GATA-6: Key Regulator of AEC Transdifferentiation

描述(申请人提供)：一般认为肺泡上皮II型(AT2)细胞是发育中的I型(AT1)细胞的前体细胞，损伤后恢复肺泡气交换表面。然而，调节肺泡上皮细胞(AEC)表型分化的转录程序几乎完全未知。我们已经开发了定义明确的原代细胞培养系统，在其中可以实验地调节AEC在体外的转分化，提供了一个很好的模型，可以用来剖析调节AT2和AT1细胞表型之间转换的分子通路。转录因子(Tf)GATA-6在AT1细胞发育过程中起着至关重要的作用。本研究的目的是通过相互激活和抑制分化相关基因，研究GATA-6在调节AT2和AT1细胞表型转换中的作用，长期目标是了解AEC分化的调节如何有助于损伤后成人肺泡上皮的维持和修复。我们假设：1)成人肺泡上皮中伴随AT1和AT2细胞表型转换的细胞类型特异性基因的激活和抑制受Tf相互上调或下调/相互作用的调节；2)GATA-6相对表达/活性的变化调节AT2和AT1细胞表型之间的转换；以及3)GATA-6与其他辅因子或细胞限制性Tf的组合作用决定了AEC的分化状态。我们将利用我们建立的体外培养系统和成功分离AT2和AT1细胞的经验，以及在鉴定AEC分化方面的专业知识，通过解决以下特定目标来探索这些假说：1)研究GATA-6表达在AEC分化表型调控中的作用；2)确定GATA-6表达对AEC分化表型的影响；以及3)通过表征GATA-6与细胞类型特异性基因和其他细胞限制性因子的相互作用来研究AEC分化的调控。GATA-6在AEC分化中的作用将通过评估其在分离和培养的AEC中的表达、调节GATA-6的表达和对AEC表型的影响以及其与AT1细胞特异性基因水通道蛋白-5在AT2和AT1细胞中的相互作用和启动子的占有率来阐明。这些研究将为AEC分化的分子编程提供新的见解，这将为进一步研究开发肺损伤恢复的新治疗策略奠定基础。

DESCRIPTION (provided by applicant): It is generally believed that alveolar epithelial type II (AT2) cells serve as progenitors of type I (AT1) cells in development and following injury to restore the alveolar gas exchange surface. However, the transcriptional programs that mediate differentiation between alveolar epithelial cell (AEC) phenotypes are almost entirely unknown. We have developed well-defined primary cell culture systems in which AEC transdifferentiation in vitro can be experimentally modulated, providing an excellent model with which to dissect molecular pathways that regulate transitions between AT2 and AT1 cell phenotypes. The transcription factor (TF) GATA-6 has been shown to be essential for AT1 cell differentiation during development. The goal of this proposal is to investigate the role of GATA-6 in regulating transitions between AT2 and AT1 cell phenotypes through reciprocal activation and repression of differentiation-related genes, with the long-range objective of understanding how regulation of AEC differentiation contributes to maintenance and repair of adult alveolar epithelium following injury. We hypothesize that: 1) activation and repression of cell type-specific genes that accompany transitions between AT1 and AT2 cell phenotypes in alveolar epithelium in adult lung are modulated by reciprocal up- or down-regulation/interactions of TF; 2) changes in the relative expression/activity of GATA-6 regulate transitions between AT2 and AT1 cell phenotypes; and 3) combinatorial interactions of GATA-6 with other cofactors or cell-restricted TF determine the state of AEC differentiation. We will capitalize on our established in vitro culture systems and success in isolating both AT2 and AT1 cells, together with expertise in characterizing AEC differentiation, to explore these hypotheses by addressing the following Specific Aims: 1) investigate the role of GATA-6 expression in regulation of AEC differentiated phenotype, 2) determine effects of modulating GATA-6 expression on AEC differentiated phenotype, and 3) investigate regulation of AEC differentiation by characterizing interactions of GATA-6 with cell-type specific genes and other cell-restricted TF. The role of GATA-6 in AEC differentiation will be elucidated by evaluating its expression in isolated and cultured AEC and in situ, modulating GATA-6 expression and assessing effects on AEC phenotype, and characterizing its interactions with and promoter occupancy of an AT1 cell-specific gene, aquaporin-5, in AT2 vs. AT1 cells. These studies will provide novel insights into molecular programming of AEC differentiation, which will form the basis for further studies to develop new therapeutic strategies for recovery from lung injury.