Role of alveolar fibroblasts in extracellular matrix organization and alveolar type 1 cell differentiation

摘要： 需要对肺泡间隔和隔壁成熟的调节机制有深入的了解。 发展早产儿支气管肺发育不良(BPD)的治疗方法和潜在的治疗方法 用于成人肺再生。我们和其他人已经开始定义PDGFRA肌、脂和基质成纤维细胞(FB)的变化 在肺泡化过程中发挥作用， 动态平衡、再生和纤维化。我们的长期目标是阐明分子 FB功能的调节者以及不同的FBS如何支持上皮细胞。我们在这里的目标是识别分子机械- 导致肺泡成纤维细胞功能改变的异常现象，调节细胞外基质组织和上皮分化。 提顿。整合转录数据集预测GATA6是PDGFRA-FB分化的调节因子。我们的预赛 数据表明，围产期PDGFRA FBS中GATA6的条件失活导致了基质的丢失和脂纤维的增加。 BLAST的功能与BPD和高氧动物模型的研究结果相当。PDGFRA中GATA6的失活 FBS还 导致胶原碎裂和AT1细胞从基底板脱落，表现为迄今尚未确定的 肺泡内FBS在细胞外基质组织中的作用GATA6失活导致LipoFB转录显著增加 因子TCF21的表达和初步的体外数据表明，GATA6抑制TCF21。中心假设是 GATA6和转录辅助因子通过抑制脂类FB分化来调节肺泡FBS中FB的功能。这个 本研究的理论基础是对矩阵FB函数的调节有了新的理解，以及这些函数是如何 不同阶段改变细胞外基质和FB-上皮细胞的串扰。目标1将检验GATA6的假设 抑制Lipo Fb功能，促进细胞外基质在肺泡FBS中沉积。这个目标将使用血统追踪来阻止- 矿物质促进肺泡纤维蛋白转分化为脂蛋白纤维蛋白。并使用体外细胞衍生基质鉴定ECM修饰- 用Lipo和矩阵FBS测定。Aim 2将验证GATA6处于调节脂类的转录网络的假设 和矩阵FB函数。为此，我们将利用GATA6和TCF21的体外获得和功能丧失来鉴定和 验证转录网络和合作转录因子。目标3将检验GATA6调节的假设 PDGFRA FBS细胞外基质合成和旁分泌信号引导AT2向AT1分化。在这方面的拟议研究 应用将确定基质FB功能的转录调节及其在细胞外基质沉积中的作用 以及肺泡龛中的细胞-细胞信号。拟议的研究在概念上是创新的，因为我们提出了问题 关于成纤维细胞可塑性和功能开关的性质。这项拟议的研究具有科学创新性， 由于肺泡成纤维细胞的收缩功能已被彻底研究，但对其功能知之甚少 矩阵组织功能或 它们在支持AT2增殖和AT1分化方面的作用 意义重大，因为它解决了1)缺乏对肺泡Fb的基质和信号功能的知识；2)识别和 验证了GATA6周围调节FB功能的转录网络。

ABSTRACT: An in-depth understanding of the mechanisms that regulate alveolar septation and septal wall maturation will be required to develop therapies for premature infants with Bronchopulmonary Dysplasia (BPD) and for developing potential therapies for adult lung regeneration. We and others have begun to define changes in PDGFRa myo, lipo and matrix fibroblast (FB) function during alveolarization, homeostasis, regeneration, and fibrosis. Our long-term goal is elucidation of molecular regulators of FB functions and how diverse FBs support epithelial cells. Our objective herein is to identify molecular mech- anisms that result in functional changes in alveolar fibroblasts that regulate ECM organization and epithelial differentia- tion. Integration of transcriptomic datasets predicted GATA6 as a regulator of PDGFRa-FB differentiation. Our preliminary data show that conditional inactivation of GATA6 in perinatal PDGFRa+ FBs resulted in loss of matrix and gain in lipo fibro- blast function comparable to findings in BPD and animal models of hyperoxia. GATA6 inactivation in PDGFRa+ FBs also resulted in fragmented collagen and detachment of AT1 cells from the basal lamina demonstrating a thus far unidentified role of alveolar FBs in ECM organization. Inactivation of GATA6 resulted in significant increase of the lipo FB transcription factor TCF21 expression and preliminary in vitro data suggest suppression of TCF21 by GATA6. The central hypothesis is that GATA6 and transcriptional cofactors regulate FB function in alveolar FBs by suppressing lipo FB differentiation. The rationale for this research is a new understanding of the regulation of matrix FB function and how these functionally different stages modify extracellular matrix and the FB-epithelial crosstalk. Aim 1 will test the hypothesis that GATA6 suppresses lipo FB function and promotes ECM deposition in the alveolar FBs. This aim will use lineage tracing to deter- mine trans-differentiation of the alveolar FB to a lipo FB. And use in vitro cell derived matrices to identify ECM modifica- tions by lipo and matrix FBs. Aim 2 will test the hypothesis that GATA6 is in a transcriptional network that regulates lipo and matrix FB function. In this aim we will use in vitro gain and loss of function of GATA6 and TCF21 and identify and validate transcriptional networks and partnering transcription factors. Aim 3 will test the hypothesis that GATA6 regulates ECM synthesis and paracrine signaling in PDGFRa+ FBs that direct AT2 to AT1 differentiation. The proposed studies in this application will identify the transcriptional regulation of matrix FB function and their role in extracellular matrix deposition and cell-cell signaling in the alveolar niche. The proposed research is conceptually innovative, because we ask questions regarding the nature of fibroblast plasticity and functional switches. The proposed research is scientifically innovative, because the contractile function of alveolar fibroblasts, has been thoroughly investigated, but little is known about their matrix organizing function or their role in supporting AT2 proliferation and AT1 differentiation This contribution will be significant because it addresses 1) lack of knowledge of matrix and signaling function of the alveolar FB; 2) identifies and validates the transcriptional network around GATA6 that regulates FB function.