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Michael J. Nolan, Tomoyo Koga, Loyal Walker, Ryan McCarty, Algis Grybauskas, Michael C. Giovingo, Kevin Skuran, Paulius V. Kuprys, Paul A. Knepper; sCD44 Internalization in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(1):592-601. doi: 10.1167/iovs.12-10627.
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To determine whether soluble CD44 (sCD44), a likely biomarker of primary open-angle glaucoma (POAG), is internalized in cultured human trabecular meshwork (TM) cells and trafficked to mitochondria.
In vitro, 32-kD sCD44 was isolated from human sera, biotinylated, and dephosphorylated. TM cells were incubated for 1 hour at 4°C with biotinylated albumin (b-albumin), biotin-labeled sCD44 (b-sCD44), or hypophosphorylated biotin-labeled sCD44 (–p b-sCD44) in the presence or absence of unlabeled sCD44, hyaluronic acid (HA), and a selected 10-mer HA binding peptide. The slides were warmed for 1 or 2 hours at 37°C, and 125 nM MitoTracker Red was added for the last 20 minutes of the incubation. The cells were washed, fixed, incubated with anti-biotin antibody and FITC-labeled goat anti-mouse antibody, and examined under a confocal microscope.
TM cell membranes were positive for b-sCD44 after 4°C incubation. When the temperature was raised to 37°C, b-sCD44 or –p b-sCD44 appeared in the cytoplasm. The internalization of b-sCD44 was blocked by excess unlabeled sCD44, HA, and a 10-mer HA-binding peptide. Double label experiments with b-sCD44 or –p b-sCD44 and MitoTracker Red indicated partial overlap. The percent co-localization of MitoTracker Red at 2 hours and FITC –p b-sCD44 was 17.4% (P < 0.001) and for FITC b-sCD44 was 11.7% (P < 0.001) compared with b-albumin. The influence of putative CD44 phosphorylation sites on mitochondrial trafficking was determined by TargetP 1.1.
sCD44 is internalized by TM cells and trafficked in part to mitochondria, which may be a factor in the toxicity of sCD44 in the POAG disease process.
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