Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes.
OBJECTIVE
Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA-mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44-phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA.


METHODS
Normal human epidermal keratinocytes (NHEKs) were treated at doses of 1 μg mL(-1) HA or HA oligosaccharides (HA4). After treatment, cell viability was checked using an MTT (3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Each differentiation marker and CD44 mRNA expression was detected by real-time PCR. Each differentiation marker and CD44-phosphorylated protein was assessed by Western blotting.


RESULTS
Hyaluronan and HA4 showed no cytotoxicity up to a dose of 1 μg mL(-1) . On day 3 after HA4 treatment, each differentiation marker mRNA and K10 protein level was higher than that of the control. On day 9, late differentiation marker mRNA and protein levels were increased with HA and HA4 treatment. In addition, HA4 treatment increased the expression of CD44 mRNA, CD44-phosphorylated protein and intracellular calcium concentrations. HA4 enhanced keratinocyte differentiation and increased CD44-phosphorylated protein levels.


CONCLUSIONS
HA4 may induce epidermal differentiation through phosphorylation of CD44.
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295, Japan.
M
M
Kage
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295, Japan.
Y
Y
Tokudome
Shiseido Research Center, 2-2-1 Hayabuchi, Tsuzuki, Yokohama, 224-8558, Japan.
Y
Y
Matsunaga
Shiseido Research Center, 2-2-1 Hayabuchi, Tsuzuki, Yokohama, 224-8558, Japan.
T
T
Hariya
Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama, 350-0295, Japan.
F
F
Hashimoto
D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009844:Oligosaccharides / Q000494:pharmacology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics
D018960:Hyaluronan Receptors; D009844:Oligosaccharides; D012333:RNA, Messenger; D006820:Hyaluronic Acid
CD44 phosphorylation; hyaluronan oligosaccharide; keratinocyte differentiation