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Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

Choi SI, Maeng YS, Kim TI, Lee Y, Kim YS, Kim EK - PLoS ONE (2015)

Bottom Line: We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation.Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp.These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul, South Korea.

ABSTRACT
Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

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Effect of inhibitors of caveolae- and clathrin-dependent endocytosis on TGFBIp internalization.A. Inhibitors of endocytosis decreased TGFBIp levels in corneal fibroblasts. Cells were left untreated (lane 1) or treated with Baf-A1 and various endocytosis inhibitors for 60 min before incubation with TGFBIp for 120 min. TGFBIp levels were determined by western blot analysis. GS; genistein, NS; nystatin, CM; chlorpromazine. Data from one representative experiment is shown. β-actin was used as a loading control. B. Densitometric quantitation of the experiment presented in A. Data represent the TGFBIp/β-actin ratio and expressed as mean ± SD of three independently treated samples from one or two experiments. ANOVA analysis of TGFBIp levels across the treatment conditions showed no significant changes. *P≤0.05 relative to controls by Student’s t-test. C. Internalization of TGFBIp in WT corneal fibroblasts and caveolin-1- cell line (3T3 MEF CAV-1 KO). 3T3 MEF CAV-1 KO cell does not express TGFBIp (lane 2 and 4). TGFBIp was internalized in corneal fibroblasts (lane 5) but not in 3T3 MEF CAV-1 KO cell lines (lanes 6 and 8). Cells were pre-incubated at 37°C for 60 min in basal medium or basal medium with CHX, and then incubated for a further 60 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium with or without CHX. Cells were washed twice with cold PBS on ice, and surface-bound TGFBIp was removed by washing three times with ice-cold acidic buffer. Cells were harvested by scraping into ice-cold PBS, pelleted by centrifugation at 1,000 × g, lysed in RIPA buffer, and 50 μg of total protein was used for western blot analysis. D. TGFBIp co-localizes with caveolin-1 in corneal fibroblasts. Cells were grown on glass slides for 12 h and then fixed using methanol at −20°C and incubated with antibodies against TGFBIp and caveolin-1. Localization of TGFBIp (green) and caveolin-1 (red) are shown. Areas of TGFBIp and caveolin-1 co-localization appear as yellow regions in the merged image. The boxed area in the third panel was magnified and is presented as the fourth panel. Arrows in the fourth panel identify regions of TGFBIp and caveolin-1 co-localization. E. Cathepsin D co-localizes with caveolin-1 in corneal fibroblasts in the absence and presence of Baf-A1. Cells were grown on glass slides for 12 h in the absence (upper panel) or presence (lower panel) of Baf-A1 (0.1 μM) and then fixed with methanol at −20°C and incubated with antibodies against cathepsin D and caveolin-1. Localization of cathepsin D (green) and caveolin-1 (red) is shown. Areas of cathepsin D and caveolin-1 co-localization appear as yellow regions in the merged image.
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pone.0119561.g003: Effect of inhibitors of caveolae- and clathrin-dependent endocytosis on TGFBIp internalization.A. Inhibitors of endocytosis decreased TGFBIp levels in corneal fibroblasts. Cells were left untreated (lane 1) or treated with Baf-A1 and various endocytosis inhibitors for 60 min before incubation with TGFBIp for 120 min. TGFBIp levels were determined by western blot analysis. GS; genistein, NS; nystatin, CM; chlorpromazine. Data from one representative experiment is shown. β-actin was used as a loading control. B. Densitometric quantitation of the experiment presented in A. Data represent the TGFBIp/β-actin ratio and expressed as mean ± SD of three independently treated samples from one or two experiments. ANOVA analysis of TGFBIp levels across the treatment conditions showed no significant changes. *P≤0.05 relative to controls by Student’s t-test. C. Internalization of TGFBIp in WT corneal fibroblasts and caveolin-1- cell line (3T3 MEF CAV-1 KO). 3T3 MEF CAV-1 KO cell does not express TGFBIp (lane 2 and 4). TGFBIp was internalized in corneal fibroblasts (lane 5) but not in 3T3 MEF CAV-1 KO cell lines (lanes 6 and 8). Cells were pre-incubated at 37°C for 60 min in basal medium or basal medium with CHX, and then incubated for a further 60 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium with or without CHX. Cells were washed twice with cold PBS on ice, and surface-bound TGFBIp was removed by washing three times with ice-cold acidic buffer. Cells were harvested by scraping into ice-cold PBS, pelleted by centrifugation at 1,000 × g, lysed in RIPA buffer, and 50 μg of total protein was used for western blot analysis. D. TGFBIp co-localizes with caveolin-1 in corneal fibroblasts. Cells were grown on glass slides for 12 h and then fixed using methanol at −20°C and incubated with antibodies against TGFBIp and caveolin-1. Localization of TGFBIp (green) and caveolin-1 (red) are shown. Areas of TGFBIp and caveolin-1 co-localization appear as yellow regions in the merged image. The boxed area in the third panel was magnified and is presented as the fourth panel. Arrows in the fourth panel identify regions of TGFBIp and caveolin-1 co-localization. E. Cathepsin D co-localizes with caveolin-1 in corneal fibroblasts in the absence and presence of Baf-A1. Cells were grown on glass slides for 12 h in the absence (upper panel) or presence (lower panel) of Baf-A1 (0.1 μM) and then fixed with methanol at −20°C and incubated with antibodies against cathepsin D and caveolin-1. Localization of cathepsin D (green) and caveolin-1 (red) is shown. Areas of cathepsin D and caveolin-1 co-localization appear as yellow regions in the merged image.

Mentions: Next, we investigated whether TGFBIp internalization occurred via clathrin- or caveolae-mediated endocytosis. We treated a corneal fibroblast cell line with the general protein tyrosine kinase inhibitor genistein, which prevents caveolae-mediated internalization [43–46]. Our results showed that treatment with genistein (100 μg/mL) significantly reduced the amount of intracellular TGFBIp (Fig 3A and 3B, lane 3). To further confirm the route of TGFBIp internalization, we treated the cells with cholesterol-chelating drug nystatin, which interferes with caveolae-mediated endocytosis [47–49], and found that nystatin (25 μg/mL) caused a significant reduction in intracellular TGFBIp levels (Fig 3A and 3B, lane 4). In contrast, an inhibitor of clathrin-mediated endocytosis, chlorpromazine (10 or 100 μg/mL), increased the level of intracellular TGFBIp (Fig 3A and 3B, lanes 5), even at higher concentrations (Fig 3A and 3B, lanes 6). To further confirm whether TGFBIp is internalized via a caveolea-mediated endocytosis, we used caveolin-1- cell line (3T3 MEF CAV-1 KO). RT-PCR and western blotting showed that 3T3 MEFs CAV-1 KO cells, which do not express TGFBI gene (S1 Table and S1 Fig), did not internalize TGFBIp (Fig 3C). These results clearly demonstrated that TGFBIp is internalized by caveolea-dependent endocytosis.


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

Choi SI, Maeng YS, Kim TI, Lee Y, Kim YS, Kim EK - PLoS ONE (2015)

Effect of inhibitors of caveolae- and clathrin-dependent endocytosis on TGFBIp internalization.A. Inhibitors of endocytosis decreased TGFBIp levels in corneal fibroblasts. Cells were left untreated (lane 1) or treated with Baf-A1 and various endocytosis inhibitors for 60 min before incubation with TGFBIp for 120 min. TGFBIp levels were determined by western blot analysis. GS; genistein, NS; nystatin, CM; chlorpromazine. Data from one representative experiment is shown. β-actin was used as a loading control. B. Densitometric quantitation of the experiment presented in A. Data represent the TGFBIp/β-actin ratio and expressed as mean ± SD of three independently treated samples from one or two experiments. ANOVA analysis of TGFBIp levels across the treatment conditions showed no significant changes. *P≤0.05 relative to controls by Student’s t-test. C. Internalization of TGFBIp in WT corneal fibroblasts and caveolin-1- cell line (3T3 MEF CAV-1 KO). 3T3 MEF CAV-1 KO cell does not express TGFBIp (lane 2 and 4). TGFBIp was internalized in corneal fibroblasts (lane 5) but not in 3T3 MEF CAV-1 KO cell lines (lanes 6 and 8). Cells were pre-incubated at 37°C for 60 min in basal medium or basal medium with CHX, and then incubated for a further 60 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium with or without CHX. Cells were washed twice with cold PBS on ice, and surface-bound TGFBIp was removed by washing three times with ice-cold acidic buffer. Cells were harvested by scraping into ice-cold PBS, pelleted by centrifugation at 1,000 × g, lysed in RIPA buffer, and 50 μg of total protein was used for western blot analysis. D. TGFBIp co-localizes with caveolin-1 in corneal fibroblasts. Cells were grown on glass slides for 12 h and then fixed using methanol at −20°C and incubated with antibodies against TGFBIp and caveolin-1. Localization of TGFBIp (green) and caveolin-1 (red) are shown. Areas of TGFBIp and caveolin-1 co-localization appear as yellow regions in the merged image. The boxed area in the third panel was magnified and is presented as the fourth panel. Arrows in the fourth panel identify regions of TGFBIp and caveolin-1 co-localization. E. Cathepsin D co-localizes with caveolin-1 in corneal fibroblasts in the absence and presence of Baf-A1. Cells were grown on glass slides for 12 h in the absence (upper panel) or presence (lower panel) of Baf-A1 (0.1 μM) and then fixed with methanol at −20°C and incubated with antibodies against cathepsin D and caveolin-1. Localization of cathepsin D (green) and caveolin-1 (red) is shown. Areas of cathepsin D and caveolin-1 co-localization appear as yellow regions in the merged image.
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Related In: Results  -  Collection

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Show All Figures
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pone.0119561.g003: Effect of inhibitors of caveolae- and clathrin-dependent endocytosis on TGFBIp internalization.A. Inhibitors of endocytosis decreased TGFBIp levels in corneal fibroblasts. Cells were left untreated (lane 1) or treated with Baf-A1 and various endocytosis inhibitors for 60 min before incubation with TGFBIp for 120 min. TGFBIp levels were determined by western blot analysis. GS; genistein, NS; nystatin, CM; chlorpromazine. Data from one representative experiment is shown. β-actin was used as a loading control. B. Densitometric quantitation of the experiment presented in A. Data represent the TGFBIp/β-actin ratio and expressed as mean ± SD of three independently treated samples from one or two experiments. ANOVA analysis of TGFBIp levels across the treatment conditions showed no significant changes. *P≤0.05 relative to controls by Student’s t-test. C. Internalization of TGFBIp in WT corneal fibroblasts and caveolin-1- cell line (3T3 MEF CAV-1 KO). 3T3 MEF CAV-1 KO cell does not express TGFBIp (lane 2 and 4). TGFBIp was internalized in corneal fibroblasts (lane 5) but not in 3T3 MEF CAV-1 KO cell lines (lanes 6 and 8). Cells were pre-incubated at 37°C for 60 min in basal medium or basal medium with CHX, and then incubated for a further 60 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium with or without CHX. Cells were washed twice with cold PBS on ice, and surface-bound TGFBIp was removed by washing three times with ice-cold acidic buffer. Cells were harvested by scraping into ice-cold PBS, pelleted by centrifugation at 1,000 × g, lysed in RIPA buffer, and 50 μg of total protein was used for western blot analysis. D. TGFBIp co-localizes with caveolin-1 in corneal fibroblasts. Cells were grown on glass slides for 12 h and then fixed using methanol at −20°C and incubated with antibodies against TGFBIp and caveolin-1. Localization of TGFBIp (green) and caveolin-1 (red) are shown. Areas of TGFBIp and caveolin-1 co-localization appear as yellow regions in the merged image. The boxed area in the third panel was magnified and is presented as the fourth panel. Arrows in the fourth panel identify regions of TGFBIp and caveolin-1 co-localization. E. Cathepsin D co-localizes with caveolin-1 in corneal fibroblasts in the absence and presence of Baf-A1. Cells were grown on glass slides for 12 h in the absence (upper panel) or presence (lower panel) of Baf-A1 (0.1 μM) and then fixed with methanol at −20°C and incubated with antibodies against cathepsin D and caveolin-1. Localization of cathepsin D (green) and caveolin-1 (red) is shown. Areas of cathepsin D and caveolin-1 co-localization appear as yellow regions in the merged image.
Mentions: Next, we investigated whether TGFBIp internalization occurred via clathrin- or caveolae-mediated endocytosis. We treated a corneal fibroblast cell line with the general protein tyrosine kinase inhibitor genistein, which prevents caveolae-mediated internalization [43–46]. Our results showed that treatment with genistein (100 μg/mL) significantly reduced the amount of intracellular TGFBIp (Fig 3A and 3B, lane 3). To further confirm the route of TGFBIp internalization, we treated the cells with cholesterol-chelating drug nystatin, which interferes with caveolae-mediated endocytosis [47–49], and found that nystatin (25 μg/mL) caused a significant reduction in intracellular TGFBIp levels (Fig 3A and 3B, lane 4). In contrast, an inhibitor of clathrin-mediated endocytosis, chlorpromazine (10 or 100 μg/mL), increased the level of intracellular TGFBIp (Fig 3A and 3B, lanes 5), even at higher concentrations (Fig 3A and 3B, lanes 6). To further confirm whether TGFBIp is internalized via a caveolea-mediated endocytosis, we used caveolin-1- cell line (3T3 MEF CAV-1 KO). RT-PCR and western blotting showed that 3T3 MEFs CAV-1 KO cells, which do not express TGFBI gene (S1 Table and S1 Fig), did not internalize TGFBIp (Fig 3C). These results clearly demonstrated that TGFBIp is internalized by caveolea-dependent endocytosis.

Bottom Line: We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation.Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp.These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Corneal Dystrophy Research Institute, Yonsei University College of Medicine, Seoul, South Korea.

ABSTRACT
Transforming growth factor-beta-induced protein (TGFBIp) is ubiquitously expressed in the extracellular matrix (ECM) of various tissues and cell lines. Progressive accumulation of mutant TGFBIp is directly involved in the pathogenesis of TGFBI-linked corneal dystrophy. Recent studies reported that mutant TGFBIp accumulates in cells; however, the trafficking of TGFBIp is poorly understood. Therefore, we investigated TGFBIp trafficking to determine the route of its internalization and secretion and to elucidate its roles in the pathogenesis of granular corneal dystrophy type 2 (GCD2). Our data indicate that newly synthesized TGFBIp was secreted via the endoplasmic reticulum/Golgi-dependent secretory pathway, and this secretion was delayed in the corneal fibroblasts of patients with GCD2. We also found that TGFBIp was internalized by caveolae-mediated endocytosis, and the internalized TGFBIp accumulated after treatment with bafilomycin A1, an inhibitor of lysosomal degradation. In addition, the proteasome inhibitor MG132 inhibits the endocytosis of TGFBIp. Co-immunoprecipitation revealed that TGFBIp interacted with integrin αVβ3. Moreover, treatment with arginine-glycine-aspartic acid (RGD) tripeptide suppressed the internalization of TGFBIp. These insights on TGFBIp trafficking could lead to the identification of novel targets and the development of new therapies for TGFBI-linked corneal dystrophy.

Show MeSH
Related in: MedlinePlus