Heparan Sulfate Proteoglycans (HSPGs) play crucial roles during development. They are associated with the cell surface and extracellular matrix of a wide range of cells of vertebrate and invertebrate tissues, and are essential cofactors in cell-matrix processes, cell-cell recognition systems and receptor-growth factor interactions. Increasing amount of data demonstrate that HSPGs play crucial roles in modulating a wide variety of signaling pathways (for a review, see (Whitelock and Iozzo, 2005
)). These large molecules influence the activity and extracellular distribution of Wnt molecules but the precise mechanism by which they act remains elusive. Their known presence in serum (Dziadek et al., 1985
) and our observation that high molecular weight serous fractions were enriched for a Wnt stabilizing activity prompted us to specifically ask whether HSPGs could stabilize Wnt protein activity in solution. Our results clearly demonstrate that purified HSPGs can maintain the activity of purified Wnt proteins in solution. This specific activity is likely to depend on intact HSPG molecules since it was sensitive to proteolysis and HS chains cleavage. Furthermore, the observed effect of HSPGs on Wnt3A could not be mimicked by heparan sulfate, D-glucuronic acid, or the control protein bovine serum albumin. HSPGs could act either by sensitizing the cells to lower levels of Wnt proteins, a mechanism used by proteins such as R-spondin (Binnerts et al., 2007
), or by stabilizing the activity of Wnt proteins at the protein level. We found the latter to be true as incubation with Wnt proteins was essential for HSPGs to exert their stabilizing function. We further demonstrated that HSPGs act by preventing the aggregation of Wnt ligands that normally occurs in an aqueous environment. This is most likely achieved through direct interaction between proteoglycans and Wnt proteins, as such an interaction has been observed between glypican1 and XWnt8 (Ai et al., 2003
). It will be interesting for future studies to address the specificity of the interactions between different HSPG family members and Wnt proteins.
The role of HSPGs in the regulation of the Wnt pathway has been extensively studied, notably during the patterning of the Drosophila
wing imaginal disc where HSPGs have been shown to influence Wg distribution (see introduction). In vertebrates, experiments performed on Xenopus
animal cap explants have shown that heparinase treatment led to inhibition of Wnt-induced mesodermal markers as well as impaired mesoderm formation, a phenotype that could be rescued by exogenous HSPGs (Itoh and Sokol, 1994
). Similarly, the membrane-anchored HSPG knypek (XGly4) has been shown to potentiate XWnt11-induced convergent extension movements during gastrulation (Topczewski et al., 2001
), and both the glycosyl transferase XEXT1 and the sulfatase XtSulf1 have been shown to be required for XWnt11-induced axis formation (Tao et al., 2005
; Freeman et al., 2008
). In mouse, knockout of glypican-3 perturbs the balance between the Wnt/β-catenin and the Wnt/planar cell polarity pathways (Song et al., 2005
While all these studies extend the observation that HSPGs modulate the Wnt signaling pathway to vertebrates, experiments performed in Xenopus
have revealed that additional layers of complexity exist in the developing embryo. For example, the syndecan XSyn4 was shown to bind directly to Frizzled-7 (xFz7) and Disheveled (xDsh) to activate the Wnt/planar cell polarity pathway (Munoz et al., 2006
), and the extracellular sulfatase XtSulf1, which has an important role in the post synthetic remodeling of the HS chains and therefore generates diversity among HSPGs, was recently shown to favor the Wnt/β-catenin signaling pathway by facilitating the interaction between XWnt11 and LRP6 (Freeman et al., 2008
). Thus, HSPGs can also regulate Wnt signaling through direct interactions with receptors and components of the Wnt signal transduction machinery.
Wnt proteins have been shown to associate with lipoprotein particles (Panakova et al., 2005
; Neumann et al., 2009
), which themselves interact with HSPGs (Eugster et al., 2007
). It is tempting to speculate that HSPGs mediate the interaction between Wnt proteins and lipoprotein particles to ensure proper spreading of Wnt ligands in the extracellular environment. Finally, Dally-like has been shown to be required for Wg to transcytose from the apical to the basolateral side of wing imaginal discs in order to permit long-range signaling (Gallet et al., 2008
). All these mechanisms are likely to act in concert with the stabilizing effect of HSPGs on Wnt ligands described in this study to ensure proper control of Wnt signaling during development.