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Adam M. Miller, Michael J. Nolan, John Choi, Tomoyo Koga, Xiang Shen, Beatrice Y. J. T. Yue, Paul A. Knepper; Lactate Treatment Causes NF-κB Activation and CD44 Shedding in Cultured Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(4):1615-1621. doi: 10.1167/iovs.06-1086.
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purpose. To challenge human trabecular meshwork (TM) cells using lactate to mimic cell stress and observe the effects on cell viability, NF-κB, and membrane type 1 matrix metalloproteinase (MT1-MMP) expression and the ectodomain shedding of soluble (s)CD44.
methods. Human TM cells grown in 10% fetal calf serum (FCS) were incubated in 0.1% FCS with 1, 10, or 40 mM lactate or PBS for 5 and 30 minutes and 1, 3, and 6 hours. Cell viability was determined with trypan blue staining. NF-κB and MT1-MMP expression was evaluated through Western blot analysis of medium and the cytoplasmic and nuclear fractions. Media sCD44 concentration was determined by enzyme-linked immunosorbent assay and Western blot analysis.
results. The TM cell viability was significantly decreased after incubation for 3 hours with 40 mM lactate (P < 0.01) and 6 hours with 10 and 40 mM lactate (P < 0.001). Western blot analysis showed an increased NF-κB p50 and MT1-MMP expression and activity by 5 minutes in lactate-treated TM cells compared with that of control cells. At 6 hours, NF-κB p65 was increased in nuclear fraction of lactate-treated compared with control cells. Treatment with 1 mM lactate caused an increase in the media concentration of both the 32 and 55 kDa sCD44 at 3 (P < 0.05) and 6 hours (P < 0.01).
conclusions. Lactate treatment resulted in dose- and time-dependent effects on human TM cell viability, translocation of NF-κB, and activation of MT1-MMP. Increased shedding of sCD44 occurred with the l mM dose of lactate. Lactate treatment of human TM cells in culture offers a useful cell model to examine the stress responses that occur in glaucoma.
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