CONSTITUTIVELY ACTIVE PTH/PTHRP RECEPTORS IN VIVO

Jansen型干骺端软骨发育不良（JMC）的特点是短 由于生长板成熟受损和严重高钙血症导致的身高 和低磷血症，尽管甲状旁腺激素（PTH）正常/检测不到 或PTH相关肽（PTHrP）。 在两个不相关的JMC患者中， PTH/PTHrP受体突变被鉴定为导致组成性 受体激活 虽然这些发现解释了矿物质的变化， 离子代谢，导致骨骼异常的机制 保持模糊。然而，生长板是最突出的 PTHrP和PTH/PTHrP受体以不同的方式表达的位点 时空关系。缺乏大部分基因的老鼠 编码PTHrP在出生后不久死于未知原因， 严重的软骨内骨形成异常，因此提供了 PTHrP在正常人中的生物学重要性的第一个直接证据 生长和骨骼发育。缺乏大部分PTH/PTHrP的小鼠 受体基因显示相似，但在子宫内死亡，表明PTHrP 对生长板的作用通过PTH/PTHrP受体介导。 由于这些发现，我们假设，组成性活跃， PTH/PTHrP受体是引起JMC特异性 生长板异常因此，我们建议开发转基因 突变型PTH/PTHrP受体表达的小鼠， 控制II型胶原启动子以指导其表达 到“休息”，增殖，和上层的肥大 软骨细胞和X型胶原启动子直接受体 表达至肥大软骨细胞。生长板分析 由此产生的动物应该确定哪些软骨细胞介导了 受体依赖性生长板异常，并将允许 解剖对软骨发育的直接和间接作用。 然后将显示JMC表型的转基因动物用于 至少部分地拯救PTHrP较少的动物。由于当地 突变型PTH/PTHrP受体的PTHrP样作用，例如杂交动物 可能显示骨骼异常的逆转/改善，而 全身PTHrP缺乏对其他器官的影响保持不变。 为了开发JMC的动物模型，将野生型PTH/PTHrP的一部分转染到小鼠的肝细胞中。 受体基因将被编码突变受体的基因所取代。 杂合子动物将用于确定如何激活受体 突变导致生长板和矿物质离子异常， 这些小鼠和PTHrP缺乏小鼠之间的差异将提供进一步确定 PTHrP的所有作用是否可能通过共同的 PTH/PTHrP受体，或是否涉及不同的受体。的 因此，拟议中的研究将利用一种罕见的人类疾病， 定义，PTHrP作用的细胞和分子机制， 其在软骨成熟和骨延长中的作用。 从这些见解 研究可能会导致JMC的治疗方案，以及更常见的形式， 代谢性骨疾病，例如骨质疏松症、肾性骨营养不良，以及 尿毒症儿童的生长障碍。

Jansen-type metaphyseal chondrodysplasia (JMC) is characterized by short stature due to impaired growth-plate maturation, and severe hypercalcemia and hypophosphatemia despite normal/undetectable parathyroid hormone (PTH) or PTH-related peptide (PTHrP). In two unrelated JMC patients, different PTH/PTHrP receptor mutations were identified that lead to constitutive receptor activation. While the findings explain the changes in mineral ion metabolism, the mechanisms that lead to the skeletal abnormalities remain obscure. The growth-plate is, however, one of the most prominent sites where PTHrP and the PTH/PTHrP receptor are expressed in a distinct temporal and spatial relationship. Mice which lack' most of the gene encoding PTHrP die shortly after birth from yet unknown causes and display severe abnormalities of endochondral bone formation, and thus provided first direct evidence for the biological importance of PTHrP in normal growth and skeletal development. Mice that lack most of the PTH/PTHrP receptor gene display a similar, but die in utero, indicating that PTHrP actions on growth-plates are mediated through the PTH/PTHrP receptor. Because of these findings, we hypothesize that constitutively active PTH/PTHrP receptors are necessary and sufficient to cause the JMC-specific growth plate abnormalities. We therefore, propose to develop transgenic mice in which the mutant PTH/PTHrP receptors are expressed under the control of either the type Il collagen promotor to direct its expression to "resting", proliferating, and the upper layer of hypertrophic chondrocytes, and the type X collagen promotor to direct receptor expression to hypertrophic chondrocytes. Growth plate analysis of the resulting animals should determine which chondrocytic cells mediate the receptor-dependent growth-plate abnormalities, and would allow the dissection of direct and indirect actions on cartilage development. Transgenic animals that display the JMC phenotype will then be used to rescue, at least partially, PTHrP-less animals. Due to the localized PTHrP-like action of the mutant PTH/PTHrP receptors, such hybrid animals may show a reversal/improvement of skeletal abnormalities, while the impact of generalized PTHrP-deficiency on other organs remains unchanged. To develop an animal model of JMC, portions of the wild-type PTH/PTHrP receptor gene will be replaced with that encoding the mutant receptor. Heterozygous animals will be used to determine how activating receptor mutations cause growth plate and mineral ion abnormalities, and matings between these and PTHrP-less mice will provide tools to determine further whether all actions of PTHrP are likely to be mediated through the common PTH/PTHrP receptor, or whether distinct receptors are involved. The proposed studies will thus take advantage of a rare human disease to define, the cellular and molecular mechanisms of PTHrP action that mediate its role in cartilage maturation and bone elongation. Insights from these studies may lead to treatment protocols of JMC, and more common forms of metabolic bone diseases, e.g. osteoporosis, renal osteodystrophy, and the growth failure in uremic children.