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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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Expression and secretion of TGFBIp in wild-type (WT) and heterozygous (HE) or homozygous (HO) mutant primary corneal fibroblasts.A. Western blot analysis of TGFBIp in cell lysates (upper panel) and conditioned media (lower panel) of WT, HE, and HO cells. β-actin was used as a loading control. Molecular weight markers (in kDa) are indicated. B. Secretion of WT and mutant TGFBIp was inhibited by treatment with brefeldin A (BFA) and monensin (MON). C. TGFBIp co-localized with markers of cis-Golgi (upper panel), medial-Golgi (middle panel), and trans-Golgi (lower panel) in cortical cells. Representative confocal images of immunofluorescence staining of TGFBIp (green) with GM130, mannosidase II, and TGN38 (all red) are shown. Overlapping areas are displayed in yellow in the merged images. Bars = 25 μm. D. Secretion of mutant TGFBIp is delayed in GCD2 corneal fibroblasts. Corneal fibroblasts from a patient with a homozygous TGFBIp mutation and a WT control were pulse-labeled for 20 min using 35S-cysteine and then incubated for 0, 15, 30, 60, 120, 180, and 240 min in unlabeled media before immunoprecipitation of TGFBIp from cell lysates and conditioned media. Phosphorimaging was performed after SDS-PAGE to detect TGFBIp. One representative experiment is shown. E. Quantitation of the experiment presented in D. Triplicate lysate samples were analyzed. *P < 0.05.
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pone.0119561.g001: Expression and secretion of TGFBIp in wild-type (WT) and heterozygous (HE) or homozygous (HO) mutant primary corneal fibroblasts.A. Western blot analysis of TGFBIp in cell lysates (upper panel) and conditioned media (lower panel) of WT, HE, and HO cells. β-actin was used as a loading control. Molecular weight markers (in kDa) are indicated. B. Secretion of WT and mutant TGFBIp was inhibited by treatment with brefeldin A (BFA) and monensin (MON). C. TGFBIp co-localized with markers of cis-Golgi (upper panel), medial-Golgi (middle panel), and trans-Golgi (lower panel) in cortical cells. Representative confocal images of immunofluorescence staining of TGFBIp (green) with GM130, mannosidase II, and TGN38 (all red) are shown. Overlapping areas are displayed in yellow in the merged images. Bars = 25 μm. D. Secretion of mutant TGFBIp is delayed in GCD2 corneal fibroblasts. Corneal fibroblasts from a patient with a homozygous TGFBIp mutation and a WT control were pulse-labeled for 20 min using 35S-cysteine and then incubated for 0, 15, 30, 60, 120, 180, and 240 min in unlabeled media before immunoprecipitation of TGFBIp from cell lysates and conditioned media. Phosphorimaging was performed after SDS-PAGE to detect TGFBIp. One representative experiment is shown. E. Quantitation of the experiment presented in D. Triplicate lysate samples were analyzed. *P < 0.05.

Mentions: Although studies have established that TGFBIp protein is secreted into the ECM and degraded via autophagy/lysosomal pathway, the underlying trafficking pathway(s) remain poorly understood. To address this issue, we examined the secretion of TGFBIp in the presence of brefaldin-A (BFA), which reversibly blocks protein transport from the ER [39] and monensin (MON), which effectively inhibits protein transport from medial to trans Golgi cisternae [40,41]. In normal culture conditions, western blot analysis of TGFBIp in cell lysates and conditioned media of wild-type (WT) and GCD2 homozygous (HO) primary fibroblasts indicated two forms of the protein at ~66 and ~68 kDa (Fig 1A). The effects of inhibitors on TGFBIp secretion were assessed by incubating cells for 6 h at 37°C in a media containing 5 μg/mL BFA or 5 μM MON. Cells were lysed and western blot analysis of TGFBIp in cell lysates was performed. As shown in Fig 1, TGFBIp secretion was nearly completely abrogated by exposure to BFA and MON (Fig 1B) in both WT and GCD2 HO corneal fibroblasts. These data demonstrate that TGFBIp is transported from the ER lumen to the Golgi apparatus and then to the ECM via the ER/Golgi-dependent secretory pathway; also termed the classical or conventional secretory pathways. In addition, our data demonstrate that TGFBIp co-localized with the Golgi proteins GM130 (a cis-Golgi matrix protein), mannosidase II (a medial-Golgi enzyme), and TGN38 (trans-Golgi network protein 38), suggesting the secretion of newly synthesized TGFBIp into the ECM via the ER/Golgi-dependent secretory pathway (Fig 1C).


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Expression and secretion of TGFBIp in wild-type (WT) and heterozygous (HE) or homozygous (HO) mutant primary corneal fibroblasts.A. Western blot analysis of TGFBIp in cell lysates (upper panel) and conditioned media (lower panel) of WT, HE, and HO cells. β-actin was used as a loading control. Molecular weight markers (in kDa) are indicated. B. Secretion of WT and mutant TGFBIp was inhibited by treatment with brefeldin A (BFA) and monensin (MON). C. TGFBIp co-localized with markers of cis-Golgi (upper panel), medial-Golgi (middle panel), and trans-Golgi (lower panel) in cortical cells. Representative confocal images of immunofluorescence staining of TGFBIp (green) with GM130, mannosidase II, and TGN38 (all red) are shown. Overlapping areas are displayed in yellow in the merged images. Bars = 25 μm. D. Secretion of mutant TGFBIp is delayed in GCD2 corneal fibroblasts. Corneal fibroblasts from a patient with a homozygous TGFBIp mutation and a WT control were pulse-labeled for 20 min using 35S-cysteine and then incubated for 0, 15, 30, 60, 120, 180, and 240 min in unlabeled media before immunoprecipitation of TGFBIp from cell lysates and conditioned media. Phosphorimaging was performed after SDS-PAGE to detect TGFBIp. One representative experiment is shown. E. Quantitation of the experiment presented in D. Triplicate lysate samples were analyzed. *P < 0.05.
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pone.0119561.g001: Expression and secretion of TGFBIp in wild-type (WT) and heterozygous (HE) or homozygous (HO) mutant primary corneal fibroblasts.A. Western blot analysis of TGFBIp in cell lysates (upper panel) and conditioned media (lower panel) of WT, HE, and HO cells. β-actin was used as a loading control. Molecular weight markers (in kDa) are indicated. B. Secretion of WT and mutant TGFBIp was inhibited by treatment with brefeldin A (BFA) and monensin (MON). C. TGFBIp co-localized with markers of cis-Golgi (upper panel), medial-Golgi (middle panel), and trans-Golgi (lower panel) in cortical cells. Representative confocal images of immunofluorescence staining of TGFBIp (green) with GM130, mannosidase II, and TGN38 (all red) are shown. Overlapping areas are displayed in yellow in the merged images. Bars = 25 μm. D. Secretion of mutant TGFBIp is delayed in GCD2 corneal fibroblasts. Corneal fibroblasts from a patient with a homozygous TGFBIp mutation and a WT control were pulse-labeled for 20 min using 35S-cysteine and then incubated for 0, 15, 30, 60, 120, 180, and 240 min in unlabeled media before immunoprecipitation of TGFBIp from cell lysates and conditioned media. Phosphorimaging was performed after SDS-PAGE to detect TGFBIp. One representative experiment is shown. E. Quantitation of the experiment presented in D. Triplicate lysate samples were analyzed. *P < 0.05.
Mentions: Although studies have established that TGFBIp protein is secreted into the ECM and degraded via autophagy/lysosomal pathway, the underlying trafficking pathway(s) remain poorly understood. To address this issue, we examined the secretion of TGFBIp in the presence of brefaldin-A (BFA), which reversibly blocks protein transport from the ER [39] and monensin (MON), which effectively inhibits protein transport from medial to trans Golgi cisternae [40,41]. In normal culture conditions, western blot analysis of TGFBIp in cell lysates and conditioned media of wild-type (WT) and GCD2 homozygous (HO) primary fibroblasts indicated two forms of the protein at ~66 and ~68 kDa (Fig 1A). The effects of inhibitors on TGFBIp secretion were assessed by incubating cells for 6 h at 37°C in a media containing 5 μg/mL BFA or 5 μM MON. Cells were lysed and western blot analysis of TGFBIp in cell lysates was performed. As shown in Fig 1, TGFBIp secretion was nearly completely abrogated by exposure to BFA and MON (Fig 1B) in both WT and GCD2 HO corneal fibroblasts. These data demonstrate that TGFBIp is transported from the ER lumen to the Golgi apparatus and then to the ECM via the ER/Golgi-dependent secretory pathway; also termed the classical or conventional secretory pathways. In addition, our data demonstrate that TGFBIp co-localized with the Golgi proteins GM130 (a cis-Golgi matrix protein), mannosidase II (a medial-Golgi enzyme), and TGN38 (trans-Golgi network protein 38), suggesting the secretion of newly synthesized TGFBIp into the ECM via the ER/Golgi-dependent secretory pathway (Fig 1C).

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