Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice.
Analysis of the transcriptional activity of endogenous NFAT5 in primary cells using transgenic NFAT-luciferase reporter mice.
复制标题
DOI：
10.1186/1471-2199-9-13
复制
发表时间：
2008-01-25
影响因子：
--
通讯作者：
Aramburu J
中科院分区：
文献类型：
--
作者：
Morancho B;Minguillón J;Molkentin JD;López-Rodríguez C;Aramburu J
转录因子NFAT5/TONEBP调节哺乳动物细胞对高张状态的反应。然而，人们对在原代细胞中触发其转录活性的生理病理张力阈值知之甚少。Wilkins等人。最近开发了一种携带荧光素酶报告基因(9xNFAT-Luc)的转基因小鼠，该报告基因由一组NFAT位点驱动，由依赖钙调神经磷酸酶的NFATc蛋白激活。由于记者的NFAT位点与最佳的NFAT5位点非常相似，我们测试了该记者是否能在转基因细胞中检测到NFAT5的激活。在不同类型的未转化转基因细胞：淋巴细胞、巨噬细胞和成纤维细胞中，9xNFAT-Luc报告基因被NFAT5依赖的高张力激活。佛波酯PMA加离子霉素对该报告的激活不依赖于NFAT5，并由NFATc蛋白介导。在360-380mOsm/kg(等渗条件为300mOsm/kg)的高渗条件下，检测到T淋巴细胞中NFAT5的转录激活，并在400mOsm/kg时强烈诱导NFAT5的转录激活。在渗透调节障碍患者和缺乏水通道蛋白和加压素受体的小鼠的血浆中也记录到了这种水平。激活NFAT5所需的高张阈值在骨髓来源的巨噬细胞(430mOsm/kg)和胚胎成纤维细胞(480mOsm/kg)中较高。P38抑制剂(SB203580和SB202190)和25μM LY294002抑制PI3K相关的激酶活性。该报告对FK506的敏感性因细胞类型而异，在原代T细胞中比在成纤维细胞和巨噬细胞中更敏感。我们的结果表明，NFAT5是对T淋巴细胞细胞外紧张性病理性增加的敏感反应。NFAT5在淋巴细胞中的高张力激活是由不同于其他类型细胞所需的信号通路的组合所介导的。我们认为，9xNFAT-Luc转基因小鼠模型可能有助于研究NFAT5和NFATc因子在原代细胞中的生理病理调节。
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. 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. 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.
登录
查看更多内容
影响因子：
14.9
作者：
Esensten, JH;Tsytsykova, AV;Lopez-Rodriguez, C;Ligeiro, FA;Rao, A;Goldfeld, AE
通讯作者：
Goldfeld, AE
影响因子：
64.8
作者：
JAIN, JN;MCCAFFREY, PG;RAO, A
通讯作者：
RAO, A
影响因子：
3.3
作者：
Loyher, ML;Mutin, M;Tappaz, ML
通讯作者：
Tappaz, ML
影响因子：
13.6
作者：
Lim, Sun Woo;Ahn, Kyung Ohk;Kwon, H. Moo
通讯作者：
Kwon, H. Moo
DOI：
10.1073/pnas.0403139101
发表时间：
2004-07-20
影响因子：
11.1
作者：
Go, WY;Liu, XB;Ho, SN
通讯作者：
Ho, SN