Effects Of Thalidomide and Stem Cell Factor On Fetal Hemoglobin Synthesis

在临床试验中，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，在一些患者中提高了总血红蛋白和胎儿血红蛋白的比例。此外，研究还表明，沙利度胺增加了类胚体中的细胞内活性氧(ROS)。沙利度胺的治疗作用机制尚不清楚。我们推测，沙利度胺诱导成人红细胞生成中的γ-珠蛋白基因表达，这一诱导可能是通过增加ROS的形成来介导的。为了验证这一假设，我们利用原代培养的人CD34+祖细胞，评估了增加沙利度胺剂量(从0.01微米到100微米)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+细胞的影响显示，在最大浓度为100uM时，细胞数量显著增加。实时定量聚合酶链式反应分析表明，沙利度胺通过依赖PKCα的Ras/Raf/ERK1/2信号通路，激活转录因子NF-Ya，抑制阻遏因子COUP-TFII，显著诱导SCF诱导的γ-珠蛋白表达。用Go6976特异性抑制PKC-α可阻断SCF诱导的ERK1/2和p38MAPK的激活，并阻断SCF对γ-珠蛋白基因表达的上调。ERK1/2的激活在SCF调控的下游转录调控中起着关键作用，参与了对γ-珠蛋白基因诱导的调控。SCF诱导的核转位需要激活ERK1/2，内源性Nrf2的磷酸化增加，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抑制子从γ-珠蛋白启动子的置换，表明PKC-α-ERK1/2 MAPK通路参与了SCF诱导的成人红细胞生成中的γ-珠蛋白基因的诱导。此外，与这一概念一致，SCF诱导的伽马珠蛋白基因诱导可通过抑制PKC-α或ERK1/2 MAPK而减弱。我们的结果提示，SCF刺激PKC/ROS/ERK1/2 MAPK信号通路，从而调节下游的抑制子COUP-TFII(通过抑制丝氨酸/苏氨酸磷酸酶2A的活性)和转录激活因子NF-Ya(通过诱导Ref-1)，并且降低COUP-TFII的表达和增加NF-Ya的表达诱导成人红细胞生成中伽玛珠蛋白的重新激活。这些观察结果提供了对应激性红细胞生成过程中调节伽马珠蛋白增强的分子途径的洞察。

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.