Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells
Role of nitric oxide signaling components in differentiation of embryonic stem cells into myocardial cells
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DOI：
10.1073/pnas.0810230105
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发表时间：
2008-12-02
影响因子：
11.1
通讯作者：
Murad, Ferid
中科院分区：
文献类型：
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作者：
Mujoo, Kalpana;Sharin, Vladislav G.;Murad, Ferid
一氧化氮(NO)参与多种生理和病理事件。我们以前的研究表明，在小鼠和人类ES细胞中，NO信号成分的表达存在差异。在这里，我们展示了NO供体和可溶性鸟苷酸环化酶(SGC)激活剂在ES细胞分化为心肌细胞过程中的作用。我们在小鼠和人ES细胞上的实验结果表明，在NO供体作用下，NKX2.5和肌球蛋白轻链(MLC2)的mRNA表达增加，使用非特异性NOS抑制剂的心脏特异基因的mRNA表达减少，sGCα(1)亚单位的mRNA水平也随之增加和降低。尽管sGC激活剂单独应用时心脏基因(MLC2和NKX2.5)的mRNA表达增加，但当不联合供体和sGC激活剂时，标记基因的mRNA和蛋白表达均有较强的诱导。对NO代谢产物的测量显示，当细胞暴露于不同浓度的NO供体时，条件培养液和细胞裂解物中的亚硝酸盐水平增加。未分化干细胞中的cGMP分析显示，在没有捐赠者的情况下，缺乏刺激。然而，分化的细胞获得了不受捐赠者刺激的能力。尽管3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3，4-b]pyridine(BAY 41-2272)单独能够刺激cGMP的积累，但NO供体和BAY 41-2272联合使用对cGMP水平的刺激作用强于单独使用这两种药物。这些研究表明，cGMP介导的NO信号在ES细胞向心肌细胞分化过程中起重要作用。
Nitric oxide (NO) is involved in number of physiological and pathological events. Our previous studies demonstrated a differential expression of NO signaling components in mouse and human ES cells. Here, we demonstrate the effect of NO donors and soluble guanylyl cyclase (sGC) activators in differentiation of ES cells into myocardial cells. Our results with mouse and human ES cells demonstrate an increase in Nkx2.5 and myosin light chain (MLC2) mRNA expression on exposure of cells to NO donors and a decrease in mRNA expression of both cardiac-specific genes with nonspecific NOS inhibitor and a concomitant increase and decrease in the mRNA levels of sGC alpha(1) subunit. Although sGC activators alone exhibited an increase in mRNA expression of cardiac genes (MLC2 and Nkx2.5), robust inductions of mRNA and protein expression of marker genes were observed when NO donors and sGC activators were combined. Measurement of NO metabolites revealed an increase in the nitrite levels in the conditioned media and cell lysates on exposure of cells to the different concentrations of NO donors. cGMP analysis in undifferentiated stem cells revealed a lack of stimulation with NO donors. Differentiated cells however, acquired the ability to be stimulated by NO donors. Although, 3-(4-amino-5-cyclopropylpyrimidin-2-yl)-1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine (BAY 41-2272) alone was able to stimulate cGMP accumulation, the combination of NO donors and BAY 41-2272 stimulated cGMP levels more than either of the agents separately. These studies demonstrate that cGMP-mediated NO signaling plays an important role in the differentiation of ES cells into myocardial cells.