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Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2.

Li C, Chen H, Hu L, Xing Y, Sasaki T, Villosis MF, Li J, Nishita M, Minami Y, Minoo P - BMC Mol. Biol. (2008)

Bottom Line: We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling.These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Division of Neonatology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA. changgon@usc.edu

ABSTRACT

Background: Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.

Results: Here we report that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.

Conclusion: The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.

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Wnt3a stimulation of STF mediated by Fzd2 and Ror2 is inhibited by Dkk1 and Krm1. H441 cells were transfected with pSV-β-gal (Promega, WI), STF, Fzd2 and Ror2WT with and without Dkk1 and Krm1 expression constructs, and then treated with conditioned media, L-CM or Wnt3aCM. Equal amounts of DNA and conditioned media were used in each assay. Luciferase values were normalized to beta-galactosidase for transfection efficiency. Values of the assay transfected with control vector and treated with L-CM alone (empty bars) were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05.
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Figure 11: Wnt3a stimulation of STF mediated by Fzd2 and Ror2 is inhibited by Dkk1 and Krm1. H441 cells were transfected with pSV-β-gal (Promega, WI), STF, Fzd2 and Ror2WT with and without Dkk1 and Krm1 expression constructs, and then treated with conditioned media, L-CM or Wnt3aCM. Equal amounts of DNA and conditioned media were used in each assay. Luciferase values were normalized to beta-galactosidase for transfection efficiency. Values of the assay transfected with control vector and treated with L-CM alone (empty bars) were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05.

Mentions: To further determine if Ror2 potentiates Wnt3a-activation of STF through activation of canonical Wnt signaling, we examined whether Lrps are required. We first performed transfection assays with siRNAs that specifically target Lrp6. Specificity of the Lrp6 siRNAs was determined by western blot analysis (Figure 10B). As shown in Figure 10A Lrp6 siRNAs almost completely abolished the Wnt3a-activation of STF by Ror2 alone and reduced the response mediated by Fzd2 alone or Fzd2 and Ror2 combination by 70% and 80%, respectively. To block the function of Lrp5/6 more broadly, we performed transfection assays with an Lrp5/6 specific inhibitor, Dkk1, and its cofactor, Kremen1 (Krm1) [24]. As shown in Figure 11, Dkk1 and Krm1 almost entirely inhibited STF activity in presence of Ror2 or Fzd2. Similar levels (folds) of inhibition by Dkk1 and Krm1 were observed for Fzd2-Ror2-mediated STF stimulation. Activation of the canonical Wnt signaling through Fzd-Lrp5/6 can also be inhibited intracellularly by GSK3beta. GSK3beta binds to APC and Axin to facilitate beta-catenin phosphorylation and subsequent degradation. When a Xenopus GSK-3, (Xgsk-3), expression plasmid was used in the cotransfection studies, Wnt3a-stimulated STF response decreased significantly in H441 cells transfected with either Ror2 alone, Fzd2 alone or Fzd2 plus Ror2 (Figure 12). Thus, the sum of these results is consistent with a model in which Ror2 potentiates Fzd2-Lrp5/6 in mediating Wnt3a stimulation of the canonical Wnt signaling.


Ror2 modulates the canonical Wnt signaling in lung epithelial cells through cooperation with Fzd2.

Li C, Chen H, Hu L, Xing Y, Sasaki T, Villosis MF, Li J, Nishita M, Minami Y, Minoo P - BMC Mol. Biol. (2008)

Wnt3a stimulation of STF mediated by Fzd2 and Ror2 is inhibited by Dkk1 and Krm1. H441 cells were transfected with pSV-β-gal (Promega, WI), STF, Fzd2 and Ror2WT with and without Dkk1 and Krm1 expression constructs, and then treated with conditioned media, L-CM or Wnt3aCM. Equal amounts of DNA and conditioned media were used in each assay. Luciferase values were normalized to beta-galactosidase for transfection efficiency. Values of the assay transfected with control vector and treated with L-CM alone (empty bars) were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254434&req=5

Figure 11: Wnt3a stimulation of STF mediated by Fzd2 and Ror2 is inhibited by Dkk1 and Krm1. H441 cells were transfected with pSV-β-gal (Promega, WI), STF, Fzd2 and Ror2WT with and without Dkk1 and Krm1 expression constructs, and then treated with conditioned media, L-CM or Wnt3aCM. Equal amounts of DNA and conditioned media were used in each assay. Luciferase values were normalized to beta-galactosidase for transfection efficiency. Values of the assay transfected with control vector and treated with L-CM alone (empty bars) were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05.
Mentions: To further determine if Ror2 potentiates Wnt3a-activation of STF through activation of canonical Wnt signaling, we examined whether Lrps are required. We first performed transfection assays with siRNAs that specifically target Lrp6. Specificity of the Lrp6 siRNAs was determined by western blot analysis (Figure 10B). As shown in Figure 10A Lrp6 siRNAs almost completely abolished the Wnt3a-activation of STF by Ror2 alone and reduced the response mediated by Fzd2 alone or Fzd2 and Ror2 combination by 70% and 80%, respectively. To block the function of Lrp5/6 more broadly, we performed transfection assays with an Lrp5/6 specific inhibitor, Dkk1, and its cofactor, Kremen1 (Krm1) [24]. As shown in Figure 11, Dkk1 and Krm1 almost entirely inhibited STF activity in presence of Ror2 or Fzd2. Similar levels (folds) of inhibition by Dkk1 and Krm1 were observed for Fzd2-Ror2-mediated STF stimulation. Activation of the canonical Wnt signaling through Fzd-Lrp5/6 can also be inhibited intracellularly by GSK3beta. GSK3beta binds to APC and Axin to facilitate beta-catenin phosphorylation and subsequent degradation. When a Xenopus GSK-3, (Xgsk-3), expression plasmid was used in the cotransfection studies, Wnt3a-stimulated STF response decreased significantly in H441 cells transfected with either Ror2 alone, Fzd2 alone or Fzd2 plus Ror2 (Figure 12). Thus, the sum of these results is consistent with a model in which Ror2 potentiates Fzd2-Lrp5/6 in mediating Wnt3a stimulation of the canonical Wnt signaling.

Bottom Line: We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling.These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, Division of Neonatology, University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA. changgon@usc.edu

ABSTRACT

Background: Wnt signaling is mediated through 1) the beta-catenin dependent canonical pathway and, 2) the beta-catenin independent pathways. Multiple receptors, including Fzds, Lrps, Ror2 and Ryk, are involved in Wnt signaling. Ror2 is a single-span transmembrane receptor-tyrosine kinase (RTK). The functions of Ror2 in mediating the non-canonical Wnt signaling have been well established. The role of Ror2 in canonical Wnt signaling is not fully understood.

Results: Here we report that Ror2 also positively modulates Wnt3a-activated canonical signaling in a lung carcinoma, H441 cell line. This activity of Ror2 is dependent on cooperative interactions with Fzd2 but not Fzd7. In addition, Ror2-mediated enhancement of canonical signaling requires the extracellular CRD, but not the intracellular PRD domain of Ror2. We further provide evidence that the positive effect of Ror2 on canonical Wnt signaling is inhibited by Dkk1 and Krm1 suggesting that Ror2 enhances an Lrp-dependent STF response.

Conclusion: The current study demonstrates the function of Ror2 in modulating canonical Wnt signaling. These findings support a functional scheme whereby regulation of Wnt signaling is achieved by cooperative functions of multiple mediators.

Show MeSH
Related in: MedlinePlus