Alveolar Basement Membrane/Cell Interactions in the Lung

描述(申请人提供)：纤维性肺疾病的发病机制涉及增殖的肺泡II型细胞(AT2)不能有效地分化为I型(AT1)细胞，导致上皮修复故障、不可逆转的损伤、功能丧失和纤维化。正常情况下控制这一过程的机制尚不完全清楚，因此在纤维化肺部疾病中可能改变的潜在调节分子或途径尚未阐明。我们认为，肺泡细胞正常分化的关键是肺泡细胞类型下的细胞外基质(ECM)微环境的相对硫酸盐化。这反过来又控制着两个重要的分化因子的表达：叉头(Fox)转录因子家族的一个成员和特定的无翼(Wnt)信号通路。这些因子与转化生长因子的表达和信号传递共同作用，增强Wnt信号转导靶点，共同驱动细胞分化过程，建立稳定的肺泡表型。在这项研究中，我们将展示在肺泡再上皮化相关的增殖事件之后，在肺泡上皮细胞和它们的ECM微环境之间有一个关键的动态平衡。这受到固定和可溶性硫酸盐化ECM的显著调节，其下游作用是特异性地增强Wnt7a和FOXA1的表达，这两种细胞与转化生长因子β一起作用，调节AT2表型的转变，控制AT1细胞的分化。需要解决的假设是：在DNA合成和胞质分裂之后，子代AT2细胞暴露在高水平的硫酸盐ECM下，触发FOXA1和WNT7a的表达增强，同时伴随着转化生长因子β的表达和信号的增加，这两个因素汇聚在一起，有效地推动AT2向AT1细胞的分化。为了解决这一假设，我们将利用从人类和正常动物分离的AT2细胞以及啮齿动物的条件性基因敲除和过度表达，在传统和改进的与人和啮齿动物成纤维细胞的共培养中--AT2细胞微环境的重要调节因素。细胞和ECM将选择性地使用特定的Fox表达增强剂或抑制剂、转化生长因子β和Wnt表达和信号转导以及ECM成分进行修饰。这些结果将作为通过蛋白质和/或基因表达方法在整个肺泡损伤和纤维化动物模型中检查靶分子的背景。这些研究的结果有望提供必要的信息，以更好地了解肺泡上皮细胞动态平衡中的基本细胞-细胞和细胞-ECM关系，以及引导肺泡损伤和/或疾病导致肺纤维化改变的发病机制。与公共健康相关：疾病或基于环境的有毒物质会损害或损害排列在肺内表面的细胞，这些细胞如何自我重新填充在很大程度上决定了肺是否痊愈。要做到这一点，特定的细胞必须分裂并分化为执行至关重要的肺特定功能的其他细胞。这一过程的中断会造成严重的后果，导致错误的修复和对肺的永久性损害，并损害功能。这笔赠款将确定控制这一过程的具体机制，并通过这样做，使新的和创新的方法能够促进受伤肺的愈合过程。

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.