Functional and structural diversity of the human Dickkopf gene family
Introduction
The orchestration of early embryonic development requires the concerted actions of many secreted signaling molecules. The Wnt gene family comprises a large class of secreted proteins related to the Int1/Wnt1 proto-oncogene and Drosophila wingless (Wg). Wnts are expressed in a variety of tissues and organs, and are required for many developmental processes, including segmentation in Drosophila, endoderm development in Caenorhabditis elegans, establishment of limb polarity, neural crest differentiation, kidney morphogenesis, sex determination and brain development in mammals (Cadigan and Nusse, 1997, Parr and McMahon, 1994, Wodarz and Nusse, 1998). Recent studies have led to the identification of several components of the Wnt signal transduction pathway in responding cells. Wnt signals are transduced by the Frizzled (Fz) family of seven transmembrane domain receptors (Bhanot et al., 1996). The resulting signal leads to the activation of the cytoplasmic protein Dishevelled (Dsh) and stabilization of the Armadillo/β-catenin protein (Perrimon, 1994). Negative regulators of the Wnt pathway include glycogen synthase kinase 3 (GSK3)/shaggy (Perrimon, 1994), the tumor suppressor gene product APC (adenomatous polyposis coli; Gumbiner, 1997) and Axin (Zeng et al., 1997). In the absence of a Wnt ligand, these regulators promote phosphorylation and consequential degradation of β-catenin. Conversely, Wnt signaling inactivates GSK3 and therefore stabilizes β-catenin, which translocates to the nucleus and forms a complex with TCF transcription factors, thereby regulating target gene expression. Deregulation of this pathway can lead to carcinogenesis, emphasizing the long-recognized connection between Wnts, normal development and cancer (Cadigan and Nusse, 1997). This connection has been strengthened recently with the identification of the c-Myc proto-oncogene as a target of Wnt signaling (He et al., 1998).
While the outcome of Wnt signaling may be influenced by multiple intracellular regulatory mechanisms, recent studies have also identified several classes of secreted factors which modulate Wnt action outside of the cell. These include a family of secreted Frizzled-related proteins (FRPs; Moon et al., 1997, Wodarz and Nusse, 1998, Zorn, 1997), Cerberus (Piccolo et al., 1999) and Wnt Inhibitory Factor-1 (WIF-1; Hsieh et al., 1999a). FRPs are structurally related to the extracellular domains of Frizzled proteins, particularly in conserved cysteine-rich domains (Moon et al., 1997). Cerberus is a multifunctional inhibitor of BMP, Nodal and Wnt signaling (Piccolo et al., 1999). WIF-1 possesses a unique structure composed of an amino terminal WIF domain and 5 carboxy terminal epidermal growth factor repeats (Hsieh et al., 1999a, Hsieh et al., 1999b). WIF-1 and certain FRPs inhibit Wnt function by direct binding of the ligand, thus preventing access to cell surface receptors (Bafico et al., 1999, Hsieh et al., 1999a, Hsieh et al., 1999b, Leyns et al., 1997, Rattner et al., 1997, Wang et al., 1997).
The Dickkopf (Dkk) family comprises another discrete class of secreted Wnt inhibitors. xDkk-1 was identified in a screen for factors capable of inducing Xenopus head formation in the context of inhibition of bone morphogenetic protein signaling (Glinka et al., 1998). xDkk-1 is expressed in the Spemann organizer, which is responsible for induction of head structures, and is therefore a candidate for the endogenous signal for head induction (Glinka et al., 1998). Dkk-1 inhibits the axis-inducing activity of Xwnt8, consistent with the suggestion that simultaneous inhibition of BMP and Wnt signaling is required for head formation. xDkk-1 does not inhibit secondary axis induction by Xdsh, however. Thus, like the FRPs and WIF-1, xDkk-1 is most likely to act as an extracellular factor that antagonizes Wnt function directly. A murine homolog of xDkk-1 also inhibits Xwnt8-mediated axis duplication in Xenopus embryos. Furthermore, Dkk-1 appears to be the prototypical member of a family of Dkk-related genes (Glinka et al., 1998). Here, we describe the characterization of a family of four human Dkk-related proteins and a unique protein, Soggy, which possesses homology to hDkk-3 but not other Dkks. Our data demonstrate structural and functional heterogeneity within this novel class of secreted proteins.
Section snippets
Isolation of mammalian Dkk cDNAs
Human Dkk-related proteins were identified by searching of internal and public expressed sequence tag (EST) databases with the sequence of hDkk-3 using the TBLASTN algorithm (WashUversion 2.0, BLOSUM62 search matrix). From this search, four distinct partial protein sequences were identified which displayed significant homology to human Dkk-3. The full-length cDNA sequence of hDkk-1 was determined by complete sequencing of an individual clone from a human fetal kidney cDNA library which was
Characterization of the human Dkk-related gene family
Fig. 1A depicts schematically the structures of the family of human Dkk-related proteins that we have characterized in the present study. The human Dkk (hDkk) family comprises hDkk-1, hDkk-2, hDkk-3, hDkk-4, and a unique Dkk-3-related protein we refer to as Soggy (Sgy). hDkk-1 is the full-length human homolog of previously reported Xenopus and mouse Dkk-1 (Glinka et al., 1998). hDkk-2 and hDkk-3 correspond to previously described partial hDkk-2 and hDkk-3 sequences (Glinka et al., 1998). hDkk-4
Note added in proof
The Genbank accession numbers of the sequences listed in this paper are: Dkk-1 — AF177394; hDkk-2 — AF177395; hDkk-3 — AF177396; hDkk-4 — AF177397; h-Soggy — AF177398; m-Soggy — AF177399; mDkk-3 — AF177400.
Acknowledgements
We thank R. Moon for plasmids. We also thank Rick Clark, John Keilty, Betty Woolf, Pam Brauer, Donna Michnick, Kevin McDonald and members of the Millennium DNA sequencing, cDNA library and Full Length cDNA Cloning Groups for skilled assistance and advice. We are grateful to Joe Weber and Bill Holmes for critical comments on the manuscript and Chuck Gray for assistance with collaborations. This work was supported in part by grants from the March of Dimes Birth Defect Foundation and the National
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Present address: Lilly Research Laboratory, Greenfield, IN 46140, USA.
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Present address: Pfizer Central Research Division, Eastern Point Rd., Groton, CT 06340, USA.