Molecular control of a novel transitional cell state in alveolar regeneration

摘要 肺泡损伤和无效的修复被认为是慢性阻塞性肺疾病发病的基础。 肺部疾病和肺纤维化。而全基因组关联研究和临床样本 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，上述途径失控的细胞状态仍然难以捉摸。 在我们最近的研究中，使用有机化合物、单细胞转录和活体肺损伤模型，我们发现 一种先前未描述的、短暂的、AEC1前的过渡细胞状态(PATS)，在AEC2和AEC1之间穿行 肺泡再生中的AEC1。有趣的是，PAT中表达的基因的路径分析显示出显著的 转录因子TP53和Sox4靶标的富集化和DNA损伤修复途径。我们还发现 这种细胞状态在立方AEC2分化为 极平而薄的AEC1。对AEC2中TP53和SOX4的条件消融显示， AEC1的数量，以及PAT的数量显著增加。这些数据表明了一个重要的角色 转录因子TP53和Sox4通过AEC1前转移期调节AEC2向AEC1分化 肺泡再生过程中的状态和DNA损伤修复。根据我们的初步数据，我们假设 AEC2前体细胞经历了一种新颖的、分子上截然不同的AEC1前过渡状态 分化为AEC1。我们还假设TP53和Sox4介导的机制是必不可少的 细胞周期停滞、细胞黏附、细胞伸展和DNA损伤修复途径 AEC2与AEC1的分化。 这项建议的主要目标是从分子和功能上表征新鉴定的前 AEC1的过渡状态，并研究这种细胞状态在肺泡再生中的调控机制。在Aim1， 我们将研究一种新的AEC1前过渡期的分子同一性、时间动力学和可塑性 肺泡再生中的状态。在AIM2中，我们将检验TP53和Sox4介导的机制的假设 在AEC1前过渡期控制细胞周期调节、细胞黏附和DNA损伤修复通路 在AEC2分化为AEC1的过程中。我们将使用有机体模型，体内遗传和药物丢失- 功能模型，以及研究这些特定目的的分子分析。这项工作增加了 重要性，因为最近的全基因组关联研究揭示了DNA成分的突变 损伤修复信号是肺气肿和肺纤维化的主要驱动因素之一。因此，我们的 在AEC1前过渡状态中发现与拉伸相关的DNA损伤使其可能容易患上 肺部疾病。因此，拟议研究的结果将具有更广泛的意义，并将为 为未来涉及人类肺泡再生和疾病的研究奠定基础。

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.