Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice

Research article Open Access Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice Beatriz Morancho1Email author, Jordi Minguillón1Email author, Jeffery D Molkentin2Email author, Cristina López-Rodríguez1Email author and Jose Aramburu1Email author BMC Molecular Biology20089:13 https://doi.org/10.1186/1471-2199-9-13 ©  Morancho et al; licensee BioMed Central Ltd. 2008 Received: 05 September 2007Accepted: 25 January 2008Published: 25 January 2008 Abstract Background The transcription factor NFAT5/TonEBP regulates the response of mammalian cells to hypertonicity. However, little is known about the physiopathologic tonicity thresholds that trigger its transcriptional activity in primary cells. Wilkins et al. recently developed a transgenic mouse carrying a luciferase reporter (9xNFAT-Luc) driven by a cluster of NFAT sites, that was activated by calcineurin-dependent NFATc proteins. Since the NFAT site of this reporter was very similar to an optimal NFAT5 site, we tested whether this reporter could detect the activation of NFAT5 in transgenic cells. Results The 9xNFAT-Luc reporter was activated by hypertonicity in an NFAT5-dependent manner in different types of non-transformed transgenic cells: lymphocytes, macrophages and fibroblasts. Activation of this reporter by the phorbol ester PMA plus ionomycin was independent of NFAT5 and mediated by NFATc proteins. Transcriptional activation of NFAT5 in T lymphocytes was detected at hypertonic conditions of 360–380 mOsm/kg (isotonic conditions being 300 mOsm/kg) and strongly induced at 400 mOsm/kg. Such levels have been recorded in plasma in patients with osmoregulatory disorders and in mice deficient in aquaporins and vasopressin receptor. The hypertonicity threshold required to activate NFAT5 was higher in bone marrow-derived macrophages (430 mOsm/kg) and embryonic fibroblasts (480 mOsm/kg). Activation of the 9xNFAT-Luc reporter by hypertonicity in lymphocytes was insensitive to the ERK inhibitor PD98059, partially inhibited by the PI3-kinase inhibitor wortmannin (0.5 μM) and the PKA inhibitor H89, and substantially downregulated by p38 inhibitors (SB203580 and SB202190) and by inhibition of PI3-kinase-related kinases with 25 μM LY294002. Sensitivity of the reporter to FK506 varied among cell types and was greater in primary T cells than in fibroblasts and macrophages. Conclusion Our results indicate that NFAT5 is a sensitive responder to pathologic increases in extracellular tonicity in T lymphocytes. Activation of NFAT5 by hypertonicity in lymphocytes was mediated by a combination of signaling pathways that differed from those required in other cell types. We propose that the 9xNFAT-Luc transgenic mouse model might be useful to study the physiopathological regulation of both NFAT5 and NFATc factors in primary cells. Keywords FK506SB203580Aldose ReductaseNucleus Pulposus CellRenal Medulla Background NFAT5/TonEBP belongs to the Rel family of transcription factors, which also comprises NF-κB and the calcineurin-dependent NFATc proteins (NFAT1/NFATc2, NFAT2/NFATc1, NFAT3/NFATc4, NFAT4/NFATc3) [1, 2]. Rel proteins have in common a conserved DNA binding domain, but do not display recognizable similarities outside of it. The DNA binding domain of NFAT5 is considered a hybrid between that of NF-κB and NFATc proteins, since it is a constitutive dimer, structurally similar to NF-κB, but has NFATc-like DNA sequence specificity, with its optimal binding site being a 5'-TGGAAA(C/A/T)A(T/A)-3' motif, in which the NFATc cognate element is 5'-(T/A/C)GGAA(A/G)-3' [2–4]. NFATc and NFAT5 differ substantially in their mechanisms of activation and biological function. NFATc proteins are characteristically activated by the phosphatase calcineurin in response to increases in intracellular calcium concentration [5, 6], whereas NFAT5 is activated by hypertonicity [1]. Activation of NFAT5 is regulated by different kinases, such as the stress-activated kinase p38, Fyn [7], PKA [8], ERK [9], the PI3-kinase-related kinase (PIKK) ATM [10, 11], and phosphoinositide 3-kinase (PI3-kinase) [11]. p38 has been shown to regulate NFAT5 in some cell types but not in others [7, 12]. NFATc proteins play fundamental roles in the immune, nervous and cardiovascular systems (reviewed in [13–15]). NFAT5 allows mammalian cells to adapt to hypertonicity [16, 17], by inducing the expression of osmoprotective proteins, such as aldose reductase (AR), Na+/Cl--coupled betaine/γ-aminobutyric acid transporter (BGT1), Na+-dependent myo-inositol transporter (SMIT), Na+ and Cl--dependent taurine transporter (TauT), UT-A urea transporter, and Hsp70 (reviewed in [18] and [19]). NFAT5-deficient mice suffer severe atrophy of the renal medulla, a naturally hypertonic environment, and impaired lymphocyte function [16, 17]. The osmoresponsive function of NFAT5 has been documented in diverse cell types, such as lymphocytes [3, 20], embryonic fibroblasts [16, 17], kidney cells [16, 21], neurons [22, 23], and cell lines of different lineages [10]. However, little is known about tonicity thresholds (physiologic or pathologic) at which NFAT5 is activated in specific types of primary cells. In this regard, a transgenic mouse model with an integrated NFAT5-responsive reporter would facilitate the analysis of its transcriptional regulation in primary cells and tissues. An NFAT-luciferase (9xNFAT-Luc) transgenic mouse carrying 9 copies of an NFAT site (5'-TGGAAAATT-3') positioned 5' to the minimal promoter of the α-myosin heavy chain gene was developed by Wilkins et al., who studied the role of the calcineurin-NFATc pathway in cardiac hypertrophy [24]. As described in the original article, luciferase activity was detectable in most organs and was highest in the brain, kidney and heart, indicating that the reporter was functional in different types of tissues. Since the NFAT site used in the reporter construct almost coincided with an optimal binding site for NFAT5 (5'-TGGAAAAAT-3'), we wondered whether it could be activated by this factor. In this work we show that the 9xNFAT-Luc reporter is activated by NFAT5 in response to hypertonicity in transgenic primary T lymphocytes, macrophages and mouse embryo fibroblasts (MEF), and by NFATc proteins in response to calcineurin activation. Activation of NFAT5 in lymphocytes was detected in response to hypertonicity levels in the range measured in plasma in patients and animal models with osmoregulatory disorders. Activation of NFAT5 transcriptional activity by hypertonicity was substantially downregulated by the p38 inhibitors SB203580 and SB202190, and by inhibition of PIKK with 25 μM LY294002. The reporter was partially sensitive to the calcineurin inhibitor FK506, the PI3-kinase inhibitor wortmannin (0.5 μM), and the protein kinase A inhibitor H89, but was not inhibited by the ERK inhibitor PD98059. These results, together with others in the l (...truncated)