Molecular control of a novel transitional cell state in alveolar regeneration

总结 肺泡损伤和无效修复被认为是慢性阻塞性肺疾病发病机制的基础。 肺疾病和肺纤维化。虽然全基因组关联研究和临床标本 提示慢性应激、炎症和DNA损伤信号的作用，潜在的机制和 在肺泡再生过程中上述途径失调的细胞状态仍然难以捉摸。 在我们最近的研究中，使用类器官，单细胞转录组学和体内肺损伤模型，我们发现 先前未表征的、短暂的、前AEC 1过渡细胞状态（PATS），在AEC 2和AEC 2之间穿越， AEC 1在肺泡再生中的作用有趣的是，对PATS中表达的基因的途径分析显示， 富集转录因子TP 53和SOX 4的靶点，以及DNA损伤修复途径。我们还发现 这种细胞状态在立方状AEC 2分化为 非常平坦和薄的AEC 1。在AEC 2中条件性消融Tp 53和Sox 4显示， AEC 1的数量，以及PATS数量的大幅增加。这些数据表明， 转录因子TP 53和SOX 4通过前AEC 1过渡期调节AEC 2向AEC 1分化 状态和DNA损伤修复。根据我们的初步数据，我们假设 AEC 2祖细胞通过一种新的和分子上不同的前AEC 1过渡状态， 区分为AEC 1。我们还假设TP 53和SOX 4介导的机制是必不可少的， 细胞周期停滞，细胞粘附，细胞拉伸和DNA损伤修复途径， AEC 2与AEC 1的区别 该提案的主要目标是从分子和功能上表征新发现的前- AEC 1过渡状态，并研究其在肺泡再生中的作用机制。在目标1中， 我们将研究一个新的前AEC 1过渡的分子身份，时间动力学和可塑性， 肺泡再生的状态。在Aim 2中，我们将检验TP 53和SOX 4介导的机制的假设。 在前AEC 1过渡状态下控制细胞周期调节、细胞粘附和DNA损伤修复途径 在AEC 2分化成AEC 1期间。我们将使用类器官模型，体内遗传和药理学损失- 功能模型和分子测定来研究这些特定目的。这项工作已经采取了额外的 重要性，因为最近的全基因组关联研究揭示了DNA组分的突变， 损伤修复信号传导是肺气肿和肺纤维化的主要驱动因素之一。所以我们的 发现在前AEC 1过渡状态中与拉伸相关的DNA损伤使其可能容易受到 肺部疾病因此，拟议研究的结果将具有更广泛的意义，并将奠定 为将来研究人类肺泡再生和疾病奠定基础。

SUMMARY Alveolar injury and ineffective repair have been hypothesized to underlie the pathogenesis of chronic obstructive pulmonary disease and pulmonary fibrosis. While genome-wide association studies and clinical specimens have suggested a role for chronic stress, inflammation and DNA damage signaling, the underlying mechanisms and the cell states in which the above pathways are dysregulated during alveolar regeneration remain elusive. In our recent studies, using organoids, single cell transcriptomics and in vivo lung injury models, we uncovered a previously uncharacterized, transient, pre-AEC1 transitional cell state (PATS), traversing between AEC2 and AEC1 in alveolar regeneration. Interestingly, pathway analysis for genes expressed in PATS showed a significant enrichment for targets of transcription factors TP53 and SOX4, and DNA damage repair pathway. We also found that this cell state is vulnerable to stretch mediated DNA damage during differentiation of cuboidal AEC2 into extremely flat and thin AEC1. Conditional ablation of Tp53 and Sox4 in AEC2s revealed a dramatic decrease in the number of AEC1, and a significant increase in the number of PATS. These data suggest an essential role for transcription factors TP53 and SOX4 in regulating AEC2 to AEC1 differentiation via pre-AEC1 transitional state and the DNA damage repair during alveolar regeneration. Based on our preliminary data, we hypothesize that the AEC2 progenitors go through a novel and molecularly distinct pre-AEC1 transitional state to differentiate into AEC1. We also hypothesize that TP53 and SOX4 -mediated mechanisms are essential for the cell cycle arrest, cell adhesion, cell stretching, and DNA damage repair pathway during differentiation of AEC2 to AEC1. The major objectives of this proposal are to molecularly and functionally characterize the newly identified pre- AEC1 transitional state and to study the mechanisms governing this cell state in alveolar regeneration. In Aim1, we will study the molecular identity, the temporal dynamics and the plasticity of a novel pre-AEC1 transitional state in alveolar regeneration. In Aim2, we will test the hypothesis that TP53 and SOX4 mediated mechanisms control cell cycle regulation, cell adhesion, and DNA damage repair pathways in pre-AEC1 transitional state during AEC2 differentiation into AEC1. We will use organoid models, in vivo genetic and pharmacological loss- of-function models, and molecular assays to study these specific aims. This work has taken on added importance, as recent genome-wide association studies revealed mutations in the components of the DNA damage repair signaling as one of the major drivers for emphysema and pulmonary fibrosis. Therefore, our finding that stretch associated DNA damage in the pre-AEC1 transitional state makes it potentially vulnerable to lung diseases. Thus, the outcomes from the proposed studies will have broader significance and will lay the foundation for future studies involving human alveolar regeneration and diseases.