Norrin attenuates protease-mediated death of transformed retinal ganglion cells.
OBJECTIVE
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.
Lin
Song
S
Eye Research Institute of Oakland University, Rochester, MI 48309, USA.
Cheng
Mei
M
Dailey
Wendelin
W
Drenser
Kimberly
K
Chintala
Shravan
S
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D010450:Endopeptidases / Q000378:metabolism*; D005136:Eye Proteins / Q000494:pharmacology*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D012165:Retinal Ganglion Cells / Q000166:cytology* / Q000187:drug effects*; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
D005136:Eye Proteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D047428:Protein Kinase Inhibitors; D051153:Wnt Proteins; D051176:beta Catenin; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator