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

不均等分裂对中性粒细胞分化的调节作用及G-CSF的参与

• 批准号: 15590091
• 负责人: YAMAGUCHI Teruhide
• 金额: $ 2.3万
• 依托单位: National Institute of Health Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590091/
• 关键词: neutrophilic differentiation; unequal division; aPKC; PKCiota; HL-60 cell; p70S6K; G-CSF; PKCι

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

我们以前曾报道过，经二甲亚砜（Me_2SO）或视黄酸处理的HL-60细胞，出现转铁蛋白受体（Trf-R）阳性（Trf-R+）和阴性（Trf-R-）细胞，并向嗜酸性细胞分化和增殖。为了阐明这两种类型细胞分化和增殖的分子机制，我们采用双向聚丙烯酰胺电泳比较了Trf-R+和Trf-R-细胞的蛋白表达谱。而55 kDa和25 kDa斑点在Trf-R+细胞中更丰富，11 kDa斑点在Trf-R-细胞中的表达明显高于Trf-R+细胞。MALDI-MS图谱分析和Edman蛋白质组学分析表明，该蛋白质为Calgranulin/S100 A8蛋白。siRNA敲低S100 A8蛋白可抑制Me_2SO诱导的HL-60细胞分化，提示S100 A8蛋白可能与非典型蛋白激酶C（aPKC）有关。 ...更多信息 本研究旨在阐明aPKC在嗜酸性粒细胞成熟分化和增殖中的作用。特别地，我们分析了aPKC、PKC 1/λ与G-CSF依赖性增殖以及S6 K1和PI 3 K活化的关系。蛋白激酶C i在HL-60细胞中大量表达，但不表达PKC β;在加入G-CSF后15 min至30 min观察到PKC i的最大刺激。使用抗-PKC 1抗体的免疫沉淀测定显示，在加入G-CSF后30分钟，PKC 1与S6 K1形成复合物。HL-60细胞加入G-CSF后，PI 3 K和S6 K1的激活之间存在一个时间滞后。在此滞后时间的5至15分钟内，发现PKC 1从细胞核易位到细胞膜，并重新易位到胞质溶胶，导致与S6 K1的缔合。PKCι的小干扰RNA抑制G-CSF诱导的S6 K1增殖和Thr-389磷酸化，并促进分化。PKCι的动态移位和激活过程可以解释为PI 3 K和S6 K1之间最大激活时间滞后的原因。这些结果表明，S100 A8在HL-60细胞分化中起重要作用。PI 3 K/PKC 1/p70 S6 K级联反应在HL-60细胞的增殖分化中起主要作用。少

项目成果

Two-dimensional electrophoresisof disease-associated proteins in human cerebrospinal fluid from patients with rheumatoid arthritis

类风湿性关节炎患者脑脊液中疾病相关蛋白的二维电泳

• 发表时间: 2005
• 期刊: J.Elecctrophoresis 49
• 作者: Yamamoto;Y.;Akita;Y.;Tai;S.;Fukasaku;S.;Yamaguchi;T.;Oshizawa;T.;Yamaoka;K.;Shimamura;M.;Hazato;T.
• 通讯作者: T.

Characterization of in vitro and in vivo gene transfer properties of adenovirus serotype 35 vector.

腺病毒血清型 35 载体的体外和体内基因转移特性的表征。

• 发表时间: 2003
• 期刊: Mol.Ther. 8
• 作者: Sakurai F.;Mizuguchi H.;Yamaguchi T.;Hayakawa T.
• 通讯作者: Hayakawa T.

Adenovirus vector-mediated doxycycline-inducible RNA interference

• DOI: 10.1089/1043034041648462
• 发表时间: 2004-08-01
• 期刊: HUMAN GENE THERAPY
• 影响因子: 4.2
• 作者: Hosono, T;Mizuguchi, H;Hayakawa, T
• 通讯作者: Hayakawa, T

HX531,RXR antagonist inhibited the 9-cis retinoic acid-induced binding with cofactor.

HX531,RXR拮抗剂抑制9-顺式视黄酸诱导的与辅因子的结合。

• 发表时间: 2005
• 期刊: J.Steroid Biochem Mol. 94
• 作者: Kanayasu-Toyoda;T.;Fujino;T.;Oshizawa;T.;Suzuki;T.;Nishimaki-Mogami;T.;Sato;Y.;Sawada;J.;Inoue;K.;Shudo;K.;Yamaguchi;T.
• 通讯作者: T.

HX531, RXR antagonist inhibited the 9-cis retinoic acid-induced binding with cofactor.

HX531，RXR 拮抗剂抑制 9-顺式视黄酸诱导的与辅因子的结合。

• 发表时间: 2005
• 期刊: J Steroid.Biochem.Molec.Biol. 94
• 作者: Kanayasu-Toyoda;T.;Fujino;T.;Oshizawa;T.;Suzuki;T.;Nishimaki-Mogami;T.;Sato;Y.;Sawada;J.;Inoue;K.;Shudo;K.;Yamaguchi;T.
• 通讯作者: T.