BIOCHEMISTRY AND MOLECULAR BIOLOGY OF ABSORPTIVE HYPERCALCIURIA

该项目的广泛和长期目标是确定是否 1，25-二羟维生素D3 [1，25（OH）2D3]合成或 这种类固醇激素的分子作用提供了维生素D的基础， 吸收性高钙尿症（AH）。具体来说，我们将 专注于维生素D受体的生物化学和分子生物学 (VDR)寻找肠道钙吸收增加的原因 总是发现在。这一目标将通过6项具体措施来实现。 目标。 目的1探讨皮肤成纤维细胞VDR的生物化学特性 （数量，亲和力）和功能（24-OHase活性） 啊 目标2也将利用皮肤成纤维细胞的研究，但将使用分子 生物技术定量VDR mRNA，测量 1，25（OH）2D3诱导VDR mRNA上调并评估VDR mRNA 合成和稳定性。在这两个目标中要检验的假设是： VDR mRNA的稳定性在初级受体中增加， 上调（由于VDR mRNA非编码区异常）或 由于易化的1-OH酶引起的次级受体上调。 目标3将继续探索VDR等位基因变异的性质 通过确定它是否是一个非编码区， 疾病的标志物，如果是，进一步检查其位置和性质 的改变。其他研究也将评估是否类似 多态性可以在5 ′-非编码区中发现。 目标4将开始探索25-OHD 3 - 1 α-OH酶的活性， 适当的探头，如果和当他们变得可用。 目的5将检查磷酸盐治疗对生化和 分子生物学参数。 Aim 6将探索其他经典维生素D靶细胞的用途， 许多上述研究，同时服务于验证 在成纤维细胞中进行观察。总体而言，这些具体目标应 描述VDR基因异常的贡献，或 该基因的过度表达，对AH的发展。

The broad, long-term objective of this project is to ascertain whether alterations in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] synthesis or in the molecular action of this steroid hormone provide the basis of vitamin D- dependent forms of absorptive hypercalciuria (AH). Specifically, we will focus on the biochemistry and molecular biology of the vitamin D receptor (VDR) to search for the cause of increased intestinal calcium absorption invariably found in AH. This objective will be approached by 6 specific aims. Aim 1 will explore the biochemistry of the VDR in skin fibroblasts (number, affinity,) and function (24-OHase activity) from patients with AH. Aim 2 will also utilize studies in skin fibroblasts but will use molecular biological techniques to quantitate VDR mRNA, measure the sensitivity of the 1,25(OH)2D3-induced upregulation of VDR mRNA and assess VDR mRNA synthesis and stability. The hypothesis to be tested in these two aims is that VDR mRNA stability would be increased in primary receptor upregulation (due to abnormality in the VDR mRNA non-coding region) or in secondary receptor upregulation due to facilitated 1-OHase. Aim 3 will continue to explore the nature of the VDR allelic variation observed in' the 3'-noncoding region by ascertaining whether it is a marker for the disease and, if so, further examine its location and nature of the alteration. Additional studies will also assess whether similar polymorphisms may be found in the 5'-noncoding region. Aim 4 will begin to explore the activity of the 25-OHD3-1alpha-OHase using appropriate probes if and when they become available. Aim 5 will examine the effect of phosphate therapy on the biochemical and molecular biological parameters outlined above. Aim 6 will explore the use of other classic vitamin D target cells for many of the above mentioned studies while serving to verify the observations made in fibroblasts. Overall, these specific aims should delineate the contribution of abnormalities in the VDR gene, or in the over-expression of this gene, to the development of AH.