BIOCHEMISTRY AND MOLECULAR BIOLOGY OF ABSORPTIVE HYPERCALCIURIA

该项目的广泛、长期目标是确定 1，25-二羟基维生素D3[1，25(OH)2D3]合成中的变化 这种类固醇激素的分子作用为维生素D- 吸入性高钙尿(AH)的依赖形式。具体来说，我们将 关注维生素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 一成不变地出现在AH中。这一目标将由6个具体的 目标。 目的1探讨皮肤成纤维细胞中维生素D受体的生物化学 (数量、亲和力)和功能(24-OHase活性) 阿。 AIM 2也将利用皮肤成纤维细胞的研究，但将使用分子 用生物技术定量检测VDR mRNA，测定其敏感性 1，25(OH)2D3诱导VDR基因表达上调及VDR基因表达 合成和稳定性。在这两个目标中要检验的假设是 VDR mRNA在初级受体中的稳定性将会增强 上调(由于VDR mRNA非编码区异常)或 易化1-羟基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 在‘3’非编码区观察，确定它是否是 疾病的标志，如果是，则进一步检查其位置和性质 改变的原因。其他研究也将评估类似的 在5‘-非编码区可能存在多态。 目标4将开始探索25-OHD3-1α-OHase的活性 适当的探头，如果它们可用的话。 目标5将检查磷酸盐疗法对生化和 上述分子生物学参数。 Aim 6将探索其他经典维生素D靶细胞的用途 上述许多研究在验证 在成纤维细胞中进行的观察。总体而言，这些具体目标应该 描述VDR基因中异常的贡献，或在 该基因的过度表达，对AH的发生发展起重要作用。

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.