Bryja V et al. (2009), The extracellular domain of Lrp5/6 inhibits non...

XB-ART-38620

Mol Biol Cell 2009 Feb 01;203:924-36. doi: 10.1091/mbc.e08-07-0711.

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The extracellular domain of Lrp5/6 inhibits noncanonical Wnt signaling in vivo.

Bryja V , Andersson ER , Schambony A , Esner M , Bryjová L , Biris KK , Hall AC , Kraft B , Cajanek L , Yamaguchi TP , Buckingham M , Arenas E .

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Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.

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Species referenced: Xenopus
Genes referenced: actl6a cdc42 ctnnb1 dvl2 dvl3 lrp5 lrp6 myc rac1 rhoa wnt11 wnt11b wnt3a wnt5a
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	Figure 1. Wnt5a can bind Lrp6, inhibit Lrp6 phosphorylation and Lrp6 inhibits Rac1 activation. (A) SN4741 cells were stimulated with 100 ng/ml Wnt3a or Wnt5a. Stimulation with either of these led to the phosphorylation of Dvl2 and Dvl3 (indicated by open arrowheads), as assessed by a mobility shift in a Western blot. Only Wnt3a led to an increase in active Î²-catenin and phosphorylation of Lrp6 at Ser1490. (B) Wnt5a inhibits Wnt3a-induced phosphorylation of Lrp6 at Ser1490, but not Dvl2 phosphorylation, in a dose-dependant manner. Total Lrp6 did not significantly change by Wnt treatment. Actin was used as loading control. The level of Lrp6 phosphorylation from three independent experiments is quantified in C. (D) Lrp6 Fc can associate with HA-Wnt5a. Lrp6 Fc was overexpressed in HA-Wnt5a expressing B1A fibroblasts (B1A fibroblasts were used as a control). Total cell lysates (TCL) or conditioned media (CM) were subjected to IgG pull-down, and HA-Wnt5a was detected only in samples also expressing Lrp6-Fc. (E) Myc-tagged Lrp6 mutants lacking either E1 and E2 (Myc-Lrp6Î”E1-E2) or E3 and E4 (Myc-Lrp6Î”E3-E4) were overexpressed in B1A, or B1A cells stably expressing HA-tagged Wnt5a. Cells lysates were immunoprecipitated using antibody directed against HA-tag. Expression of Myc-Lrp6 and HA-Wnt5a in immunoprecipitates was determined by Western blotting. (F) HEK cells were transfected with Myc-Rac1 and indicated Lrp6 constructs. The activation of Rac1 was determined using Rac1 activation assay, and Western blot for Myc-Rac1. The signal ratio for GTP-Rac1/Rac1 was quantified and demonstrates that only full length Lrp6, but not mutants in any of the extracellular domains of Lrp6, inhibited Rac1 activity.
	Figure 2. Lrp5/6 are crucial regulators of convergent extension (CE) movements in Xenopus. (A and B) Injection of Lrp5 or Lrp6 mRNA or XLRP5/6 MOs all inhibit convergent extension of Keller explants from stage 10.5 that are rescued by mLrp5/6 but cannot be rescued by Î²-catenin coinjection (, significant difference from control; , significant rescue of MO, p > 0.95). (C and D) The CE defects induced by XLRP5 MO or XLRP6 MO are not rescued by Wnt5a or Wnt11 overexpression or constitutively active (ca) RhoA and Rac1. However, down-regulation of noncanonical signaling by XWnt5a or XWnt11 MO rescued XLRP5 and XLRP6 depletion phenotypes. XLRP5 MO induced inhibition of elongation was also rescued by dn RhoA and Rac1 (*, significant rescue of MO; p > 0.95). (E) Typical morphology of Keller explants injected with XLRP5 and XLRP6 MOs. The numbers under the graphs indicate number of injected embryos/number of independent experiments.
	Figure 3. (A) Embryos injected with mLRP5 or XLRP5 MO were lysed and analyzed for the activity of small GTPases Rac1 and Cdc42. (B) XWnt5a- or XWnt11-induced CE defects can be partially rescued by coinjection of Lrp5 or Lrp6 (, significant rescue of XWnt-11 and XWnt-5a, respectively; p > 0.95). (C) Effects of FL and mutant hLRP6 on CE defects induced by XLRP5 MOs. hLRP6 and also hLRP6 lacking cytoplasmic domain (hLRP6ÎC) can efficiently rescue XLRP5 MO defects, whereas hLRP6 lacking the extracellular domains E1-E4 cannot (, significant rescue of MO; p > 0.95). (D) Lrp6 lacking cytoplasmic domain (hLRP6ÎC) can rescue elongation defects caused by overexpression of XWnt-11 or XWnt-5a. The numbers under the graphs indicate number of injected embryos/number of independent experiments.
	Figure 4. XLRP5/6 regulate heart development. (A) Knockdown of XLrp5 and XLrp6 affects heart development. Embryos injected with XLRP5 and XLRP6 MO in one or both dorsal blastomeres were analyzed by whole mount ISH for the expression of heart markers Nkx2.5 and Troponin lc (Tnlc) at stage 28. Injected side shown by blue circle in single-blastomere injections, open triangle shows Tnlc/Nkx2.5 on injected side, closed triangle shows TnIc/Nkx2.5 on uninjected side. (B) XLRP5 MO-induced reduction in heart markers can be rescued with mLrp5 and XWnt-11 MO (Tnlc) and only partially rescued with Î²-catenin overexpression (Nkx2.5). (C) Quantification of ISH analysis of cardiac markers (, significantly differs from Î²-galactosidase controls; p > 0.95); *, significantly differs from XLRP5MO; p > 0.95).

References [+] :

Andersson, Wnt5a regulates ventral midbrain morphogenesis and the development of A9-A10 dopaminergic cells in vivo. 2008, Pubmed

	Figure 1. Wnt5a can bind Lrp6, inhibit Lrp6 phosphorylation and Lrp6 inhibits Rac1 activation. (A) SN4741 cells were stimulated with 100 ng/ml Wnt3a or Wnt5a. Stimulation with either of these led to the phosphorylation of Dvl2 and Dvl3 (indicated by open arrowheads), as assessed by a mobility shift in a Western blot. Only Wnt3a led to an increase in active Î²-catenin and phosphorylation of Lrp6 at Ser1490. (B) Wnt5a inhibits Wnt3a-induced phosphorylation of Lrp6 at Ser1490, but not Dvl2 phosphorylation, in a dose-dependant manner. Total Lrp6 did not significantly change by Wnt treatment. Actin was used as loading control. The level of Lrp6 phosphorylation from three independent experiments is quantified in C. (D) Lrp6 Fc can associate with HA-Wnt5a. Lrp6 Fc was overexpressed in HA-Wnt5a expressing B1A fibroblasts (B1A fibroblasts were used as a control). Total cell lysates (TCL) or conditioned media (CM) were subjected to IgG pull-down, and HA-Wnt5a was detected only in samples also expressing Lrp6-Fc. (E) Myc-tagged Lrp6 mutants lacking either E1 and E2 (Myc-Lrp6Î”E1-E2) or E3 and E4 (Myc-Lrp6Î”E3-E4) were overexpressed in B1A, or B1A cells stably expressing HA-tagged Wnt5a. Cells lysates were immunoprecipitated using antibody directed against HA-tag. Expression of Myc-Lrp6 and HA-Wnt5a in immunoprecipitates was determined by Western blotting. (F) HEK cells were transfected with Myc-Rac1 and indicated Lrp6 constructs. The activation of Rac1 was determined using Rac1 activation assay, and Western blot for Myc-Rac1. The signal ratio for GTP-Rac1/Rac1 was quantified and demonstrates that only full length Lrp6, but not mutants in any of the extracellular domains of Lrp6, inhibited Rac1 activity.
	Figure 2. Lrp5/6 are crucial regulators of convergent extension (CE) movements in Xenopus. (A and B) Injection of Lrp5 or Lrp6 mRNA or XLRP5/6 MOs all inhibit convergent extension of Keller explants from stage 10.5 that are rescued by mLrp5/6 but cannot be rescued by Î²-catenin coinjection (, significant difference from control; , significant rescue of MO, p > 0.95). (C and D) The CE defects induced by XLRP5 MO or XLRP6 MO are not rescued by Wnt5a or Wnt11 overexpression or constitutively active (ca) RhoA and Rac1. However, down-regulation of noncanonical signaling by XWnt5a or XWnt11 MO rescued XLRP5 and XLRP6 depletion phenotypes. XLRP5 MO induced inhibition of elongation was also rescued by dn RhoA and Rac1 (*, significant rescue of MO; p > 0.95). (E) Typical morphology of Keller explants injected with XLRP5 and XLRP6 MOs. The numbers under the graphs indicate number of injected embryos/number of independent experiments.
	Figure 3. (A) Embryos injected with mLRP5 or XLRP5 MO were lysed and analyzed for the activity of small GTPases Rac1 and Cdc42. (B) XWnt5a- or XWnt11-induced CE defects can be partially rescued by coinjection of Lrp5 or Lrp6 (, significant rescue of XWnt-11 and XWnt-5a, respectively; p > 0.95). (C) Effects of FL and mutant hLRP6 on CE defects induced by XLRP5 MOs. hLRP6 and also hLRP6 lacking cytoplasmic domain (hLRP6ÎC) can efficiently rescue XLRP5 MO defects, whereas hLRP6 lacking the extracellular domains E1-E4 cannot (, significant rescue of MO; p > 0.95). (D) Lrp6 lacking cytoplasmic domain (hLRP6ÎC) can rescue elongation defects caused by overexpression of XWnt-11 or XWnt-5a. The numbers under the graphs indicate number of injected embryos/number of independent experiments.
	Figure 4. XLRP5/6 regulate heart development. (A) Knockdown of XLrp5 and XLrp6 affects heart development. Embryos injected with XLRP5 and XLRP6 MO in one or both dorsal blastomeres were analyzed by whole mount ISH for the expression of heart markers Nkx2.5 and Troponin lc (Tnlc) at stage 28. Injected side shown by blue circle in single-blastomere injections, open triangle shows Tnlc/Nkx2.5 on injected side, closed triangle shows TnIc/Nkx2.5 on uninjected side. (B) XLRP5 MO-induced reduction in heart markers can be rescued with mLrp5 and XWnt-11 MO (Tnlc) and only partially rescued with Î²-catenin overexpression (Nkx2.5). (C) Quantification of ISH analysis of cardiac markers (, significantly differs from Î²-galactosidase controls; p > 0.95); *, significantly differs from XLRP5MO; p > 0.95).