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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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Related in: MedlinePlus

Fzd2 and Fzd7, but not Fzd6 mediate Wnt3a stimulation of canonical Wnt signaling. H441 cells were transfected with pSV-β-gal (Promega, WI) and STF, plus expression constructs for Fzd2, Fzd6, or Fzd7. An equal quantity of control vector was used as negative control. Twenty-four hours after transfection, cells were treated with L-CM and Wnt3aCM. Two dilutions of Wnt3aCM (1/8 or 1/3) were applied. Equal amounts of conditioned media were used in each assay. Luciferase values representing STF activities were normalized to beta-galactosidase for transfection efficiency. Values of the assays treated with L-CM alone were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05 when compared with data of bar 2. ‡ indicates p < 0.05 when compared with data of bar 3.
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Figure 3: Fzd2 and Fzd7, but not Fzd6 mediate Wnt3a stimulation of canonical Wnt signaling. H441 cells were transfected with pSV-β-gal (Promega, WI) and STF, plus expression constructs for Fzd2, Fzd6, or Fzd7. An equal quantity of control vector was used as negative control. Twenty-four hours after transfection, cells were treated with L-CM and Wnt3aCM. Two dilutions of Wnt3aCM (1/8 or 1/3) were applied. Equal amounts of conditioned media were used in each assay. Luciferase values representing STF activities were normalized to beta-galactosidase for transfection efficiency. Values of the assays treated with L-CM alone were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05 when compared with data of bar 2. ‡ indicates p < 0.05 when compared with data of bar 3.

Mentions: The latter observations suggested that low level expression of endogenous Fzd receptors might be responsible for the low-responsiveness of H441 cells to Wnt3a and Wnt5a signaling. To validate this possibility, we transiently expressed Fzd 2 and Fzd7, two receptors shown to mediate Wnt3a signaling in 293 cells [5], along with STF in H441 cells. Transfected cells were then treated with Wnt3a- or Wnt5a-conditioned media. In comparison to controls, transfected H441 cells responded strongly to Wnt3a stimulation as demonstrated by the magnitude of STF activation (Figure 3). In contrast, transient transfection of a Fzd6 expression construct failed to establish or enhance Wnt3a responsiveness in H441 cells. None of the transfected Fzds activated STF by Wnt5a (data not shown). Ror2 is known to mediate the non-canonical Wnt signaling and the antagonistic effect of Wnt5a on canonical Wnt signaling. However, in Figure 1 we found that Ror2 enhanced the Wnt3a-stimulated STF activation in both L and H441 cell lines. To confirm this observation, we treated H441 cells transfected with Ror2 expression constructs with Wnt3a-conditioned medium for different time periods. As shown in Figure 4, Ror2 moderately potentiated stimulation of STF when H441 cells were treated with Wnt3a for durations from 4 hrs to 16 hrs. Ror2 slightly enhanced STF stimulation when H441 cells were treated for 2 hours.


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)

Fzd2 and Fzd7, but not Fzd6 mediate Wnt3a stimulation of canonical Wnt signaling. H441 cells were transfected with pSV-β-gal (Promega, WI) and STF, plus expression constructs for Fzd2, Fzd6, or Fzd7. An equal quantity of control vector was used as negative control. Twenty-four hours after transfection, cells were treated with L-CM and Wnt3aCM. Two dilutions of Wnt3aCM (1/8 or 1/3) were applied. Equal amounts of conditioned media were used in each assay. Luciferase values representing STF activities were normalized to beta-galactosidase for transfection efficiency. Values of the assays treated with L-CM alone were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05 when compared with data of bar 2. ‡ indicates p < 0.05 when compared with data of bar 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Fzd2 and Fzd7, but not Fzd6 mediate Wnt3a stimulation of canonical Wnt signaling. H441 cells were transfected with pSV-β-gal (Promega, WI) and STF, plus expression constructs for Fzd2, Fzd6, or Fzd7. An equal quantity of control vector was used as negative control. Twenty-four hours after transfection, cells were treated with L-CM and Wnt3aCM. Two dilutions of Wnt3aCM (1/8 or 1/3) were applied. Equal amounts of conditioned media were used in each assay. Luciferase values representing STF activities were normalized to beta-galactosidase for transfection efficiency. Values of the assays treated with L-CM alone were adjusted to unity to normalize all other experimental values in the same group. * indicates p < 0.05 when compared with data of bar 2. ‡ indicates p < 0.05 when compared with data of bar 3.
Mentions: The latter observations suggested that low level expression of endogenous Fzd receptors might be responsible for the low-responsiveness of H441 cells to Wnt3a and Wnt5a signaling. To validate this possibility, we transiently expressed Fzd 2 and Fzd7, two receptors shown to mediate Wnt3a signaling in 293 cells [5], along with STF in H441 cells. Transfected cells were then treated with Wnt3a- or Wnt5a-conditioned media. In comparison to controls, transfected H441 cells responded strongly to Wnt3a stimulation as demonstrated by the magnitude of STF activation (Figure 3). In contrast, transient transfection of a Fzd6 expression construct failed to establish or enhance Wnt3a responsiveness in H441 cells. None of the transfected Fzds activated STF by Wnt5a (data not shown). Ror2 is known to mediate the non-canonical Wnt signaling and the antagonistic effect of Wnt5a on canonical Wnt signaling. However, in Figure 1 we found that Ror2 enhanced the Wnt3a-stimulated STF activation in both L and H441 cell lines. To confirm this observation, we treated H441 cells transfected with Ror2 expression constructs with Wnt3a-conditioned medium for different time periods. As shown in Figure 4, Ror2 moderately potentiated stimulation of STF when H441 cells were treated with Wnt3a for durations from 4 hrs to 16 hrs. Ror2 slightly enhanced STF stimulation when H441 cells were treated for 2 hours.

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