NF-κB-regulated transcriptional control of CLCA in a differentiated mouse keratinocyte line.
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.


CONCLUSION
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.
['Hiromatsu|Ryo|R|', 'Hatta|Mitsutoki|M|', 'Okamura|Kazuhiko|K|', 'Sakagami|Ryuji|R|', 'Yamazaki|Jun|J|']
D000818:Animals D002118:Calcium D002478:Cells, Cultured D018118:Chloride Channels D005786:Gene Expression Regulation D015603:Keratinocytes D051379:Mice D016328:NF-kappa B D011401:Promoter Regions, Genetic D014158:Transcription, Genetic
CLCA Differentiation Keratinocyte NF-κB