酪氨酸蛋白磷酸酶Shp-2和白血病抑制因子影响HL-60细胞增殖与分化过程中的相互关系
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摘要
酪氨酸蛋白磷酸酶Shp-2是胞质中的一个作用广泛的酪氨酸磷酸酶，参与细胞生长、分化、移动、死亡的调控，是细胞因子、抗原、细胞外基质等的下游信号分子。它与多种R-PTKs存在的交互作用是通过特异性识别SH2结构域的磷酸化酪氨酸位点介导。Shp-2对生长因子信号传导有正向调节作用并刺激发育相关路径、阻滞抑制生长的信号传导。Shp-2突变可导致Fak去磷酸化和磷酸化循环中断，使迁移过程中的细胞粘附性增高和移动性降低，在胚胎中则显示胚层发育、四肢分化和上皮发育障碍。Shp-2与受体蛋白质酪氨酸激酶(R-PTK)间的作用还参与胞内-胞间并行的信号传导，使细胞因子与单个细胞的结合可以引发一批相邻细胞的响应并得到精确执行。白血病抑制因子（Leukemia inhibitory factor, LIF）是一种多功能、作用广泛的细胞因子，与IL-11是在结构和功能上都比较接近，它能够抑制白血病细胞，如HL-60、U937、M1等的增殖，并促进其分化。LIF的生物学效应是通过结合靶细胞膜上的LIF受体完成的。LIF首先与LIF受体(亚基(gp190)结合，并使之与另一(亚基(gp130)结合形成异源性二聚体，从而启动细胞内信号转导使胞外的信号传入核内，调控特定基因的表达，使细胞产生一系列的生理和病理变化。Shp-2 SH2结构域通过配体作用可以刺激磷酸酶活性，那么，在可以激活JAK1-STAT途径的LIF刺激下，Shp-2对细胞的作用会受到什么样的影响呢？ 因而，有必要对Shp-2和LIF在对白血病细胞作用做进一步的研究。研究两者的相互影响，对了解白血病细胞的分化、增殖异常机理及其调控机制，为白血病的临床治疗提供基础理论依据有重要意义。
本研究采用Shp-2（c＞s）作为对照（Shp-2突变体），Shp-2（c＞s）以半胱氨酸替代N端SH2结构域上的丝氨酸，而SH2结构域分别介导激酶和磷酸酶的相互作用，这使得Shp-2（c＞s）即使与受体结合，也不能发挥其生物学活性。
然后，将Shp-2和Shp-2（c＞s）分别转染进白血病细胞HL-60，给予LIF刺激，检测其增殖和分化指标。Shp-2（c＞s）的存在不但可以使其与Shp-2受体的结合不会产生生物学活性，还可以通过竞争部分阻滞细胞胞质中的Shp-2结合受体。
细胞核增殖抗原(PCNA)免疫组化、聚合酶链式反应和免疫印迹杂交法结果分别显示：HL-60组、IL-11刺激HL-60组、LIF刺激HL-60组细胞表达水平高于相应的Shp-2、Shp-2（c＞s）转染细胞；而LIF刺激HL-60组、Shp-2、Shp-2（c＞s）转染细胞组则低于非LIF刺激细胞组；IL-11刺激HL-60组、IL-11刺激野生组、IL-11刺激Shp-2突变组前后区别不显著。细胞增殖抑制实验可见Shp-2和Shp-2（c＞s）两组增殖量比HL-60组明显为低， Shp-2（c＞s）比Shp-2略高；LIF刺激后各组细胞增殖水平亦明显低于刺激前。结果表明，LIF刺激后的各组细胞PCNA表达明显下降；Shp-2和Shp-2（c＞s）转染后影响HL-60细胞的增殖效应。
CD15免疫组化和流式细胞仪结果显示：Shp-2野生组、突变组的染色程度均高于相应的HL-60组；LIF刺激Shp-2野生组后明显升高，LIF刺激突变组和LIF刺激HL-60组后升高不明显。可见Shp-2和Shp-2（c＞s）两组CD15表达水平比HL-60组明显为高；LIF刺激前Shp-2和Shp-2（c＞s）之间差别并不大，但LIF刺激后的Shp-2组明显大于Shp-2（c＞s）组。结果表明，LIF刺激后的各组细胞CD15表达明显升高；Shp-2 和Shp-2（c＞s）转染后细胞CD15表达升高，但LIF刺激Shp-2（c＞s）转染细胞时，CD15表达受到抑制。
结论：在胞质内Shp-2加强表达的情况下，细胞增殖受到明显抑制。然而，即便是胞质内存在能够竞争受体的N端SH2结构域突变体-Shp-2（c＞s），细胞增殖情况仍受到相当抑制。在LIF的刺激下Shp-2可以促进细胞抑制作用，但Shp-2组与Shp-2突变组无明显差别，说明Shp-2与LIF的抑制增殖信号的调节没有直接的关联。同样，Shp-2和Shp-2（c＞s）组的细胞都有促进分化的现象，后者程度较轻，但在LIF刺激下，Shp-2组分化明显加强，而Shp-2（c＞s）组与刺激前相比亦有增幅，但远不如Shp-2组显著，说明Shp-2 N-SH2的突变能够影响LIF刺激细胞分化的作用，或者是Shp-2的加强表达可以很大的增强LIF促进细胞分化。
Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two src-homology 2(SH2) domains,has received much attention in the signal transduction field recently.Shp-2 was found to interact physically with a variety of ligand-actived receptor protein tyrosine kinases,R-PTKs(5-7).This interaction is apparently mediated through specific recognition of a phosphotyrosine (pTyr) site on areceptor by the SH2 domains of the phosphatase.Shp-2 might be positively required for signal transduction of growth factor , stimulate differentiation and block proliferation.Since Fak is involved in the dynamics rather than the formation of focal adhesion,disruption of Fak dephosphorylation by the Shp-2 mutation should give rise to increased focal adhesion numbers.Physical interaction of Shp-2 with a R-PTK may not only serve to receive a signal to its cellular neighbors.As such,a localized cellular response could be precisely executed. Leukemia inhibitory factor (LIF), a member of the family of multifunctional cytokines, can inhibit the proliferation of leukemia cells, and accelerate the differentiation, such as HL-60, U937 and M1. LIF receptor is multimeric --- shares a (subunit, the low-affinity LIF receptor (gp190), and an additional subunit, IL-6 related signal transducer-gp130. LIF receptor initiates signaling by inducing heterodimerization of gp130 with LIF receptor (component LIFR).
In our study,Shp-2（c＞s）is control, in which cypress instead of cysteine of the N-terminal SH2 domain of Shp-2. The SH2 domain shared by kinases and phosphatases directs opposite biochemical reactions,therefor, Shp-2（c＞s）could bind ligand and does not have activation of catalysis.After Shp-2 and Shp-2（c＞s）were
separately transfected into human leukemia line HL-60 cells with LipofectAmine,Shp-2 and Shp-2（c＞s）were expressed on the membrane of HL-60.Then,to investigate differentiation and proliferation functions of the cell with LIF stimulation.
we detected the level of expression of PCNA by polymerase chain reaction,western blotting and immunobiochemistry.we found that higher level of expression of PCNA was determined in the group of HL-60,the group of HL-60 with IL-11 stimulation and the group of HL-60 with LIF stimulation compared with the groups of Shp-2 and Shp-2（c＞s） with same stimulation.the group of HL-60 with LIF stimulation,the group of Shp-2 with LIF stimulation and the group of Shp-2（c＞s） with LIF stimulation were lower than the other group of HL-60,Shp-2 and Shp-2（c＞s）.It suggests that the lower level of expression of PCNA was determined in the groups of Shp-2 and Shp-2（c＞s） compared with the groups of HL-60,and it is lowest in the groups with LIF stimulation.
Meanwhile,expression level of CD15 was also detected by immunobiochemistry and flow cytometry.It indicates that the level of the groups of Shp-2 and Shp-2（c＞s） were higher than the corresponding group of HL-60.thereinto,the group of Shp-2 with LIF stimulation was signally higher than the groups of Shp-2 without LIF stimulation,the groups of Shp-2（c＞s） and HL-60 with LIF stimulation were lightly higher than the corresponding group of Shp-2（c＞s） and HL-60.It suggested that signally higher expression level of CD15 in the group of Shp-2 with LIF stimulation was signally than the groups of Shp-2 without LIF stimulation.
the above results demonstrated:when the expression of Shp-2 strengthen in the cytoplasm,the proliferation of HL-60 was signally inhibited.the groups of HL-60,Shp-2 and Shp-2（c＞s） with LIF stimulation were more restrained,but the difference in the groups of HL-60,Shp-2 and Shp-2（c＞s） were same.It suggests that the connection between Shp-2 and LIF in HL-60 proliferation may not be evidenced.in the groups of Shp-2 and Shp-2（c＞s） the differentiation could be accelerate,the level of accelerating in the Shp-2（c＞s） is light than in Shp-2.But with LIF stimulation,the differentiation in the Shp-2 was signally strengthen.It suggests
that the mutation of the N-terminal SH2 domain of Shp-2 could affect the
本研究采用Shp-2（c＞s）作为对照（Shp-2突变体），Shp-2（c＞s）以半胱氨酸替代N端SH2结构域上的丝氨酸，而SH2结构域分别介导激酶和磷酸酶的相互作用，这使得Shp-2（c＞s）即使与受体结合，也不能发挥其生物学活性。
然后，将Shp-2和Shp-2（c＞s）分别转染进白血病细胞HL-60，给予LIF刺激，检测其增殖和分化指标。Shp-2（c＞s）的存在不但可以使其与Shp-2受体的结合不会产生生物学活性，还可以通过竞争部分阻滞细胞胞质中的Shp-2结合受体。
细胞核增殖抗原(PCNA)免疫组化、聚合酶链式反应和免疫印迹杂交法结果分别显示：HL-60组、IL-11刺激HL-60组、LIF刺激HL-60组细胞表达水平高于相应的Shp-2、Shp-2（c＞s）转染细胞；而LIF刺激HL-60组、Shp-2、Shp-2（c＞s）转染细胞组则低于非LIF刺激细胞组；IL-11刺激HL-60组、IL-11刺激野生组、IL-11刺激Shp-2突变组前后区别不显著。细胞增殖抑制实验可见Shp-2和Shp-2（c＞s）两组增殖量比HL-60组明显为低， Shp-2（c＞s）比Shp-2略高；LIF刺激后各组细胞增殖水平亦明显低于刺激前。结果表明，LIF刺激后的各组细胞PCNA表达明显下降；Shp-2和Shp-2（c＞s）转染后影响HL-60细胞的增殖效应。
CD15免疫组化和流式细胞仪结果显示：Shp-2野生组、突变组的染色程度均高于相应的HL-60组；LIF刺激Shp-2野生组后明显升高，LIF刺激突变组和LIF刺激HL-60组后升高不明显。可见Shp-2和Shp-2（c＞s）两组CD15表达水平比HL-60组明显为高；LIF刺激前Shp-2和Shp-2（c＞s）之间差别并不大，但LIF刺激后的Shp-2组明显大于Shp-2（c＞s）组。结果表明，LIF刺激后的各组细胞CD15表达明显升高；Shp-2 和Shp-2（c＞s）转染后细胞CD15表达升高，但LIF刺激Shp-2（c＞s）转染细胞时，CD15表达受到抑制。
结论：在胞质内Shp-2加强表达的情况下，细胞增殖受到明显抑制。然而，即便是胞质内存在能够竞争受体的N端SH2结构域突变体-Shp-2（c＞s），细胞增殖情况仍受到相当抑制。在LIF的刺激下Shp-2可以促进细胞抑制作用，但Shp-2组与Shp-2突变组无明显差别，说明Shp-2与LIF的抑制增殖信号的调节没有直接的关联。同样，Shp-2和Shp-2（c＞s）组的细胞都有促进分化的现象，后者程度较轻，但在LIF刺激下，Shp-2组分化明显加强，而Shp-2（c＞s）组与刺激前相比亦有增幅，但远不如Shp-2组显著，说明Shp-2 N-SH2的突变能够影响LIF刺激细胞分化的作用，或者是Shp-2的加强表达可以很大的增强LIF促进细胞分化。
Shp-2, a widely expressed cytoplasmic tyrosine phosphatase with two src-homology 2(SH2) domains,has received much attention in the signal transduction field recently.Shp-2 was found to interact physically with a variety of ligand-actived receptor protein tyrosine kinases,R-PTKs(5-7).This interaction is apparently mediated through specific recognition of a phosphotyrosine (pTyr) site on areceptor by the SH2 domains of the phosphatase.Shp-2 might be positively required for signal transduction of growth factor , stimulate differentiation and block proliferation.Since Fak is involved in the dynamics rather than the formation of focal adhesion,disruption of Fak dephosphorylation by the Shp-2 mutation should give rise to increased focal adhesion numbers.Physical interaction of Shp-2 with a R-PTK may not only serve to receive a signal to its cellular neighbors.As such,a localized cellular response could be precisely executed. Leukemia inhibitory factor (LIF), a member of the family of multifunctional cytokines, can inhibit the proliferation of leukemia cells, and accelerate the differentiation, such as HL-60, U937 and M1. LIF receptor is multimeric --- shares a (subunit, the low-affinity LIF receptor (gp190), and an additional subunit, IL-6 related signal transducer-gp130. LIF receptor initiates signaling by inducing heterodimerization of gp130 with LIF receptor (component LIFR).
In our study,Shp-2（c＞s）is control, in which cypress instead of cysteine of the N-terminal SH2 domain of Shp-2. The SH2 domain shared by kinases and phosphatases directs opposite biochemical reactions,therefor, Shp-2（c＞s）could bind ligand and does not have activation of catalysis.After Shp-2 and Shp-2（c＞s）were
separately transfected into human leukemia line HL-60 cells with LipofectAmine,Shp-2 and Shp-2（c＞s）were expressed on the membrane of HL-60.Then,to investigate differentiation and proliferation functions of the cell with LIF stimulation.
we detected the level of expression of PCNA by polymerase chain reaction,western blotting and immunobiochemistry.we found that higher level of expression of PCNA was determined in the group of HL-60,the group of HL-60 with IL-11 stimulation and the group of HL-60 with LIF stimulation compared with the groups of Shp-2 and Shp-2（c＞s） with same stimulation.the group of HL-60 with LIF stimulation,the group of Shp-2 with LIF stimulation and the group of Shp-2（c＞s） with LIF stimulation were lower than the other group of HL-60,Shp-2 and Shp-2（c＞s）.It suggests that the lower level of expression of PCNA was determined in the groups of Shp-2 and Shp-2（c＞s） compared with the groups of HL-60,and it is lowest in the groups with LIF stimulation.
Meanwhile,expression level of CD15 was also detected by immunobiochemistry and flow cytometry.It indicates that the level of the groups of Shp-2 and Shp-2（c＞s） were higher than the corresponding group of HL-60.thereinto,the group of Shp-2 with LIF stimulation was signally higher than the groups of Shp-2 without LIF stimulation,the groups of Shp-2（c＞s） and HL-60 with LIF stimulation were lightly higher than the corresponding group of Shp-2（c＞s） and HL-60.It suggested that signally higher expression level of CD15 in the group of Shp-2 with LIF stimulation was signally than the groups of Shp-2 without LIF stimulation.
the above results demonstrated:when the expression of Shp-2 strengthen in the cytoplasm,the proliferation of HL-60 was signally inhibited.the groups of HL-60,Shp-2 and Shp-2（c＞s） with LIF stimulation were more restrained,but the difference in the groups of HL-60,Shp-2 and Shp-2（c＞s） were same.It suggests that the connection between Shp-2 and LIF in HL-60 proliferation may not be evidenced.in the groups of Shp-2 and Shp-2（c＞s） the differentiation could be accelerate,the level of accelerating in the Shp-2（c＞s） is light than in Shp-2.But with LIF stimulation,the differentiation in the Shp-2 was signally strengthen.It suggests
that the mutation of the N-terminal SH2 domain of Shp-2 could affect the
引文
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