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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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Effects of CD9 on Wnt/β-catenin signaling and exosome release in vivo. (A) Reduction of BMDC-derived exosomes from CD9 knockout mice. Exosomes were purified from BMDCs derived from CD9 wild-type and knockout mice as described in Materials and methods and analyzed by Western blotting using the indicated antibodies. BMDC lysates were immunoblotted with anti-CD9 as shown in the bottom panel. (B) CD82 expression inhibits β-catenin signaling and facilitates the exosome release of β-catenin in PC3 cells. PC3 cells were transiently cotransfected with the indicated plasmids and with the TOPflash and the Renilla luciferase vectors. Activity of the β-catenin signaling pathway was quantified by measuring relative firefly luciferase activity units (RLUs) normalized to Renilla luciferase and expressed as fold change in the luciferase signal (top panel). In the bottom panel, Western blots of total lysates and exosome fractions were purified from PC3 cells transiently transfected with indicated plasmids. CD82 expression increased the exosome release of β-catenin by a factor of 3. (C) A novel role for tetraspanins in the regulation of Wnt/β-catenin signaling. At steady state, tetraspanins are organized in a signaling complex with E-cadherin at the plasma membrane. This signaling complex, including tetraspanins, E-cadherin, and β-catenin, is internalized and delivered to early endosomes. Exosome biogenesis begins with outward vesicle budding at the limiting membrane of endosomes, generating intraluminal vesicles (ILVs). These exosome-containing endosomes eventually mature into late endosomes, also known as mutivesicular bodies (MVBs). These MVBs then fuse with plasma membrane and release their intraluminal vesicles, referred to as exosomes, which contain β-catenin. The exosome targeting of β-catenin causes a reduction in the intracellular pool of β-catenin and therefore reduces Wnt/β-catenin signaling.
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fig7: Effects of CD9 on Wnt/β-catenin signaling and exosome release in vivo. (A) Reduction of BMDC-derived exosomes from CD9 knockout mice. Exosomes were purified from BMDCs derived from CD9 wild-type and knockout mice as described in Materials and methods and analyzed by Western blotting using the indicated antibodies. BMDC lysates were immunoblotted with anti-CD9 as shown in the bottom panel. (B) CD82 expression inhibits β-catenin signaling and facilitates the exosome release of β-catenin in PC3 cells. PC3 cells were transiently cotransfected with the indicated plasmids and with the TOPflash and the Renilla luciferase vectors. Activity of the β-catenin signaling pathway was quantified by measuring relative firefly luciferase activity units (RLUs) normalized to Renilla luciferase and expressed as fold change in the luciferase signal (top panel). In the bottom panel, Western blots of total lysates and exosome fractions were purified from PC3 cells transiently transfected with indicated plasmids. CD82 expression increased the exosome release of β-catenin by a factor of 3. (C) A novel role for tetraspanins in the regulation of Wnt/β-catenin signaling. At steady state, tetraspanins are organized in a signaling complex with E-cadherin at the plasma membrane. This signaling complex, including tetraspanins, E-cadherin, and β-catenin, is internalized and delivered to early endosomes. Exosome biogenesis begins with outward vesicle budding at the limiting membrane of endosomes, generating intraluminal vesicles (ILVs). These exosome-containing endosomes eventually mature into late endosomes, also known as mutivesicular bodies (MVBs). These MVBs then fuse with plasma membrane and release their intraluminal vesicles, referred to as exosomes, which contain β-catenin. The exosome targeting of β-catenin causes a reduction in the intracellular pool of β-catenin and therefore reduces Wnt/β-catenin signaling.

Mentions: To investigate the role of tetraspanins in exosome release in vivo, bone marrow dendritic cells (BMDCs) were generated from wild-type and CD9 knockout mice as described in Materials and methods. BMDC-derived exosomes were purified from culture medium on d 7 and analyzed by Western blotting using anti-flotillin antibody. As shown in Fig. 7 A, BMDCs generated from CD9 knockout mice secreted a smaller quantity of exosomes as compared with BMDCs generated from wild-type mice. Quantitation of transmission electron micrographs of these exosome fractions confirmed these results, demonstrating that the quantity of morphologically recognizable exosomes released from the CD9 cells is one third of that released from wild-type cells (Fig. S5 C). These data support the concept that tetraspanins play a central role in exosome release.


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)

Effects of CD9 on Wnt/β-catenin signaling and exosome release in vivo. (A) Reduction of BMDC-derived exosomes from CD9 knockout mice. Exosomes were purified from BMDCs derived from CD9 wild-type and knockout mice as described in Materials and methods and analyzed by Western blotting using the indicated antibodies. BMDC lysates were immunoblotted with anti-CD9 as shown in the bottom panel. (B) CD82 expression inhibits β-catenin signaling and facilitates the exosome release of β-catenin in PC3 cells. PC3 cells were transiently cotransfected with the indicated plasmids and with the TOPflash and the Renilla luciferase vectors. Activity of the β-catenin signaling pathway was quantified by measuring relative firefly luciferase activity units (RLUs) normalized to Renilla luciferase and expressed as fold change in the luciferase signal (top panel). In the bottom panel, Western blots of total lysates and exosome fractions were purified from PC3 cells transiently transfected with indicated plasmids. CD82 expression increased the exosome release of β-catenin by a factor of 3. (C) A novel role for tetraspanins in the regulation of Wnt/β-catenin signaling. At steady state, tetraspanins are organized in a signaling complex with E-cadherin at the plasma membrane. This signaling complex, including tetraspanins, E-cadherin, and β-catenin, is internalized and delivered to early endosomes. Exosome biogenesis begins with outward vesicle budding at the limiting membrane of endosomes, generating intraluminal vesicles (ILVs). These exosome-containing endosomes eventually mature into late endosomes, also known as mutivesicular bodies (MVBs). These MVBs then fuse with plasma membrane and release their intraluminal vesicles, referred to as exosomes, which contain β-catenin. The exosome targeting of β-catenin causes a reduction in the intracellular pool of β-catenin and therefore reduces Wnt/β-catenin signaling.
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Related In: Results  -  Collection

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fig7: Effects of CD9 on Wnt/β-catenin signaling and exosome release in vivo. (A) Reduction of BMDC-derived exosomes from CD9 knockout mice. Exosomes were purified from BMDCs derived from CD9 wild-type and knockout mice as described in Materials and methods and analyzed by Western blotting using the indicated antibodies. BMDC lysates were immunoblotted with anti-CD9 as shown in the bottom panel. (B) CD82 expression inhibits β-catenin signaling and facilitates the exosome release of β-catenin in PC3 cells. PC3 cells were transiently cotransfected with the indicated plasmids and with the TOPflash and the Renilla luciferase vectors. Activity of the β-catenin signaling pathway was quantified by measuring relative firefly luciferase activity units (RLUs) normalized to Renilla luciferase and expressed as fold change in the luciferase signal (top panel). In the bottom panel, Western blots of total lysates and exosome fractions were purified from PC3 cells transiently transfected with indicated plasmids. CD82 expression increased the exosome release of β-catenin by a factor of 3. (C) A novel role for tetraspanins in the regulation of Wnt/β-catenin signaling. At steady state, tetraspanins are organized in a signaling complex with E-cadherin at the plasma membrane. This signaling complex, including tetraspanins, E-cadherin, and β-catenin, is internalized and delivered to early endosomes. Exosome biogenesis begins with outward vesicle budding at the limiting membrane of endosomes, generating intraluminal vesicles (ILVs). These exosome-containing endosomes eventually mature into late endosomes, also known as mutivesicular bodies (MVBs). These MVBs then fuse with plasma membrane and release their intraluminal vesicles, referred to as exosomes, which contain β-catenin. The exosome targeting of β-catenin causes a reduction in the intracellular pool of β-catenin and therefore reduces Wnt/β-catenin signaling.
Mentions: To investigate the role of tetraspanins in exosome release in vivo, bone marrow dendritic cells (BMDCs) were generated from wild-type and CD9 knockout mice as described in Materials and methods. BMDC-derived exosomes were purified from culture medium on d 7 and analyzed by Western blotting using anti-flotillin antibody. As shown in Fig. 7 A, BMDCs generated from CD9 knockout mice secreted a smaller quantity of exosomes as compared with BMDCs generated from wild-type mice. Quantitation of transmission electron micrographs of these exosome fractions confirmed these results, demonstrating that the quantity of morphologically recognizable exosomes released from the CD9 cells is one third of that released from wild-type cells (Fig. S5 C). These data support the concept that tetraspanins play a central role in exosome release.

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