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H. Cai, T.H. Tezel, L.V. Del Priore; Aging of Bruch’s Membrane Decreases Phosphorylation of Retinal Pigment Epithelium Proteins . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2320.
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We have shown previously that aging of human Bruch’s membrane(BM) alters numerous functions of the overlying RPE, including RPE attachment to basement membrane, proliferation, apoptosis and outer segment phagocytosis. Herein we determine the effects of Bruch’s membrane aging on phosphorylation and dephosphorylation of RPE proteins.
Immortalized human ARPE–19 cells were seeded onto human BM from young (donor age = 30– 46 years; 5 specimens) and older (age = 80–90 years; 5 specimens) human eye bank eyes (5 specimens each). ARPE–19 were harvested 10 days after seeding, a cell lysate was generated using cell lysis buffer, and specimens were analyzed by Western blot using antibodies against specific phosphorylation sites of known proteins. The quantity of phosphorylated proteins was estimated using densitometry of 49 phosphorylation sites of 38 known proteins.
Culturing RPE onto older BM caused a marked decrease in the number of proteins phosphorylated within ARPE–19. Aging of human BM caused a decrease in phosphorylation of docking protein 2 [tyrosine 142], cyclin–dependent protein–serine kinase [threonine 14+ tyrosine 15], MAPK/ERK protein–serine kinase 1 [serine 297], extracellular regulated protein–serine kinase 2 [threonine 185+ tyrosine 187], eukaryotic translation initiation factor 2 alpha [serine 51], mitogen–activated protein–serine kinase p38 alpha [threonine180+ tyrosine 182] and glycogen synthase–serine kinase 3 beta [tyrosine 279] in RPE.
Our data indicates that aging of human BM cause a significant decrease in phosphorylation of proteins in RPE cultured onto the explant surface, which is consistent with prior studies suggesting that senescence of pigmented cells, such as melanocytes, may be accompanied by repression of tyrosine–phosphorylation of the extracellular signal–regulated kinase. The role of protein phosphorylation, which is a manifestation of RPE senescence, on the pathogenesis of age–related macular degeneration remains to be determined.
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