Abstract
Purpose: :
To identify families of stress kinases activated by retinal detachment and their relationship to Fas-receptor signaling.
Methods: :
Retinal-RPE separation was created in Brown Norway rats by injection of 1% hyaluronic acid into the subretinal space. Met12, a competitive inhibitor of the Fas-receptor, mutant-Met12, or vehicle was injected into the subretinal space at the time of separation. Retinas were harvested 3 days after detachment, protein was extracted and stress-kinase activation was assayed by immunoblotting. Attached retinas served as controls. Fas-dependence of kinase activation was further tested in 661W cells, a cone-derived photoreceptor cell line, by using a Fas-receptor activating antibody. Cell lysates were collected at several time points after antibody administration, and kinase activation was assayed with immunoblotting.
Results: :
In the in vivo model, there were increased levels of several stress kinases following retinal detachment, including p42/44, p38 and JNK, all of which were partially blocked by the Fas-receptor inhibitor. In some pathways, such as p42/44, phosphorylation of the kinases to their active form was independent of Fas-activation, while in other cascades, such as those involving JNK, the activation was Fas-dependent. Similar results were seen in the 661W cells treated with the Fas-activating antibody for 24-48 hours. However, slight variations were found between the two models, with p38 phosphorylated after cells were exposed to Fas-antibody in the in vitro model, but not phosphorylated after retinal detachment in vivo.
Conclusions: :
Retinal detachment induced the activation of several types of stress kinases by increasing their protein levels and phosphorylation. Fas-signaling played a significant role in detachment-induced stress kinase activation by differentially regulating protein production and phosphorylation.
Keywords: retinal detachment • signal transduction