Regulation of bone growth by hetero-oligomerization of Cx43 and Cx40.8

Cx43 和 Cx40.8 异源寡聚化对骨生长的调节

• 批准号: 8039261
• 负责人: Mary Kathryn Iovine
• 金额: $ 7.14万
• 依托单位: LEHIGH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-05 至 2012-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8039261
• 关键词: Alleles; Biological Assay; Bone Growth; Cell Cycle; Cell Proliferation; Cell membrane; Cells; Chimeric Proteins; Communication; Connexin 43; Connexins; Coupling; Defect; Disease; Dominant-Negative Mutation; Dyes; Event; Exhibits; Gap Junctions; Genes; Goals; Golgi Apparatus; Growth; Homeostasis; Human; Immunofluorescence Immunologic; In Vitro; Lead; Length; Missense Mutation; Molecular; Mutation; Natural regeneration; Normal tissue morphology; Phenotype; Play; Population; Property; Proteins; Regulation; Role; Signal Transduction; Stem cells; System; Testing; Work; Zebrafish; disease phenotype; human disease; in vivo; insight; knock-down; malformation; mutant; novel; oculodentodigital dysplasia; skeletal; tissue culture; zebrafish genome

DESCRIPTION (Provided by Applicant): Mutations in connexin genes lead to human disease, suggesting that gap junctional communication is required for normal tissue homoeostasis and function. For example, missense mutations in human CX43 cause skeletal malformations and other pleiotropic phenotypes associated with the autosomal dominant oculodentodigital dysplasia (ODDD). It is unclear how missense mutations might cause disease phenotypes. One possibility is that the mutations act in a "dominant-negative" manner, effectively modifying or inhibiting the normal coupling properties of the Cx43 gap junctions. However, it has been difficult to correlate directly the function of particular missense alleles with particular phenotypes using mammalian systems. Mutations in zebrafish cx43 cause the short fin phenotype, characterized by defects in the length of bony fin ray segments and reduced levels of cell proliferation. Using the relatively simple system of zebrafish fin growth, it has been possible to correlate defects in fin length, segment length, and cell proliferation using three missense alleles of zebrafish cx43. The zebrafish genome also contains a cx43-like gene called cx40.8. Interestingly, Cx40.8 is expressed in the same cells as Cx43 during fin regeneration. Unlike Cx43, Cx40.8 appears to localize to the Golgi apparatus rather than primarily at the plasma membrane. Yet, knock-down of Cx40.8 causes similar defects in cell proliferation and segment length as reduced Cx43 function, indicating that Cx40.8 plays an active role in regulating fin growth. One possibility is that Cx40.8 oligomerizes with Cx43 and directly modifies the activity of Cx43 gap junctions. Thus, zebrafish Cx43 and Cx40.8 might represent a natural system to examine Cx43 hetero-oligomerization and its effects on gap junction assembly and coupling properties. Specific Aims 1 and 2 of this application are to determine if Cx40.8 physically interacts with Cx43 in vivo, and if Cx40.8/Cx43 gap junctions exhibit distinct electrical coupling properties. Evidence for this interaction and regulation may reveal an endogenous mechanism that regulates gap junctional coupling. Specific Aim 3 is to identify the molecular mechanism that allows Cx40.8 to be retained in the Golgi. Indeed, retention may be developmentally regulated so that under certain conditions Cx40.8 is excluded from gap junctions, while under different conditions Cx40.8 is permitted to hetero-oligomerize with Cx43. Results from these aims will provide novel insights into the underlying mechanism of how Cx43 missense alleles cause ODDD disease phenotypes. In humans, missense alleles in CX43 are associated with oculodentodigital dysplasia. This disease is transmitted as autosomal dominant, indicating that the interaction of both wild-type and missense forms of the proteins contributes to the disease-related phenotypes. RELEVANCE: This application seeks to evaluate the interactions of zebrafish cx43 and a zebrafish cx43-like protein, cx40.8. The goal is to reveal if the co-expression of closely related connexin proteins can modify gap junctional properties in vitro, and cx43-related phenotypes in vivo.

描述（由申请人提供）：连接蛋白基因的突变导致人类疾病，这表明正常组织同位抑制剂和功能需要间隙连接性通信。 例如，人类CX43中的错义突变引起骨骼畸形和其他与常染色体显性眼牙性眼牙性异常发育不良相关的多效性表型（ODDD）。 目前尚不清楚错义突变如何引起疾病表型。 一种可能性是该突变以“显性阴性”方式起作用，从而有效地修改或抑制CX43间隙连接的正常耦合特性。 但是，很难直接将特定错位等位基因的功能与使用哺乳动物系统的特定表型相关联。 斑马鱼CX43中的突变导致短鳍表型，其特征是骨鳍射线段的缺陷和细胞增殖水平降低。 使用相对简单的斑马鱼鳍生长系统，可以使用斑马鱼CX43的三个错位等位基因将鳍长度，段长度和细胞增殖的缺陷相关联。 斑马鱼基因组还包含一个称为CX40.8的CX43样基因。 有趣的是，在FIN再生过程中，CX40.8在与CX43相同的细胞中表达。 与CX43不同，CX40.8似乎定位于高尔基体设备，而不是主要在质膜上。 然而，CX40.8的敲除在细胞增殖和段长度上与CX43功能降低导致相似的缺陷，这表明CX40.8在调节FIN生长中起着积极作用。 一种可能性是CX40.8用CX43寡聚，并直接修改CX43间隙连接的活性。 因此，斑马鱼CX43和CX40.8可能代表一个自然系统，用于检查CX43杂极化及其对间隙连接组装和耦合特性的影响。 本应用的具体目的1和2是确定CX40.8在体内是否与CX43物理相互作用，并且CX40.8/CX43间隙连接符号是否具有独特的电耦合特性。 这种相互作用和调节的证据可能揭示了一种内源性机制，该机制可以调节间隙连接耦合。 具体目标3是确定允许将CX40.8保留在高尔基体中的分子机制。 实际上，保留率可能受到发展的调节，以便在某些条件下CX40.8被排除在间隙连接中，而在不同条件下，允许CX40.8与CX43异构型。 这些目标的结果将提供有关CX43错位等位基因如何引起奇数疾病表型的基本机制的新见解。 在人类中，CX43中的错义等位基因与眼义异发育不良有关。 该疾病以常染色体的优势传播，表明蛋白质的野生型和错义形式的相互作用有助于与疾病相关的表型。 相关性：该应用程序旨在评估斑马鱼CX43和斑马鱼CX43样蛋白的相互作用，CX40.8。 目的是揭示密切相关的连接蛋白的共表达是否可以在体外修改间隙连接性特性，以及在体内与CX43相关的表型。