Role of the P2X7 nucleotide receptor in intestinal epithelial cell proliferation and differentiation

细胞外核苷酸对P2 X配体门控离子通道的刺激与细胞增殖和分化、蛋白质合成、细胞分泌和吸收的调节以及细胞凋亡的调节等多种生理效应有关。肠腺-绒毛轴是一个动态的自我更新系统，其受涉及细胞增殖、分化和凋亡的不同细胞和分子机制的调节。我们的假设是，P2受体可能在这些生理过程的稳态中发挥重要作用。免疫组化结果显示P2 X受体在小肠绒毛轴上沿着有不同的表达模式，提示不同的P2 X受体在肠上皮细胞（IEC）的成熟过程中发挥不同的作用。P2 X7受体（P2 X7 R）是P2 X家族的一个独特成员。它响应于配体刺激而形成孔，导致膜去极化和细胞凋亡。 我们和其他人已经表明，瞬时P2 X7 R刺激导致MAP激酶激活。根据这些数据，我们假设，P2 X7 R不仅应该被视为“自杀受体”，而且还具有调节细胞成熟的受体。在这种情况下，该研究计划将重点阐明肠上皮细胞（IEC）成熟过程中依赖于P2 X7 R活性的功能分子和细胞机制。首先，我们提出了与IEC成熟相关的P2 X7 R信号转导通路的特征。 这将通过使用不同的细胞和分子方法进行研究，包括干扰RNA（siRNA），Western印迹和体外激酶测定。在第二个目标中，我们将鉴定P2 X7 R启动子调节元件和相关的转录因子，它们对于维持成熟细胞中的受体表达很重要。将通过siRNA和染色质免疫沉淀试验（ChIP）在体外测定鉴定的转录因子的重要性的验证。这项研究计划将确定的分子和细胞机制的P2 X7 R表达的调控和其在IEC分化的假定作用。

Stimulation of P2X ligand-gated ion channels by extracellular nucleotides is linked to multiple physiological effects ranging from cellular proliferation and differentiation, protein synthesis, regulation of cell secretion and absorption as well as modulation of apoptosis. The intestinal crypt-villus axis is a dynamic self-renewal system that is regulated by different cellular and molecular mechanisms involved in cell proliferation, differentiation and apoptosis. Our hypothesis is that P2 receptors could play an important role in the homeostasis of these physiological process. Immunohistochemistry studies showed a differential pattern of P2X receptors expression along the crypt-villus axis, suggesting that different P2X receptors play different role in the maturation of intestinal epithelial cells (IEC). The P2X7 receptor (P2X7R) is a unique member of the P2X family. It forms a pore in response to ligand stimulation leading to membrane depolarization and apoptosis.   We and others have shown that transient P2X7R stimulation lead to MAP kinases activation. In lights of those data, we hypothese, that P2X7R should not only be viewed as a "suicidal receptor" but also has a receptor regulating cell maturation. In that context, this research program will focus to unravel the functional molecular and cellular mechanisms dependant on P2X7R activity during intestinal epithelial cells (IEC) maturation. First, we propose to characterize the P2X7R signal transduction pathways associated with IEC maturation.  This will be investigated with the use of diverse cellular and molecular approaches, including interference RNA (siRNA), Western blotting and in vitro kinase assays. In a second aim, we will identify the P2X7R promoter regulatory elements and associated transcription factors important in sustaining receptor expression in maturating cells. Validation of the importance of the identified transcription factors will be assayed in vitro by siRNA and by chromatin immunoprecipitation assays (ChIP). This research program will define the molecular and cellular mechanisms governing the regulation of P2X7R expression and its putative role in IEC differentiation.