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Int. J. Mol. Sci. 2018, 19(2), 605; https://doi.org/10.3390/ijms19020605

Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization

Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
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Received: 20 December 2017 / Revised: 11 February 2018 / Accepted: 14 February 2018 / Published: 18 February 2018
(This article belongs to the Special Issue Calcium Binding Proteins)
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Abstract

NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization. View Full-Text
Keywords: ALG-2; calcium-binding protein; calcineurin; calcium signaling; carbachol; nanoluciferase; NFAT; PDCD6; reporter gene assay; SOCE; transcription factor ALG-2; calcium-binding protein; calcineurin; calcium signaling; carbachol; nanoluciferase; NFAT; PDCD6; reporter gene assay; SOCE; transcription factor
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Zhang, W.; Takahara, T.; Achiha, T.; Shibata, H.; Maki, M. Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization. Int. J. Mol. Sci. 2018, 19, 605.

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