and lysosomes in spite of the significant activation of basal autophagy in GCD2 corneal fibroblasts. These results suggest that an insufficient autophagy–lysosome pathway might be responsible for the intracellular accumulation of mutant-TGFBIp during the pathogenesis of GCD2.