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Published on: May 12, 2011
Norrin attenuates protease-mediated death of transformed retinal ganglion cells
Song Lin1, Mei Cheng, Wendelin Dailey
1Eye Research Institute of Oakland University, Rochester, MI 48309, USA.
Purpose:
To investigate the effects of norrin, a nonconventional ligand for Wingless-Int (Wnt)-beta-catenin signaling pathway, on protease-mediated death of transformed rat retinal ganglion cells (RGC-5).
Methods:
Transformed RGC-5 cells were treated with 2.0 microM staurosporine (SS), a broad-spectrum protein kinase-C inhibitor, to induce growth arrest, differentiation, and elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). RGC-5 cells were also treated with 2.0 microM SS and varying doses of recombinant norrin (3.125 to 100 ng/ml). Activation of Wnt pathway was assessed by nuclear translocation of beta-catenin. Proteolytic activity of tPA and uPA was determined by zymography assays and cell viability was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assays. Expression and phosphorylation of the low-density lipoprotein-related receptor-1 (LRP-1), a cell surface receptor for tPA and uPA, was determined by immunoprecipitation and western blot analysis.
Results:
Compared to RGC-5 cells left untreated, cells treated with either SS alone or SS and norrin secreted elevated levels of tPA and uPA. A significant number of RGC-5 cells treated with only SS underwent cell death, whereas cells treated with SS and norrin did not, even though RGC-5 cells secreted elevated levels of tPA and uPA under both treatment conditions. Although norrin activated the Wnt pathway, Dickkopf related protein 1 (Dkk1), an inhibitor of Wnt/beta-catenin pathway, failed to completely block norrin's neuroprotective effects. Assays for expression and phosphorylation of LRP-1 indicated that tPA and uPA cause RGC-5 cell death, in part, by reducing phosphorylation of LRP-1, whereas norrin attenuated tPA and uPA-mediated RGC cell death, in part, by restoring phosphorylation of LRP-1.
Conclusions:
Our results suggest that norrin attenuates tPA- and uPA-mediated death of RGC-5 cells by activating Wnt/beta-catenin pathway and by regulating phosphorylation of LRP-1.
Insights
Norrin protects rat retinal ganglion cells from protease-mediated death by activating the Wnt pathway and regulating LRP-1 phosphorylation. This finding offers potential therapeutic strategies for retinal cell protection.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Retinal ganglion cells (RGCs) are crucial for vision.
- Protease-mediated cell death pathways contribute to RGC loss in various retinal diseases.
- The Wingless-Int (Wnt)-beta-catenin signaling pathway plays a role in cell survival and development.
Purpose of the Study:
- To investigate the neuroprotective effects of norrin, a nonconventional Wnt pathway ligand.
- To determine norrin's impact on protease-mediated death in transformed rat retinal ganglion cells (RGC-5).
- To elucidate the molecular mechanisms underlying norrin's protective action.
Main Methods:
- RGC-5 cells were treated with staurosporine (SS) to induce cell death and elevated levels of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA).
- Cells were co-treated with SS and varying doses of recombinant norrin.
- Wnt pathway activation, protease activity, cell viability, and low-density lipoprotein-related receptor-1 (LRP-1) phosphorylation were assessed.
Main Results:
- Norrin treatment prevented SS-induced RGC-5 cell death despite elevated tPA and uPA levels.
- Norrin activated the Wnt pathway, and its protective effects were partially independent of Dickkopf related protein 1 (Dkk1).
- Norrin attenuated protease-mediated cell death by restoring LRP-1 phosphorylation.
Conclusions:
- Norrin exhibits significant neuroprotective effects against protease-mediated death in RGC-5 cells.
- Activation of the Wnt/beta-catenin pathway and regulation of LRP-1 phosphorylation are key mechanisms of norrin's action.
- These findings suggest norrin as a potential therapeutic agent for conditions involving RGC death.
