Defining the molecular mechanisms underlying human RPE plasticity

描述（由申请人提供）：在本提案中，我们试图了解人原代视网膜色素上皮细胞（RPE）如何分化为间充质谱系细胞。我们已经表明，RPE来源于各种来源，包括成人，胎儿，ESC衍生，ARPE-19和牛，获得间充质表型。此外，当置于各自的分化培养基中时，从单个细胞克隆扩增的原代成人RPE产生脂肪细胞、软骨细胞和骨。这个主题很重要，因为RPE通常是稳定的上皮，但会发生对视力有害的增殖和化生变化。视网膜色素上皮化生为间充质相关细胞一直与视网膜疾病有关，但这些变化的机制仍然是一个谜。在目标1中，我们将研究这种可塑性是如何在表观基因组水平上编码的。最近的研究已经确定了启动子和增强子上的表观遗传特征，这些特征表明了活性基因和稳定基因（如果给予适当的提示，可以激活的基因）。我们将询问与间充质谱系相关的基因是否在人RPE细胞中处于平衡构象，以及它们是否在分化成骨细胞、软骨细胞和脂肪细胞途径时转变为活性形式。在目标2中，我们将确定刺激这种分化的外源性因素，通过测试已知在向成骨细胞、软骨细胞和脂肪细胞命运发育期间作用于间充质干细胞（MSC）的因素，包括TGF-β超家族的成员，以及通过使用小分子抑制剂，这将有助于鉴定减弱该过程的治疗剂。与RPE化生相关的一个突出的异常细胞命运是骨化，因此了解RPE细胞如何进行成骨特别重要。在目标3中，我们将重点关注RPE在向这种命运过渡时所经历的步骤，评估是否遵循了正常成骨生成的转录因子序列，或者是否激活了异常途径。初步研究表明，在向骨转化的RPE中，必需的成骨转录因子Runx 2显著上调。我们将确定使用shRNA方法敲低Runx 2是否会抑制RPE成骨。这三个目标共同解决了中心机制，从外源因素到基因调控和RPE表型可塑性基础的表观遗传变化。已知RPE稳定性的破坏发生在几种重要的视网膜疾病中，例如视网膜前膜形成、黄斑变性和眼球萎缩，并且理解这种可塑性将为开发这些病症的治疗提供新的方法。这些发现也将与理解维持RPE作为稳定的，色素沉着的，鹅卵石极化的上皮层的因素直接相关，这是特别及时的，因为来自人类胚胎干细胞的RPE细胞正在进入视网膜疾病的临床试验，必须采取措施尽量减少化生变化，以确保安全移植条件的发展。 公共卫生相关性：RPE化生和异常命运（如骨形成）的获得与许多视网膜疾病（如增殖性玻璃体视网膜病变）和疼痛病症（如肺结核眼）相关。在这里，我们将讨论这种情况发生的机制，从确定导致这些变化的因素，了解这些因素如何改变基因表达。这项研究与RPE疾病直接相关，但也有超越 视网膜，以及影响其他上皮的疾病，这些疾病同样可以表现出与病理学相关的化生变化。

DESCRIPTION (provided by applicant): In this proposal we seek to understand how human primary retinal pigment epithelial cells (RPE) differentiate into cells of the mesenchymal lineage. We have shown that RPE derived from a variety of sources, including human adult, fetal, ESC-derived, ARPE-19 and bovine, acquire mesenchymal phenotypes. Moreover, primary adult human RPE that were clonally expanded from a single cell produced adipocytes, chondrocytes and bone when placed in the respective differentiation media. This topic is important because RPE, while normally a stable epithelium, can undergo proliferative and metaplastic changes detrimental to vision. RPE metaplasia into mesenchymal-related cells has been long -associated with retinal disease, yet the mechanism underlying these changes remains a mystery. In aim 1 we will study how this plasticity is encoded at the level of the epigenome. Recent studies have identified epigenetic signatures at promoters and enhancers that indicate active genes and poised genes (those that could be activated if given appropriate cues). We will ask whether genes associated with mesenchymal lineages are in a poised conformation in human RPE cells, and whether they transit to an active form upon differentiation into osteo-, chondro- and adipocyte pathways. In aim 2 we will determine exogenous factors stimulating this differentiation, by testing factors known to act on mesenchymal stem cells (MSCs) during development towards osteogenic, chondrocyte and adipocyte fates, including members of the TGF-beta superfamily, and by use of small molecule inhibitors, which will help identify therapeutics to attenuate this process. One of the prominent abnormal cell fates associated with RPE metaplasia is ossification, hence it is particularly important to understand how RPE cells undergo osteogenesis. In aim 3 we will focus on the steps RPE move through when transitioning towards this fate, evaluating whether the transcription factor sequence known for normal osteogenesis generation is followed, or whether abnormal pathways are activated. Preliminary studies show dramatic upregulation of the essential osteogenic transcription factor Runx2 in RPE undergoing transformation towards bone. We will determine whether knocking down Runx2 using a shRNA approach will inhibit RPE osteogenesis. Together, these three aims address the central mechanisms, from exogenous factor through to gene regulation and epigenetic changes that underlie RPE phenotypic plasticity. Disruption of RPE stability is known to occur in several important retinal diseases such as epiretinal membrane formation, macular degeneration and phthsis bulbii, and understanding this plasticity will provide novel approaches toward developing treatments of these conditions. The findings will also have direct relevance to understanding the factors that maintain the RPE as a stable, pigmented, cobblestone polarized epithelial layer, which is especially timely given that RPE cells derived from human embryonic stem cells are entering clinical trials for retinal disease, and steps must be taken to minimize metaplastic changes to ensure development of safe transplantation conditions. PUBLIC HEALTH RELEVANCE: RPE metaplasia and acquisition of abnormal fates such as bone formation is associated with a number of retinal diseases such as proliferative vitreo-retinopathy and painful conditions such as phthisical eye. Here we will address the mechanism by which this occurs, from identifying the factors that cause these changes, to understanding how those factors alter gene expression. This study has direct relevance to RPE disease, but also has implications beyond the retina, to diseases affecting other epithelia that similarly can exhibit metaplastic changes associated with pathology.