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

描述（由申请人提供）：Pumilio蛋白是进化高度保守的rna结合蛋白家族，在胚胎发育和细胞命运规范中起翻译抑制作用。基于它们在无脊椎动物和低等脊椎动物中的已知功能，我们提出Pumilio （Pum）蛋白的一个原始功能是维持干细胞的增殖和维持。我们之前已经证明，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）依赖的多能造血祖细胞系EML中过表达Pum2的rna结合结构域，EML在体外可以分化为红系、髓系和淋巴系细胞。Pum2-RBD的过表达导致EML细胞的维持不依赖于SCF，并在缺乏SCF的情况下抑制其多谱系分化。EML细胞中生存和分化信号的解耦伴随着(a)全长c-kit和新型截断c-kit受体tr-kit的表达增加，以及(b)细胞内在的、不依赖于scf的c-kit及其下游MAPK、PI3K和PLCy信号通路的激活。我们观察到，tr-kit的表达仅限于HSC和多能祖细胞，而tr-kit蛋白表达的增加与EML细胞的SCF独立维持有关，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可能发挥重要作用。例如，tr-kit与全长c-kit受体的相互作用可能导致不依赖于scf的c-kit激活，并可能在EML细胞的不依赖于scf的维持和抑制其多谱系分化中发挥重要作用。由于Pum2和tr-kit蛋白在造血干细胞和早期多能祖细胞富集的骨髓细胞中表达，但在后来的造血祖细胞中不表达，我们假设造血干细胞和多能祖细胞利用不同的scf依赖和scf独立的c-kit信号通路来调节它们的维持和分化。相比之下，缺乏自我更新能力且不表达tr-kit的分化程度更高的祖细胞仅利用典型的scf诱导的c-kit信号。在这个假设模型中，HSC和多能祖细胞的存活和维持是通过独立于scf的c-kit信号传导介导的，而它们的分化依赖于典型的scf诱导的c-kit信号传导。在这个项目中，我们想要验证以下假设：tr-kit介导scf独立的c-kit信号通路，以及对HSC和多能祖细胞的维持和分化的调节涉及不同的scf依赖和scf独立的c-kit信号通路。这些研究可以为干细胞自我更新、抑制分化和诱导增殖两个关键因素的分子调控提供重要见解，并可能与衰老HSC和多能祖细胞的研究相关。

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.