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M.J. Nolan, B.Y. J. T. Yue, J.R. Samples, P.A. Knepper; Oxidative Stress Induces Secretion of Hypophosphorylated Soluble CD44 by Trabecular Meshwork Cells in Culture . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1840.
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Protein phosphorylation is a regulatory feature of several neurodegenerative diseases. Recently, we reported that a 32–kDa ectodomain fragment of CD44, i.e., soluble CD44 (sCD44), is hypo–phosphorylated in primary open angle glaucoma (POAG) aqueous humor and is cytotoxic to trabecular meshwork (TM) and retinal ganglion cells in cell culture. This study examined whether metabolic stress of TM cells results in hypophosphorylation of the ectodomain of sCD44.
Human TM cells were grown in Eagles mimimum essential medium (MEM) containing 10% fetal calf serum (FCS) until confluent. The cells were washed twice with PBS and incubated in MEM containing 0.1% FCS with 1, 10 mM lactate or PBS. The media was aspirated and replenished with fresh media containing 1, 10 mM lactate or PBS at selected time points ––3, 6, 12, 24, 36 and 48 hours. The concentration of sCD44 in the media at each time point was determined by ELISA. Aliquots of the media at each time point were immunoprecipitated, electrophoresed, and subjected to western blot analysis using anti–CD44 and phospho–specific serine/threonine antibodies. The blots were analyzed by densitometry to determine the ratio of phosphorylation to sCD44 protein.
Lactate treatment resulted in a time– and dose–dependent release of sCD44 into the media. sCD44 concentration in the media increased with 1 mM or 10 mM lactate after 3 hours of treatment (P<0.04) and the sCD44 concentration remained elevated in the 10 mM lactate treated cells. Western blot analysis of the 32–kDa sCD44 phosphorylation status in the 10 mM lactate treated media showed a progressive decrease in serine/threonine phosphorylation after 6 hours of lactate treatment. Densitometry indicated that the control media showed an increased in phosphorylation of sCD44 whereas 10 mM lactate treated cell media showed a three–fold decrease in phosphorylation after 12, 24, 36, and 48 hours.
Metabolic stress, as exemplified by lactate treatment of TM cells, caused the release of sCD44 and decreased the phosphorylation of sCD44. Lactate treatment of TM cells is a useful cell model of cell stress and may be applicable to cell stress responses in POAG.
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