Effects Of Thalidomide and Stem Cell Factor On Fetal Hemoglobin Synthesis

沙利度胺已被证明对一些骨髓增生异常综合征（MDS）患者有效，在一些临床试验中，沙利度胺增加了总血红蛋白和胎儿血红蛋白的比例。此外，已经证明沙利度胺增加了胚状体的细胞内活性氧（ROS）。沙利度胺治疗效果的机制仍在研究中。我们假设沙利度胺诱导成人红细胞生成中的γ -珠蛋白基因表达，这种诱导可能是通过增加ROS形成介导的。为了验证这一假设，我们利用培养的原代人CD34+祖细胞，评估了增加沙利度胺剂量（0.01uM至100uM）对细胞生长、珠蛋白基因表达和ROS生成的影响。不同浓度的沙利度胺对培养的CD34+细胞的影响表明，当沙利度胺的最大浓度为100uM时，细胞数量显著增加。γ -和β -珠蛋白基因表达的实时定量PCR分析表明，沙利度胺通过pkc - α依赖的Ras/Raf/Erk1/2信号通路显著诱导SCF诱导的γ -珠蛋白，该信号通路涉及转录因子NF-Ya的激活和抑制因子Coup-TFII的抑制。Go6976特异性抑制pkc - α阻断了SCF诱导的Erk1/2和p38 MAPK的激活，并消除了SCF增加的γ -珠蛋白基因表达。Erk1/2的激活在SCF调控的下游转录调控中起关键作用，包括γ -珠蛋白基因诱导的调控。SCF诱导的NF-Ya核易位需要激活Erk1/2内源性Nrf2磷酸化增加，这涉及硫氧还蛋白的上调和Coup-TFII的下调。抑制pkc - α或Erk1/2均可阻止SCF诱导的NF-Ya、RNA聚合酶II的募集和Coup-TFII抑制因子从γ -球蛋白启动子的位移，这表明pkc - α -Erk1/2 MAPK通路有助于SCF诱导成人红细胞生成的γ -球蛋白基因诱导。此外，与这一概念一致的是，SCF通过抑制pkc - α或Erk1/2 MAPK而诱导γ -珠蛋白基因的诱导减弱。我们的数据表明，SCF刺激PKC/ROS/Erk1/2 MAPK信号通路，从而调节下游抑制因子COUP-TFII（通过抑制丝氨酸/苏氨酸磷酸酶2A活性）和转录激活因子NF-Ya（通过诱导Ref-1），并且在成人红细胞生成中，COUP-TFII表达的降低和NF-Ya表达的增加诱导γ -球蛋白再激活。这些观察结果提供了对应激红细胞生成过程中调节γ -球蛋白增加的分子途径的深入了解。

Thalidomide has been shown to be effective in some patients with myelodysplastic syndromes (MDS), increasing both the total hemoglobin and in the proportion of the fetal hemoglobin in some patients in clinical trials. Also, it has been demonstrated that thalidomide increases the intracellular reactive oxygen species (ROS) in embryoid bodies. The mechanisms of thalidomides therapeutic effect are still being defined. We hypothesize that thalidomide induces the gamma-globin gene expression in adult erythropoiesis, and that this induction may be mediated by increased ROS formation. To investigate this hypothesis, we assessed the effect of increasing dosages of thalidomide (0.01uM to 100uM) on cell growth, globin gene expression and ROS generation using cultured primary human CD34+ progenitor cells. The effects of varying concentrations of thalidomide on the cultured CD34+ cells, demonstrate a significant increase in cell number at maximum thalidomide concentration of 100uM. Real time quantitative PCR analysis of gamma- and & beta-globin gene expression demonstrated that thalidomide significantly induces gamma-globin induced by SCF is through a PKC-alpha-dependent Ras/Raf/Erk1/2 signaling pathway involving activation of the transcription factor NF-Ya and inhibition of the repressor Coup-TFII. Specific inhibition of PKC-alpha with Go6976 blocked both activation of Erk1/2 and p38 MAPK induced by SCF, and abrogated the SCF increased gamma-globin gene expression. Activation of Erk1/2 plays a critical role in SCF modulated down-stream transcriptional regulators, involving regulation of gamma-globin gene induction. SCF induced nuclear translocation of NF-Ya is required to activate Erk1/2 increased phosphorylation of endogenous Nrf2, which involves up-regulation of thioredoxin, and down-regulation of Coup-TFII. Inhibition of either PKC-alpha or Erk1/2 prevented SCF induced recruitment of NF-Ya, RNA polymerase II and displacement of Coup-TFII repressor from gamma-globin-promoter, indicating that the PKC-alpha-Erk1/2 MAPK pathway contributes to SCF induced the gamma-globin gene induction in adult erythropoiesis. Furthermore, consistent with this concept, SCF induced the gamma-globin gene induction attenuated by inhibition of PKC-alpha or Erk1/2 MAPK. Our data suggest that SCF stimulates the PKC/ROS/Erk1/2 MAPK signaling pathway, which regulates the downstream repressor COUP-TFII (by inhibiting serine/threonine phosphatase 2A activity) and the transcriptional activator NF-Ya (by inducing Ref-1), and that decreased COUP-TFII expression and increased NF-Ya expression induce gamma-globin reactivation in adult erythropoiesis. These observations provide insight into the molecular pathways that regulate gamma-globin augmentation during stress erythropoiesis.