Ryan D. McCarty

sCD44 Internalization in Human Trabecular Meshwork Cells

PURPOSE 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.