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A.S. Hackam, D. Nolan, C. Wang, D. Otteson, D.J. Zack; Functional Characterization of Dickkopf 3 (Dkk3), a Wnt Pathway Gene that is Differentially Expressed During Retinal Degeneration . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1677.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Wnt signaling mediates numerous processes in embryonic and adult tissues. Although several Wnt–related genes are associated with retinal diseases, the role of Wnt during photoreceptor degeneration is unknown. Our previous microarray studies identified altered expression of multiple Wnt pathway genes during retinal degeneration in the rd1 mouse, including the Dickkopf 3 (Dkk3) gene. To develop a hypothesis of how elevated Dkk3 may be associated with retinal degeneration, we characterized the expression distribution and function of Dkk3. Methods: In situ hybridization was performed on retinas from rd1 and control (C57/Bl6) mice using probes generated against the antisense strand of the mouse Dkk3 gene. Stable cell lines expressing Dkk3 were created in HEK293 cells and protein levels were confirmed by Western blotting. Induction of Wnt signaling was measured by the TOP–flash luciferase reporter assay, using CMV–LacZ for normalization. Cell viability was measured by the mitochondrial function Wst–1 assay on HEK293 cells expressing Dkk3 or control genes. Results: Dkk3 transcripts were localized to the inner nuclear layer of normal and degenerating retina, potentially to Muller glia, the cells involved in photoreceptor protection in various retina damage situations. We demonstrated a dramatic 25–fold increase of luciferase activity in cultured cells expressing Dkk3, indicating potent activation of Wnt signaling. Additionally, Dkk3 had a significant pro–survival effect against H2O2–induced oxidative stress (p<0.001). The protection by Dkk3 was equivalent to the anti–apoptotic gene BclXL used as the positive control. Conclusions: We have shown that over–expression of Dkk3 activates the Wnt signaling pathway and protects against oxidative stress in vitro. Together with its putative expression in glia, these data suggest a hypothesis in which Dkk3 and Wnt signaling are activated in the retina as part of the glial response to photoreceptor injury.
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