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Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

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Proposed Model for Regulation of ClC-3 on Recycling of β1 IntegrinClC-3-mediated β1 integrin recycling via K18 reorganization by promoting Ser-52 phosphorylation. Under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalized β1 integrin transport to membrane ruffles along the paved way by binding to phosphorylated K18. Insets representative confocal photomicrograph of ClC-3 immunofluorescence in a ruffling HeLa cell induced by EGF stimulation.
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Figure 8: Proposed Model for Regulation of ClC-3 on Recycling of β1 IntegrinClC-3-mediated β1 integrin recycling via K18 reorganization by promoting Ser-52 phosphorylation. Under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalized β1 integrin transport to membrane ruffles along the paved way by binding to phosphorylated K18. Insets representative confocal photomicrograph of ClC-3 immunofluorescence in a ruffling HeLa cell induced by EGF stimulation.

Mentions: How does ClC-3 regulate β1 integrin recycling? Upon phosphorylation by Akt, ACAP1 directly binds to β1 integrin on endosomal membranes to promote integrin recycling [46]. ClC-3 may directly bind to β1 integrin to regulate its trafficking. But the Co-IP results showed that ClC-3 did not directly interact with β1 integrin. β1 integrin trafficking in the cytoplasm occurs along two traditional routes (a Rab4-mediated or a Rab11-regulated route) [47] or non-conventional pathway [48-50]. We found that ClC-3 did not colocalize with Rab4 or Rab11 in the cytoplasm after internalization of β1 integrin. This indicated that ClC-3 may regulate recycling of β1 integrin via a non-traditional route. We next focused on the roles of cytoskeleton on β1 integrin recycling mediated by ClC-3. After excluding several cytoskeletal proteins (tubulin, actin and vimentin), we finally determined that keratin 18 (K18) functioned as a bridge in ClC-3-mediated regulation of β1 integrin recycling. Researchers have found that PKC-mediated phosphorylation of cytoskeleton vimentin is a key process in integrin trafficking through the cell [49]. Consistent with this, our further results demonstrated that knockdown of ClC-3 abated the increase in K18 Ser52 phosphorylation induced by EGF and that K18 Ser52→Ala mutant prevented the binding of ClC-3 to K18. These findings indicated that ClC-3-dependent Ser52 phosphorylation of cytoskeleton K18 was necessary for β1 integrin trafficking to membrane ruffles. Ser52 phosphorylation can trigger K18 reorganization [36]. Together, our data paint a picture for the mechanism of ClC-3-mediated β1 integrin recycling. In this picture, under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalizes β1 integrin for transport to membrane ruffles along the paved way by binding to phosphorylated K18 (Figure 8).


Chloride channel-3 promotes tumor metastasis by regulating membrane ruffling and is associated with poor survival.

Xu B, Jin X, Min L, Li Q, Deng L, Wu H, Lin G, Chen L, Zhang H, Li C, Wang L, Zhu J, Wang W, Chu F, Shen J, Li H, Mao J - Oncotarget (2015)

Proposed Model for Regulation of ClC-3 on Recycling of β1 IntegrinClC-3-mediated β1 integrin recycling via K18 reorganization by promoting Ser-52 phosphorylation. Under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalized β1 integrin transport to membrane ruffles along the paved way by binding to phosphorylated K18. Insets representative confocal photomicrograph of ClC-3 immunofluorescence in a ruffling HeLa cell induced by EGF stimulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385862&req=5

Figure 8: Proposed Model for Regulation of ClC-3 on Recycling of β1 IntegrinClC-3-mediated β1 integrin recycling via K18 reorganization by promoting Ser-52 phosphorylation. Under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalized β1 integrin transport to membrane ruffles along the paved way by binding to phosphorylated K18. Insets representative confocal photomicrograph of ClC-3 immunofluorescence in a ruffling HeLa cell induced by EGF stimulation.
Mentions: How does ClC-3 regulate β1 integrin recycling? Upon phosphorylation by Akt, ACAP1 directly binds to β1 integrin on endosomal membranes to promote integrin recycling [46]. ClC-3 may directly bind to β1 integrin to regulate its trafficking. But the Co-IP results showed that ClC-3 did not directly interact with β1 integrin. β1 integrin trafficking in the cytoplasm occurs along two traditional routes (a Rab4-mediated or a Rab11-regulated route) [47] or non-conventional pathway [48-50]. We found that ClC-3 did not colocalize with Rab4 or Rab11 in the cytoplasm after internalization of β1 integrin. This indicated that ClC-3 may regulate recycling of β1 integrin via a non-traditional route. We next focused on the roles of cytoskeleton on β1 integrin recycling mediated by ClC-3. After excluding several cytoskeletal proteins (tubulin, actin and vimentin), we finally determined that keratin 18 (K18) functioned as a bridge in ClC-3-mediated regulation of β1 integrin recycling. Researchers have found that PKC-mediated phosphorylation of cytoskeleton vimentin is a key process in integrin trafficking through the cell [49]. Consistent with this, our further results demonstrated that knockdown of ClC-3 abated the increase in K18 Ser52 phosphorylation induced by EGF and that K18 Ser52→Ala mutant prevented the binding of ClC-3 to K18. These findings indicated that ClC-3-dependent Ser52 phosphorylation of cytoskeleton K18 was necessary for β1 integrin trafficking to membrane ruffles. Ser52 phosphorylation can trigger K18 reorganization [36]. Together, our data paint a picture for the mechanism of ClC-3-mediated β1 integrin recycling. In this picture, under EGF stimulation, cytoplasmic ClC-3 first binds to K18 and initiates its reorganization to pave roads by promoting Ser52 phosphorylation, and then internalizes β1 integrin for transport to membrane ruffles along the paved way by binding to phosphorylated K18 (Figure 8).

Bottom Line: High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients.We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis.ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling.

View Article: PubMed Central - PubMed

Affiliation: Guangdong Key Laboratory for Bioactive Drugs Research, Guangdong Pharmaceutical University, Guangzhou, China.

ABSTRACT
The chloride channel-3 (ClC-3) protein is known to be a component of Cl- channels involved in cell volume regulation or acidification of intracellular vesicles. Here, we report that ClC-3 was highly expressed in the cytoplasm of metastatic carcinomatous cells and accelerated cell migration in vitro and tumor metastasis in vivo. High-grade expression of cytoplasmic ClC-3 predicted poor survival in cancer patients. We found that independent of its volume-activated Cl- channel properties, ClC-3 was able to promote cell membrane ruffling, required for tumor metastasis. ClC-3 mediated membrane ruffling by regulating keratin 18 phosphorylation to control β1 Integrin recycling. Therefore, cytoplasmic ClC-3 plays an active and key role in tumor metastasis and may be a valuable prognostic biomarker and a therapeutic target to prevent tumor spread.

Show MeSH
Related in: MedlinePlus