The Wnt proteins constitute a large family of extracellular signaling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. This paper describes Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. Xenopus embryos are used to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8, produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, these results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity (Hsieh, 1999).

The expression patterns of the Wnt antagonists of the Dickkopf (Dkk) family were characterized in the developing mouse forebrain. In situ hybridisation on sections from E12 embryos showed an expression of dkk2 in the thalamus and dkk3 in the cortical hem and thalamus. At later developmental stages (E15.5, E17.5, and P0), little or no expression of dkk1, dkk2, and dkk4 was found in the forebrain, while dkk3 expression was detected in the ventricular zone (VZ) of the lateral and III ventricles, cortical neurons, migrating cells of the primary and secondary dentate migration, and the neuroblastic layer of the eye. In the adult forebrain, dkk3 expression was detected in the lateral VZ, pyramidal neurons of the hippocampus, and cortical neurons. Evidence is provided indicating that only dkk1 and dkk4, along with two other Wnt antagonists axin2 and wif1, but not dkk2 and dkk3, are involved in a feedback mechanism to restrain Wnt signalling in transgenic mice carrying a conditional augmentation of beta-catenin in the forebrain (Diep, 2004).

Components of the extracellular matrix (ECM) modulate neuronal development. Additional ECM elements were sought that might play roles in retinal histogenesis and a secreted glycoprotein was identified that is heavily expressed in the retina. This molecule, named by others Wnt Inhibitory Factor-1 (WIF-1), is expressed during and after the period of rod photoreceptor morphogenesis in the mouse. A potential WIF-1 ligand, Wnt4, as well as a potential Wnt4 receptor, fzd4, and a potential Wnt4 coreceptor, LRP6, are expressed in the region of, and at the time of, rod photoreceptor genesis. WIF-1 and Wnt4 are coexpressed during retinal development and bind to each other; therefore, they are likely to interact during rod production. WIF-1 protein inhibits rod production, and anti-WIF-1 antibodies increase rod production; in contrast, Wnt4 promotes rod production. Together, these data suggest that WIF-1 and Wnt4, both components of the ECM, regulate mammalian photoreceptor development (Hunter, 2004).

Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist of Wnt signaling and functions by directly binding to Wnt ligands in the extracellular space. The 5' promoter region (approximately 1.5 kb) of the human WIF-1 gene has been identified. Functional analysis of this region shows that a whole fragment displays high basal promoter activity in different cell lines, while the truncated forms do not, indicating that integrity of the WIF-1 promoter region may be important for WIF-1 activity. Moreover, the expression level of beta-catenin in cancer cell lines was found to correlate with the WIF-1 promoter activity, suggesting that the WIF-1 promoter may be regulated by the Wnt/beta-catenin pathway and may function in a negative feedback manner. These results also suggest that a methylated CpG island, which was observed in human lung cancer, lies within the functional WIF-1 promoter region and therefore bears the importance of the methylation-status of this CpG island as an important key in Wnt activation in human cancer (Reguart, 2004).

NMR structure of the WIF domain of the human Wnt-inhibitory factor-1

The human Wnt-binding protein Wnt-inhibitory factor-1 (WIF-1) comprises an N-terminal WIF module followed by five EGF-like repeats. This study reports the three-dimensional structure of the WIF domain of WIF-1 determined by NMR spectroscopy. The fold consists of an eight-stranded ß-sandwich reminiscent of the immunoglobulin fold. Residual detergent (Brij-35) used in the refolding protocol was found to bind tightly to the WIF domain. The binding site was identified by intermolecular nuclear Overhauser effects observed between the WIF domain and the alkyl chain of the detergent. The results point to a possible role of WIF domains as a recognition motif of Wnt and Drosophila Hedgehog proteins that are activated by palmitoylation (Liepinsh, 2006).

WIF-1 is a protein of 379 amino acid residues, composed of a signal sequence, a WIF domain, five EGF repeats and a hydrophilic C terminus. In an assay for Wnt inhibition, the WIF domain of human WIF-1 was as effective as the full-length protein, suggesting that the WIF domain is sufficient for Wnt binding. Based on sequence conservation between different organisms, the WIF domain comprises about 140 residues (Liepinsh, 2006).

WIF domains also constitute the main part of the extracellular domains of Ryk (related-to-tryosine-kinase) receptor tyrosine kinases, suggesting that Ryk receptors bind to Wnt proteins and participate in Wnt signaling. This hypothesis was verified by experiments that showed that mammalian Ryk is a Wnt coreceptor required for stimulation of neurite outgrowth (Liepinsh, 2006).

The identification of Wnt proteins as natural Ryk ligands underlines the functional importance of mammalian Ryk receptors. Previous experiments have shown that human Ryk is overexpressed in epithelial ovarian cancer, its overexpression in mouse fibroblasts confers transforming ability, and Ryk-deficient mice develop abnormal craniofacial structures. In agreement with Wnt proteins acting as Ryk ligands, the Caenorhabditis elegans Ryk ortholog LIN-18 has also been shown to participate in Wnt signaling (Liepinsh, 2006).

Ryk receptors homologous to the mammalian proteins are expressed by the Drosophila genes derailed, derailed-2 and doughnut, and while shifted encodes the only Drosophila ortholog of WIF-1. Unexpectedly, however, the protein Shifted (Shf), was not found to bind to Wnt proteins. Instead, it was found to bind to lipid-modified Hedgehog. The lipid modification of Hh involves a palmitoyl group at its N-terminal and a cholesterol molecule at its C-terminal end (Liepinsh, 2006).

In conclusion, the WIF domain presents an independent folding unit in receptor molecules that bind to palmitoylated signaling proteins. The 3D structure of the WIF domain of human WIF-1 will allow the informed design of site-directed mutagenesis experiments to verify the functional importance of Wnt and Hedgehog palmitoylation for recognition by WIF-1 and Ryk receptors and to explore the specificity of recognition of different Wnts (Liepinsh, 2006).

Sfrp5 coordinates foregut specification and morphogenesis by antagonizing both canonical and noncanonical Wnt11 signaling

Cell identity and tissue morphogenesis are tightly orchestrated during organogenesis, but the mechanisms regulating this are poorly understood. This study shows that interactions between Wnt11 and the secreted Wnt antagonist secreted frizzled-related protein 5 (Sfrp5) coordinate cell fate and morphogenesis during Xenopus foregut development. sfrp5 is expressed in the surface cells of the foregut epithelium, whereas wnt11 is expressed in the underlying deep endoderm. Depletion of Sfrp5 results in reduced foregut gene expression and hypoplastic liver and ventral pancreatic buds. In addition, the ventral foregut cells lose adhesion and fail to form a polarized epithelium. The cell fate and epithelial defects are due to inappropriate Wnt/β-catenin and Wnt/PCP signaling, respectively, both mediated by Wnt11. Evidence is provided that Sfrp5 locally inhibits Wnt11 to maintain early foregut identity and to allow an epithelium to form over a mass of tissue undergoing Wnt-mediated cell movements. This novel mechanism coordinating canonical and noncanonical Wnt signaling may have broad implications for organogenesis and cancer (Li, 2009).

Wnt inhibitory factor-1 and tumors

To detect novel Wnt-pathway genes involved in tumorigenesis, this study analysed the RNA expression levels of 40 genes of the Wnt pathway by chip hybridization of microdissected matched pairs of 54 primary prostate carcinomas. Eleven genes showed greater than two-fold differential expression in at least 10% of prostate cancers. Three of these genes encode extracellular components of the Wnt pathway (WNT2, WIF1, SFRP4); two are receptors (FZD4, FZD6); two belong to the intracellular signal cascade (DVL1, PPP2CB); one regulates transcription (TCF4); and three represent genes regulated by this pathway (CCND2, CD44, MYC). While SFRP4, FZD4, FZD6, DVL1, TCF4, and MYC are up-regulated, WIF1, WNT2, PPP2CB, CCND2, and CD44 are down-regulated in certain prostate cancer patients. Wnt inhibitory factor 1 (WIF1) and secreted frizzled related protein (SFRP4) showed the most significant aberrant expression at the RNA level. WIF1 is down-regulated in 64% of primary prostate cancers, while SFRP4 is up-regulated in 81% of the patients. Immunohistochemical analysis using a polyclonal antibody revealed strong cytoplasmic perinuclear WIF1 expression in normal epithelial cells of the prostate, breast, lung, and urinary bladder. Strong reduction of WIF1 protein expression was found in 23% of prostate carcinomas, but also in 60% of breast, 75% of non-small cell lung (NSCLC), and 26% of bladder cancers analysed. No significant association between WIF1 down-regulation and tumor stage or grade was observed for prostate, breast or non-small cell lung carcinomas, indicating that loss of WIF1 expression may be an early event in tumorigenesis in these tissues. However, down-regulation of WIF1 correlates with higher tumor stage in urinary bladder tumors (Wissmann, 2003).

Using cDNA-Representational Difference Analysis it was found that expression of Opg, Ctse, Krt2-4, Fut-2, 24p3 and Wif-1 genes was elevated in intestinal adenomas as compared to normal epithelial cells of Apc(Min/+) mutant mice. Expression of Wif-1, which encodes Wnt inhibitory factor-1 was also detected in a number of tumor cell lines of epithelial cell origin including two human colon adenocarcinoma cell lines. The possible role of Wif-1 over-expression in the etiology of colorectal cancer is discussed (Cebrat, 2004).

Aberrant activation of the Wingless-type (Wnt) signaling pathway is associated with a variety of human cancers, and the importance of aberrant Wnt signaling in lung cancer has been reported. In contrast, inhibition of Wnt signaling suppresses growth in numerous cell types. Wnt inhibitory factor-1 (WIF-1) is a secreted antagonist that can bind Wnt in the extracellular space and inhibit Wnt signaling. Down-regulation of WIF-1 has been reported in several human cancers. To discover the mechanism of WIF-1 silencing in lung cancer, the human WIF-1 promoter was identified and the methylation status was examined in the CpG islands. By using methylation-specific PCR and sequence analysis after bisulfite treatment, frequent CpG island hypermethylation was identified in the functional WIF-1 promoter region. This hypermethylation correlates with its transcriptional silencing in human lung cancer cell lines. Moreover, treatment with 5-aza-2'-deoxycytidine restores WIF-1 expression. WIF-1 expression was examined in 18 freshly resected lung cancers, and a down-regulation was shown in 15 of them (83%). This silencing also correlates with WIF-1 promoter methylation. These results suggest that methylation silencing of WIF-1 is a common and likely important mechanism of aberrant activation of the Wnt signaling pathway in lung cancer pathogenesis, raising its therapeutic interest (Mazieres, 2004).

Aberrant Wnt signaling, mainly through mutations of APC and in some cases of CTNNB1 or AXIN2, has been found in the majority of colorectal cancers. Recently, frequent promoter hypermethylation was identified to cause silencing of the secreted frizzled-related protein (sFRP) family in colorectal cancer. Restoration of sFRP in colorectal cancer cells attenuates Wnt signaling even in the presence of downstream mutations. Wnt inhibitory factor-1 (WIF-1), a different secreted antagonist of Wnt signaling, is also silenced by promoter hypermethylation in colorectal cancer cells. Restoration of WIF-1 function, Wnt-1 siRNA, or a monoclonal anti-Wnt-1 antibody, attenuates Wnt-1 signaling and induces significant apoptosis in these cells containing downstream mutations and expressing Wnt-1. In addition, this monoclonal anti-Wnt-1 antibody shows synergistic effects with docetaxel in treating these colorectal cancer cells and great efficacy in treating primary colorectal cancer cultures freshly prepared from patients. Therefore, these data support the hypothesis that constitutive Wnt signaling may be required to complement downstream mutations in the evolution of colorectal cancer. Furthermore, these results suggest that blockade of the Wnt signal may have a therapeutic role in the treatment of colorectal cancer (He, 2005).

shifted: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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