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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 of caveolin-1 and -2 in established cell lines and caveolae formation in WT, HE, and HO mutant TGFBIp-expressing primary cultured corneal fibroblasts.A. Total cellular protein (50 μg) from the specified cell lines was subjected to western blot analysis with anti-caveolin-1 (first panel), anti-caveolin-1 (second panel, LE: longer exposure), anti-caveolin-2 (third panel), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, fourth panel). GAPDH was used as a loading control. B. Western blot analysis of caveolin-1 and -2 expressions in WT, HE, and HO TGFBIp-expressing corneal fibroblasts. β-actin was used as a loading control. C. Densitometric quantitation of the experiment presented in B. D. Transmission electron microscopy (TEM) of corneal fibroblasts reveals formation of caveolae in corneal fibroblasts. Cells were grown in basal media, fixed, and then prepared for scanning TEM as described in Materials and Methods. ① Caveolae are detected on the inner surface of the plasma membrane of corneal fibroblasts. Cells contain caveolae in their apical membranes that are characterized by coat-free flask-shaped invaginations (asterisks in ③) with a diaphragm at the neck (arrows in ③). ② Note the characteristic clustering of caveolae into racemose structures, or caveosomes, on the basal side (④ asterisks). Bar = 500 nm. E. At steady state, TGFBIp is localized in the caveolae both at the plasma membrane and inside the cell. TGFBIp was immunogold-labeled on ultrathin cryosections of WT corneal fibroblasts using the TGFBIp monoclonal antibody. ① and ② plasma membrane caveolae-like structures (arrowhead) appeared as flask-shaped invaginations on the plasma membrane (p). ③ and ④ Ultrathin cryosections were labeled with anti-TGFBIp antibodies. TGFBIp-gold signal (arrow) accumulated in the lysosomes of WT corneal fibroblasts. ④ Caveolae-like structures also appeared in the lysosomes (circle). The scale bars in all panels are 100 nm.
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pone.0119561.g004: Expression of caveolin-1 and -2 in established cell lines and caveolae formation in WT, HE, and HO mutant TGFBIp-expressing primary cultured corneal fibroblasts.A. Total cellular protein (50 μg) from the specified cell lines was subjected to western blot analysis with anti-caveolin-1 (first panel), anti-caveolin-1 (second panel, LE: longer exposure), anti-caveolin-2 (third panel), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, fourth panel). GAPDH was used as a loading control. B. Western blot analysis of caveolin-1 and -2 expressions in WT, HE, and HO TGFBIp-expressing corneal fibroblasts. β-actin was used as a loading control. C. Densitometric quantitation of the experiment presented in B. D. Transmission electron microscopy (TEM) of corneal fibroblasts reveals formation of caveolae in corneal fibroblasts. Cells were grown in basal media, fixed, and then prepared for scanning TEM as described in Materials and Methods. ① Caveolae are detected on the inner surface of the plasma membrane of corneal fibroblasts. Cells contain caveolae in their apical membranes that are characterized by coat-free flask-shaped invaginations (asterisks in ③) with a diaphragm at the neck (arrows in ③). ② Note the characteristic clustering of caveolae into racemose structures, or caveosomes, on the basal side (④ asterisks). Bar = 500 nm. E. At steady state, TGFBIp is localized in the caveolae both at the plasma membrane and inside the cell. TGFBIp was immunogold-labeled on ultrathin cryosections of WT corneal fibroblasts using the TGFBIp monoclonal antibody. ① and ② plasma membrane caveolae-like structures (arrowhead) appeared as flask-shaped invaginations on the plasma membrane (p). ③ and ④ Ultrathin cryosections were labeled with anti-TGFBIp antibodies. TGFBIp-gold signal (arrow) accumulated in the lysosomes of WT corneal fibroblasts. ④ Caveolae-like structures also appeared in the lysosomes (circle). The scale bars in all panels are 100 nm.

Mentions: Expression of exogenous caveolin-1 induces the formation of caveolae on the surface of cells that normally do not form them [51]. Furthermore, knockout of the gene encoding caveolin-1 in mice also reduces the expression of caveolin-2 and leads to the loss of morphologically defined caveolae [52]. These studies suggest a possible correlation between the expression of caveolins and the efficiency of TGFBIp endocytosis. Therefore, we examined the expression of caveolin-1 and -2 in cells that have different internalization levels of TGFBIp (Fig 4A). Caveolin-1 and -2 proteins showed cell-specific expression pattern (Fig 4A). Specifically, caveolin-1 was more abundant in SK-N-MC cells than in the NIH3T3 cells. Longer exposure of the blots showed a very low level of caveolin-1 expression in ZW13-1 cells. Caveolin-2 also showed differential expression with a moderate level of expression in ZW13-1 cells and a higher level in the NIH3T3 and SK-N-MC cell lines (Fig 4A). However, HEK293T cells had no detectable expression of caveolin-1 and caveolin-2 (Fig 4A), consistent with previously published data [53]. However, the low-level expressions of CAV-1 were detected at mRNA level by RT-PCR (S1 Fig). Taken together, these results demonstrate that the expression of caveolin-1 protein closely resembled that of caveolin-2 and that there was a clear correlation between the cellular expression levels of caveolins and the endocytosis of TGFBIp in these cell lines. These results also suggest that the expression levels of caveolin-1 and -2 might influence TGFBIp endocytosis in corneal fibroblasts. Therefore, we analyzed the expression levels of caveolin-1 and -2 in WT and GCD2 corneal fibroblasts. Fig 3B shows that corneal fibroblasts differentially express caveolin-1 and -2, but the levels of expression did not differ significantly between WT and GCD2 corneal fibroblasts (Fig 4B and 4C).


Lysosomal trafficking of TGFBIp via caveolae-mediated endocytosis.

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

Expression of caveolin-1 and -2 in established cell lines and caveolae formation in WT, HE, and HO mutant TGFBIp-expressing primary cultured corneal fibroblasts.A. Total cellular protein (50 μg) from the specified cell lines was subjected to western blot analysis with anti-caveolin-1 (first panel), anti-caveolin-1 (second panel, LE: longer exposure), anti-caveolin-2 (third panel), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, fourth panel). GAPDH was used as a loading control. B. Western blot analysis of caveolin-1 and -2 expressions in WT, HE, and HO TGFBIp-expressing corneal fibroblasts. β-actin was used as a loading control. C. Densitometric quantitation of the experiment presented in B. D. Transmission electron microscopy (TEM) of corneal fibroblasts reveals formation of caveolae in corneal fibroblasts. Cells were grown in basal media, fixed, and then prepared for scanning TEM as described in Materials and Methods. ① Caveolae are detected on the inner surface of the plasma membrane of corneal fibroblasts. Cells contain caveolae in their apical membranes that are characterized by coat-free flask-shaped invaginations (asterisks in ③) with a diaphragm at the neck (arrows in ③). ② Note the characteristic clustering of caveolae into racemose structures, or caveosomes, on the basal side (④ asterisks). Bar = 500 nm. E. At steady state, TGFBIp is localized in the caveolae both at the plasma membrane and inside the cell. TGFBIp was immunogold-labeled on ultrathin cryosections of WT corneal fibroblasts using the TGFBIp monoclonal antibody. ① and ② plasma membrane caveolae-like structures (arrowhead) appeared as flask-shaped invaginations on the plasma membrane (p). ③ and ④ Ultrathin cryosections were labeled with anti-TGFBIp antibodies. TGFBIp-gold signal (arrow) accumulated in the lysosomes of WT corneal fibroblasts. ④ Caveolae-like structures also appeared in the lysosomes (circle). The scale bars in all panels are 100 nm.
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Related In: Results  -  Collection

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pone.0119561.g004: Expression of caveolin-1 and -2 in established cell lines and caveolae formation in WT, HE, and HO mutant TGFBIp-expressing primary cultured corneal fibroblasts.A. Total cellular protein (50 μg) from the specified cell lines was subjected to western blot analysis with anti-caveolin-1 (first panel), anti-caveolin-1 (second panel, LE: longer exposure), anti-caveolin-2 (third panel), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, fourth panel). GAPDH was used as a loading control. B. Western blot analysis of caveolin-1 and -2 expressions in WT, HE, and HO TGFBIp-expressing corneal fibroblasts. β-actin was used as a loading control. C. Densitometric quantitation of the experiment presented in B. D. Transmission electron microscopy (TEM) of corneal fibroblasts reveals formation of caveolae in corneal fibroblasts. Cells were grown in basal media, fixed, and then prepared for scanning TEM as described in Materials and Methods. ① Caveolae are detected on the inner surface of the plasma membrane of corneal fibroblasts. Cells contain caveolae in their apical membranes that are characterized by coat-free flask-shaped invaginations (asterisks in ③) with a diaphragm at the neck (arrows in ③). ② Note the characteristic clustering of caveolae into racemose structures, or caveosomes, on the basal side (④ asterisks). Bar = 500 nm. E. At steady state, TGFBIp is localized in the caveolae both at the plasma membrane and inside the cell. TGFBIp was immunogold-labeled on ultrathin cryosections of WT corneal fibroblasts using the TGFBIp monoclonal antibody. ① and ② plasma membrane caveolae-like structures (arrowhead) appeared as flask-shaped invaginations on the plasma membrane (p). ③ and ④ Ultrathin cryosections were labeled with anti-TGFBIp antibodies. TGFBIp-gold signal (arrow) accumulated in the lysosomes of WT corneal fibroblasts. ④ Caveolae-like structures also appeared in the lysosomes (circle). The scale bars in all panels are 100 nm.
Mentions: Expression of exogenous caveolin-1 induces the formation of caveolae on the surface of cells that normally do not form them [51]. Furthermore, knockout of the gene encoding caveolin-1 in mice also reduces the expression of caveolin-2 and leads to the loss of morphologically defined caveolae [52]. These studies suggest a possible correlation between the expression of caveolins and the efficiency of TGFBIp endocytosis. Therefore, we examined the expression of caveolin-1 and -2 in cells that have different internalization levels of TGFBIp (Fig 4A). Caveolin-1 and -2 proteins showed cell-specific expression pattern (Fig 4A). Specifically, caveolin-1 was more abundant in SK-N-MC cells than in the NIH3T3 cells. Longer exposure of the blots showed a very low level of caveolin-1 expression in ZW13-1 cells. Caveolin-2 also showed differential expression with a moderate level of expression in ZW13-1 cells and a higher level in the NIH3T3 and SK-N-MC cell lines (Fig 4A). However, HEK293T cells had no detectable expression of caveolin-1 and caveolin-2 (Fig 4A), consistent with previously published data [53]. However, the low-level expressions of CAV-1 were detected at mRNA level by RT-PCR (S1 Fig). Taken together, these results demonstrate that the expression of caveolin-1 protein closely resembled that of caveolin-2 and that there was a clear correlation between the cellular expression levels of caveolins and the endocytosis of TGFBIp in these cell lines. These results also suggest that the expression levels of caveolin-1 and -2 might influence TGFBIp endocytosis in corneal fibroblasts. Therefore, we analyzed the expression levels of caveolin-1 and -2 in WT and GCD2 corneal fibroblasts. Fig 3B shows that corneal fibroblasts differentially express caveolin-1 and -2, but the levels of expression did not differ significantly between WT and GCD2 corneal fibroblasts (Fig 4B and 4C).

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