April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Identification of Dkk3 Interacting Proteins and Target Genes in the Wnt Pathway
Author Affiliations & Notes
  • R. E. Nakamura
    Ophthalmology, University of Miami, Miami, Florida
  • H. Yi
    Ophthalmology, University of Miami, Miami, Florida
  • A. S. Hackam
    Ophthalmology, University of Miami, Miami, Florida
  • Footnotes
    Commercial Relationships  R.E. Nakamura, None; H. Yi, None; A.S. Hackam, None.
  • Footnotes
    Support  Karl Kirchgessner Foundation NEI core grant P30EY014801 RPB Career Dev Award RPB Unrestricted grant (BPEI) 1R01EY017837
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 3722. doi:
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      R. E. Nakamura, H. Yi, A. S. Hackam; Identification of Dkk3 Interacting Proteins and Target Genes in the Wnt Pathway. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3722.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : The Wnt pathway is a critical cellular communication pathway involved in retinal development and degeneration. Recently, we showed that Wnt signaling protects photoreceptors from oxidative stress by acting on Muller glia. Muller glia secrete Dkk3, which is an anti-apoptotic Wnt pathway protein that positively modulates Wnt signaling. The purpose of this study is to identify interacting proteins of Dkk3 and its target genes in an effort to understand how Dkk3 regulates cellular survival in the retina.

Methods: : Interactions between Dkk3 and the Wnt pathway receptors V5-Krm1, V5-Krm2 and LRP6, and the Wnt3a ligand were performed on transfected SH-SY5Y whole cell lysates by co-immunoprecipitation (co-IP) using anti-FLAG antibody, followed by immunoblotting with anti-V5, anti-LRP6 and anti-Wnt3a antibodies. Mass spectrometry (Midwest Bioservice) was used to identify novel Dkk3 interacting proteins from co-IP complexes isolated from HEK293 stably expressing Dkk3. Primary retinal cultures from PN8 C57/Bl6 mice were treated with Wnt3a (100 ng/ml) plus Dkk3 (50 ng/ml) or Wnt3a (100 ng/ml) alone at 3 days in vitro, for 24 hr, and total RNA was then harvested. cDNA was synthesized and used to test the expression of 89 Wnt signaling genes using QPCR arrays (SA Bioscience).

Results: : Dkk3 interacted with Krm1 and Krm2 but not the LRP6 receptor or the Wnt3a ligand (n=3). Mass spectrometry revealed that Dkk3 interacts with glucose response protein 78 (GRP78), which was confirmed by co-IP (n=3) and immunolocalization. Additional interactors found by mass spectrometry in the Dkk3-containing precipitate included HSP70 and ATP synthetase. Finally, our preliminary data indicated that Dkk3 upregulated multiple Wnt ligands and downregulated intracellular signaling molecules by at least 2 fold.

Conclusions: : These data suggest that Dkk3 may mediate its pro-survival pathway by increasing Wnt ligand levels and/or by interacting with Krm receptors and GRP78, but not by interacting with the positive regulators LRP6 and Wnt3a. Future studies will determine whether Dkk3-mediated signaling contributes to photoreceptor neuroprotection.

Keywords: Muller cells • neuroprotection • signal transduction 

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