p38磷酸化与K562细胞红系分化的关系

Relationship between Phosphorylation of p38 and Erythroid Differentiation of Human K562 Erythroleukemia Cells

目的探讨p38丝裂原活化的蛋白激酶(p38MAPKs)信号通路直接持续活化在γ-珠蛋白基因转录激活和胎儿血红蛋白(HbF)合成中的作用,为阐明p38磷酸化与K562细胞红系分化的关系提供直接依据。方法经脂质体介导将pCDNA 3.1-MKK3(Glu)和pCDNA3.1-MKK3(Ala)重组质粒转染人红白血病细胞株K562,经G418筛选,反转录(RT)-PCR和Western blot鉴定获得p38持续高磷酸化和低磷酸化的稳定细胞株K562-MKK3(Glu)和K562-MKK3(Ala)。通过RT-PCR和联苯胺染色观察不同细胞模型中γ-珠蛋白基因的表达和HbF的合成。采用SPSS 11.5软件进行统计学分析。结果不同细胞模型中p38mRNA和蛋白水平表达均无明显变化。但与K562亲本细胞、K562-vect和K562-MKK3(Ala)细胞比较,K562-MKK3(Glu)细胞中p38蛋白磷酸化水平、γ-珠蛋白表达均显著增加。联苯胺染色结果显示,K562亲本细胞、K562-vect、K562-MKK3(Ala)、K562-MKK3(Glu)和SB203580处理的K562-MKK3(Glu)细胞中联苯胺阳性细胞百分率分别为(3.2±1.4)%、(3.7±1.2)%、(2.8±0.9)%、(32.6±5.3)%和(7.8±2.3)%(q=7.56P<0.01)。结论p38MAPKs信号通路直接持续活化可激活γ-珠蛋白基因的转录,并促进HbF的合成。p38磷酸化在K562细胞红系分化中起重要作用。

Objective To investigate the role of directly constitutive activation of p38 mitogen - activated protein kinases（p38MAPKs） signaling in γ-globin gene expression and fetal hemoglobin （HbF） induction, and provide direct data for the relationship between phosphorylation of p38 and erythroid differentiation of human K562 erythroleukemia cells. Methods The human K562 erythroleukemia cells were transfected with pCDNA 3.1 - MKK3 （Glu） and pCDNA 3.1 - MKK3 （Ala） recombinant plasmids by lipofectamine^TM 2000. Then, the stable cell lines overexpressing constitutively active p38 and constitutively inhibitive p38 activation were established by the addition of G418 to select single cell G418 - resistant clones and identification with reverse transcriptase - polymerase chain reaction（ RT - RCR） and Western blot assays, named K562 - M KK3 （Glu）and K562 - MKK3 （Ala）cells, respectively. Furthermore, the direct effects of constitutively active p38 on the γ- globin gene expression and HbF induction were analyzed by RT - PCR and benzidine staining,respectively. Results The results of RT - PCR and Western blot showed that there were no evident changes in the mRNA and protein levels of p38 for various cell models, but compared with K562, K562 - vect, and K562 - MKK3 （Ala） cells, the phosphorylation of p38 and expression of γ- globin levels in K562 - MKK3 （Glu） cells were significantly up - regulated. The results of benzidine staining displayed that the mean percentages of positive cells stained by benzidine in K562, K562 - vect, K562 - MKK3 （ Ala ）, K562 - MKK3 （ Glu ） cells, and K562 - MKK3. （ Glu ） cells treated with SB203580 were （ 3.2 ± 1.4）%,（3.7±1.2）%,（2.8±0.9）%,（32.6±5.3）%,and（7.8± 2.3）%（q = 7.56 P〈0.01）,respectlvely. Conclusions Constitutive activation of p38MAPKs signaliag can directly activate γ- globin gene expression and HbF induction. Therefore, hyperphosphorylation of p38 plays an important role in erythroid differentiation of human K562 erythroleukemia cells.