Wnt ligands are secreted glycoproteins that control a wide range of processes in the developing embryo and in adult tissues. Aberrant Wnt signaling is increasingly being implicated in numerous diseases, including malignancies, Alzheimers disease, retinal degenerations and abnormal development of the eye, limbs and skeleton [1–3]. Characterizing proteins that regulate the Wnt pathway have revealed important insights into Wnt-dependent processes and potential directions towards novel therapies .
The best understood of the major Wnt pathways is the canonical/β-catenin pathway. In the absence of Wnt ligands, β-catenin levels are suppressed by the APC-axin-GSK3β protein complex via phosphorylation and subsequent degradation by the proteosome . β-catenin is a transcriptional cofactor and is also an essential component of cell-cell adhesion complexes. Wnt ligands bind to the cell surface receptors Frizzled and LDL receptor related proteins 5 and 6 (LRP5/6), leading to Disheveled activation and ultimately reducing β-catenin degradation. Stabilized β-catenin is transported into the nucleus where it binds to Tcf/Lef type transcription factors and initiates transcription of Wnt target genes.
The Dickkopf (Dkk) family of proteins, Dkk1, 2, 3 and 4 and Soggy, are secreted regulators of Wnt signaling [6–8]. The five Dkk proteins share 37–50% protein identity and contain two conserved cysteine-rich regions separated by a variable linker region . Dkk1, Dkk2 and Dkk4 inhibit Wnt signaling by binding to LRP5/6 and the transmembrane protein Kremen which results in LRP5/6 internalization and prevents Wnt and Frizzled from forming an active complex with LRP5/6 [9, 10]. Dkk2 can also activate the Wnt pathway in certain situations, depending on the cell type, the presence of Wnt ligands and levels of LRP5/6 [11–13].
Unlike its related family members, characterizing Dkk3 activity has been elusive. Dkk3 did not regulate Wnt signaling in various activity assays, including Wnt-dependent secondary axis induction in Xenopus embryos and Wnt1/Fz8 signaling in cultured cells [8, 11, 12]. Dkk3 also did not physicallyinteract with LRP5/6 or Kremen [9, 14]. However, Caricasole et al demonstrated that Dkk3 was a weak inhibitor of Wnt7A signaling in PC12 cells although co-expression of LRP5 or LRP6 was required to uncover this activity . Dkk3 displayed Wnt inhibitor activity in the osteocarcinoma Saos-2 cell line, measured by decreased cytoplasmic levels of β-catenin , but did not inhibit Wnt reporter Tcf/Lef luciferase activity assays in a prostate cancer cell line . Therefore, the relationship between Dkk3 and Wnt signaling is unclear despite its sequence similarity to the other Dkk genes.
Dkk3 is expressed during embryonic development in many organs, including neural epithelium, limb bud, bone and heart, particularly in regions of epithelial-mesenchyme transformation . Dkk3 is also widely expressed in adult tissues, with the highest levels found in the heart and brain . Dkk3-deficient mice develop normally, are fertile and have a mild phenotype that includes hyperactivity, increased immunological and hematological markers and a slight decrease in lung ventilation . The absence of severe phenotypes in Dkk3 knock-out mice may be due to compensation from the Dkk3 homolog soggy . Alternatively, physiological stress or injury may be required for the appearance of a Dkk3-dependent phenotype.
In this study, we investigated the activity of Dkk3 in Wnt signaling and cell death. We demonstrated two novel functions for Dkk3. First, Dkk3 is a cell-specific positive regulator of the canonical Wnt signaling pathway in primary cell culture and cell lines. Second, Dkk3 protected transfected cells from apoptotic stress. We also characterized the distribution of Dkk3 in the retina and found that Dkk3 is expressed highly in Müller glia and ganglion cells during retinal development and in adult retina. Müller glia are the principle supportive glia in the retina and are believed to protect photoreceptors during retinal injury by secreting growth factors [20–23]. We previously demonstrated that Dkk3 transcripts were increased in a mouse model of retinal degeneration, particularly during cone photoreceptor death . Furthermore, Wnt signaling is upregulated during retinal degeneration in Müller glia and Wnt activators protect primary photoreceptor cultures from apoptosis . Together, these results identify for the first time that Dkk3 is a secreted pro-survival signaling protein, and suggest that Dkk3 may play a role in Müller glia activity in the retina.