Notch in hematopoiesis

【目的】本研究的目的是了解(1)Notch受体信号是如何产生的，(2)Notch诱导的造血细胞分化抑制机制。【方法】(1)利用Notch2特异性抗体研究Notch2分子的切割、易位和磷酸化。(2)研究了Notch1 （aN1）活化形式对造血细胞分化的影响。接下来，我们检测了参与造血细胞分化和生长的造血转录因子（HTF）的表达变化。最后，在表达aN1的细胞系中表达GATA的显性阴性形式（DN-GATA）和PU.1，并利用所得到的转染物研究其对分化刺激的反应。[结果与讨论](1-1)Notch2分子的跨膜亚基被切割。由此产生的分子组成细胞内区域，然后很快进入细胞核。（1-2）非定位Notch2分子被磷酸化。（1-3） Notch配体在靶细胞中启动转录控制。（2-1）均能抑制红系分化。（2-2）分化刺激可改变各种HTF的表达水平，除GATA2外，其余HTF均未受aN1的影响。野生型32D细胞在G-CSF作用下分化时，GATA2的表达水平下降，而在引入aN1后，GATA2的表达水平保持不变（aN1/32D）。进一步外源表达DN-GATA或PU.1（已知可阻断GATA因子的功能），逆转了an1诱导的分化阻断。这些结果表明aN1通过维持GATA2的表达水平和功能来阻断造血细胞的分化。[结论]揭示了Notch信号启动的部分机制。Notch的激活通过维持GATA2的表达水平和功能来阻断造血细胞的分化。少

[Purpose] Aims of this study were to understand (1) how the signal through the Notch receptor is generated and (2) the mechanism of Notch-induced inhibition of differentiation in hematopoietic cells.[Methods](1) Cleavage, translocation and phosphorylation of the Notch2 molecule were studied using specific antibodies against Notch2.(2) Effect of activated form of Notch1 (aN1) on differentiation of hematopoietic cells was studied. Next, changes in expression of hematopoietic transcription factors (HTF), which were involved in the differentiation and growth of hematopoietic cells, were examined. Finally, dominant-negative form of GATA (DN-GATA) and PU.1 were expressed in a cell line expressing aN1, and the resulting trasnfectants were used to study the response to differentiation stimulation.[Results and Discussions](1-1) The transmembrane subunit of the Notch2 molecule was cleaved. Resulting molecules comprising the intracellular region were then shortly entered the nucleus.(1-2) The tra … More nslocated Notch2 molectules were phosphorylated.(1-3) Notch ligands initiated transcriptional control in the tarxet cells.(2-1) all inhibited mveloid and erythroid differentiation.(2-2) Expression level of various HTF was changed by the differentiation stimulation and such changes were not modified by aN1 in all the HTF except for GATA2. The expression level of GATA2 was decreased when wild-type 32D cells differentiate responding to G-CSF, while it was maintained when aN1 was introduced into 32D (aN1/32D). Further exogenous expression of DN-GATA or PU.1, which is known to block the function of GATA factors, reverted the aN1-induced differentiation block. These results indicated that aN1 blocks differentiation of hematopoietic cells through sustaining the expression level and function of GATA2.[Conclusion] We revealed a part of mechanisms of initiation of Notch signaling. Activation of Notch blocks differentiation of hematopoietic cells, through sustaining the expression level and function of GATA2. Less