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: 7268018
• 负责人: Roland Jurecic
• 金额: $ 18.27万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7268018
• 关键词: Activation Analysis; Affect; Aging; Antibodies; B-Lymphocytes; Blood Cells; Bone Marrow Cells; Caenorhabditis elegans; Cell Line; Cell Lineage; Cell Maintenance; Cells; Commit; Development; Dimerization; Drosophila genus; Elements; Embryonic Development; Engineering; Equilibrium; Erythroid; Family; Genes; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; In Vitro; Invertebrates; Lead; Length; Lymphoid Cell; MAP Kinase Gene; MATK gene; Maintenance; Mediating; Modeling; Molecular; Multipotent Stem Cells; Multiprotein Complexes; Mus; Myelogenous; Pattern; Phenotype; Phosphorylation; Play; Precipitation; Protein Analysis; Protein Overexpression; Proteins; Proto-Oncogene Protein c-kit; RNA Recognition Motif; RNA-Binding Proteins; Regulation; Reporting; Research Personnel; Role; SRC-associated p68 protein; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Stem Cell Factor; Stem cells; Testing; Transplantation; Tyrosine; Tyrosine Phosphorylation; Vertebrates; Xenopus; Yeasts; attenuation; base; congenic; cytokine; in vitro Assay; insight; kit protein; mutant; novel; progenitor; programs; protein function; receptor; research study; self-renewal

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（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规范过程中充当翻译抑制因子。基于其在无脊椎动物和低等脊椎动物中的已知功能，有人提出 Pumilio (Pum) 蛋白的原始功能是维持干细胞的增殖和维持。我们之前已经证明，小鼠 Pum1 和 Pum2 基因优先在造血干细胞 (HSC) 中转录。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) 依赖性多能造血祖细胞系 EML 中过表达 Pum2 的 RNA 结合结构域，该细胞系可以在体外分化为红细胞、骨髓细胞和淋巴细胞谱系。 Pum2-RBD 的过度表达导致 EML 细胞的不依赖于 SCF 的维持，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这种解偶联伴随着 (a) 全长 c-kit 和称为 tr-kit 的新型截短 c-kit 受体的表达增加，以及 (b) c-kit 及其下游 MAPK、PI3K 和 PLCy 信号通路的细胞内在、不依赖于 SCF 的激活。 tr-kit 的表达仅限于 HSC 和多能祖细胞，并且 tr-kit 蛋白表达增加与 EML 细胞的 SCF 独立维持相关，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不依赖于 SCF 的 c-kit 激活，并且可能在不依赖于 SCF 的 EML 细胞维持和抑制其多谱系分化中发挥重要作用。由于 Pum2 和 tr-kit 蛋白在富含 HSC 和早期多能祖细胞的骨髓细胞中表达，但在后期定型祖细胞中不表达，因此我们假设 HSC 和多能祖细胞利用不同的 SCF 依赖性和 SCF 独立性 c-kit 信号通路来调节其维持和分化。相比之下，缺乏自我更新能力且不表达 tr-kit 的分化程度较高的祖细胞仅利用典型的 SCF 诱导的 c-kit 信号传导。在此假设模型中，HSC 和多能祖细胞的存活和维持是通过不依赖于 SCF 的 c-kit 信号传导介导的，而它们的分化则取决于典型的 SCF 诱导的 c-kit 信号传导。在这个项目中，我们想要测试这样的假设：tr-kit 介导不依赖于 SCF 的 c-kit 信号传导，并且 HSC 和多能祖细胞的维持和分化的调节涉及不同的 SCF 依赖性和 SCF 独立性 c-kit 信号传导途径。这些研究可以为干细胞自我更新、分化抑制和增殖诱导这两个关键要素的分子调控提供重要见解，并且可能与衰老 HSC 和多能祖细胞的研究相关。