So Young Kim1, Areum Yeo1, Hyemi Noh1, Yong Woo Ji1, Jong Suk Song2, Hyeon Chang Kim3, Lark Kyun Kim4, Hyung Keun Lee1,5. 1. Institute of Vision Research, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea. 2. Department of Ophthalmology, Korea University College of Medicine, Seoul, Korea. 3. Department of Preventive Medicine, Yonsei University College of Medicine, Seoul, Korea. 4. Severance Biomedical Science Institute and BK21 PLUS Project to Medical Sciences, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea. 5. Corneal Dystrophy Research Institute, Department of Ophthalmology, Yonsei University College of Medicine, Seoul, Korea.
Abstract
Purpose: Granular corneal dystrophy type 2 (GCD2) is caused by a point mutation (R124H) in the TGF-β-induced gene (TGFBI). However, the mechanisms underlying the accumulation of TGF-β-induced protein (TGFBIp) are poorly understood. Therefore, we evaluated the signaling cascade affecting the expression of TGFBIp using patient-derived cells. Methods: Keratocyte primary cultures were prepared from corneas from the eye bank or from heterozygous or homozygous patients with GCD2 after penetrating or lamellar keratoplasty. GCD2 diagnoses were based on the results of a DNA analysis for the R124H TGFβI mutation. Keratocytes were treated with various cytokines and then analyzed using quantitative PCR (qPCR) array, qPCR, flow cytometry, ELISA, and Western blotting. Results: TGFBI expression was counterregulated by IL-7 in corneal fibroblasts. IL-7 expression was significantly reduced in corneal fibroblasts from patients with GCD2. TGF-β and TGFBI expression were reduced on IL-7 treatment in corneal fibroblasts. Interestingly, the interplay between TGF-β and IL-7 was regulated by the RANKL/RANK signaling cascade. Also, IL-7 regulates the expression of a membrane-type matrix metalloproteinase (MT-MMP), which plays a crucial role in migration and neovascularization in the cornea. Conclusions: These studies demonstrate that impaired IL-7 expression in patients with GCD2 affects disease pathogenesis via a failure to control TGF-β expression. The RANKL/RANK axis regulates TGF-β and TGFBI expression via IL-7-mediated MT-MMP regulation in corneal fibroblasts. These findings improve our understanding of the pathogenesis of GCD2.
Purpose: Granular corneal dystrophy type 2 (GCD2) is caused by a point mutation (R124H) in the TGF-β-induced gene (TGFBI). However, the mechanisms underlying the accumulation of TGF-β-induced protein (TGFBIp) are poorly understood. Therefore, we evaluated the signaling cascade affecting the expression of TGFBIp using patient-derived cells. Methods: Keratocyte primary cultures were prepared from corneas from the eye bank or from heterozygous or homozygous patients with GCD2 after penetrating or lamellar keratoplasty. GCD2 diagnoses were based on the results of a DNA analysis for the R124H TGFβI mutation. Keratocytes were treated with various cytokines and then analyzed using quantitative PCR (qPCR) array, qPCR, flow cytometry, ELISA, and Western blotting. Results:TGFBI expression was counterregulated by IL-7 in corneal fibroblasts. IL-7 expression was significantly reduced in corneal fibroblasts from patients with GCD2. TGF-β and TGFBI expression were reduced on IL-7 treatment in corneal fibroblasts. Interestingly, the interplay between TGF-β and IL-7 was regulated by the RANKL/RANK signaling cascade. Also, IL-7 regulates the expression of a membrane-type matrix metalloproteinase (MT-MMP), which plays a crucial role in migration and neovascularization in the cornea. Conclusions: These studies demonstrate that impaired IL-7 expression in patients with GCD2 affects disease pathogenesis via a failure to control TGF-β expression. The RANKL/RANK axis regulates TGF-β and TGFBI expression via IL-7-mediated MT-MMP regulation in corneal fibroblasts. These findings improve our understanding of the pathogenesis of GCD2.