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Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling.

Chairoungdua A, Smith DL, Pochard P, Hull M, Caplan MJ - J. Cell Biol. (2010)

Bottom Line: Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin-mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels.These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin.Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
CD82 and CD9 are tetraspanin membrane proteins that can function as suppressors of tumor metastasis. Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin-mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels. Inhibition of Wnt/β-catenin signaling is independent of glycogen synthase kinase-3β and of the proteasome- and lysosome-mediated protein degradation pathways. CD82 and CD9 expression induces β-catenin export via exosomes, which is blocked by a sphingomyelinase inhibitor, GW4869. CD82 fails to induce exosome release of β-catenin in cells that express low levels of E-cadherin. Exosome release from dendritic cells generated from CD9 knockout mice is reduced compared with that from wild-type dendritic cells. These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin. Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways.

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β-catenin is secreted into exosomes. (A) Electron microscopy of extracellular exosomes secreted by HEK 293T cells. Exosomes were isolated by sequential centrifugation steps as described in Materials and methods from HEK 293T cells transfected with the indicated plasmids. Control cells were transfected with empty vector. Bar, 200 nm. (B) β-Catenin, E-cadherin, and tetraspanins are secreted into exosomes. Total lysate (T) and exosome (E) fractions purified from HEK 293T cells transfected with the indicated plasmids were analyzed by Western blotting using the indicated antibodies. EEA1, calnexin, and γ-adaptin are membrane markers for early endosomes, ER, and TGN, respectively. Cytochrome c was used as a marker for apoptotic cell fragments. (C) Exosome release of β-catenin is dependent on CD9 and CD82. HEK 293T cells were transiently transfected with a cDNA encoding CD9 and CD82 under control of the Tet-Off promoter or empty vector as control. After being cultured in the presence (DOX (+)) and absence (DOX (−)) of 100 ng/ml of doxycycline for 48 h, total lysate (T) and exosome (E) fractions were analyzed by Western blotting using the indicated antibodies. The exosomal protein, flotillin, was used as a loading control. (D) Exosome release of β-catenin is reduced after treatment with a sphingomyelinase (nSMase) inhibitor. HEK 293T cells were transfected with CD82 or with empty vector as control and then treated with 5 µM GW4869 or DMSO for 16 h. Total lysate (T) and exosome (E) fractions were blotted with anti–β-catenin and anti–flotillin-1 antibodies.
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fig5: β-catenin is secreted into exosomes. (A) Electron microscopy of extracellular exosomes secreted by HEK 293T cells. Exosomes were isolated by sequential centrifugation steps as described in Materials and methods from HEK 293T cells transfected with the indicated plasmids. Control cells were transfected with empty vector. Bar, 200 nm. (B) β-Catenin, E-cadherin, and tetraspanins are secreted into exosomes. Total lysate (T) and exosome (E) fractions purified from HEK 293T cells transfected with the indicated plasmids were analyzed by Western blotting using the indicated antibodies. EEA1, calnexin, and γ-adaptin are membrane markers for early endosomes, ER, and TGN, respectively. Cytochrome c was used as a marker for apoptotic cell fragments. (C) Exosome release of β-catenin is dependent on CD9 and CD82. HEK 293T cells were transiently transfected with a cDNA encoding CD9 and CD82 under control of the Tet-Off promoter or empty vector as control. After being cultured in the presence (DOX (+)) and absence (DOX (−)) of 100 ng/ml of doxycycline for 48 h, total lysate (T) and exosome (E) fractions were analyzed by Western blotting using the indicated antibodies. The exosomal protein, flotillin, was used as a loading control. (D) Exosome release of β-catenin is reduced after treatment with a sphingomyelinase (nSMase) inhibitor. HEK 293T cells were transfected with CD82 or with empty vector as control and then treated with 5 µM GW4869 or DMSO for 16 h. Total lysate (T) and exosome (E) fractions were blotted with anti–β-catenin and anti–flotillin-1 antibodies.

Mentions: Exosomes are small (30–100 nm in diameter) membrane-bound vesicles released by a variety of cell types. As mentioned above, tetraspanins are enriched in the membranes of exosomes. To test the possibility that tetraspanins reduce cellular levels of β-catenin by facilitating its export via exosomes, a number of experiments were performed. Standard protocols were used to isolate exosomes from media that was bathing cultured cells (Thery et al., 2006) throughout 48 h after their transfection. After several centrifugation steps as outlined in Materials and methods, exosomes were pelleted and analyzed by electron microscopy and Western blotting. Electron microscopy of a negatively stained exosome preparation revealed “cup-shaped” membrane vesicles with diameters of 30–100 nm (Fig. 5 A), which corresponds to the previously characterized ultrastructural morphology of exosomes (Février and Raposo, 2004).Interestingly, vesicles were detected in culture medium harvested from all conditions. However, more exosomes were purified from CD9- and CD82-transfected HEK 293T cells. These results suggest that CD9 and CD82 expression enhances exosome production by these cells. Aliquots of protein extracted from cells (10 µg) and of protein extracted from pelleted exosomes (2 µg) were subjected to Western blot analysis using antibodies specific for the known exosomal proteins HSP70 and flotillin-1, or for markers specific to other subcellular compartments, including early endosomes (EEA1), endoplasmic reticulum (calnexin), TGN (γ-adaptin), and a marker of apoptotic cells (cytochrome c). Western blot analysis (Fig. 5 B) shows the enrichment of tetraspanins in exosome fractions. In addition, ER, TGN, and early endosomal proteins were not detected in the exosome fraction, which did contain the two exosomal proteins, HSP70 and flotillin. The exosome fraction also did not contain apoptotic vesicles, because the apoptotic marker cytochrome c was not detected in the exosome fractions. These results suggest that the contamination of the exosome fraction with material derived from these compartments or from apoptotic cells is minimal.


Exosome release of β-catenin: a novel mechanism that antagonizes Wnt signaling.

Chairoungdua A, Smith DL, Pochard P, Hull M, Caplan MJ - J. Cell Biol. (2010)

β-catenin is secreted into exosomes. (A) Electron microscopy of extracellular exosomes secreted by HEK 293T cells. Exosomes were isolated by sequential centrifugation steps as described in Materials and methods from HEK 293T cells transfected with the indicated plasmids. Control cells were transfected with empty vector. Bar, 200 nm. (B) β-Catenin, E-cadherin, and tetraspanins are secreted into exosomes. Total lysate (T) and exosome (E) fractions purified from HEK 293T cells transfected with the indicated plasmids were analyzed by Western blotting using the indicated antibodies. EEA1, calnexin, and γ-adaptin are membrane markers for early endosomes, ER, and TGN, respectively. Cytochrome c was used as a marker for apoptotic cell fragments. (C) Exosome release of β-catenin is dependent on CD9 and CD82. HEK 293T cells were transiently transfected with a cDNA encoding CD9 and CD82 under control of the Tet-Off promoter or empty vector as control. After being cultured in the presence (DOX (+)) and absence (DOX (−)) of 100 ng/ml of doxycycline for 48 h, total lysate (T) and exosome (E) fractions were analyzed by Western blotting using the indicated antibodies. The exosomal protein, flotillin, was used as a loading control. (D) Exosome release of β-catenin is reduced after treatment with a sphingomyelinase (nSMase) inhibitor. HEK 293T cells were transfected with CD82 or with empty vector as control and then treated with 5 µM GW4869 or DMSO for 16 h. Total lysate (T) and exosome (E) fractions were blotted with anti–β-catenin and anti–flotillin-1 antibodies.
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fig5: β-catenin is secreted into exosomes. (A) Electron microscopy of extracellular exosomes secreted by HEK 293T cells. Exosomes were isolated by sequential centrifugation steps as described in Materials and methods from HEK 293T cells transfected with the indicated plasmids. Control cells were transfected with empty vector. Bar, 200 nm. (B) β-Catenin, E-cadherin, and tetraspanins are secreted into exosomes. Total lysate (T) and exosome (E) fractions purified from HEK 293T cells transfected with the indicated plasmids were analyzed by Western blotting using the indicated antibodies. EEA1, calnexin, and γ-adaptin are membrane markers for early endosomes, ER, and TGN, respectively. Cytochrome c was used as a marker for apoptotic cell fragments. (C) Exosome release of β-catenin is dependent on CD9 and CD82. HEK 293T cells were transiently transfected with a cDNA encoding CD9 and CD82 under control of the Tet-Off promoter or empty vector as control. After being cultured in the presence (DOX (+)) and absence (DOX (−)) of 100 ng/ml of doxycycline for 48 h, total lysate (T) and exosome (E) fractions were analyzed by Western blotting using the indicated antibodies. The exosomal protein, flotillin, was used as a loading control. (D) Exosome release of β-catenin is reduced after treatment with a sphingomyelinase (nSMase) inhibitor. HEK 293T cells were transfected with CD82 or with empty vector as control and then treated with 5 µM GW4869 or DMSO for 16 h. Total lysate (T) and exosome (E) fractions were blotted with anti–β-catenin and anti–flotillin-1 antibodies.
Mentions: Exosomes are small (30–100 nm in diameter) membrane-bound vesicles released by a variety of cell types. As mentioned above, tetraspanins are enriched in the membranes of exosomes. To test the possibility that tetraspanins reduce cellular levels of β-catenin by facilitating its export via exosomes, a number of experiments were performed. Standard protocols were used to isolate exosomes from media that was bathing cultured cells (Thery et al., 2006) throughout 48 h after their transfection. After several centrifugation steps as outlined in Materials and methods, exosomes were pelleted and analyzed by electron microscopy and Western blotting. Electron microscopy of a negatively stained exosome preparation revealed “cup-shaped” membrane vesicles with diameters of 30–100 nm (Fig. 5 A), which corresponds to the previously characterized ultrastructural morphology of exosomes (Février and Raposo, 2004).Interestingly, vesicles were detected in culture medium harvested from all conditions. However, more exosomes were purified from CD9- and CD82-transfected HEK 293T cells. These results suggest that CD9 and CD82 expression enhances exosome production by these cells. Aliquots of protein extracted from cells (10 µg) and of protein extracted from pelleted exosomes (2 µg) were subjected to Western blot analysis using antibodies specific for the known exosomal proteins HSP70 and flotillin-1, or for markers specific to other subcellular compartments, including early endosomes (EEA1), endoplasmic reticulum (calnexin), TGN (γ-adaptin), and a marker of apoptotic cells (cytochrome c). Western blot analysis (Fig. 5 B) shows the enrichment of tetraspanins in exosome fractions. In addition, ER, TGN, and early endosomal proteins were not detected in the exosome fraction, which did contain the two exosomal proteins, HSP70 and flotillin. The exosome fraction also did not contain apoptotic vesicles, because the apoptotic marker cytochrome c was not detected in the exosome fractions. These results suggest that the contamination of the exosome fraction with material derived from these compartments or from apoptotic cells is minimal.

Bottom Line: Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin-mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels.These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin.Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06510, USA.

ABSTRACT
CD82 and CD9 are tetraspanin membrane proteins that can function as suppressors of tumor metastasis. Expression of CD9 and CD82 in transfected cells strongly suppresses β-catenin-mediated Wnt signaling activity and induces a significant decrease in β-catenin protein levels. Inhibition of Wnt/β-catenin signaling is independent of glycogen synthase kinase-3β and of the proteasome- and lysosome-mediated protein degradation pathways. CD82 and CD9 expression induces β-catenin export via exosomes, which is blocked by a sphingomyelinase inhibitor, GW4869. CD82 fails to induce exosome release of β-catenin in cells that express low levels of E-cadherin. Exosome release from dendritic cells generated from CD9 knockout mice is reduced compared with that from wild-type dendritic cells. These results suggest that CD82 and CD9 down-regulate the Wnt signaling pathway through the exosomal discharge of β-catenin. Thus, exosomal packaging and release of cytosolic proteins can modulate the activity of cellular signaling pathways.

Show MeSH
Related in: MedlinePlus