Alveolar Basement Membrane/Cell Interactions in the Lung

描述（由申请人提供）：纤维化肺病的发病机制涉及增殖的肺泡II型细胞（AT 2）无法有效分化为I型细胞（AT 1），导致上皮修复缺陷、不可逆损伤、功能丧失和纤维化。通常控制这一过程的机制尚未完全了解，因此可能在纤维化肺病中改变的潜在调节分子或途径尚未阐明。我们提出，正常肺泡细胞分化的关键是肺泡细胞类型下的细胞外基质（ECM）微环境的相对硫酸化。这反过来又控制两个重要的分化因子的表达：叉头（Fox）转录因子家族的成员和特定的无翅（Wnt）信号通路。这些因子与转化生长因子（TGF）的表达和信号传导结合作用，其增强Wnt信号传导靶点，共同驱动细胞分化过程并建立稳定的肺泡表型。在这个提议中，我们将表明，在与肺泡再上皮化相关的增殖事件之后，肺泡上皮细胞与其ECM微环境之间存在关键的动态平衡。这是由固定和可溶性硫酸化ECM显着调节，其下游效应是特异性增强Wnt 7a和Foxa 1表达，其与TGF β一起调节从AT 2表型的转变并控制AT 1细胞分化。要解决的假设是：在DNA合成和胞质分裂之后，子AT 2细胞暴露于高水平的硫酸化ECM触发Foxa 1和Wnt 7a的表达增强，同时TGF？表达和信号传导增加，这有效地促使AT 2细胞分化为AT 1细胞。为了解决这一假设，我们将利用来自人类的分离的AT 2细胞和来自啮齿动物的正常以及条件性基因敲除和过表达子，与人类和啮齿动物成纤维细胞-AT 2细胞微环境的重要调节因子-进行传统和改良的共培养。细胞和ECM将用Fox表达、TGF β和Wnt表达和信号传导以及ECM组成的特异性增强剂或抑制剂选择性地修饰。这些结果将作为一个上下文的背景下，在整个动物模型的肺泡损伤和纤维化的蛋白质和/或基因表达方法的目标分子的检查。这些研究的结果预计将提供必要的信息，需要更好地了解基本的细胞-细胞和细胞-细胞外基质的关系，在肺泡上皮细胞的稳态，以及机制，引导肺纤维化的变化，肺泡损伤和/或疾病的结果的发病机制。公共卫生相关性：疾病或基于环境的有毒物质会损害或损伤肺内表面的细胞，这些细胞如何自我繁殖在很大程度上决定了肺是否愈合。为了实现这一点，特定的细胞必须分裂并分化成其他细胞，这些细胞执行至关重要的肺特异性功能。这一过程的中断会产生严重后果，导致修复错误和肺的永久性损伤，并损害功能。这笔赠款将定义控制这一过程的具体机制，并在这样做的过程中，使新的和创新的方法，以促进受伤的肺部愈合过程的发展。

DESCRIPTION (provided by applicant): The pathogenesis of fibrotic lung disease involves the inability of proliferating alveolar type II cells (AT2) to differentiate effectively into type I (AT1) cells, leading to faulty epithelial repair, irreversible damage, loss of function, and fibrosis. The mechanisms that normally control this process are not fully understood, so potential regulatory molecules or pathways that may be altered in fibrotic pulmonary diseases have not been elucidated. We propose that the key to normal alveolar cell differentiation is the relative sulfation of the extracellular matrix (ECM) microenvironment underlying alveolar cell types. This in turn controls expression of two important differentiation factors: a member of the forkhead (Fox) family of transcription factors and specific wingless (Wnt) signaling pathways. These factors act in conjunction with expression and signaling of transforming growth factor ¿ (TGF¿), which enhances Wnt signaling targets, to collectively drive the cell differentiation process and establish stable alveolar phenotypes. In this proposal, we will show that following proliferative events associated with re-epithelialization in the alveolus, there is a critical, dynamic balance between alveolar epithelial cells and their ECM microenvironment. This is significantly modulated by both fixed and soluble sulfated ECMs, whose downstream effects are to specifically enhance Wnt7a and Foxa1 expression, which act together with TGF¿ to regulate the shift from the AT2 phenotype and control AT1 cell differentiation. The hypothesis to be addressed is: Following DNA synthesis and cytokinesis, exposure of the daughter AT2 cell to high levels of sulfated ECMs triggers enhanced expression of Foxa1 and Wnt7a in parallel with increased TGF¿ expression and signaling, which converge to effectively drive differentiation of AT2 to AT1 cells. To address this hypothesis, we will utilize isolated AT2 cells from humans and normal as well as conditional gene knockouts and overexpressors from rodents in traditional and modified co-culture with human and rodent fibroblasts - important regulators of the AT2 cell microenvironment. Cells and ECMs will be selectively modified with specific enhancers or inhibitors of Fox expression, and TGF¿ and Wnt expression and signaling, and ECM composition. These results will serve as a contextural backdrop for examination of targeted molecules by protein and/or gene expression methods in a whole animal model of alveolar injury and fibrosis. Results of these studies are expected to provide essential information needed to better understand basic cell-cell and cell-ECM relationships in alveolar epithelial homeostasis as well as the mechanisms that steer the pathogenesis of fibrogenic change in the lung as a consequence of alveolar injury and/or disease. PUBLIC HEALTH RELEVANCE: Disease or environmentally-based toxic agents can damage or injure cells that line the internal surfaces of the lung, and how these cell repopulate themselves in large part determines whether the lung heals or not. To accomplish this, specific cells must divide and differentiate into other cells that carry out critically important lung-specific functions. Interruption of this process has serious consequences resulting in faulty repair and permanent damage to the lung and compromises function. This grant will define the specific mechanisms that control this process, and in doing so, enable the development of new and innovative ways to promote the healing process in injured lungs.