Characterization of Wise Protein and Its Molecular Mechanism to Interact with both Wnt and BMP Signals

Katherine B Lintern,Sonia Guidato,Alison Rowe,José W Saldanha,Nobue Itasaki

doi:10.1074/jbc.m109.025478

Katherine B Lintern, Sonia Guidato + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.025478

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Abstract

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.

Highlights

Wise appears to have a dual role in modulating the Wnt pathway
All of these findings indicate that USAG-1/Wise/Ectodin has a clear antagonistic effect on BMP signaling, where it binds BMP2, -4, -6, and -7 [3, 14] and presumably prevents BMP binding to its receptors
Full-length Wise protein was found to be sensitive to endo-␤-N-acetylglucosaminidase D but insensitive to endo-␤-N-acetylglucosaminidase H (Endo H; Fig. 2E), showing that Wise protein has a complex oligosaccharide processed by Golgi Mannosidase II in the Golgi apparatus

Summary

Introduction

Wise appears to have a dual role in modulating the Wnt pathway. Injection of Wnt RNA into a ventral vegetal blastomere of Xenopus embryos at the four-cell stage induces a full secondary axis to form, and this is blocked by the addition of Wise RNA as well as other Wnt inhibitors [1]. Activation of the Wnt/␤-catenin pathway in hair follicles triggers regeneration of hair growth, and expression of Wise appears to have a defined role to inhibit this [15]. Osteoblast differentiation of MC3T3-E1 cells, as measured by alkaline phosphatase activity, can be induced by a wide range of BMP molecules In this assay, Ectodin, the mouse ortholog of Wise, was shown to inhibit differentiation induced by BMP2, -4, -6, or -7 in a dose-dependent manner [3]. In mouse adult kidneys, the ability of BMP7 to repair established renal injury is blocked by USAG-1 [13] All of these findings indicate that USAG-1/Wise/Ectodin has a clear antagonistic effect on BMP signaling, where it binds BMP2, -4, -6, and -7 [3, 14] and presumably prevents BMP binding to its receptors. It aims to reveal the molecular nature of the protein in view of possible glycosylation, secretion, and association with extracellular matrix

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