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. 2005 Jun;25(12):5022-30.
doi: 10.1128/MCB.25.12.5022-5030.2005.

Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5

Zhishan Wang  1 , Weiguo Shu, Min Min Lu, Edward E Morrisey
Affiliations

Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5

Zhishan Wang et al. Mol Cell Biol. 2005 Jun.

Abstract

Wnt7b is a Wnt ligand that has been demonstrated to play critical roles in several developmental processes, including lung airway and vascular development and chorion-allantois fusion during placental development. Wnt signaling involves the binding of Wnt ligands to cell surface receptors of the frizzled family and coreceptors of the LRP5/6 family. However, little is known of the ligand-receptor specificity exhibited by different Wnts, Fzds, and LRPs in Wnt signaling. Expression analysis of Fzds and LRP5/6 in the developing lung and vasculature showed that Fzd1, -4, -7, and -10 and LRP5/6 are expressed in tissue-specific patterns during lung development. Fzd1, -4, and -7 are expressed primarily in the developing lung mesenchyme, and Fzd10 is expressed in airway epithelium. LRP5 and LRP6 are expressed in airway epithelium during lung development, whereas LRP5 but not LRP6 expression is observed in the muscular component of large blood vessels, including the aorta. Cell transfection studies demonstrate that Wnt7b can activate the canonical Wnt pathway but not the noncanonical Wnt pathway in a cell-specific manner. Biochemical analysis demonstrates that Wnt7b can bind to Fzd1 and -10 on the cell surface and cooperatively activate canonical Wnt signaling with these receptors in the presence of LRP5. Together, these data demonstrate that Wnt7b signals through Fzd1 and -10 and LRP5 and implicate these Wnt coreceptors in the regulation of lung airway and vascular development.

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Figures

FIG. 1.
FIG. 1.
Expression of Fzd genes during lung development. In situ hybridization was performed on E12.5, E14.5, and E16.5 mouse embryos using probes specific for Fzd1, Fzd4, and Fzd7. Immunohistochemistry was performed using a rabbit polyclonal antibody to Fzd10. Expression of Fzd1 (A to C) and Fzd7 (G to I) is observed in the aorta, bronchial smooth muscle, and pulmonary vessels. Expression of Fzd4 (D to F) is observed diffusely throughout the lung mesenchyme. Fzd10 expression (J to L) is observed primarily in distal airway epithelium from E12.5 to E14.5. Expression of all four genes decreases after E14.5 (C, F, I and L). ao, aorta; bsm, bronchial smooth muscle; pv, pulmonary vessels; ai, distal airway epithlium. Bars, 200 μm.
FIG. 2.
FIG. 2.
Expression of LRP5 and LRP6 during lung and heart development. In situ hybridization was performed on E12.5 (A and B), E14.5 (C, D, and G to J), and E18.5 (E, F, K, and L) mouse embryos using specific probes for LRP5 (A, C, E, G, I, and K) and LRP6 (B, D, F, H, J, and L). Expression of LRP5 and LRP6 is observed in distal airway epithelium at E12.5 (A and B) and E14.5 (C and D). Expression decreases significantly in distal airway epithelium by E18.5 (E and F). Expression of LRP5 and LRP6 is observed in upper airways at E14.5 (G and H), but only LRP6 expression is observed in upper airways at E18.5 (F). LRP5 but not LRP6 expression is observed in large blood vessels, including the aorta and pulmonary vessels, from E12.5 (A) through E18.5 (K). LRP5 but not LRP6 is also expressed in the developing outflow tract heart valves at E14.5 (I and J, arrow). ao, aorta; bsm, bronchial smooth muscle; pv, pulmonary vessels; ai, distal airway epithelium; ua, upper airway. Bars, 150 μm (A and B) and 200 μm (C to L).
FIG. 3.
FIG. 3.
Wnt7b activates canonical signaling in vascular smooth muscle cells. The indicated cell lines were transfected with either TOPFLASH or FOPFLASH luciferase reporter plasmids along with Wnt7b expression plasmid or the β-catenin/4145 expression plasmid. All values are adjusted against TOPFLASH transfected with an empty expression plasmid (data not shown). (A) Expression of Wnt7b activates TOPFLASH approximately eightfold in PAC-1 and A7r5 cells. (B) Expression of β-catenin/4145 robustly activates TOPFLASH in all cell lines tested. Data represent the mean of three assays ± standard error of the mean. (C) RT-PCR shows that Fzd1 (low levels) and Fzd7 (high levels) are expressed in 293 cells while Fzd4 and Fzd7 are expressed at high levels and Fzd1 and Fzd10 are expressed at lower levels in PAC-1 cells. GAPDH is shown as a positive control for amplification.
FIG. 4.
FIG. 4.
Wnt7b does not activate JNK dependent Wnt signaling. The PathDetect reporter system was used to measure JNK activity in both 293 and PAC-1 cells. (A) The GAL4 DNA binding domain (dbd) alone or in combination with pcDNA3 (pCD3) did not activate JNK. Expression of Wnt7b, in the presence or absence of LRP5 and LRP6, did not activate JNK, while expression of the DEP domain of dishevelled did. Data represent the means of three assays ± standard errors of the means. (B) Western blots showing Jun phosphorylation in response to pcDNA3 expression (lane 1), pcDNA3.1/Wnt7b expression (lane 2), or pcDNA3.1/DEP expression (lane 3). The total amount of c-Jun protein was used to control for loading.
FIG. 5.
FIG. 5.
Fzd1 and Fzd10 bind Wnt7b at the cell surface. 293 cells were transfected with an expression plasmid encoding a HA-tagged Wnt7b protein. Conditioned medium from these cells was used to determine binding of Wnt7b to different Fzd receptors. (A) Western blots of control transfected 293 cells (lane 1), culture supernatant from Wnt7b-HA-expressing 293 cells (lane 2), and cell lysates from Wnt7b-HA-expressing 293 cells (lane 3). Concentrated Wnt7b-HA containing supernatant was incubated with untransfected 293 cells (B) or cells transfected with either pcDNA3 (C) or expression plasmids encoding Fzd1 (D), Fzd4 (E), Fzd7 (F), and Fzd10 (G). Only cells expressing Fzd1 and Fzd10 bound the HA-tagged Wnt7b protein at detectable levels. (H) 293 cells were transfected with a HA-tagged Wnt7b expression plasmid and FLAG-tagged Fzd1, Fzd4, Fzd7, and Fzd10 expression plasmids. Cell lysates were immunoprecipitated (co-IP) with a monoclonal antibody to FLAG, resolved on SDS-PAGE gels, Western blotted (WB), and probed with anti-HA epitope monoclonal antibody.
FIG. 6.
FIG. 6.
Wnt7b activates canonical Wnt signaling cooperatively through Fzd1, Fzd10, and LRP5. (A) Coexpression of Wnt7b and Fzd1, -4, -7, and -10 does not activate the TOPFLASH reporter, while β-catenin/4145 does. (B) Coexpression of Wnt7b and LRP5 but not LRP6 activates TOPFLASH reporter. (C) Coexpression of Wnt7b/Fzd1/LRP5 and Wnt7b/Fzd10/LRP5 activates the TOPFLASH reporter. Data represent the mean of three assays ± standard error of the mean. (D) Western blots (WB) show that coexpression of Wnt7b/Fzd1/LRP5 and Wnt7b/Fzd10/LRP5 results in the highest levels of β-catenin stabilization. Note that expression of Wnt7b alone or with only Fzd1, Fzd4, and Fzd10 did result in a detectable but less-dramatic increase in β-catenin stabilization.
FIG. 7.
FIG. 7.
Wnt7b/Fzd1/LRP5 and Wnt7b/Fzd10/LRP5 cooperatively activate canonical Wnt activity in PAC-1 cells. PAC-1 cells were transfected with expression plasmids encoding the indicated proteins. Wnt7b alone activated the TOPFLASH reporter approximately 7-fold, and coexpression of LRP5 increased this activation to 12-fold. However, coexpression of Wnt7b/Fzd1/LRP5 and Wnt7b/Fzd10/LRP5 resulted in a significant increase in TOPFLASH activity to more than 20-fold. Data represent the mean of three assays ± standard error of the mean.
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