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Background Hyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive. Objective We examined the interaction of increased HA production mediated by 1-ethyl-β-N-acetylglucosaminide (β-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation. Methods The amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in β-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined. Results β-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that β-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in β-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by β-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of β-NAG2 to UDP-NAG abolished β-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes. Conclusion These results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.

The incidence of sunlight-induced skin changes (photoaged skin, skin carcinogenesis) increases with increasing age and it is thought to be associated with an accumulation of mutations in skin cells. These mutations are mainly caused by UV exposure. The reactive oxygen species produced in UV-exposed skin can cause various kinds of DNA damages e.g., 8-oxoguanine, which are primarily repaired by the base excision repair (BER) system. In addition, UV can directly cause DNA damages; cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP), both of which can be repaired by the nucleotide excision repair (NER) system. There have been several reports showing an age-related reduction in the DNA repair capacity in the NER, BER, and other repair systems, which contributes to the phenotypes of aging. To clarify the mechanism of skin aging, we examined the NER of skin fibroblasts from healthy donors of different ages. In a host cell reactivation assay, the cells from elderly donors exhibited a significant decline in the ability to restore transfected reporter DNA damaged by UV. In contrast, the ability to remove CPD and 6-4PP declined little with age, as assessed by an enzyme-linked immunosorbent assay. The mRNA expression of DNA repair synthesis-related genes was markedly decreased in the cells from elderly subjects as compared with those from young subjects. These results imply that the age-sensitive step took place after the damage excision in the NER, and that there is an impairment of the latter step of the NER in aging. Based on our data, as well as other reports, the reduced post-UV DNA repair capacity in aging resulting in an accumulation of UV-induced DNA damage is thus considered to be associated with the phenotypes of photoaged skin.