INTRACELLULAR HYALURONAN IN EPIDERMAL KERATINOCYTES

Rat epidermal keratinocyte monolayer cultures (REKs) actively synthesize hyaluronan (HA) most of which is retained on the cell surface or released into culture medium. However, a small proportion of HA also resides in an intracellular compartment (IC-HA). We characterized IC-HA localization and processing in REKs using specific staining with HA-binding probe, and its size by gel filtration of metabolically labeled HA. About 3% of REKs exhibited abundant IC-HA, while half of the cells lacked any microscopically demonstrable IC-HA. IC-HA was localized in 200–600 nm cytoplasmic vesicles. Dual staining of IC-HA with markers for lysosomes showed no colocalization. When REKs were treated with HA10 (decasaccharide), all IC-HA disappeared, while HA6, HA4, and sulfated glycosaminoglycans had no effect. All IC-HA was cleared 10 min after addition of HA10, and a 50%-reduction was reached in 5 min. An anti-CD44 mab, that increases HA on the cell surface, also increased IC-HA. Inhibiton of endocytosis via coated pits and caveolae did not reduce the amount of IC-HA. Perturbation of lysosomal activity caused accumulation of IC-HA. Most of the IC-HA had low MW ( 2000 kDa). The data demonstrate a rapid uptake and lysosomal degradation of IC-HA in REKs via a receptor dependent route separate from coated pits and caveolae, that involves a receptor with HA10 specificity, identified as CD44 or functionally dependent on CD44. The IC-HA consists of small HA fragments which may have a specific role in cellular homeostasis.