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

描述(申请人提供)：连接蛋白基因突变导致人类疾病，表明缝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遗传性眼齿指发育不良(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。一种可能是这些突变以“显性-负性”的方式起作用，有效地改变或抑制了Cx43缝隙连接的正常耦合特性。然而，使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相关。斑马鱼Cx43的突变导致短鳍表型，其特征是骨鳍射线段长度缺陷和细胞增殖水平降低。利用相对简单的斑马鱼鳍生长系统，已经有可能利用斑马鱼Cx43的三个错义等位基因将鳍长度、片段长度和细胞增殖的缺陷联系起来。斑马鱼基因组还包含一个与Cx43类似的基因，称为cx40.8。有趣的是，在鳍再生过程中，Cx40.8和Cx43在同一个细胞中表达。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基因的敲除导致了与Cx43功能降低类似的细胞增殖和片段长度的缺陷，表明Cx40.8在调节鳍的生长中起着积极的作用。一种可能性是Cx40.8与Cx43寡聚，并直接改变Cx43缝隙连接的活性。因此，斑马鱼Cx43和Cx40.8可能是研究Cx43异源齐聚及其对缝隙连接组装和耦合性质影响的一个自然系统。本申请的具体目的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缝隙连接是否表现出明显的电耦合特性。这种相互作用和调控的证据可能揭示了调节缝隙连接耦合的内源机制。具体目的3是确定使Cx40.8保留在高尔基体中的分子机制。事实上，保留可以被发育调节，使得在某些条件下Cx40.8被排除在缝隙连接之外，而在不同的条件下Cx40.8被允许与Cx43进行异位齐聚。这些目标的结果将为Cx43错义等位基因如何导致ODD疾病表型的潜在机制提供新的见解。在人类中，Cx43的错义等位基因与眼指发育不良有关。这种疾病是以常染色体显性遗传的方式传播的，这表明野生型和错义蛋白的相互作用导致了与疾病相关的表型。 相关性：本申请旨在评估斑马鱼Cx43和类似斑马鱼Cx43的蛋白cx40.8之间的相互作用。我们的目标是揭示密切相关的连接蛋白的共同表达是否可以在体外改变缝隙连接特性，以及在体内是否可以改变Cx43相关的表型。

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.