TARGETED OVEREXPRESSION OF PTH RELATED PEPTIDE

小鼠的基因操作实验表明，甲状旁腺激素相关蛋白(PTHrP)是一种发育调节分子。例如，PTHrP在角质形成细胞、乳腺上皮细胞和软骨细胞中的过度表达会导致每个病例的发育表型，而该基因的敲除会导致软骨营养不良，这在出生时是致命的。我们已经通过一种遗传策略挽救了PTHrP缺失，这只小鼠提供了一个窗口，揭示了在具有共同的上皮-间充质形态发生的组织中以前未被认识的PTHrP效应。这种表型包括乳腺上皮发育障碍、牙齿萌出和皮肤的多发性异常。通过转角蛋白-14-甲状旁腺素rP基因将甲状旁腺素转移到这些上皮部位，可以挽救这些发育不良的特征。我们最近还在PTHrP缺失的中枢神经系统中发现了一个与年龄相关的神经退行性过程，似乎有兴奋性毒性作为其基础。在这里，我们建议建立一系列的小鼠模型，以便能够在体内更详细地研究这些调节效应。目的1.我们将在角蛋白-14启动子的控制下建立“tet-off”和“tet-on”系统，为增殖的上皮细胞提供可调控的PTHrP表达。这将使我们能够研究PTHrP在乳房发育的胚胎期和青春期对分支形态发生的影响，试图将PTHrP与牙齿移动/破骨细胞吸收的多次迭代联系起来，并探索皮肤PTHrP缺乏是否与过早衰老有关的问题。目的2.我们将利用同源重组将LacZ报告基因插入到PTHrP等位基因中，并将其作为PTHrP缺失等位基因之一产生拯救的PTHrP缺失小鼠。这将使得对该基因的时空表达的研究具有以前不可能的敏感性和保真度，一个合适的例子是在目标3中建议使用这种小鼠。目标3.这里我们的工作假设是PTHrP在一个自我保护反馈环中发挥作用(去极化产生PTHrP产生1型受体产生L类型的钙通道抑制)，从而神经元可以对抗长潜伏期的兴奋性毒性。LacZ记者将能够对LacZ/PTHrP缺失双杂合子的神经退化过程进行高分辨率研究，我们将尝试通过泛神经元启动子(神经元特异性烯醇化酶)控制下的PTHrP转基因来从遗传上挽救/逆转这一过程。

Gene manipulation experiments in mice indicate that parathyroid hormone-related protein (PTHrP) functions as a developmental regulatory molecule. For example, overexpression of PTHrP in keratinocytes, mammary epithelial cells and chondrocytes results in a developmental phenotype in each case, while knockout of the gene leads to a chondrodystrophy that is lethal at birth. We have rescued the PTHrP-null via a genetic strategy, and this mouse provides a window on previously unappreciated PTHrP effects in tissues that share a common epithelial-mesenchymal morphogenesis. This phenotype includes failures in mammary epithelial development and tooth eruption and multiple abnormalities in skin. Delivery of PTHrP to these epithelial sites via a keratin-14-PTHrP transgene rescues these dysplastic features. We have also recently identified a neurodegenerative process in the PTHrP-null CNS that is age-related and seems to have excitotoxicity as its basis. Here we propose to create a series of mouse models to enable more detailed study of these regulatory effects in vivo. Aim 1. We will create "tet-off" and "tet-on" systems under the control of the keratin-14 promoter to provide regulated PTHrP expression in proliferative epithelia. This will allow us to study PTHrP effects on branching morphorgenesis during both embryonic and adolescent phases of mammary development, attempt to implicate PTHrP in multiple iterations of tooth movement/osteoclastic resorption, and explore the question of whether PTHrP deficiency in skin is associated with premature aging. Aim 2. We will use homologous recombination to insert a lacZ reporter gene into a PTHrP allele and will generate rescued PTHrP-null mice bearing the reporter as one of the PTHrP-null alleles. This will enable the study of the temporospatial expression of the gene with a sensitivity and fidelity not previously possible, an apt example being the proposed use of this mouse in Aim 3. Aim 3. It is our working hypothesis here that PTHrP functions in an autoprotective feedback loop (depolarization yields PTHrP yields type 1 receptor yields inhibition of L-type calcium channels) whereby neurons can combat long-latency excitotoxicity. The lacZ reporter will enable high resolution study of the neurodegenerative process in lacZ/PTHrP-null double heterozygotes, and we will attempt to rescue/reverse this process genetically via a PTHrP transgene under the control of a pan-neuronal promoter (neuron-specific enolase).