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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 Wnt3A and Rspo1 in C2C12 cells.(A) C2C12 cells in 6-well plates were treated with Wnt3A CM (5%) in the absence or presence of mouse Mesd (mMesd) (0.5 µM) or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations for 6 h. The level of phosphorylated LRP6 was analyzed. (B) C2C12 cells in 6-well plates were incubated with Wnt3A CM (5%) in the presence or absence of mMesd (0.5 µM) or mMesd (155–191) at the indicated concentrations for 48 h. The levels of total cellular OPG were analyzed by Western blotting. (C) C2C12 cells in 6-well plates were incubated with Rspo1 (20 ng/ml) and/or Wnt3A CM (2%) in the absence or presence of mMesd (0.5 µM) or mMesd (155–191) (2 µM) for 48 h. The levels of total cellular OPG were analyzed by Western blotting. All the samples were also probed with the anti-actin antibody to verify equal loading.
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pone-0058102-g004: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by Wnt3A and Rspo1 in C2C12 cells.(A) C2C12 cells in 6-well plates were treated with Wnt3A CM (5%) in the absence or presence of mouse Mesd (mMesd) (0.5 µM) or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations for 6 h. The level of phosphorylated LRP6 was analyzed. (B) C2C12 cells in 6-well plates were incubated with Wnt3A CM (5%) in the presence or absence of mMesd (0.5 µM) or mMesd (155–191) at the indicated concentrations for 48 h. The levels of total cellular OPG were analyzed by Western blotting. (C) C2C12 cells in 6-well plates were incubated with Rspo1 (20 ng/ml) and/or Wnt3A CM (2%) in the absence or presence of mMesd (0.5 µM) or mMesd (155–191) (2 µM) for 48 h. The levels of total cellular OPG were analyzed by Western blotting. All the samples were also probed with the anti-actin antibody to verify equal loading.

Mentions: C2C12 cells are uncommitted mouse mesenchymal progenitor cells that can be differentiated into osteoblasts upon the activation of Wnt/β-catenin signaling [9], [25], [26]. We employed C2C12 cells to further examine the effect of Mesd peptide on Wnt3A-induced Wnt/β-catenin signaling. LRP6 phosphorylation is critical for Wnt/β-catenin signaling induced by Wnt proteins [3], and OPG is a direct target gene of Wnt/β-catenin signaling in osteoblasts [27], [28]. We found that mouse Mesd C-terminal region peptide mMesd (155–191), like Mesd protein, was able to suppress Wnt3A-induced endogenous LRP6 phosphorylation and OPG expression in C2C12 cells (Figure 4A and 4B).


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 Wnt3A and Rspo1 in C2C12 cells.(A) C2C12 cells in 6-well plates were treated with Wnt3A CM (5%) in the absence or presence of mouse Mesd (mMesd) (0.5 µM) or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations for 6 h. The level of phosphorylated LRP6 was analyzed. (B) C2C12 cells in 6-well plates were incubated with Wnt3A CM (5%) in the presence or absence of mMesd (0.5 µM) or mMesd (155–191) at the indicated concentrations for 48 h. The levels of total cellular OPG were analyzed by Western blotting. (C) C2C12 cells in 6-well plates were incubated with Rspo1 (20 ng/ml) and/or Wnt3A CM (2%) in the absence or presence of mMesd (0.5 µM) or mMesd (155–191) (2 µM) for 48 h. The levels of total cellular OPG were analyzed by Western blotting. All the samples were also probed with the anti-actin antibody to verify equal loading.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585277&req=5

pone-0058102-g004: Mesd C-terminal region peptide blocks Wnt/β-catenin signaling induced by Wnt3A and Rspo1 in C2C12 cells.(A) C2C12 cells in 6-well plates were treated with Wnt3A CM (5%) in the absence or presence of mouse Mesd (mMesd) (0.5 µM) or mouse Mesd C-terminal region peptide mMesd (155–191) at the indicated concentrations for 6 h. The level of phosphorylated LRP6 was analyzed. (B) C2C12 cells in 6-well plates were incubated with Wnt3A CM (5%) in the presence or absence of mMesd (0.5 µM) or mMesd (155–191) at the indicated concentrations for 48 h. The levels of total cellular OPG were analyzed by Western blotting. (C) C2C12 cells in 6-well plates were incubated with Rspo1 (20 ng/ml) and/or Wnt3A CM (2%) in the absence or presence of mMesd (0.5 µM) or mMesd (155–191) (2 µM) for 48 h. The levels of total cellular OPG were analyzed by Western blotting. All the samples were also probed with the anti-actin antibody to verify equal loading.
Mentions: C2C12 cells are uncommitted mouse mesenchymal progenitor cells that can be differentiated into osteoblasts upon the activation of Wnt/β-catenin signaling [9], [25], [26]. We employed C2C12 cells to further examine the effect of Mesd peptide on Wnt3A-induced Wnt/β-catenin signaling. LRP6 phosphorylation is critical for Wnt/β-catenin signaling induced by Wnt proteins [3], and OPG is a direct target gene of Wnt/β-catenin signaling in osteoblasts [27], [28]. We found that mouse Mesd C-terminal region peptide mMesd (155–191), like Mesd protein, was able to suppress Wnt3A-induced endogenous LRP6 phosphorylation and OPG expression in C2C12 cells (Figure 4A and 4B).

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