BACKGROUND

Recent studies have demonstrated that the production of reactive oxygen species (ROS) itself plays an indispensable role in the process of differentiation in various tissues. However, it is unclear whether ROS have an effect on the differentiation of keratinocytes essential for the development of the epidermal permeability barrier.

OBJECTIVE

The aim of the study is to determine a major H2O2-generating source by ionomycin in normal human keratinocytes (NHKs), and elucidate the physiological role of H2O2 generated by identified dual oxidase 1 (DUOX1) on differentiation markers of NHKs.

METHODS

To detect H2O2 level generated by ionomycin in NHKs, luminal-HRP assays are performed. To examine the effects of DUOX1 on differentiation markers of NHKs, analysis of Q-RT-PCR, siRNA knockdown, and Western blot analysis were performed.

RESULTS

We found that levels of H2O2 generated by ionomycin, a Ca(2+) signal inducer, showed Ca(2+) dependence manner. In addition, DPI, an inhibitor of NOXes, significantly reversed the ionomycin-induced H2O2 level, and inhibited the mRNA expression levels of keratin 1, keratin 10, and filaggrin compared with other ROS generating system inhibitors. Interestingly, we demonstrated that extracellular Ca(2+) markedly up-regulated mRNA expression levels of DUOX1 among NADPH oxidase (NOX) isoforms. Knockdown of DUOX1 by RNA interference (RNAi) in NHKs significantly antagonized an increase of ionomycin-induced H2O2 level, and specifically decreased the expressions of several keratinocyte differentiation markers such as keratin 1, transglutaminase 3, desmoglein 1, and aquaporin 9. In addition, we also found that formation of cornified envelope was significantly reduced in DUOX1-knockdown NHKs.

CONCLUSION

These results suggest that DUOX1 is the major H2O2-producing source in NHKs stimulated with Ca(2+), and plays a significant role in regulating the expression of specific markers necessary for the normal differentiation of keratinocytes.