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: 7772229
• 负责人: Mary Kathryn Iovine
• 金额: $ 7.45万
• 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/7772229
• 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，表明间隙连接通讯是正常组织稳态和功能所必需的。 例如，人类 CX43 的错义突变会导致骨骼畸形和其他与常染色体显性眼齿指发育不良 (ODDD) 相关的多效性表型。 目前尚不清楚错义突变如何导致疾病表型。 一种可能性是突变以“显性失活”方式起作用，有效地改变或抑制 Cx43 间隙连接的正常耦合特性。 然而，利用哺乳动物系统很难将特定错义等位基因的功能与特定表型直接关联起来。 斑马鱼 cx43 突变导致短鳍表型，其特征是骨鳍条长度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 的三个错义等位基因将鳍长度、节段长度和细胞增殖的缺陷关联起来。 斑马鱼基因组还包含一个类似 cx43 的基因，称为 cx40.8。 有趣的是，在鳍再生过程中，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，而不是主要定位于质膜。 然而，Cx40.8 的敲除会导致与 Cx43 功能降低类似的细胞增殖和节段长度缺陷，表明 Cx40.8 在调节鳍生长中发挥着积极作用。 一种可能性是 Cx40.8 与 Cx43 寡聚并直接修饰 Cx43 间隙连接的活性。 因此，斑马鱼 Cx43 和 Cx40.8 可能代表检查 Cx43 异源寡聚化及其对间隙连接组装和耦合特性的影响的自然系统。 本申请的具体目标 1 和 2 是确定 Cx40.8 是否在体内与 Cx43 发生物理相互作用，以及 Cx40.8/Cx43 间隙连接是否表现出不同的电耦合特性。 这种相互作用和调节的证据可能揭示调节间隙连接耦合的内源机制。 具体目标 3 是确定 Cx40.8 保留在高尔基体中的分子机制。 事实上，保留可能在发育上受到调节，使得在某些条件下Cx40.8被排除在间隙连接之外，而在不同条件下Cx40.8被允许与Cx43异源寡聚化。 这些目标的结果将为 Cx43 错义等位基因如何导致 ODDD 疾病表型的潜在机制提供新的见解。 在人类中，CX43 中的错义等位基因与眼齿指发育不良有关。 这种疾病以常染色体显性遗传的方式传播，表明野生型和错义形式的蛋白质的相互作用导致了疾病相关的表型。 相关性：本申请旨在评估斑马鱼 cx43 和斑马鱼 cx43 样蛋白 cx40.8 的相互作用。 目的是揭示密切相关的连接蛋白的共表达是否可以改变体外间隙连接特性以及体内 cx43 相关表型。