Haloui Mounsif, Taurin Sebastien, Akimova Olga A, Guo Deng-Fu, Tremblay Johanne, Dulin Nickolai O, Hamet Pavel, Orlov Sergei N
Centre de recherche, Centre hospitalier de l'Université de Montréal (CHUM) - Technopôle ANGUS, Montreal, PQ, Canada Department of Medicine, University of Chicago, Chicago, IL, USA.
FEBS J. 2007 Jul;274(14):3557-3567. doi: 10.1111/j.1742-4658.2007.05885.x. Epub 2007 Jun 12.
In vascular smooth muscle cells and several other cell types, inhibition of Na(+)/K(+)-ATPase leads to the expression of early response genes, including c-Fos. We designed this study to examine whether or not a putative Na(+) (i)/K(+) (i)-sensitive element is located within the c-Fos 5'-UTR from - 650 to + 103 containing all known response elements activated by 'classic' stimuli, such as growth factors and Ca(2+) (i)-raising compounds. In HeLa cells, the highest increment of c-Fos mRNA content was noted after 6 h of Na(+)/K(+)-ATPase inhibition with ouabain that was abolished by actinomycin D, an inhibitor of RNA synthesis. c-Fos protein accumulation in ouabain-treated cells correlated with a gain of Na(+) (i) and loss of K(+) (i). Augmented c-Fos expression was also observed under inhibition of Na(+)/K(+)-ATPase in K(+)-free medium and in the presence of the Na(+) ionophore monensin. The effect of ouabain on c-Fos expression was sharply attenuated under dissipation of the transmembrane Na(+) gradient, but was preserved in the presence of Ca(2+) chelators and the extracellular regulated kinase inhibitor PD98059, thus indicating an Na(+) (i)-mediated, Ca(2+) (i)- and extracellular regulated kinase-independent mechanism of gene expression. In contrast to massive c-Fos expression, we failed to detect any effect of ouabain on accumulation of luciferase driven by the c-Fos 5'-UTR. Negative results were also obtained in ouabain-treated vascular smooth muscle cells and C11 Madin-Darby canine kidney cells possessing augmented c-Fos expression. Our results reveal that Na(+) (i)-induced c-Fos expression is not mediated by the 5'-UTR containing transcriptional elements activated by growth factors and other 'classic stimuli'.
在血管平滑肌细胞和其他几种细胞类型中，抑制钠钾ATP酶会导致早期反应基因的表达，包括c-Fos。我们设计本研究以检测在c-Fos 5'-非翻译区（5'-UTR）从-650至+103的区域内是否存在一个假定的对细胞内钠（Na⁺(i)）/钾（K⁺(i)）敏感的元件，该区域包含所有已知的由“经典”刺激（如生长因子和升高细胞内钙（Ca²⁺(i)）的化合物）激活的反应元件。在HeLa细胞中，用哇巴因抑制钠钾ATP酶6小时后，观察到c-Fos mRNA含量最高增幅，而放线菌素D（一种RNA合成抑制剂）可消除这种增幅。在哇巴因处理的细胞中，c-Fos蛋白积累与细胞内钠增加和钾减少相关。在无钾培养基中以及存在钠离子载体莫能菌素的情况下，抑制钠钾ATP酶时也观察到c-Fos表达增强。当跨膜钠梯度消失时，哇巴因对c-Fos表达的影响急剧减弱，但在存在钙螯合剂和细胞外调节激酶抑制剂PD98059的情况下仍存在，这表明基因表达是由细胞内钠介导的、不依赖细胞内钙和细胞外调节激酶的机制。与大量c-Fos表达相反，我们未能检测到哇巴因对由c-Fos 5'-UTR驱动的荧光素酶积累有任何影响。在用哇巴因处理的血管平滑肌细胞和c-Fos表达增强的C11 Madin-Darby犬肾细胞中也得到了阴性结果。我们的结果表明，细胞内钠诱导的c-Fos表达不是由包含由生长因子和其他“经典刺激”激活的转录元件的5'-UTR介导的。
