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A.M. Miller, J. Choi, B.Y. Yue, X. Shen, J.R. Samples, E.R. Doherty, P.A. Knepper; Metabolic Stress Causes NF-B Activation and CD44 Shedding in Cultured TM Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1138.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Signaling cascades respond to extrinsic factors by activation of intrinsic pathways. NF-ΚB, a prime example of a transcription factor involved in signal transduction, is up-regulated in the trabecular meshwork (TM) in glaucoma and targets several genes, including those involved with extracellular matrix remodeling and stabilization, notably CD44. The purpose of this study was to challenge TM cells with lactate and observe the effect on cell viability, NF-ΚB protein expression and nuclear translocation, and the release of soluble CD44 (sCD44). Methods: Human TM cells were grown in minimum essential medium Eagle(MEM) containing 10% fetal calf serum (FCS) and 5% calf serum until confluent. The cells were washed twice with PBS, and incubated in MEM containing 0.1% FCS with 1, 10, or 40 mM lactate or PBS for 5 and 30 minutes, 1, 3, and 6 hours. Cell viability was determined using trypan blue staining, and supernatant sCD44 concentration and molecular weights were determined by enzyme-linked immunosorbent assay and western blot analysis, respectively. NF-ΚB protein expression and nuclear translocation were revealed through western blot analysis of cytoplasmic and nuclear fractions using anti-NF-ΚB antibodies specific for p50 and p65. Results: Western blot analysis showed that p50 was constitutively present in the cytoplasmic fraction in both control and lactate-treated TM cells. Nuclear lysates were characterized by an absence of p50 in control TM cells, whereas those in 1, 10, and 40 mM lactate-treated TM cells were immunopositive for p50 by 5 minutes. At 6 hours, p65 was increased in the cytoplasmic fraction of 40 mM lactate-treated cells as well as in the nuclear fraction of 1, 10, and 40 mM lactate-treated TM cells as compared to control cells. 1 mM lactate caused an increase in the supernatant concentration of both the 32 and 55 kDa sCD44 at 3 (n=3, p<0.05) and 6 hours (n=3, p<0.03). Lactate significantly decreased TM cell viability at 3 hours using 40 mM lactate (n=3, p<0.01) and 6 hours with 10 and 40 mM lactate (n=3, p<0.001). Conclusions: Lactate treatment caused NF-ΚB to translocate to the nucleus within 5 minutes, increased shedding of sCD44 by 3 hours, and at doses of 10 and 40 mM, decreased TM cell viability by 3 hours. TM cell NF-ΚB activation and the subsequent sCD44 release suggest that TM cells respond to stress through the activation of inside-out signaling pathways. The increased concentration of sCD44 in glaucomatous aqueous humor may occur through an NF-ΚB-mediated mechanism.
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