Abstract
Purpose: :
The Wnt pathway is a critical cellular communication pathway involved in retinal development and retinal degeneration. Previously, we demonstrated that activation of the Wnt signaling pathway in Muller glia protects photoreceptors from oxidative stress (Yi, et al, 2007). Muller glia secrete Dkk3, a Wnt pathway protein that potentiates Wnt3a-mediated signaling and protects several cell lines from exogenous insults (Nakamura et al, 2007). The purpose of this study is to identify the interacting proteins that mediate these properties of Dkk3 to further understand the role of Dkk3 during retinal degeneration.
Methods: :
GST-pull down, co-immunoprecipitation, and bis[sylfosuccinimidyl]suberate (BS3) cross linking were performed to test Dkk3 interaction with the Wnt regulators Krm1 and Krm2. FLAG-Dkk3 pull down or blot overlay followed by MS/MS analysis were carried out using human Muller glia cell line, mouse retina and brain lysates to identify novel interacting proteins. The interactions were verified via confocal analysis and TOP-FLASH luciferase Wnt reporter assays.
Results: :
Our data showed that Dkk3 interacts with both Krm1 and Krm2 but not with low density related lipoprotein 6 receptor (LRP6) when these genes were co-expressed (n=5). Interestingly, co-expression of these genes did not modulate Wnt signaling activity measured via luciferase assay (n=5, p<NS). Furthermore, we demonstrated by blot overlay that Dkk3 interacts with cysteine and glycine rich protein 2 (CRP2) and co-expression of these genes enhanced Wnt3a-mediated Wnt signaling (n=3, p<0.01).
Conclusions: :
Dkk3 interacts with the known Wnt regulators Krm1 and Krm2, and the non-Wnt pathway related protein CRP2. Since CRP2 is reported to interact with protein inhibitor of activated STAT1 (PIAS1), Dkk3 may mediate its pro-survival properties via a non-canonical pathway, most likely through the STAT pathway.
Keywords: Muller cells • protein purification and characterization • neuroprotection