Cloning And Characterization Of A Hydroxyurea-inducible

羟基脲（HU）是一种治疗镰状细胞病的有效药物，被认为通过干扰红系前体的成熟间接促进胎儿血红蛋白（Hb F）的产生。hu介导的γ -珠蛋白表达调控的分子机制目前尚不清楚。通过差异显示，我们在成年红细胞中鉴定了HU诱导的小gtp结合蛋白、分泌相关蛋白和ras相关蛋白（SAR）。在K562细胞中稳定的SAR表达导致巨细胞增生和未成熟细胞的出现，这与γ -珠蛋白mRNA的增加有关。sar介导的γ -珠蛋白基因的诱导也抑制了K562细胞的生长，包括G1/S期阻滞、凋亡和成熟延迟，这些细胞变化与先前已知的HU对红细胞的影响一致。同样，SAR也增强了骨髓原代CD34+细胞中γ -珠蛋白和β -珠蛋白的转录，但其对γ -珠蛋白诱导的影响比对β -珠蛋白的影响更深远。虽然在sar表达细胞和hu处理的K562细胞中均观察到GATA-2和p21的上调，但在sar表达细胞中PI3激酶和磷酸化的ERK被特异性抑制。我们还研究了SAR在原代骨髓干细胞中的作用。利用pMSCV逆转录病毒表达系统产生了SAR-GFP融合构建体。细胞分选后获得了感染sars的CD34+干细胞的纯群体。都有?-然后呢？实时荧光定量PCR动态检测-珠蛋白mRNA转录物。SAR都增强了？-球蛋白和？-珠蛋白mRNA在CD34+干细胞中的表达，但对？-球蛋白的影响比-球蛋白的影响更大，更持久。虽然这些发现强烈表明SAR和？-珠蛋白基因的表达，以及SAR（过）表达足以在原代细胞和细胞系上复制HU的作用，但它们并没有回答SAR是否是该途径的必要成分。为了了解HU介导的是否需要SAR ？通过sRNAi技术，我们降低了K562和Hek293细胞中SAR的表达。SAR sRNAi能够抑制40%的SAR表达，并相应下调？-珠蛋白在K562细胞中占60%在Hek293细胞中，SAR RNAi部分阻断HU介导的s期细胞阻滞。这些数据揭示了SAR不同于其先前已知的蛋白质运输功能的新作用。我们认为SAR可能通过调节pi3激酶/ERK和GATA-2/p21依赖的信号转导途径参与红细胞生长和γ -珠蛋白的产生。

Hydroxyurea (HU), a drug effective in the treatment of sickle cell disease, is thought to indirectly promote fetal hemoglobin (Hb F) production by perturbing the maturation of erythroid precursors. The molecular mechanisms involved in HU-mediated regulation of gamma-globin expression currently remain unclear. We identified a HU induced small GTP-binding protein, secretion-associated and ras-related (SAR) in adult erythroid cells by differential display. Stable SAR expression in K562 cells resulted in macrocytosis and immature cells appearance associated with increased gamma-globin mRNA. SAR-mediated induction of the gamma-globin gene also inhibited K562 cell growth including arrest in G1/S phase, apoptosis and delay of maturation, cellular changes consistent with the previously known effects of HU on erythroid cells. Similarly, SAR also enhanced both gamma-and beta-globin transcription in bone marrow primary CD34+ cells, but its effects on gamma-globin induction is more profound than that on beta-globin. Though upregulation of GATA-2 and p21 were observed both in SAR-expressing cells and HU-treated K562 cells, the PI3 kinase and phosphorylated ERK were inhibited specifically in SAR-expressing cells. We also investigated the effects of SAR in primary bone marrow stem cells. A SAR-GFP fusion construct was produced using the pMSCV retroviral expression system. A pure population of SAR-infected CD34+ stem cells was obtained after cell sorting. Both ?- and ?-globin mRNA transcripts were measured dynamically using real-time PCR. SAR enhanced both ?-globin and ?-globin mRNA expression in the CD34+ stem cells, but the effects on ? -globin were larger and more sustained than the effects on ?-globin. While these findings strongly indicate a tight correlation between SAR and ?-globin gene expression, and that SAR (over)expression is sufficient to replicate the HU effects on primary cells and cell lines, they do not answer whether SAR is a necessary component to the pathway. To understand whether SAR is needed for HU mediated ?-globin induction, we knocked- down SAR expression in K562 and Hek293 cells with sRNAi techniques. SAR sRNAi was able to inhibit SAR expression by 40% with a corresponding down regulation ?-globin 60% in K562 cells. In Hek293 cells, SAR RNAi partly block HU mediated S-phase cells arrest. These data reveal a novel role of SAR distinct from its previously known protein trafficking function. We suggest that SAR may participate in both erythroid cell growth and gamma-globin production by regulating PI3-kinase/ERK and GATA-2/p21 dependent signal transduction pathways.