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

Wei Zhang,Takuya Achiha,Masatoshi Maki,Hideki Shibata,Terunao Takahara

doi:10.3390/ijms19020605

Wei Zhang, Takuya Achiha + Show 3 more

Open Access

https://doi.org/10.3390/ijms19020605

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Journal: International Journal of Molecular Sciences	Publication Date: Feb 18, 2018
Citations: 5	License type: CC BY 4.0

Affiliation: Nagoya University

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.

Highlights

NFAT, a name after “a nuclear factor of activated T cells”, is a transcription factor containing a REL-homology region (RHR)
Subsequent to Ca2+ mobilization upon cell stimulation, NFAT is dephosphorylated by a Ca2+-calmodulin (CaM)-activated protein phosphatase, calcineurin [6,7], and exposes a nuclear localization signal to translocate from the cytoplasm to the nucleus [8]
To indirectly monitor effects of increases in the intracellular Ca2+ concentrations, we developed a series of Ca2+-inducible NanoLuc reporters based on the Ca2+-dependent activation of dimeric NFAT (Figure 1A)

Summary

Introduction

NFAT, a name after “a nuclear factor of activated T cells”, is a transcription factor containing a REL-homology region (RHR). Subsequent to Ca2+ mobilization upon cell stimulation, NFAT is dephosphorylated by a Ca2+-calmodulin (CaM)-activated protein phosphatase, calcineurin (the reaction inhibited by FK506 and cyclosporine A) [6,7], and exposes a nuclear localization signal to translocate from the cytoplasm to the nucleus [8]. The PKC activation leads to induction of Fos and Jun as well as phosphorylation of Jun by Jun-N-terminal kinase (JNK) activation and enhances transcription driven by TPA response element (TRE, alias AP1-RE) [12,13]. Concomitant use of PMA in addition to Ca2+-mobilizing agents in experimental conditions complicates the interpretation of obtained results and restricts the application of the IL2 NFAT-RE reporter system to the Ca2+ signaling studies

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Conclusion