Kosiorek Michalina, Podszywalow-Bartnicka Paulina, Zylinska Ludmila, Pikula Slawomir
Department of Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland; Department of Neurodegenerative Disorders, Laboratory of Neurogenetics, Mossakowski Medical Research Centre PAS, Warsaw, Poland.
Laboratory of Cytometry, Nencki Institute of Experimental Biology, Warsaw, Poland.
PLoS One. 2014 Jun 6;9(6):e99118. doi: 10.1371/journal.pone.0099118. eCollection 2014.
The bulk of human genes undergo alternative splicing (AS) upon response to physiological stimuli. AS is a great source of protein diversity and biological processes and is associated with the development of many diseases. Pheochromocytoma is a neuroendocrine tumor, characterized by an excessive Ca2+-dependent secretion of catecholamines. This underlines the importance of balanced control of calcium transport via regulation of gene expression pattern, including different calcium transport systems, such as plasma membrane Ca2+-ATPases (PMCAs), abundantly expressed in pheochromocytoma chromaffin cells (PC12 cells). PMCAs are encoded by four genes (Atp2b1, Atp2b2, Atp2b3, Atp2b4), whose transcript products undergo alternative splicing giving almost 30 variants.
In this scientific report, we propose a novel mechanism of regulation of PMCA alternative splicing in PC12 cells through cooperation of the nuclear factor of activated T-cells (NFAT) and histone deacetylases (HDACs). Luciferase assays showed increased activity of NFAT in PC12 cells, which was associated with altered expression of PMCA. RT-PCR experiments suggested that inhibition of the transcriptional activity of NFAT might result in the rearrangement of PMCA splicing variants in PC12 cells. NFAT inhibition led to dominant expression of 2x/c, 3x/a and 4x/a PMCA variants, while in untreated cells the 2w,z/b, 3z,x/b,c,e,f, and 4x/b variants were found as well. Furthermore, chromatin immunoprecipitation experiments showed that NFAT1-HDAC4 or NFAT3-HDAC4 complexes might be involved in regulation of PMCA2x splicing variant generation.
We suggest that the influence of NFAT/HDAC on PMCA isoform composition might be important for altered dopamine secretion by PC12 cells.
大部分人类基因在对生理刺激作出反应时会发生可变剪接（AS）。AS是蛋白质多样性和生物过程的重要来源，并且与许多疾病的发生发展相关。嗜铬细胞瘤是一种神经内分泌肿瘤，其特征是儿茶酚胺的分泌过多依赖于钙离子。这凸显了通过调节基因表达模式来平衡控制钙转运的重要性，这些基因表达模式包括不同的钙转运系统，如质膜钙ATP酶（PMCAs），在嗜铬细胞瘤嗜铬细胞（PC12细胞）中大量表达。PMCA由四个基因（Atp2b1、Atp2b2、Atp2b3、Atp2b4）编码，其转录产物经过可变剪接产生近30种变体。
在本科学报告中，我们提出了一种通过活化T细胞核因子（NFAT）和组蛋白去乙酰化酶（HDACs）的协同作用来调节PC12细胞中PMCA可变剪接的新机制。荧光素酶检测显示PC12细胞中NFAT的活性增加，这与PMCA表达的改变有关。逆转录-聚合酶链反应（RT-PCR）实验表明，抑制NFAT的转录活性可能导致PC12细胞中PMCA剪接变体的重排。NFAT抑制导致2x/c、3x/a和4x/a PMCA变体的显性表达，而在未处理的细胞中也发现了2w,z/b、3z,x/b,c,e,f和4x/b变体。此外，染色质免疫沉淀实验表明，NFAT1-HDAC4或NFAT3-HDAC4复合物可能参与PMCA2x剪接变体产生的调节。
我们认为NFAT/HDAC对PMCA异构体组成的影响可能对PC12细胞多巴胺分泌的改变很重要。
