THE ROLE OF TR-KIT RECEPTOR IN STEM CELL MAINTENANCE AND DIFFERENTIATION

TR-Kit 受体在干细胞维持和分化中的作用

• 批准号: 7149857
• 负责人: Roland Jurecic
• 金额: $ 15.64万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149857
• 关键词: RNA binding protein; RNA interference; binding sites; biological signal transduction; biotechnology; cell differentiation; cell line; gene expression; gene induction /repression; gene targeting; genetic translation; hematopoiesis; mitogen activated protein kinase; phosphatidylinositol 3 kinase; phosphorylation; polymerase chain reaction; protein isoforms; protooncogene; stem cells; transfection /expression vector; translation factor

DESCRIPTION (provided by applicant): Pumilio proteins are evolutionary highly conserved family of RNA-binding proteins that function as translational repressers during embryo development and cell fate specification. Based on their known functions in invertebrates and lower vertebrates it was proposed that a primordial function of Pumilio (Pum) proteins is to sustain proliferation and maintenance of stem cells. We have shown previously that mouse Pum1 and Pum2 genes are transcribed preferentially in hematopoietic stem cells (HSC). To start analyzing their role during blood cell development we have over-expressed the RNA-binding domain of Pum2 in a stem cell factor (SCF)-dependent multipotent hematopoietic progenitor cell line EML, which can differentiate into erythroid, myeloid and lymphoid cell lineages in vitro. The over-expression of Pum2-RBD leads to SCF-independent maintenance of EML cells, and is suppressing their mutilineage differentiation in the absence of SCF. This uncoupling of the survival and differentiation signals in EML cells is accompanied by (a) an increased expression of the full-length c-kit and a novel truncated c-kit receptor called tr-kit, and (b) cell intrinsic, SCF-independent activation of the c-kit, and its downstream MAPK, PI3K and PLCy signaling pathways. The observations that the expression of tr-kit is restricted to HSC and multipotent progenitors, and that an increased expression of tr-kit protein is associated with SCF- independent maintenance of EML cells, suggest a potentially important role for tr-kit in the regulation of the balance between maintenance (self-renewal) and differentiation of HSC and multipotent progenitors. For example, an interaction of tr-kit with the full-length c-kit receptor could lead to SCF-independent c-kit activation, and could play an important role in the SCF-independent maintenance of EML cells, and suppression of their multilineage differentiation. Since the Pum2 and tr-kit proteins are expressed in bone marrow cells enriched for HSC and early multipotent progenitors, but not in later committed progenitors, we hypothesize that HSC and multipotent progenitors utilize distinct SCF-dependent and SCF-independent c-kit signaling pathways that could regulate their maintenance and differentiation. In contrast, more differentiated progenitors that lack self-renewal ability and do not express tr-kit, utilize only the canonical SCF-induced c-kit signaling. In this hypothetical model, the survival and maintenance of HSC and multipotent progenitors is mediated through SCF-independent c-kit signaling, whereas their differentiation depends on the canonical SCF-induced c- kit signaling. In this project we want to test the hypothesis that tr-kit mediates SCF-independent c-kit signaling, and that the regulation of maintenance and differentiation of HSC and multipotent progenitors involves distinct SCF-dependent and SCF-independent c-kit signaling pathways. These studies could provide important insights into the molecular regulation of two critical elements of stem cell self-renewal, inhibition of differentiation and induction of proliferation, and could be relevant for the study of aging HSC and multipotent progenitors.

描述(申请人提供)：Pumilio蛋白是进化上高度保守的RNA结合蛋白家族，在胚胎发育和细胞命运指定过程中作为翻译抑制因子发挥作用。基于它们在无脊椎动物和低等脊椎动物中的已知功能，有人认为Pum蛋白的原始功能是维持干细胞的增殖和维持。我们以前已经证明，小鼠Pum1和Pum2基因在造血干细胞(HSC)中优先转录。为了分析它们在血细胞发育中的作用，我们在干细胞因子(SCF)依赖的多能造血祖细胞系EML中过表达了Pum2的RNA结合域，该细胞系在体外可以分化为红系、髓系和淋巴系。Pum2-RBD的过表达导致EML细胞对SCF的非依赖性维持，并在没有SCF的情况下抑制其多系分化。EML细胞生存和分化信号的这种解偶联伴随着(A)全长c-kit和一种新的被截短的c-kit受体tr-kit的表达增加，以及(B)c-kit及其下游的MAPK、PI3K和PLCy信号通路的细胞内非SCF依赖的激活。研究发现，TRKIT的表达仅限于HSC和多能祖细胞，并且其蛋白表达的增加与EML细胞的SCF非依赖性维持有关，这提示了TRKIT在调节HSC和多能祖细胞的维持(自我更新)和分化之间的平衡方面具有潜在的重要作用。例如，tr-kit与全长c-kit受体的相互作用可能导致SCF非依赖性c-kit激活，并在维持EML细胞的SCF非依赖性和抑制其多系分化中发挥重要作用。由于Pum2和tr-kit蛋白在HSC和早期多能祖细胞中表达，但在晚期定向祖细胞中不表达，我们推测HSC和多能祖细胞利用不同的SCF依赖和SCF非依赖的c-kit信号通路来调节它们的维持和分化。相反，缺乏自我更新能力和不表达tr-kit的更多分化的祖细胞，只利用典型的SCF诱导的c-kit信号。在这个假想的模型中，HSC和多能祖细胞的生存和维持是通过SCF非依赖的c-kit信号介导的，而它们的分化依赖于SCF诱导的典型的c-kit信号。在这个项目中，我们想要检验一种假设，即TR-KIT介导了SCF非依赖的c-kit信号，并且调节HSC和多能祖细胞的维持和分化涉及不同的SCF依赖和SCF非依赖的c-Kit信号通路。这些研究可以为干细胞自我更新的两个关键因素--抑制分化和诱导增殖--的分子调控提供重要的见解，并可能对衰老的HSC和多能祖细胞的研究具有重要意义。