CONSTITUTIVELY ACTIVE PTH/PTHRP RECEPTORS IN VIVO

Jansen型干骺端软骨发育不良症(JMC)的特点是短 由于生长板成熟受损而导致的身高，以及严重的高钙血症 和低磷血症，尽管甲状旁腺激素(PTH)正常/检测不到 或甲状旁腺素相关肽(PTHrP)。在两名无关的JMC患者中，不同的 甲状旁腺激素/甲状旁腺素受体突变被鉴定为导致结构性 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 仍然默默无闻。然而，生长板是最突出的 PTHrP和PTH/PTHrP受体在不同的 时间和空间关系。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和表现 严重的软骨内骨形成异常，因此提供 甲状旁腺素相关蛋白在正常人群中生物学意义的首次直接证据 生长和骨骼发育。缺乏大部分甲状旁腺素/甲状旁腺素rP的小鼠 受体基因表现类似，但在子宫中死亡，表明PTHrP 对生长板的作用是通过PTH/PTHrP受体介导的。 由于这些发现，我们假设，体质活动 甲状旁腺素/甲状旁腺素受体是引起JMC特异性的必要条件和充分条件。 生长板异常。因此，我们建议开发转基因 突变的甲状旁腺激素/甲状旁腺激素受体在 控制任一Il型胶原启动子以指导其表达 到“静止期”、增殖期和肥厚期 软骨细胞和X型胶原启动子直接受体 向肥大的软骨细胞表达。成长板分析 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 对软骨发育的直接和间接作用的剖析。 表现JMC表型的转基因动物将被用来 至少部分拯救PTHrP含量较低的动物。由于本地化的 突变的甲状旁腺素/甲状旁腺素受体的PTHrP样作用 可能显示骨骼异常的逆转/改善，而 泛发性PTHrP缺乏对其他器官的影响没有改变。 建立JMC动物模型，部分野生型PTH/PTHrP 受体基因将被编码突变受体的基因取代。 杂合子动物将被用来确定如何激活受体 突变会导致生长板和矿物质离子异常，以及配对 在这些和PTHrP缺乏的小鼠之间将提供工具来进一步确定 甲状旁腺激素受体的所有作用是否可能通过共同的 甲状旁腺激素/甲状旁腺素受体，或是否涉及不同的受体。这个 因此，拟议的研究将利用一种罕见的人类疾病 确定甲状旁腺素相关蛋白作用的细胞和分子机制 它在软骨成熟和骨延长中的作用。从这些方面获得的见解 研究可能导致JMC的治疗方案，以及更常见的形式 代谢性骨病，如骨质疏松症、肾性骨营养不良，以及 尿毒症儿童的生长发育障碍。

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.