EpCAM (CD326) Function in Epithelial Cell Biology and Immunophysiology

EpCAM (CD326) 在上皮细胞生物学和免疫生理学中的功能

• 批准号: 8937903
• 负责人: Mark Udey
• 金额: $ 114.74万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937903
• 关键词: Adhesions; Adhesives; Alleles; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antigens; Avidity; Binding; Biochemistry; Biological Models; CCR; Cell Adhesion Molecules; Cell surface; Cells; Cellular biology; Collaborations; Conceptus; Contact Dermatitis; Core Facility; Cutaneous; Dendritic Cells; Development; Developmental Delay Disorders; Electrical Resistance; Embryo; Epidermis; Epithelial; Epithelial Cells; Epithelium; Exhibits; Female; Giant Cells; Head; Human; Immune; Immune system; Immunobiology; Immunotherapy; Implant; In Vitro; Intestines; Investigation; Journals; Knockout Mice; Langerhans cell; Leukocytes; Link; Lysosomes; Malignant Neoplasms; Manuscripts; Mus; Mutation; Organ; Organoids; Parietal; Partner in relationship; Phenotype; Physiology; Placenta; Play; Proteins; Publications; Publishing; Reaction; Reporting; Role; Series; Site; Skin; Structure; Surface; Syndrome; TACSTD2 gene; Thymic epithelial cell; Tight Junctions; Time; Tissues; Tumor Antigens; United States National Academy of Sciences; Work; beta-Galactosidase; cancer stem cell; cell motility; claudin-1 protein; embryonic stem cell; in vivo; insight; interest; intestinal epithelium; keratinocyte; lymph nodes; male; migration; novel; pregnant; pup; recombinase; research study; selective expression; transmission process; trophoblast; tumor; vector

More than a decade ago, we reported that among dendritic cells, EpCAM was selectively expressed by epidermal Langerhans cells. In the past few years, we have revisited the utility of EpCAM as Langerhans cell surface marker and have extended our earlier studies and demonstrated that EpCAM expression, in conjunction with other surface markers, differentiates Langerhance cells from all other dendritic cells, including several recently described novel cutaneous dendritic cell subsets. In an effort to elucidate important aspects of EpCAM function in vivo, we identified a pre-existing targeted mouse embryonic stem cell generated by BayGenomics and, working with the CCR Mouse Knockout Core Facility headed by Dr. Lino Tessarollo, generated EpCAM +/- mice in which beta-galactosidase was inserted into one EpCAM allelle. EpCAM +/- mice were viable, fertile and indistinguishable from wild type littermates. Examination of beta-galactosidase expression as a surrogate for EpCAM revealed that EpCAM was expressed in a variety of developing epitehelial structures in skin and other organs. Mating of EpCAM +/- male and female mice gave rise to only wild type and heterozygous animals. No viable EpCAM-deficient pups were obtained. Assessment of embryos in timed pregnant females revealed that EpCAM-deficient embryos implanted and were indistinguishable from EpCAM-sufficient embryos until EGA 8.5 when they began to exhibit developmental delay. EpCAM-deficient embryos became nonviable and were resorbed soon thereafter. We subsequently determined that EpCAM was transiently expressed in conceptus-derived placentas with maximal expression at EGA 8.5-9.5. Detailed studies of placentas associated with EpCAM-deficient embryos revealed that they were small and thin with incompletely developed and poorly vascularized labyrinthine layers. EpCAM-deficient placentas also contained markedly decreased numbers of parietal trophoblast giant cells, a phenotype that has previously been associated with embryonic lethality. The findings were reported in PLoS One in 2009. Thus, although mechanistic aspects of EpCAM function remain to be elucidated, EpCAM clearly has one or more nonredundant roles in normal physiology. To gain additional insights into EpCAM function, we have generated mice with an EpCAM allele that can be conditionally deleted in a lineage-specific fashion. Working with Dr. Tessarollo and using recombineering, we developed a targeting vector that allowed loxp sites to be inserted into the EpCAM locus. Embryonic stem cells with a targeted EpCAM allelle were generated and identified, and then utilized to generate the corresponding mice. Germline transmission of the targeted allele has been confirmed. We have now crossed mice with targeted EpCAM alleles with mice that express the recombinase cre in cells of various lineages to ask and answer relevant questions. Tissues or cells of interest include Langerhans cells, keratinocytes, thymic epithelial cells and intestinal epithelia. The latter tissue is of interest because EpCAM mutations have been causally linked to congential tufting enteropathy, a rare congenital diarrheal syndrome. Experiments that have been completed to date clearly indicate that the targeted EpCAM allele efficiently recombines in several lineages and that this results in phenotypes in several tissues. We are characterizing these phenotypes and are carrying out complementary in vitro studies to gain additional insights into mechanistic aspects of EpCAM function. An initial series of investigations regarding EpCAM function in Langerhans cells has been completed and the findings were published in the Proceedings of the National Academy of Science in 2012. We determined that Langerhans cells that fail to express EpCAM exhibit decreased motility in epidermis and exit epidermis more slowly than EpCAM-expressing Langerhans cells after antigen-induced activation. This results in decreased migration of Langerhans cells from skin to regional lymph nodes and enhanced contact sensitivity reactions. These results definitively linking Langerhans cell migration to function and support the concept that Langerhans cells have a anti-inflammatory function as suggested by others. The results also suggest that EpCAM may be anti-adhesive, rather than pro-adhesive as previously suggested. In studies that are ongoing, we are additionally characterizing the role of EpCAM in Langerhans cell-keratinocyte interactions and in additional immune-related functions. We have also explored mechanisms by which EpCAM regulates intercellular adhesion using human intestinal epithelial cells as a model system. We have determined that EpCAM acts in part by binding tightly to the tight junction-associated protein claudin-7. This interaction sequesters claudin-7 distinctly away from tight junctions and protects claudin-7 and associated claudin-1 from degradation in lysosomes. In the absence of EpCAM, whole cell levels of claudin-7 and claudin-1 decrease dramatically but remaining claudin-7 and claudin-1 becomes tight junction-associated. This results in increased tight junction avidity and increased trans-epithelial electrical resistance. These findings have been described in a manuscript that has been submitted for publication. These results have recently been published in the Journal of Biological Chemistry. Studies intended to delineate the mechanism(s) by which EpCAM stabilizes selected claudins are in progress. We have also begun to propagate mouse intestinal organoids and are assessing EpCAM-function in the this model system. In an effort to explore EpCAM function in other tissues, we are involved in collaborations with several developmental biologists with relevant expertise.

十多年前，我们报告说，在树突状细胞中，EPCAM由表皮兰格汉细胞有选择地表达。在过去的几年中，我们重新审视了EPCAM作为Langerhans细胞表面标记的实用性，并扩展了我们的早期研究，并证明Epcam的表达与其他表面标记相结合，将Langerhance细胞与所有其他树突状细胞区分开，包括最近描述的几个新颖的皮肤病性皮肤病细胞集。为了阐明体内EPCAM功能的重要方面，我们确定了由Baygenomics产生的预先存在的靶向小鼠胚胎干细胞，并与Lino Tessarollo博士领导的CCR小鼠基因敲除核心设施一起生成了Epcam +/-鼠标，生成了beta-Galactosidase inter Beta-Galactosidase inter inte beta-galactosidase inte Epcam Allelle intecam Allelle。 EPCAM +/-小鼠与野生型同窝仔相可行，肥沃和无法区分。对β-半乳糖苷酶表达作为EPCAM的替代物的研究表明，EPCAM在皮肤和其他器官中的各种发育中的EpiteHelial结构中表达。 EPCAM +/-雄性和雌性小鼠的交配仅引起野生型和杂合动物。未获得可行的EPCAM缺陷幼崽。对定时怀孕女性的胚胎的评估表明，植入的EPCAM缺陷胚胎与Epcam失去的胚胎无法区分，直到EGA 8.5开始表现出发育延迟。 EPCAM缺陷型胚胎变得不可行，此后很快被吸收。随后，我们确定EPCAM在概念衍生的胎盘中瞬时表达，在EGA 8.5-9.5处最大表达。与EPCAM缺陷胚胎相关的胎盘的详细研究表明，它们又小又薄，具有不完全发育且血管化的迷宫层不完全。 EPCAM缺陷型胎盘还包含明显减少的顶叶滋养细胞巨细胞，这种表型以前与胚胎致死性有关。这些发现在2009年的PLOS中报道了。因此，尽管EPCAM功能的机械方面仍有待阐明，但EPCAM显然在正常生理学中具有一个或多种非冗余作用。为了获得对EPCAM功能的更多见解，我们已经生成了具有EPCAM等位基因的小鼠，该等位基因可以以特定于谱系的方式有条件地删除。与Tessarollo博士合作并使用重新组合，我们开发了一个靶向向量，该向量允许LOXP位点插入EPCAM基因座。生成并鉴定出具有靶向EPCAM等位基因的胚胎干细胞，然后用来生成相应的小鼠。靶向等位基因的生殖线传播已得到证实。现在，我们已经与靶向EPCAM等位基因的小鼠与在各种谱系细胞中表达重组酶CRE的小鼠越过小鼠，以询问和回答相关问题。感兴趣的组织或细胞包括Langerhans细胞，角质形成细胞，胸腺上皮细胞和肠上皮细胞。后一种组织很感兴趣，因为EPCAM突变与聚集的腹膜疗法（一种罕见的先天性腹泻综合征）有因果关系。迄今已完成的实验清楚地表明，靶向的EPCAM等位基因有效地重组了几个谱系，这会导致几种组织中的表型。我们正在表征这些表型，并正在进行互补的体外研究，以获得对Epcam功能机械方面的更多见解。关于Langerhans细胞中EPCAM功能的一系列研究已经完成，发现在2012年美国国家科学院会议录中发表了。我们确定，未能表达EPCAM的Langerhans细胞表现出在表皮中表现出的运动性降低，并且在表达Epcam的表皮比表达较慢的langerhans细胞比表达langerhans细胞的表现更为缓慢。这导致Langerhans细胞从皮肤到区域淋巴结的迁移降低，并增强了接触敏感性反应。这些结果确定地将Langerhans细胞迁移联系起来，并支持Langerhans细胞具有抗炎功能的概念。结果还表明，如先前建议的那样，EPCAM可能具有抗粘性，而不是促粘性。在正在进行的研究中，我们还表征了EPCAM在Langerhans细胞 - 甲状腺细胞相互作用以及其他免疫相关功能中的作用。我们还探索了EPCAM使用人肠上皮细胞作为模型系统来调节细胞间粘附的机制。我们已经确定EPCAM部分通过与紧密相关的蛋白Claudin-7紧密结合。这种相互作用隔离了Claudin-7明显远离紧密连接，并保护Claudin-7和相关的Claudin-1免受溶酶体降解。在没有EPCAM的情况下，Claudin-7和Claudin-1的整个细胞水平急剧下降，但剩余的Claudin-7和Claudin-1与连接相关。这导致紧密的连接率增加并增加了跨上皮电阻。这些发现已在已提交出版的手稿中描述。这些结果最近发表在《生物化学杂志》上。旨在描述EPCAM稳定选定Claudins的机制的研究。我们还开始传播小鼠肠道器官，并正在评估该模型系统中的EPCAM功能。为了探索其他组织中的EPCAM功能，我们参与了与具有相关专业知识的几位发展生物学家的合作。