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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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Internalization of TGFBIp in various cell lines.A. TGFBIp was internalized in NIH3T3, SK-N-MC, and ZW13-1 cell lines but not in HEK293T. Cells were pre-incubated at 4°C for 30 min in basal medium, and then incubated for a further 120 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium. 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 lysate was used for western blot analysis. B. Internalization of mutant TGFBIp and WT TGFBIp was similar in NIH3T3 cells. Cells were treated as in A and analyzed by western blotting. C. Internalization of WT- and Mut-TGFBIp in the NIH3T3 cell line. Cells were incubated at 4°C for 30 min and were then shifted to 37°C in the continuous presence of TGFBIp. At the indicated time points, cells were analyzed for the amount of internalized TGFBIp. The experiment was repeated three times independently. There were no statistically significant differences between the rates of WT- and Mut-TGFBIp internalization (p > 0.05). D. Visualization of TGFBIp internalization in NIH3T3 cells by confocal microscopy. Cells were grown on glass coverslips and treated as in A, before fixation in methanol at −20°C for 3 min. Immunocytochemical staining was performed with monoclonal anti-TGFBI antibody, as described in Materials and Methods. E. TGFBIp co-localizes with Lamp-2. NIH3T3 cells grown on glass slides were subjected to immunocytochemical staining with monoclonal anti-TGFBIp and anti-Lamp-2 antibodies as described in Materials and Methods. Coverslips were mounted on the glass slides with mounting medium, and the cells were viewed using a Leica TCS SP5 confocal microscope.
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pone.0119561.g002: Internalization of TGFBIp in various cell lines.A. TGFBIp was internalized in NIH3T3, SK-N-MC, and ZW13-1 cell lines but not in HEK293T. Cells were pre-incubated at 4°C for 30 min in basal medium, and then incubated for a further 120 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium. 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 lysate was used for western blot analysis. B. Internalization of mutant TGFBIp and WT TGFBIp was similar in NIH3T3 cells. Cells were treated as in A and analyzed by western blotting. C. Internalization of WT- and Mut-TGFBIp in the NIH3T3 cell line. Cells were incubated at 4°C for 30 min and were then shifted to 37°C in the continuous presence of TGFBIp. At the indicated time points, cells were analyzed for the amount of internalized TGFBIp. The experiment was repeated three times independently. There were no statistically significant differences between the rates of WT- and Mut-TGFBIp internalization (p > 0.05). D. Visualization of TGFBIp internalization in NIH3T3 cells by confocal microscopy. Cells were grown on glass coverslips and treated as in A, before fixation in methanol at −20°C for 3 min. Immunocytochemical staining was performed with monoclonal anti-TGFBI antibody, as described in Materials and Methods. E. TGFBIp co-localizes with Lamp-2. NIH3T3 cells grown on glass slides were subjected to immunocytochemical staining with monoclonal anti-TGFBIp and anti-Lamp-2 antibodies as described in Materials and Methods. Coverslips were mounted on the glass slides with mounting medium, and the cells were viewed using a Leica TCS SP5 confocal microscope.

Mentions: In spite of its extensive distribution throughout the ECM, lysosomal degradation of TGFBIp suggests the possibility of TGFBIp internalization through endocytosis. Therefore, we investigated the endocytosis of TGFBIp in four cell lines: HEK293T, NIH3T3, ZW13-1 [42], and SK-N-MC. The cells were incubated with exogenous TGFBIp for 2 h at 37°C to allow internalization. Quantification of the results showed that HEK293T cells, which do not express TGFBIp, did not internalize the protein. However, NIH3T3 and ZW13-1 cells, which also do not express TGFBIp, clearly demonstrated TGFBIp internalization. Furthermore, we detected a higher level of internalized TGFBIp in SK-N-MC cells, which endogenously express TGFBIp (Fig 2A).


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Internalization of TGFBIp in various cell lines.A. TGFBIp was internalized in NIH3T3, SK-N-MC, and ZW13-1 cell lines but not in HEK293T. Cells were pre-incubated at 4°C for 30 min in basal medium, and then incubated for a further 120 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium. 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 lysate was used for western blot analysis. B. Internalization of mutant TGFBIp and WT TGFBIp was similar in NIH3T3 cells. Cells were treated as in A and analyzed by western blotting. C. Internalization of WT- and Mut-TGFBIp in the NIH3T3 cell line. Cells were incubated at 4°C for 30 min and were then shifted to 37°C in the continuous presence of TGFBIp. At the indicated time points, cells were analyzed for the amount of internalized TGFBIp. The experiment was repeated three times independently. There were no statistically significant differences between the rates of WT- and Mut-TGFBIp internalization (p > 0.05). D. Visualization of TGFBIp internalization in NIH3T3 cells by confocal microscopy. Cells were grown on glass coverslips and treated as in A, before fixation in methanol at −20°C for 3 min. Immunocytochemical staining was performed with monoclonal anti-TGFBI antibody, as described in Materials and Methods. E. TGFBIp co-localizes with Lamp-2. NIH3T3 cells grown on glass slides were subjected to immunocytochemical staining with monoclonal anti-TGFBIp and anti-Lamp-2 antibodies as described in Materials and Methods. Coverslips were mounted on the glass slides with mounting medium, and the cells were viewed using a Leica TCS SP5 confocal microscope.
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pone.0119561.g002: Internalization of TGFBIp in various cell lines.A. TGFBIp was internalized in NIH3T3, SK-N-MC, and ZW13-1 cell lines but not in HEK293T. Cells were pre-incubated at 4°C for 30 min in basal medium, and then incubated for a further 120 min at 37°C in basal or TGFBIp-supplemented (~1 μg/mL) medium. 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 lysate was used for western blot analysis. B. Internalization of mutant TGFBIp and WT TGFBIp was similar in NIH3T3 cells. Cells were treated as in A and analyzed by western blotting. C. Internalization of WT- and Mut-TGFBIp in the NIH3T3 cell line. Cells were incubated at 4°C for 30 min and were then shifted to 37°C in the continuous presence of TGFBIp. At the indicated time points, cells were analyzed for the amount of internalized TGFBIp. The experiment was repeated three times independently. There were no statistically significant differences between the rates of WT- and Mut-TGFBIp internalization (p > 0.05). D. Visualization of TGFBIp internalization in NIH3T3 cells by confocal microscopy. Cells were grown on glass coverslips and treated as in A, before fixation in methanol at −20°C for 3 min. Immunocytochemical staining was performed with monoclonal anti-TGFBI antibody, as described in Materials and Methods. E. TGFBIp co-localizes with Lamp-2. NIH3T3 cells grown on glass slides were subjected to immunocytochemical staining with monoclonal anti-TGFBIp and anti-Lamp-2 antibodies as described in Materials and Methods. Coverslips were mounted on the glass slides with mounting medium, and the cells were viewed using a Leica TCS SP5 confocal microscope.
Mentions: In spite of its extensive distribution throughout the ECM, lysosomal degradation of TGFBIp suggests the possibility of TGFBIp internalization through endocytosis. Therefore, we investigated the endocytosis of TGFBIp in four cell lines: HEK293T, NIH3T3, ZW13-1 [42], and SK-N-MC. The cells were incubated with exogenous TGFBIp for 2 h at 37°C to allow internalization. Quantification of the results showed that HEK293T cells, which do not express TGFBIp, did not internalize the protein. However, NIH3T3 and ZW13-1 cells, which also do not express TGFBIp, clearly demonstrated TGFBIp internalization. Furthermore, we detected a higher level of internalized TGFBIp in SK-N-MC cells, which endogenously express TGFBIp (Fig 2A).

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