title: NF-κB-regulated transcriptional control of CLCA in a differentiated mouse keratinocyte line.
abstract: BACKGROUND
  CLCA was postulated to be a calcium-activated chloride channel accessory protein. Recent reports indicate that CLCA isoforms are likely to be expressed in different layers of the stratified epithelium of the skin.
  
  
  OBJECTIVE
  The present study investigated the transcriptional mechanism by which murine CLCA2 (mCLCA2) is expressed in the transformed keratinocyte line Pam212 that can differentiate.
  
  
  METHODS
  A luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay, reverse transcription-PCR, and immunocytochemistry were performed using Pam212 cells.
  
  
  RESULTS
  Promoter activity of mCLCA2 was inhibited profoundly by site-directed mutagenesis of a putative nuclear factor-κB (NF-κB) binding site and by treatment with siRNA against p65. ChIP and transcription factor assays showed the specific association of endogenously activated p65 protein with the NF-κB binding domain. As confirmed by the nuclear translocation of p65, tumor necrosis factor α and caffeic acid phenethyl ester (CAPE) increased and decreased mCLCA2 promoter activity, respectively, but exhibited modest effects on endogenous mCLCA2 expression in cells in culture medium containing 0.05 mM Ca(2+). When the Ca(2+) concentration was raised to 1.0mM, the mRNA and protein levels of mCLCA2 increased as well as those of the differentiation markers keratin 1 (K1) and K10. CAPE profoundly suppressed only the Ca(2+)-triggered expression of mCLCA2, not K1 or K10. Immunohistochemistry of native skin and organotypic 3D cultures confirmed the distribution of the CLCA2 homolog in differentiated cells.
  
  
  CONCLUSIONS
  The present study revealed for the first time that basal NF-κB activity is involved in the Ca(2+)-dependent regulation of mCLCA2 expression in a mouse keratinocyte line.
authors.affiliation: Department of Odontology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Hiromatsu
authors.affiliation: Department of Physiological Science & Molecular Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan.
authors.forename: Mitsutoki
authors.initials: M
authors.lastname: Hatta
authors.affiliation: Department of Morphological Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Okamura
authors.affiliation: Department of Odontology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan.
authors.forename: Ryuji
authors.initials: R
authors.lastname: Sakagami
authors.affiliation: Department of Physiological Science & Molecular Biology, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan. Electronic address: junyama@college.fdcnet.ac.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Yamazaki
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000235:genetics* / Q000502:physiology; D005786:Gene Expression Regulation; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
chemical_list: D018118:Chloride Channels; C492299:Clca2 protein, mouse; D016328:NF-kappa B; D002118:Calcium
keywords: CLCA; Differentiation; Keratinocyte; NF-κB