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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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Integrin-dependent endocytosis of TGFBIp in corneal fibroblasts.A. Endocytosis of TGFBIp was blocked by RGD peptide in a dose-dependent manner. Corneal fibroblasts were pre-incubated for 30 min in the absence (lane 1) or presence (lanes 2–4) of RGD or RAD peptides. TGFBIp (~1 μg/mL) was added to the medium and the cells were incubated for 120 min at 37°C. TGFBIp levels were measured by western blot analysis. B. TGFBIp interacts with integrin αVβ3 and αV. Cells were lysed with RIPA buffer and the lysate was immunoprecipitated with anti-integrin αVβ3 (left-hand panel) or anti-integrin αV (right-hand panel) antibody as indicated. Immunoprecipitates were resolved on 10% SDS-PAGE gels and immunoblotted with anti-TGFBIp polyclonal antibody. C. Co-localization of integrin αV with TGFBIp was visualized by confocal immunofluorescence microscopy. The merged images show TGFBIp as red, integrin αV as green, and areas of co-localization as yellow. The boxed area in the lower left-hand panel was magnified and is presented as the lower right-hand panel. Arrows identify regions of TGFBIp and integrin αV co-localization. Scale bars, 5 μm. D. Western blot analysis of HEK293T, NIH3T3, SK-N-MC, and ZW13-1 cell lines with monoclonal antibody against integrin αV. GAPDH was used as a loading control.
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pone.0119561.g006: Integrin-dependent endocytosis of TGFBIp in corneal fibroblasts.A. Endocytosis of TGFBIp was blocked by RGD peptide in a dose-dependent manner. Corneal fibroblasts were pre-incubated for 30 min in the absence (lane 1) or presence (lanes 2–4) of RGD or RAD peptides. TGFBIp (~1 μg/mL) was added to the medium and the cells were incubated for 120 min at 37°C. TGFBIp levels were measured by western blot analysis. B. TGFBIp interacts with integrin αVβ3 and αV. Cells were lysed with RIPA buffer and the lysate was immunoprecipitated with anti-integrin αVβ3 (left-hand panel) or anti-integrin αV (right-hand panel) antibody as indicated. Immunoprecipitates were resolved on 10% SDS-PAGE gels and immunoblotted with anti-TGFBIp polyclonal antibody. C. Co-localization of integrin αV with TGFBIp was visualized by confocal immunofluorescence microscopy. The merged images show TGFBIp as red, integrin αV as green, and areas of co-localization as yellow. The boxed area in the lower left-hand panel was magnified and is presented as the lower right-hand panel. Arrows identify regions of TGFBIp and integrin αV co-localization. Scale bars, 5 μm. D. Western blot analysis of HEK293T, NIH3T3, SK-N-MC, and ZW13-1 cell lines with monoclonal antibody against integrin αV. GAPDH was used as a loading control.

Mentions: Integrins are known to be constitutively endocytosed and recycled [54,55]. Previous studies demonstrated that TGFBIp interacts directly with several integrins, including αVβ3, through mechanisms dependent and independent of the RGD binding motif [56,57]. These data suggest that the RGD motif mediates the internalization of TGFBIp through interaction with integrins. Therefore, we evaluated whether RGD-mediated interactions of TGFBIp with integrins are involved in its internalization. Exogenous human TGFBIp was incubated with corneal fibroblasts in the presence of either RGD peptide or control RAD peptide for 2 h. In the presence of the RGD peptide, the amount of internalized TGFBIp was reduced in a dose-dependent manner (Fig 6A, left panel). However, intracellular TGFBIp levels did not change in cells incubated with the control RAD peptide (Fig 6A, right panel). These results suggest that RGD peptides disrupt TGFBIp internalization by preventing its endocytosis from the ECM.


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Integrin-dependent endocytosis of TGFBIp in corneal fibroblasts.A. Endocytosis of TGFBIp was blocked by RGD peptide in a dose-dependent manner. Corneal fibroblasts were pre-incubated for 30 min in the absence (lane 1) or presence (lanes 2–4) of RGD or RAD peptides. TGFBIp (~1 μg/mL) was added to the medium and the cells were incubated for 120 min at 37°C. TGFBIp levels were measured by western blot analysis. B. TGFBIp interacts with integrin αVβ3 and αV. Cells were lysed with RIPA buffer and the lysate was immunoprecipitated with anti-integrin αVβ3 (left-hand panel) or anti-integrin αV (right-hand panel) antibody as indicated. Immunoprecipitates were resolved on 10% SDS-PAGE gels and immunoblotted with anti-TGFBIp polyclonal antibody. C. Co-localization of integrin αV with TGFBIp was visualized by confocal immunofluorescence microscopy. The merged images show TGFBIp as red, integrin αV as green, and areas of co-localization as yellow. The boxed area in the lower left-hand panel was magnified and is presented as the lower right-hand panel. Arrows identify regions of TGFBIp and integrin αV co-localization. Scale bars, 5 μm. D. Western blot analysis of HEK293T, NIH3T3, SK-N-MC, and ZW13-1 cell lines with monoclonal antibody against integrin αV. GAPDH was used as a loading control.
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Related In: Results  -  Collection

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pone.0119561.g006: Integrin-dependent endocytosis of TGFBIp in corneal fibroblasts.A. Endocytosis of TGFBIp was blocked by RGD peptide in a dose-dependent manner. Corneal fibroblasts were pre-incubated for 30 min in the absence (lane 1) or presence (lanes 2–4) of RGD or RAD peptides. TGFBIp (~1 μg/mL) was added to the medium and the cells were incubated for 120 min at 37°C. TGFBIp levels were measured by western blot analysis. B. TGFBIp interacts with integrin αVβ3 and αV. Cells were lysed with RIPA buffer and the lysate was immunoprecipitated with anti-integrin αVβ3 (left-hand panel) or anti-integrin αV (right-hand panel) antibody as indicated. Immunoprecipitates were resolved on 10% SDS-PAGE gels and immunoblotted with anti-TGFBIp polyclonal antibody. C. Co-localization of integrin αV with TGFBIp was visualized by confocal immunofluorescence microscopy. The merged images show TGFBIp as red, integrin αV as green, and areas of co-localization as yellow. The boxed area in the lower left-hand panel was magnified and is presented as the lower right-hand panel. Arrows identify regions of TGFBIp and integrin αV co-localization. Scale bars, 5 μm. D. Western blot analysis of HEK293T, NIH3T3, SK-N-MC, and ZW13-1 cell lines with monoclonal antibody against integrin αV. GAPDH was used as a loading control.
Mentions: Integrins are known to be constitutively endocytosed and recycled [54,55]. Previous studies demonstrated that TGFBIp interacts directly with several integrins, including αVβ3, through mechanisms dependent and independent of the RGD binding motif [56,57]. These data suggest that the RGD motif mediates the internalization of TGFBIp through interaction with integrins. Therefore, we evaluated whether RGD-mediated interactions of TGFBIp with integrins are involved in its internalization. Exogenous human TGFBIp was incubated with corneal fibroblasts in the presence of either RGD peptide or control RAD peptide for 2 h. In the presence of the RGD peptide, the amount of internalized TGFBIp was reduced in a dose-dependent manner (Fig 6A, left panel). However, intracellular TGFBIp levels did not change in cells incubated with the control RAD peptide (Fig 6A, right panel). These results suggest that RGD peptides disrupt TGFBIp internalization by preventing its endocytosis from the ECM.

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