Is POAG an Adaptive Senescence Phenomenon?

Abstract

Abstract: : Purpose: CD44 is involved in inflammation, regulation of cytokine gene expression, and aging. The ectodomain of CD44 (sCD44) is shed in response to stress and is cytotoxic to trabecular meshwork (TM) cells and neurotoxic to retinal ganglion cells (RGC) in cell culture. The purpose of this study was to further characterize sCD44, a 32 kDa ectodomain protein, by determining the phosphorylation of sCD44 in normal, primary open-angle glaucoma (POAG), and secondary glaucoma aqueous. Methods: Aqueous samples were subjected to CD44 enzyme-linked immunosorbent assay (ELISA) and 2-dimensional gel electrophoresis (2-D) followed by western blot analysis using anti-CD44 and anti-serine/threonine/tyrosine phospho-specific antibodies to determine sCD44 concentration and isoelectric point (pI), respectively. Age-matched samples included normal, POAG, pigmentary glaucoma (PG), pseudoexfoliation glaucoma (PXG), traumatic glaucoma (TG), and normotensive glaucoma (NTG). As a control, highly purified sCD44 was phosphorylated with casein kinase II and ATP and dephosphorylated with alkaline phosphatase to verify that phosphorylation state alters the pI of sCD44. Results: The ELISA revealed that POAG aqueous contains increased sCD44 (16.32 + 4.08 ng/ml; n=53) compared to normal (9.37+ 1.80 ng/ml; n=65, p<0.000001) and secondary glaucoma aqueous (11.41 + 3.18 ng/ml; n=20, p<0.000007). Linear regression analysis showed that sCD44 increased ~0.06 ng/ml/yr in normal aqueous and ~0.09 ng/ml/yr in POAG aqueous. Western blot analysis revealed that the representative pI (intensity 0-4 scale) of the 32 kDa sCD44 isoforms from 50-80 yo donors was 6.6 (trace) in normal, 5.9 (2+) and 6.7 (2+) in PG, 6.6 (1+) in NTG, 6.8 (1+) in TG, and 6.9 (2+) and 8.6 (2+) in POAG. The 32 kDa sCD44 was positive for serine/threonine phosphorylation, and dephosphorylation of control sCD44 resulted in a basic shift in pI. Conclusions: POAG aqueous is characterized by both an increased amount of sCD44 and an increased pI as compared to normal or secondary glaucoma aqueous. The basic shift in pI suggests that POAG aqueous is characterized by a hypophosphorylated sCD44 that may influence its shedding and toxic effects on TM and RGC. As a consequence of adaptive senescence, the pI of sCD44 in POAG is altered and clearly distinguished POAG from other types of glaucoma, suggesting a specific pathophysiology and a biochemical hallmark of the disease.

Keywords: protein modifications-post translational • phosphorylation • extracellular matrix