The Role of Glypicans in Wnt Inhibitory Factor-1 Activity and the Structural Basis of Wif1's Effects on Wnt and Hedgehog Signaling

Abstract

Proper assignment of cellular fates relies on correct interpretation of Wnt and Hedgehog (Hh) signals. Members of the Wnt Inhibitory Factor-1 (WIF1) family are secreted modulators of these extracellular signaling pathways. Vertebrate WIF1 binds Wnts and inhibits their signaling, but its Drosophila melanogaster ortholog Shifted (Shf) binds Hh and extends the range of Hh activity in the developing D. melanogaster wing. Shf activity is thought to depend on reinforcing interactions between Hh and glypican HSPGs. Using zebrafish embryos and the heterologous system provided by D. melanogaster wing, we report on the contribution of glypican HSPGs to the Wnt-inhibiting activity of zebrafish Wif1 and on the protein domains responsible for the differences in Wif1 and Shf specificity. We show that Wif1 strengthens interactions between Wnt and glypicans, modulating the biphasic action of glypicans towards Wnt inhibition; conversely, glypicans and the glypican-binding “EGF-like” domains of Wif1 are required for Wif1's full Wnt-inhibiting activity. Chimeric constructs between Wif1 and Shf were used to investigate their specificities for Wnt and Hh signaling. Full Wnt inhibition required the “WIF” domain of Wif1, and the HSPG-binding EGF-like domains of either Wif1 or Shf. Full promotion of Hh signaling requires both the EGF-like domains of Shf and the WIF domains of either Wif1 or Shf. That the Wif1 WIF domain can increase the Hh promoting activity of Shf's EGF domains suggests it is capable of interacting with Hh. In fact, full-length Wif1 affected distribution and signaling of Hh in D. melanogaster, albeit weakly, suggesting a possible role for Wif1 as a modulator of vertebrate Hh signaling.

The WIF domain of the human and Drosophila Wif-1 secreted factors confers specificity for Wnt or Hedgehog

“…S2B,BЈ). The long-range dispersion of Shf is in agreement with its reported dependence on Hh movement through the ECM in the wing disc (Glise et al, 2005;Gorfinkiel et al, 2005;Avanesov et al, 2012).…”

“…In agreement with these data, overexpression of Shf in the P compartment of the wing disc results in an extension of the Hh gradient (see Fig. 6A; supplementary material Table S2) without affecting Wg signaling (Glise et al, 2005;Gorfinkiel et al, 2005;Avanesov et al, 2012) (Fig. 3B,BЈ).…”

“…1D), were tested in the wing disc, both in HAtagged and untagged form. We also tested the possible effect of the N-terminal part (NT), as Shf has a much longer NT than WIF1 (NT The spreading properties of WIF1, Shf and chimeric Shf/WIF1 proteins It has been proposed that Wif proteins are secreted factors that spread through the ECM and interact with HSPGs (Glise et al, 2005;Gorfinkiel et al, 2005;Avanesov et al, 2012). Shf is present throughout the wing primordium, with higher levels in the anteriormost part of the A compartment, lower levels near the A/P border (supplementary material Fig.…”

“…Therefore, we undertook a detailed functional analysis of these two proteins as examples of arthropod and chordate sequences. It has been reported previously that both the WD and the EGFs of Shf are crucial for its function, as the expression of just the WD or EGF repeats in Drosophila does not rescue the shf phenotype (Glise et al, 2005;Gorfinkiel et al, 2005;Avanesov et al, 2012). We generated chimeric constructs by exchanging domains between the Drosophila and human proteins (Fig.…”

Differential expression of glypican-3 (GPC3) in lung squamous cell carcinoma and lung adenocarcinoma and its clinical significance

“…The differential expression of GPC3 can be used to differentiate and diagnose lung squamous cell carcinoma and lung adenocarcinoma. Basic research has indicated that GPC3 regulates the proliferation, differentiation, adhesion, and migration of tumor cells by modulating the insulin-like growth factor and wingless/integrated signaling pathways (Avanesov et al, 2012;Sakane et al, 2012;Pez et al, 2013;Dwivedi et al, 2013;Avanesov and Blair, 2013), thus playing important roles in tumor growth and metastasis. Stronger GPC3 expression has been correlated with increased growth and metastatic potential (Tanaka et al, 2011;Gao and Ho, 2011).…”