Josefsen D, Myklebust J H, Lømo J, Sioud M, Blomhoff H K, Smeland E B
Department of Immunology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway.
Stem Cells. 2000;18(4):261-72. doi: 10.1634/stemcells.18-4-261.
The Bcl-2 family of proteins has been shown to play a central role in the regulation of apoptosis. We have examined the expression of several Bcl-2 homologs upon stimulation of CD34(+) human hematopoietic progenitor cells. CD34(+) cells were induced to differentiate into predominantly erythroid cells in the presence of erythropoietin (Epo) and stem cell factor (SCF), while the addition of G-CSF and SCF led to differentiation predominantly into granulocytic cells, as demonstrated by immunophenotyping and morphological examination of cultured cells. In Epo- and SCF-stimulated cells, we found a marked increase in the level of Bcl-x(L) protein expression and downregulation of Bax expression, apparent from day 4 and more pronounced on days 8 and 21. In contrast, Bcl-x(L) protein expression was downregulated in G-CSF- and SCF-stimulated cells compared with cells cultured in medium alone, whereas there was no sign of change in the level of Bax. Mcl-1 expression showed a biphasic expression pattern in both early erythropoiesis and early granulopoiesis, but with an inverse regulation. Thus, Mcl-1 levels initially decreased in granulocytic progenitor cells and increased in erythroid progenitor cells. Finally, Bcl-2 expression was significantly downregulated in both Epo and SCF and G-CSF- and SCF-stimulated cells. The role of the distinct upregulation of Bcl-x(L) in early erythroid differentiation was further examined by use of specific ribozymes against Bcl-x(L). Addition of Bcl-x(L) ribozymes promoted a clear increase in cell death of Epo- and SCF-stimulated cells, while erythroid differentiation was not affected. In conclusion, we found a distinct regulation of several Bcl-2 family members in CD34(+) cells dependent on the cytokine stimulation given. The use of Bcl-x(L)-specific ribozymes suggested that Bcl-x(L) is important for survival but not for differentiation of erythroid progenitor cells.
已证明Bcl-2蛋白家族在细胞凋亡调控中起核心作用。我们检测了几种Bcl-2同源物在CD34(+)人造血祖细胞受刺激后的表达情况。在促红细胞生成素（Epo）和干细胞因子（SCF）存在的情况下，CD34(+)细胞被诱导主要分化为红细胞，而添加粒细胞集落刺激因子（G-CSF）和SCF则导致主要分化为粒细胞，这通过对培养细胞的免疫表型分析和形态学检查得以证实。在Epo和SCF刺激的细胞中，我们发现Bcl-x(L)蛋白表达水平显著增加，而Bax表达下调，从第4天开始明显，在第8天和第21天更显著。相比之下，与仅在培养基中培养的细胞相比，G-CSF和SCF刺激的细胞中Bcl-x(L)蛋白表达下调，而Bax水平没有变化迹象。Mcl-1表达在早期红细胞生成和早期粒细胞生成中均呈现双相表达模式，但调控相反。因此，Mcl-1水平在粒细胞祖细胞中最初下降，在红细胞祖细胞中上升。最后，在Epo和SCF以及G-CSF和SCF刺激的细胞中，Bcl-2表达均显著下调。通过使用针对Bcl-x(L)的特异性核酶进一步研究了Bcl-x(L)在早期红细胞分化中独特上调的作用。添加Bcl-x(L)核酶促进了Epo和SCF刺激细胞的细胞死亡明显增加，而红细胞分化未受影响。总之，我们发现CD34(+)细胞中几种Bcl-2家族成员的调控因所给予的细胞因子刺激而异。使用Bcl-x(L)特异性核酶表明，Bcl-x(L)对红细胞祖细胞的存活很重要，但对其分化不重要。
