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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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Effects of proteasome inhibitors on TGFBIp internalization.A. Corneal fibroblasts were treated with CHX (to inhibit translation), and MG132 (proteasome inhibitors) or Baf-A1 (lysosomal inhibitors) for 60 min at 37°C, and then incubated with or without TGFBIp for 120 min at 37°C as indicated. The TGFBIp level was analyzed by western blotting. B. RT-PCR analysis of the effect of the specified inhibitors on the mRNA levels of TGFBI.
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pone.0119561.g007: Effects of proteasome inhibitors on TGFBIp internalization.A. Corneal fibroblasts were treated with CHX (to inhibit translation), and MG132 (proteasome inhibitors) or Baf-A1 (lysosomal inhibitors) for 60 min at 37°C, and then incubated with or without TGFBIp for 120 min at 37°C as indicated. The TGFBIp level was analyzed by western blotting. B. RT-PCR analysis of the effect of the specified inhibitors on the mRNA levels of TGFBI.

Mentions: Ubiquitination plays a role not only in proteasome-mediated protein degradation, but also in receptor-mediated endocytosis. One of the non-proteasome functions of ubiquitination is its implication in the process of endocytosis [58]. Therefore, we assessed whether ubiquitination has any effect on TGFBIp endocytosis. To examine the endocytosis of TGFBIp in corneal fibroblasts, we measured TGFBIp internalization after treatment with cycloheximide (CHX), which inhibits translation. As expected, CHX treatment inhibited the expression of TGFBIp (Fig 7A, lane 2). The level of intracellular TGFBIp increased in corneal fibroblasts treated with exogenous TGFBIp, unlike that in the non-treated cells (Fig 7A, lane 4 compared to lane 1). Furthermore, treatment with a chemical inhibitor of the 26S proteasome, MG132, dramatically decreased the level of intracellular TGFBIp after treatment with exogenous TGFBIp (Fig 7A, lane 4 compared to lane 7), suggesting that TGFBIp internalization is facilitated by the ubiquitin-proteasome activity. The addition of MG132 to cells treated with Baf-A1 in the presence of CHX further reduced the level of intracellular TGFBIp (Fig 7A, lane 8 compared to lane 5), indicating that ubiquitination is necessary for TGFBIp intracellular trafficking to lysosomes.


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Effects of proteasome inhibitors on TGFBIp internalization.A. Corneal fibroblasts were treated with CHX (to inhibit translation), and MG132 (proteasome inhibitors) or Baf-A1 (lysosomal inhibitors) for 60 min at 37°C, and then incubated with or without TGFBIp for 120 min at 37°C as indicated. The TGFBIp level was analyzed by western blotting. B. RT-PCR analysis of the effect of the specified inhibitors on the mRNA levels of TGFBI.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4390356&req=5

pone.0119561.g007: Effects of proteasome inhibitors on TGFBIp internalization.A. Corneal fibroblasts were treated with CHX (to inhibit translation), and MG132 (proteasome inhibitors) or Baf-A1 (lysosomal inhibitors) for 60 min at 37°C, and then incubated with or without TGFBIp for 120 min at 37°C as indicated. The TGFBIp level was analyzed by western blotting. B. RT-PCR analysis of the effect of the specified inhibitors on the mRNA levels of TGFBI.
Mentions: Ubiquitination plays a role not only in proteasome-mediated protein degradation, but also in receptor-mediated endocytosis. One of the non-proteasome functions of ubiquitination is its implication in the process of endocytosis [58]. Therefore, we assessed whether ubiquitination has any effect on TGFBIp endocytosis. To examine the endocytosis of TGFBIp in corneal fibroblasts, we measured TGFBIp internalization after treatment with cycloheximide (CHX), which inhibits translation. As expected, CHX treatment inhibited the expression of TGFBIp (Fig 7A, lane 2). The level of intracellular TGFBIp increased in corneal fibroblasts treated with exogenous TGFBIp, unlike that in the non-treated cells (Fig 7A, lane 4 compared to lane 1). Furthermore, treatment with a chemical inhibitor of the 26S proteasome, MG132, dramatically decreased the level of intracellular TGFBIp after treatment with exogenous TGFBIp (Fig 7A, lane 4 compared to lane 7), suggesting that TGFBIp internalization is facilitated by the ubiquitin-proteasome activity. The addition of MG132 to cells treated with Baf-A1 in the presence of CHX further reduced the level of intracellular TGFBIp (Fig 7A, lane 8 compared to lane 5), indicating that ubiquitination is necessary for TGFBIp intracellular trafficking to lysosomes.

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