Document Detail

sCD44 internalization in human trabecular meshwork cells.
MedLine Citation:
PMID:  23287794     Owner:  NLM     Status:  MEDLINE    
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.
Michael J Nolan; Tomoyo Koga; Loyal Walker; Ryan McCarty; Algis Grybauskas; Michael C Giovingo; Kevin Skuran; Paulius V Kuprys; Paul A Knepper
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2013-01-17
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  54     ISSN:  1552-5783     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2013 Jan 
Date Detail:
Created Date:  2013-01-18     Completed Date:  2013-03-19     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  592-601     Citation Subset:  IM    
Department of Ophthalmology and Visual Sciences, University of Illinois-Chicago, Chicago, Illinois, USA.
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MeSH Terms
Antigens, CD44 / immunology*,  metabolism
Aqueous Humor / immunology,  metabolism
Cell Line
Enzyme-Linked Immunosorbent Assay
Glaucoma, Open-Angle / immunology*,  metabolism,  pathology
Microscopy, Confocal
Trabecular Meshwork / immunology*,  metabolism,  pathology
Grant Support
Reg. No./Substance:
0/Antigens, CD44

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

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