Hyaluronan Enters Keratinocytes by a Novel Endocytic Route for Catabolism

Abstract

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (<90 kDa). Approximately 25% of newly synthesized hyaluronan was endocytosed by the keratinocytes and had a half-life of 2-3 h. A biotinylated aggrecan G 1 domain/link protein probe demonstrated hyaluronan in small vesicles of ϳ100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nm diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH 4 Cl or chloroquine, or treating the cells with the hyaluronidase inhibitor apigenin increased intracellular hyaluronan staining, suggesting that it resided in prelysosomal endosomes. Competitive displacement of hyaluronan from surface receptors using hyaluronan decasaccharides, resulted in a rapid disappearance of this endosomal hyaluronan (t1 ⁄2 ϳ5 min), indicating its transitory nature. The ultrastructure of the hyaluronancontaining vesicles, co-localization with marker proteins for different vesicle types, and application of specific uptake inhibitors demonstrated that the formation of hyaluronan-containing vesicles did not involve clathrincoated pits or caveolae. Treatment of rat epidermal keratinocytes with the OX50 monoclonal antibody against the hyaluronan receptor CD44 increased endosomal hyaluronan. However, no CD44-hyaluronan co-localization was observed intracellularly unless endosomal trafficking was retarded by monensin, or cultivation at 20°C, suggesting CD44 recycling. Rat epidermal keratinocytes thus internalize a large proportion of their newly synthesized hyaluronan into non-clathrin-coated endosomes in a receptor mediated way, and rapidly transport it to slower degradation in the endosomal/lysosomal system.

BMP-7 Modulates Hyaluronan-Mediated Proximal Tubular Cell-Monocyte Interaction

“…The cell-associated fraction did contain a smaller peak of lower-M W HA consistent with a pool of intracellular, partially degraded HA as previously reported (28). After addition of BMP7-, an increase in the amount of radiolabeled HA was seen in the supernatant (CM fraction), the pericellular protein bound PC fractions, and the cell-associated (CA) fractions.…”

Development of a fluorescent substrate to measure hyaluronidase activity

“…Techniques for measuring and analyzing FRET signals in living cells have been reported [22]. Previous investigations have indicated that exogenous HA is transported to the endosomal/ lysosomal system for degradation [23]. Thus, the FRET-HA substrate may be a new tool for investigating hyaluronidase activity in intact living cells.…”

Intracellular trafficking of nonviral vectors

“…96 EM imaging studies and inhibition of cell entry by multiple inhibitors led the authors to conclude that magnetofectins entered cells through both clathrin-and caveolae-mediated pathways. Endocytic cell entry was inhibited by nystatin, which blocks caveolae formation, 97,98 and antimycin A, which generally depletes ATP, indicating that uptake occurred by energy-dependent endocytosis. Transfection was also inhibited by chloroquine, suggesting that complexes were trapped in early endosomes, and required acidification to facilitate endosomal release.…”