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

描述（由申请人提供）：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特化过程中充当翻译抑制因子。基于它们在无脊椎动物和低等脊椎动物中的已知功能，提出Pumilio（Pum）蛋白的原始功能是维持干细胞的增殖和维持。我们以前已经表明，小鼠Pum 1和Pum 2基因的转录优先在造血干细胞（HSC）。为了开始分析它们在血细胞发育过程中的作用，我们在干细胞因子（SCF）依赖性多能造血祖细胞系EML中过表达Pum 2的RNA结合结构域，该细胞系可以在体外分化成红系、髓系和淋巴系细胞系。Pum 2-RBD的过表达导致EML细胞的SCF非依赖性维持，并且在SCF不存在的情况下抑制它们的多谱系分化。EML细胞中存活和分化信号的这种解偶联伴随着（a）全长c-kit和称为tr-kit的新型截短的c-kit受体的表达增加，和（B）c-kit及其下游MAPK、PI 3 K和PLC γ信号传导途径的细胞内在的SCF非依赖性活化。观察到tr-kit的表达限于HSC和多能祖细胞，并且tr-kit蛋白的表达增加与EML细胞的SCF非依赖性维持相关，这表明tr-kit在调节HSC和多能祖细胞的维持（自我更新）和分化之间的平衡中具有潜在的重要作用。例如，tr-kit与全长c-kit受体的相互作用可导致SCF非依赖性c-kit活化，并且可在EML细胞的SCF非依赖性维持和其多谱系分化的抑制中发挥重要作用。由于Pum 2和tr-kit蛋白在富含HSC和早期多能祖细胞的骨髓细胞中表达，但在后来的定向祖细胞中不表达，因此我们假设HSC和多能祖细胞利用不同的SCF依赖性和SCF非依赖性c-kit信号通路来调节其维持和分化。相反，缺乏自我更新能力且不表达tr-kit的更分化的祖细胞仅利用典型SCF诱导的c-kit信号传导。在该假设模型中，HSC和多能祖细胞的存活和维持通过SCF非依赖性c-kit信号传导介导，而它们的分化取决于典型SCF诱导的c-kit信号传导。在这个项目中，我们想测试的假设，tr-kit介导的SCF非依赖性c-kit信号，以及调控的维持和分化的HSC和多能祖细胞涉及不同的SCF依赖性和SCF非依赖性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要素的分子调控提供重要的见解，抑制分化和诱导增殖，并可能与衰老HSC和多能祖细胞的研究有关。

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.