title: Hydrogen peroxide generated by DUOX1 regulates the expression levels of specific differentiation markers in normal human keratinocytes.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors: Bioscience Research Institute, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do 446-729, Republic of Korea.
authors: Hyun
authors: H
authors: Choi
authors: Bioscience Research Institute, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do 446-729, Republic of Korea.
authors: Ju-Yearl
authors: JY
authors: Park
authors: Bioscience Research Institute, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do 446-729, Republic of Korea.
authors: Hyoung-June
authors: HJ
authors: Kim
authors: College of Pharmacy, Seoul University, Seoul 151-742, Republic of Korea.
authors: Minsoo
authors: M
authors: Noh
authors: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Republic of Korea.
authors: Takehiko
authors: T
authors: Ueyama
authors: Department of Life Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea.
authors: Yunsoo
authors: Y
authors: Bae
authors: Bioscience Research Institute, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Electronic address: TRLee@amorepacific.com.
authors: Tae Ryong
authors: TR
authors: Lee
authors: Bioscience Research Institute, Amorepacific Corporation R&D Center, Yongin-si, Gyeonggi-do 446-729, Republic of Korea. Electronic address: biopang@amorepacific.com.
authors: Dong Wook
authors: DW
authors: Shin
mesh_terms: D002454:Cell Differentiation; D002478:Cells, Cultured; D000074623:Dual Oxidases; D000091344:Filaggrin Proteins; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry*; D015759:Ionomycin / Q000737:chemistry; D015603:Keratinocytes / Q000166:cytology*; D019255:NADPH Oxidases / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010539:Permeability; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D015759:Ionomycin; D006861:Hydrogen Peroxide; D000074623:Dual Oxidases; D019255:NADPH Oxidases; C431513:DUOX1 protein, human; C431515:DUOX2 protein, human
keywords: Differentiation; Dual oxidase1 (DUOX1); Normal human keratinocytes (NHKs); Reactive oxygen species (ROS)