Sphingosine-1-phosphate signaling in branching morphogenesis

1-磷酸鞘氨醇信号传导在分支形态发生中的作用

基本信息

批准号：
7241788
负责人：
LOIS J AREND
金额：
$ 23.4万
依托单位：
UNIVERSITY OF CINCINNATI
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-05-01 至 2009-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7241788
关键词：
Address Adult Animals Antibodies Apoptosis Apoptotic Biological Assay Biological Models Biology Cardiovascular system Cell Proliferation Cell Surface Receptors Cells Chronic Clinical Complex Congenital Abnormality Cultured Cells DNA Sequence Rearrangement Data Defect Development Developmental Biology Developmental Process Disruption Embryo Embryonic Development Environment Enzymes Epidermal Growth Factor Receptor Epithelial Cell Proliferation Epithelial Cells Epithelium Event Exhibits Failure Feedback Functional disorder GDNF gene GTP-Binding Proteins Genes Goals Grant Growth Growth Factor Hypertension Immune system Immunohistochemistry In Situ Hybridization In Vitro Institution Intervention Intracellular Second Messenger Invaded Kidney Kidney Failure Knock-in Mouse Knowledge Laboratories LacZ Genes Ligands Link Lipids Localized Mediating Mediator of activation protein Mesenchyme Metanephric Diverticulum Modeling Morphogenesis Mus Nephrons Organ Organ Culture Techniques Outcome Paracrine Communication Pathologist Pathway interactions Polycystic Kidney Diseases Process Production Range Receptor Protein-Tyrosine Kinases Receptor Signaling Regulation Renal Hypertension Renal function Reporter Research Research Personnel Resources Reverse Transcriptase Polymerase Chain Reaction Role Second Messenger Systems Signal Pathway Signal Transduction Site Sorting - Cell Movement Specificity Sphingolipids Sphingosine Sphingosine-1-Phosphate Receptor System Testing Time Tissues Tyrosine Kinase Receptor Inhibition Universities Work autocrine base cell motility enzyme activity experience feeding improved in vitro Model in vivo inhibitor/antagonist innovation insight malformation migration nephrogenesis novel prevent programs receptor relating to nervous system spatiotemporal sphingosine 1-phosphate sphingosine kinase

项目摘要

DESCRIPTION (provided by applicant): Branching morphogenesis of the embryonic kidney occurs by a tightly regulated interaction between the ureteric bud epithelium and metanephric mesenchyme. Disruption of mechanisms regulating this process can result in defects ranging from complete agenesis to a variety of malformations compromising adult renal function. Sphingosine-1-phosphate (S1P) is a potent bioactive lipid influencing cellular proliferation, apoptosis, migration, and differentiation. S1P regulatory enzymes and S1P receptors exhibit developmentally regulated expression and are critical to developmental processes in other systems. The innovative aspect of this proposal lies in the fact that S1P signaling is closely integrated with growth factors known to influence branching morphogenesis, yet the role of S1P in this process has not been addressed. Our laboratory has focused its research efforts on determining the influence of S1P signaling on kidney development. The results of this work have led us to an understanding that sphingosine kinase activity and S1P receptor signaling are essential for tubulogenesis and that S1P signaling provides a previously unrecognized mechanism contributing to the elaborate process of renal morphogenesis. The long- term goal of this research is to define mechanisms by which S1P signaling mediates kidney development. In this proposal, we will: 1) link activity of S1P regulatory enzymes and spatiotemporal expression of S1P receptors to early morphogenic events of kidney development using enzyme activity assays, real-time RT-PCR, and immunohistochemistry; and 2) demonstrate interaction with growth factors known to regulate branching of the ureteric bud using in vitro model systems including the metanephric kidney organ culture system and cell culture based systems of both ureteric bud epithelia and metanephric mesenchyme. Results from these studies will elucidate the basis of mechanisms that regulate S1P signaling in the developing kidney and contribute to the fundamental understanding of mechanisms regulating kidney development. Defective mechanisms regulating kidney morphogenesis can result in congenital abnormalities and nephron deficiency leading to hypertension and renal failure. Characterization of S1P signaling in the developing kidney will provide novel mechanistic insight to the process of branching morphogenesis, linking activities of previously identified factors with S1P into an integrated model that will advance our understanding of kidney development and elucidate potential sites of dysfunction responsible for developmental abnormalities. Significance: This is a highly significant project, as the sphingosine pathway is increasingly being acknowledged to be of great importance, yet has been minimally explored in organ development. Approach: Preliminary data include demonstrations that an inhibitor of sphingosine kinase (SK) inhibited branching in organ culture, addition of S1P could ameliorate the inhibition of SK effect on branching. These results provide sufficient justification for an R21 level grant aimed at further dissecting the role of sphingosine signaling in kidney development. Specific Aim 1 will further characterize the levels and spatial localization of SK and S1Prs in the developing kidney. These studies are appropriate. The only feasibility issue is whether appropriate antibodies are available, but if not, this can be done by in situ hybridization. For some of the genes in question, LacZ reporter knock-in mice are available as well. Additional studies will use selective receptor inhibitory agents to study the effect on branching. While pharmacological inhibitors are always problematic with regard to specificity, it is also true that the specificity of many of these agents has been dramatically improved in recent years, so that these sorts of studies can no longer be categorically dismissed. In this case they seem well justified. Specific Aim 2 will examine the possibility that known growth factor signaling pathways in the kidney, such as GDNF/c-Ret, are modified by Sphingosines. This is also a novel possibility that is worth examining. Both in vivo and in vitro studies are proposed, and seem feasible and appropriate. Investigators: Dr. Arend is a renal pathologist at the University of Cincinnati, which is a great center for developmental biology research. She has pioneered the study of sphingoisines in kidney development and is ideally suited to carry out the proposed research. Environment: The University of Cincinnati is an outstanding scientific environment, especially for developmental biology. Innovation: These studies are highly innovative.

描述（由申请人提供）：胚胎肾脏的分支形态发生是由于输尿管芽上皮和跨词间质之间的紧密调节的相互作用而发生的。调节此过程的机制的破坏可能导致从完全发育不全到各种畸形损害成人肾功能的缺陷。鞘氨酸1-磷酸盐（S1P）是一种有效的生物活性脂质，影响细胞增殖，凋亡，迁移和分化。 S1P调节酶和S1P受体表现出发育调节的表达，对其他系统的发育过程至关重要。该提案的创新方面在于一个事实，即S1P信号与已知影响分支形态发生的生长因子紧密整合在一起，但是S1P在这一过程中的作用尚未解决。我们的实验室将其研究工作集中在确定S1P信号对肾脏发育的影响上。这项工作的结果使我们了解到，鞘氨醇激酶活性和S1P受体信号对于微管发生至关重要，并且S1P信号传导提供了一种先前未认识到的机制，这有助于肾脏形态发生的精心生成。这项研究的长期目标是定义S1P信号介导肾脏发育的机制。在此提案中，我们将：1）使用酶活性测定，实时RT-PCR和免疫组织化学，将S1P调节酶和S1P受体的时空表达连接到肾脏发育的早期形态发生事件； 2）证明了与已知的生长因子的相互作用，这些生长因子使用体外模型系统（包括肾上腺肾脏器官培养系统和基于输尿管芽上皮和元ephephric间质的细胞培养系统）的生长因子进行了分支。这些研究的结果将阐明调节发育中肾脏中S1P信号传导的机制的基础，并有助于对调节肾脏发育的机制的基本理解。调节肾脏形态发生的有缺陷的机制会导致先天性异常和肾脏缺乏，从而导致高血压和肾衰竭。 S1P信号在发育中的肾脏中的表征将为分支形态发生的过程提供新的机械洞察力，将先前鉴定的因子与S1P的活动联系起来，将S1P与综合模型联系起来，这将提高我们对肾脏发育的理解，并阐明负责发育异常的功能障碍的潜在位置。意义：这是一个非常重要的项目，因为鞘氨醇途径越来越被公认为非常重要，但在器官开发中已被最少探索。方法：初步数据包括证明鞘氨醇激酶（SK）抑制器官培养物中分支的抑制剂，添加S1P可以改善SK对分支的抑制作用。这些结果为R21级赠款提供了足够的理由，旨在进一步剖析鞘氨酸信号在肾脏发育中的作用。特定的目标1将进一步表征SK和S1PR在发育中的肾脏中的水平和空间定位。这些研究是合适的。唯一的可行性问题是是否有适当的抗体可用，但是如果没有，则可以通过原位杂交来完成。对于某些基因，也可以使用LACZ记者敲门小鼠。其他研究将使用选择性受体抑制剂来研究对分支的影响。尽管药理学抑制剂在特异性方面总是有问题的，但近年来，许多这些药物的特异性也得到了显着改善，因此不再能够被绝对地被驳回。在这种情况下，他们似乎很合理。具体目标2将检查肾脏中已知的生长因子信号通路（例如GDNF/c-RET）的可能性。这也是值得检查的新颖可能性。都提出了体内和体外研究，并且似乎可行且合适。研究人员：Arend博士是辛辛那提大学的肾脏病理学家，这是一个出色的发育生物学研究中心。她率先研究了肾脏发育中的鞘氨酸酶的研究，非常适合进行拟议的研究。环境：辛辛那提大学是一个杰出的科学环境，尤其是对于发育生物学而言。创新：这些研究具有很高的创新性。