Hyaluronan enters keratinocytes by a novel endocytic route catabolism

被引:201
作者
Tammi, R
Rilla, K
Pienimäki, JP
MacCallum, DK
Hogg, M
Luukkonen, M
Hascall, VC
Tammi, M
机构
[1] Univ Kuopio, Dept Anat, FIN-70211 Kuopio, Finland
[2] Cleveland Clin Fdn, Lerner Res Inst, Dept Biomed Engn ND20, Cleveland, OH 44195 USA
[3] Univ Michigan, Dept Anat & Cell Biol, Ann Arbor, MI 48109 USA
关键词
D O I
10.1074/jbc.M103481200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 <similar to>100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nn diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH4Cl 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 (t(1/2) similar to5 min), indicating its transitory nature. The ultrastructure of the hyaluronan-containing 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 clathrin-coated 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 degreesC, 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.
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页码:35111 / 35122
页数:12
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