This work demonstrates that LRP5 is required for the development of retinal vasculature, and strongly suggests that LRP5 plays a novel role in the maturation and lumen formation of capillaries in the inner and outer plexiform layers. The Lrp5r18 mutation results in a truncated LRP5 protein that lacks the last three PPP(S/T)P repeats in the C-terminus. We further confirm that LRP5 knockout mice also develop similar defects of retinal vasculature. Thus, our findings suggest that the Lrp5r18 mutation is a loss-of-function allele and that LRP5 plays important roles in the development of retinal vasculature as well as the lumen formation of capillaries.
mutants and LRP5 knockout mice recapitulate the incomplete vascularization of the peripheral retina observed in human FEVR patients. FEVR is a genetically heterogeneous disease. Three inheritance forms (X-linked recessive, autosomal dominant and autosomal recessive) have been described in humans. The X-linked recessive FEVR has been associated with mutations in the Norrie disease gene (17
); autosomal dominant FEVR has been linked to mutations of the Wnt receptor FZD4 and its coreceptor LRP5 (14
); LRP5 mutations have also been reported to cause autosomal recessive FEVR (16
). Norrin, the protein product of the Norrie disease gene, has been suggested to act as a ligand for FZD4 (21
). Knockout mice of either FZD4 (21
) or Norrie (22
) develop defective intraretinal vasculature. Several in vitro
studies have suggested that LRP5 acts as a co-receptor for FZD4 (2
). Therefore, FEVR is probably caused by altered signaling downstream of Norrie, FZD4 and LRP5.
Previous studies have shown that the N-terminal extracellular domain of LRP5 binds to Wnt ligands while its C-terminal intracellular domain is important for signaling events (2
). The C-terminal intracellular domain of LRP5 has 207 amino acid residues with five PPP(S/T)P motifs that are presumably essential for its signaling (2
). It has been suggested that LRP5 interacts with Axin through its C-terminal intracellular domain. In the Wnt/β-catenin signaling pathway, Axin has been proposed to function as a scaffold that forms a complex with GSK-3β and β-catenin, and to promote GSK-3β-dependent phosphorylation of β-catenin (27
). Mutant LRP5 proteins lacking the extracellular domain constitutively bind to Axin and induce TCF/LEF-1 activation by destabilizing Axin and stabilizing β-catenin in vitro
). The intracellular domains of both LRP5 and LRP6 have been shown to increase free β-catenin levels constitutively and activate TCF/LEF-1 (28
). Mutant LRP5 proteins without the last three PPP(S/T)P motifs are unable to bind to Axin and fail to activate LEF-1 (28
). Studies of LRP6 have also suggested the importance of PPP(S/T)P motif in the Wnt signaling. The transfer of a PPP(S/T)P motif from LRP6 to the LDL receptor fully activates the Wnt pathway (30
), and phosphorylated PPP(S/T)P motif provides a docking site for Axin (30
). Thus, the PPP(S/T)P motifs of LRP5 and LRP6 are essential for mediating β-catenin signaling in vitro
The Lrp5r18 mutation disrupts the C-terminal 39 amino acids of LRP5, which include the last three PPP(S/T)P repeats. The absence of these PPP(S/T)P repeats probably eliminates the docking sites for Axin. Thus, LRP5 truncated mutant proteins are probably unable to bind Axin to promote subsequent stabilization of β-catenin, thereby suppressing the activation of downstream transcriptional factors and the expression of genes needed for the development of retinal vasculature. However, it is also possible that the C-terminal truncation perturbs the stability or intracellular trafficking of LRP5. Future studies of the Lrp5r18 mutant will clarify the specific role(s) of the C-terminal region and the PPP(S/T)P motifs of LRP5.
In summary, this study provides the first direct in vivo evidence of the importance of LRP5 in the development of retinal vasculature. The extreme C-terminus is essential for the function of LRP5 in vivo. We believe that the Lrp5r18 mutant will be an appropriate animal model for further elucidating the molecular mechanism that controls the development of retinal vasculature, as well as for understanding the underlying molecular mechanism of FEVR.