The role of un-equal division on regulation of neutrophilic differentiation and the participation of G-CSF

我们之前报道了转铁蛋白受体（Trf-R）阳性（Trf-R+）和阴性（Trf-R-）细胞在二甲亚砜（Me2SO）或视黄酸处理后出现，并致力于中性粒细胞分化和增殖。为了阐明这两种类型细胞分化和增殖的分子机制，我们使用二维聚丙烯酰胺电泳比较了Trf-R+和Trf-R-细胞的蛋白表达谱。而55kDa和25kDa位点在Trf-R+细胞中更为丰富，11kDa位点在Trf-R-细胞中的表达明显高于Trf-R+细胞。MALDI-MS和Edman分析表明，11kDa蛋白为Calgranulin/S100A8蛋白。siRNA敲除S100A8蛋白可抑制Me_2SO诱导HL-60细胞的分化，提示S100A8。由于非典型PKC（aPKC）已被报道在细胞极性中起重要作用，因此我们更关注于阐明aPKC在中性粒细胞成熟分化和增殖中的作用。特别是，我们分析了aPKC、PKCι/λ与g - csf依赖性增殖和S6K1和PI3K激活的关系。HL-60细胞中蛋白激酶Cι表达丰富，PKCζ不表达；在加入G-CSF后15分钟至30分钟观察到pkc1的最大刺激。使用抗PKCι抗体的免疫沉淀试验显示，在加入G-CSF 30分钟后，PKCι与S6K1形成复合物。在HL-60细胞中加入G-CSF后，PI3K和S6K1的激活存在时间差。在这段延迟时间的5 - 15分钟内，PKCι被发现从细胞核转移到膜，并重新转移到细胞质，导致与S6K1的关联。PKCι小干扰RNA抑制g - csf诱导的S6K1增殖和Thr-389磷酸化，促进分化。PKCι的动态易位和激活过程可以解释为PI3K和S6K1之间最大激活时间滞后的原因。这些结果表明，S100A8在HL-60细胞的分化中起重要作用。PI3K/PKCι/p70S6K级联在HL-60分化细胞的增殖中起主要作用。少

We, previously, reported that the transferrin receptor (Trf-R) positive (Trf-R+) and negative (Trf-R-) cells appeared after treatment with dimethyl sulfoxide (Me2SO) or retinoic acid, and commitment to neutrophilic differentiation and proliferation in Me_2SO-treated HL-60 cells. To clarify the molecular mechanism of commitment to differentiation and proliferation in both-type cells, we compared the protein expression profiles in Trf-R+ and Trf-R- cells using two-dimensional polyacrylamide electrophoresis. While 55kDa and 25kDa spots were more abundant in Trf-R+ cells, the expression of 11kDa spot is apparently increased in Trf-R- cells than that in Trf-R+ cells. The MALDI-MS spectrum analysis and Edman analysis revealed that the 11kDa protein is Calgranulin/S100A8 protein. Knock-down of S100A8 protein using siRNA inhibited the differentiation of HL-60 cells induced by Me_2SO, suggesting that S100A8.Since atypical PKC(aPKC) has been reported to play an important role on cell polarity, w … More e focused to clarify the role of aPKC on differentiation and proliferation of neutrophilic maturation. Particularly, we analyzed the participation of aPKC, PKCι/λ in relation to G-CSF-dependent proliferation and S6K1 and PI3K activation. Protein kinase Cι was abundantly expressed in HL-60 cells, but not PKCζ ; the maximum stimulation of PKCι, was observed from 15 min to 30 min after the addition of G-CSF. An immuno-precipitation assay using an anti-PKCι antibody revealed that PKCι formed a complex with S6K1 30 min after the addition of G-CSF. There was a time lag between the activation of PI3K and S6K1 when G-CSF was added to the HL-60 cells. Within 5 to 15 min during this lag time, PKCι was found to translocate from the nucleus to the membrane, and re-translocate to the cytosol, resulting in the association with S6K1. Small interfering RNA for PKCι inhibited G-CSF-induced proliferation and phosphorylation of Thr-389 of S6K1,and promoted differentiation. The dynamic translocation and activation processes of PKCι can be explained as the reason for the time lag in maximum activation between PI3K and S6K1.These results indicate that while S100A8 plays an important role on differentiation of HL-60 cells., PI3K/PKCι/p70S6K cascade contribute main role on the proliferation of differentiating HL-60 cells. Less