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M. J. Nolan, T. Koga, J. R. Samples, B. Y. J. T. Yue, P. A. Knepper; Internalization of sCD44 in Trabecular Meshwork Cells in Culture. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2069.
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© ARVO (1962-2015); The Authors (2016-present)
To determine whether the hyaluronic acid (HA) binding protein soluble CD44 (sCD44) is internalized in trabecular meshwork (TM) cells and trafficked to mitochondria. A common concept in neurodegenerative diseases is that a toxic protein is associated with the cause of the disease process, e.g., ß-amyloid in Alzheimer’s disease. Accumulation and misfolding of the toxic protein is often linked to anomalies of mitochondrial function. Exactly why certain HA binding proteins are trafficked into the mitochondria and become toxic is unclear. One putative biomarker of primary open-angle glaucoma (POAG) is sCD44 which has a mitochondrial tethering domain. Thus, sCD44 may compete for other mitochondrial tethering proteins, displace them and interfere with normal mitochondrial function.
32-kDa sCD44 purified from human sera was labeled with biotin. TM cells plated on chamber slides were incubated with 0.5 ng/ml biotin-labeled sCD44 in the presence and absence of unlabeled sCD44, HA, and a selected HA peptide for 1 hr at 4°C. The slides were slowly warmed to 37°C, 125 nM MitoTracker was added and the cells were incubated at 37°C for 20 min. The cells were washed, fixed, incubated with mouse anti-biotin antibody and FITC-labeled anti-mouse antibody and examined under a confocal microscope to determine if sCD44 trafficks to mitochondria. As a further test of sCD44 association with mitochondria, TM cells were stained with a fluorescent dye, JC-1. In normal healthy cells, JC-1 fluoresces red and shifts to green in cells with mitochondria with decreased membrane potential.
Confocal microscopy of TM cells indicated that cell membranes were immunopositive for biotin-labeled sCD44 at 4°C, and the cell membranes were immunonegative in the presence of HA or the selected HA peptide. When the temperature was raised to 37°C, biotin labeled sCD44 dissociated from the cell membrane and appeared in the cytoplasm, particularly in the perinuclear region, however excess unlabeled sCD44 appeared to block the internalization of biotinylated sCD44. Double label experiments with both biotin-labeled sCD44 and MitoTracker indicated partial overlap, suggesting that sCD44 was transported to mitochondria.
sCD44 is internalized in TM cells and was blocked by unlabeled sCD44, HA, and HA binding peptide. sCD44 is trafficked in part to mitochondria and this may be a factor in the toxicity of sCD44 in certain cells, such as the TM and retinal ganglion cells in the POAG disease process.
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