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The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

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Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by LRP5, LRP6, Wnt3A and Rspo1 in HEK293 cells.HEK293 cells in 24-well plates were transiently transfected with the LRP5 plasmid (A), the LRP6 plasmid (B) or the corresponding control vector, along with the TOPFlash luciferase construct and the β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with Wnt3A CM (5%), Rspo1 (40 ng/ml), mouse Mesd (mMesd) (2 µM), or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to the control cells without Mesd and its peptide treatment.
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pone-0058102-g003: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by LRP5, LRP6, Wnt3A and Rspo1 in HEK293 cells.HEK293 cells in 24-well plates were transiently transfected with the LRP5 plasmid (A), the LRP6 plasmid (B) or the corresponding control vector, along with the TOPFlash luciferase construct and the β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with Wnt3A CM (5%), Rspo1 (40 ng/ml), mouse Mesd (mMesd) (2 µM), or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to the control cells without Mesd and its peptide treatment.

Mentions: Wnt3A is one of the 19 vertebrate members of the Wnt family. There are four members in the Rspo family. Both Wnt3A and Rspo1 are able to activate Wnt/β-catenin signaling through LRP5 or LRP6 [5], [8], [9]. Therefore, we performed Wnt/β-catenin signaling reporter assay to test whether the Mesd C-terminal region peptide mimics Mesd protein to act as an inhibitor of Wnt/β-catenin signaling induced by Wnt3A and Rspo1 in LRP5- or LRP6-expressing cells. HEK293 cells were transiently transfected with LRP5 or LRP6 along with Wnt/β-catenin signaling reporter TOPFLash, and treated with Wnt3A CM or Rspo1 protein in the presence or absence of mouse Mesd protein or its C-terminal region peptide mMesd (155–191). As expected, TOPFlash activity was greatly increased after HEK293 cells were transiently transfected with LRP5 or LRP6 and treated with Wnt3A or Rspo1 (Figure 3). Importantly, the increased TOPFlash activity induced by LRP5, LRP6, LRP5 plus Wnt3A, LRP6 plus Wnt3A, LRP5 plus Rspo1, or LRP6 plus Rspo1 was blocked not only by Mesd protein but also by its peptide mMesd (155–191) in a dose dependent manner (Figure 3).


The C-terminal region Mesd peptide mimics full-length Mesd and acts as an inhibitor of Wnt/β-catenin signaling in cancer cells.

Lin C, Lu W, Zhang W, Londoño-Joshi AI, Buchsbaum DJ, Bu G, Li Y - PLoS ONE (2013)

Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by LRP5, LRP6, Wnt3A and Rspo1 in HEK293 cells.HEK293 cells in 24-well plates were transiently transfected with the LRP5 plasmid (A), the LRP6 plasmid (B) or the corresponding control vector, along with the TOPFlash luciferase construct and the β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with Wnt3A CM (5%), Rspo1 (40 ng/ml), mouse Mesd (mMesd) (2 µM), or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to the control cells without Mesd and its peptide treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g003: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by LRP5, LRP6, Wnt3A and Rspo1 in HEK293 cells.HEK293 cells in 24-well plates were transiently transfected with the LRP5 plasmid (A), the LRP6 plasmid (B) or the corresponding control vector, along with the TOPFlash luciferase construct and the β-galactosidase-expressing vector in each well. After 24 h incubation, cells were treated with Wnt3A CM (5%), Rspo1 (40 ng/ml), mouse Mesd (mMesd) (2 µM), or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations. The luciferase activity was then measured 24 h later with normalization to the activity of the β-galactosidase. Values are the average of triple determinations with the s.d. indicated by error bars. *P<0.05, **P<0.01 compared to the control cells without Mesd and its peptide treatment.
Mentions: Wnt3A is one of the 19 vertebrate members of the Wnt family. There are four members in the Rspo family. Both Wnt3A and Rspo1 are able to activate Wnt/β-catenin signaling through LRP5 or LRP6 [5], [8], [9]. Therefore, we performed Wnt/β-catenin signaling reporter assay to test whether the Mesd C-terminal region peptide mimics Mesd protein to act as an inhibitor of Wnt/β-catenin signaling induced by Wnt3A and Rspo1 in LRP5- or LRP6-expressing cells. HEK293 cells were transiently transfected with LRP5 or LRP6 along with Wnt/β-catenin signaling reporter TOPFLash, and treated with Wnt3A CM or Rspo1 protein in the presence or absence of mouse Mesd protein or its C-terminal region peptide mMesd (155–191). As expected, TOPFlash activity was greatly increased after HEK293 cells were transiently transfected with LRP5 or LRP6 and treated with Wnt3A or Rspo1 (Figure 3). Importantly, the increased TOPFlash activity induced by LRP5, LRP6, LRP5 plus Wnt3A, LRP6 plus Wnt3A, LRP5 plus Rspo1, or LRP6 plus Rspo1 was blocked not only by Mesd protein but also by its peptide mMesd (155–191) in a dose dependent manner (Figure 3).

Bottom Line: In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface.We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too.Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery Division, Southern Research Institute, Birmingham, Alabama, United States of America.

ABSTRACT
While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.

Show MeSH
Related in: MedlinePlus