Cloning And Characterization Of A Hydroxyurea-inducible

羟基脲诱导剂的克隆和表征

• 批准号: 7336241
• 负责人: GRIFFIN P. RODGERS
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7336241
• 关键词: Cloning; Characterization; Hydroxyurea; inducible

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.

羟基脲（HU）是一种有效治疗镰状细胞病的药物，被认为通过干扰红细胞前体的成熟间接促进胎儿血红蛋白（Hb F）的产生。涉及HU介导的γ-珠蛋白表达调节的分子机制目前仍不清楚。我们通过差异显示技术在成人红系细胞中鉴定了一种HU诱导的小GTP结合蛋白，该蛋白与分泌相关和ras相关（SAR）。SAR在K562细胞中的稳定表达导致与γ-珠蛋白mRNA增加相关的大红细胞和未成熟细胞的出现。SAR介导的γ-珠蛋白基因的诱导也抑制K562细胞生长，包括停滞在G1/S期，凋亡和成熟延迟，细胞变化与先前已知的HU对红系细胞的作用一致。类似地，SAR也增强了骨髓原代CD 34+细胞中γ-和β-珠蛋白的转录，但其对γ-珠蛋白诱导的影响比对β-珠蛋白的影响更深刻。尽管在SAR表达细胞和HU处理的K562细胞中观察到加塔-2和p21的上调，但在SAR表达细胞中特异性地抑制PI 3激酶和磷酸化ERK。我们还研究了SAR在原代骨髓干细胞中的作用。使用pMSCV逆转录病毒表达系统产生SAR-GFP融合构建体。细胞分选后获得了SAR感染的CD 34+干细胞的纯群体。两个？然后呢？使用实时PCR动态测量珠蛋白mRNA转录物。SAR增强了两者？珠蛋白和？珠蛋白mRNA表达的CD 34+干细胞，但对？- 珠蛋白的影响比对？珠蛋白虽然这些发现强烈表明SAR和？之间存在密切相关性，尽管SAR（过）表达足以复制HU对原代细胞和细胞系的作用，但它们没有回答SAR是否是该途径的必要组分。了解HU介导是否需要SAR？在K562和Hek 293细胞中，我们用sRNAi技术敲低了SAR的表达。SAR sRNAi能够抑制SAR表达40%，并相应下调？K562细胞珠蛋白含量为60%。在Hek 293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PI 3-激酶/ERK和加塔-2/p21依赖的信号转导通路参与红系细胞生长和γ-珠蛋白的产生。