Signal transduction mechanisms through granulocyte colony-stimulating factor receptor.

为了阐明G-CSF诱导中性粒细胞分化过程中生长抑制的信号转导机制，采用Northern杂交技术检测了粒细胞分化相关的细胞周期调控蛋白和转录因子的基因表达。G-CSF刺激后，细胞周期蛋白依赖性激酶抑制因子p21；lt；WAF1&gt；基因表达无明显变化，而p27&lt；KIP1&gt；和p19^&lt；INK4D&gt；基因表达水平升高。与粒细胞分化相关的转录因子C/EBPα和C/EBPε基因的表达受G-CSF刺激诱导，而PU1mRNA的表达不受影响。因此，在G-CSF依赖的中性粒细胞分化过程中，p27^&lt；KIP1&gt；和p19^&lt；INK4D&gt；的表达似乎阻止了细胞周期从G1向S的进展。C/eBPα和/或C/eBPε转录因子也可能控制基因表达…我们一直在试图确定哪些基因在能够对G-CSF诱导中性粒细胞分化做出反应的细胞中表达，但在G-CSF受体突变的细胞中不表达，这些细胞无法对分化做出反应，从而参与中性粒细胞的分化。利用基于聚合酶链式反应的消减杂交技术，获得了几个基因，包括STAT3和ERO1-L基因。G-CSF刺激诱导STAT3的磷酸化和二聚化，然后转移到细胞核，在那里STAT3激活其靶基因的转录。已知STAT3的激活是G-CSF依赖的中性粒细胞分化所必需的。我们的数据显示，激活的STAT3启动了自身基因的表达，从而产生了更多的STAT3蛋白。这一机制似乎促进了G-CSF依赖的中性粒细胞分化。此外，ERO_1-L还参与了内质网蛋白质二硫键的形成和新生多肽三级结构的形成。在中性粒细胞分化过程中，ERO_1-L基因的G-CSF依赖性表达似乎控制了STAT3的激活。因此，ERO_1-L似乎有助于在中性粒细胞分化过程中将MPO、弹性蛋白酶等杀菌蛋白合成为内质网。较少

In order to clarify the signal transduction mechanisms of growth suppression during G-CSF induced neutrophil differentiation, gene expressions of cell-cycle regulatory proteins and transcription factors which are involved in granulocyte differentiation were examined in neutrophil progenitor cells GM-162M and 32Dcl3 by Northern blot hybridization. Gene expression of cyclin dependent kinase inhibitor p21^<WAF1> was not increased by G-CSF stimulation, while levels of mRNA for p27^<KIP1> and p19^<INK4D> were elevated. On the other hand, expression of transcription factors C/EBPα and C/EBPε genes, which were possibly involved in the granulocyte differentiation, was induced by G-CSF stimulation, while quantity of PU.1 mRNA was unaffected. Therefore, expression of p27^<KIP1> and p19^<INK4D> appeared to prevent the cell-cycle progression from G1 to S during G-CSF dependent neutrophil differentiation. It's also possible that C/EBPα and/or C/EBPε transcription factors control the gene expression … More of these CDK inhibitors.We have been trying to identify genes which express in cells capable of responding to G-CSF for neutrophil differentiation but not in the cells with mutant G-CSF receptor unable to respond for the differentiation, thereby being involved in neutrophil differentiation. Using PCR-based subtraction-hybridization technique, several genes were identified including genes for Stat3 and ERO1-L.G-CSF stimulation induces phosphorylation and dimerization of Stat3 which is then transferred to nucleus where Stat3 activates transcription of its target genes. Stat3 activation is known to be necessary for G-CSF dependent neutrophil differentiation. Our data showed that activated Stat3 turns on the expression of its own genes, which produces more Stat3 protein. This mechanism seems to accelerate G-CSF dependent neutrophil differentiation. Moreover, ERO1-L is a enzyme involved in the protein disulfide-bond formation in ER and in formation of tertiary structure of nascent polypeptide. G-CSF dependent expression of ERO1-L gene appeared to be in control of Stat3 activation during neutrophil differentiation. Therefore, ERO1-L seems to help synthesizing bacteriocidal proteins such as MPO and elastase into ER during neutrophil differentiation. Less