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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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Internalized TGFBIp is transported to lysosomes.A. NIH3T3 and ZW13-1 cells were pre-incubated for 60 min in the absence (-) or presence (+) of Baf-A1. After incubation, cells were incubated for 120 min in normal media containing TGFBIp and western blotting was performed for TGFBIp. B. Densitometric quantitation of the experimental results presented in A. A Student’s t-test was performed to determine the significance of differences between treatments with and without Baf-A1. Data analysis showed that the p-value was less than 0.05, indicating statistical significance. The experiment was repeated three times independently. C. Co-localization of TGFBIp with GRP94, ER marker, and Lamp-2, lysosome marker, in NIH3T3 cells. Cells were subjected to immunocytochemical staining as described in Materials and Methods. Areas of 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. D. Co-localization of TGFBIp with caveolin-1 in the absence (upper panel) and presence (lower panel) of Baf-A1. NIH3T3 cells were grown on glass slides and treated with vehicle or Baf-A1 (0.1 μM) for 60 min before incubation for 30 min at 4°C in medium containing ~1 μg/mL TGFBIp. The cells were subjected to immunocytochemical staining as described in Materials and Methods. The boxed area in the third panel was magnified and is presented as the fourth panel.
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pone.0119561.g005: Internalized TGFBIp is transported to lysosomes.A. NIH3T3 and ZW13-1 cells were pre-incubated for 60 min in the absence (-) or presence (+) of Baf-A1. After incubation, cells were incubated for 120 min in normal media containing TGFBIp and western blotting was performed for TGFBIp. B. Densitometric quantitation of the experimental results presented in A. A Student’s t-test was performed to determine the significance of differences between treatments with and without Baf-A1. Data analysis showed that the p-value was less than 0.05, indicating statistical significance. The experiment was repeated three times independently. C. Co-localization of TGFBIp with GRP94, ER marker, and Lamp-2, lysosome marker, in NIH3T3 cells. Cells were subjected to immunocytochemical staining as described in Materials and Methods. Areas of 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. D. Co-localization of TGFBIp with caveolin-1 in the absence (upper panel) and presence (lower panel) of Baf-A1. NIH3T3 cells were grown on glass slides and treated with vehicle or Baf-A1 (0.1 μM) for 60 min before incubation for 30 min at 4°C in medium containing ~1 μg/mL TGFBIp. The cells were subjected to immunocytochemical staining as described in Materials and Methods. The boxed area in the third panel was magnified and is presented as the fourth panel.

Mentions: To determine whether internalized TGFBIp is degraded within the lysosomes, we assessed the level of TGFBIp and its co-localization with lysosomes in the NIH3T3 and ZW13-1 cells treated with Baf-A1. We detected a significant increase in TGFBIp in both the cell types after treatment with the inhibitor (Fig 5A and 5B), indicating that TGFBIp is degraded by lysosome-mediated proteolysis or autophagy after internalization. Confocal microscopy revealed that internalized TGFBIp co-localized with the lysosomal marker Lamp-2, but not the ER marker GRP94, in NIH3T3 cells (Fig 5C). Furthermore, increased co-localization of internalized TGFBIp with caveolin-1 was observed in the cells treated with Baf-A1 (Fig 5D). These data provide additional supportive evidence that extracellular TGFBIp is internalized by cells and then targeted to the lysosomes for degradation.


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Internalized TGFBIp is transported to lysosomes.A. NIH3T3 and ZW13-1 cells were pre-incubated for 60 min in the absence (-) or presence (+) of Baf-A1. After incubation, cells were incubated for 120 min in normal media containing TGFBIp and western blotting was performed for TGFBIp. B. Densitometric quantitation of the experimental results presented in A. A Student’s t-test was performed to determine the significance of differences between treatments with and without Baf-A1. Data analysis showed that the p-value was less than 0.05, indicating statistical significance. The experiment was repeated three times independently. C. Co-localization of TGFBIp with GRP94, ER marker, and Lamp-2, lysosome marker, in NIH3T3 cells. Cells were subjected to immunocytochemical staining as described in Materials and Methods. Areas of 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. D. Co-localization of TGFBIp with caveolin-1 in the absence (upper panel) and presence (lower panel) of Baf-A1. NIH3T3 cells were grown on glass slides and treated with vehicle or Baf-A1 (0.1 μM) for 60 min before incubation for 30 min at 4°C in medium containing ~1 μg/mL TGFBIp. The cells were subjected to immunocytochemical staining as described in Materials and Methods. The boxed area in the third panel was magnified and is presented as the fourth panel.
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pone.0119561.g005: Internalized TGFBIp is transported to lysosomes.A. NIH3T3 and ZW13-1 cells were pre-incubated for 60 min in the absence (-) or presence (+) of Baf-A1. After incubation, cells were incubated for 120 min in normal media containing TGFBIp and western blotting was performed for TGFBIp. B. Densitometric quantitation of the experimental results presented in A. A Student’s t-test was performed to determine the significance of differences between treatments with and without Baf-A1. Data analysis showed that the p-value was less than 0.05, indicating statistical significance. The experiment was repeated three times independently. C. Co-localization of TGFBIp with GRP94, ER marker, and Lamp-2, lysosome marker, in NIH3T3 cells. Cells were subjected to immunocytochemical staining as described in Materials and Methods. Areas of 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. D. Co-localization of TGFBIp with caveolin-1 in the absence (upper panel) and presence (lower panel) of Baf-A1. NIH3T3 cells were grown on glass slides and treated with vehicle or Baf-A1 (0.1 μM) for 60 min before incubation for 30 min at 4°C in medium containing ~1 μg/mL TGFBIp. The cells were subjected to immunocytochemical staining as described in Materials and Methods. The boxed area in the third panel was magnified and is presented as the fourth panel.
Mentions: To determine whether internalized TGFBIp is degraded within the lysosomes, we assessed the level of TGFBIp and its co-localization with lysosomes in the NIH3T3 and ZW13-1 cells treated with Baf-A1. We detected a significant increase in TGFBIp in both the cell types after treatment with the inhibitor (Fig 5A and 5B), indicating that TGFBIp is degraded by lysosome-mediated proteolysis or autophagy after internalization. Confocal microscopy revealed that internalized TGFBIp co-localized with the lysosomal marker Lamp-2, but not the ER marker GRP94, in NIH3T3 cells (Fig 5C). Furthermore, increased co-localization of internalized TGFBIp with caveolin-1 was observed in the cells treated with Baf-A1 (Fig 5D). These data provide additional supportive evidence that extracellular TGFBIp is internalized by cells and then targeted to the lysosomes for degradation.

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