VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

这份续签申请建议继续调查 1，25-二羟基维生素D3(1，25(OH)2D3)在细胞内的激素作用 文化。我们计划扩大已发表的观察结果。 这项资助的前三年在小鼠成纤维细胞(3T6)，大鼠 骨肉瘤(ROS 17/2.8)成骨样细胞，猪(LLC- PK1)和猴(LLC-MK2)肾细胞，人白血病(HL- 60)来自维生素D患者的细胞和人成纤维细胞。 依赖型软骨病II.生物反应将是 监测包括1，25(OH)2D3受体上调和 通过磷酸化修饰，Calbindin D28k(CABP) 诱导，25(OH)维生素D3-240Hase分解代谢酶活性， 以及细胞的生长和分化。需要检验的假设 1，25(OH)2D3的核受体，在其占据和 磷酸化形式，结合DNA中的增强子序列 关联触发维生素D诱导基因的转录 最终编码的蛋白质影响着无数的 生物反应。我们最近筛选出了一种表达载体 用我们的受体单抗构建文库，获得一个 与禽类1，25(OH)2D3受体结合，并提议利用这一受体 通过核选择哺乳动物1，25(OH)2D3受体cDNA 酸杂交筛选合适的文库。如果 成功，我们建议使用哺乳动物的1，25(OH)2D3受体 筛选小鼠、大鼠和人类基因组文库 各自的天然受体基因。这些试剂，如果获得了 即使在一定程度上，也将促进以下新颖的实验 培养的细胞。I)1，25(OH)2D3受体的特性 通过识别自然界中的增强子区域进行自动调节 受体基因。二)阐明受体的作用 通过转染受体cDNA实现的磷酸化 有各种缺失的。III)CABP阴性的转染法 而含CABP启动子的富含受体的3T6细胞构建。四) 1，25(OH)2D3受体DNA导入受体的研究 患者LLC-MK2缺陷肾细胞和成纤维细胞 通过对1，25(OH)2D3的抗性来确定生物反应性 被修复了。V)探讨HL-60细胞分化的机制 通过将耐药的HL-60原始细胞与人 1，25(OH)2D3受体。最后，针对受体的反义mRNA 将被加入以确定其对1，25(OH)2D3的影响 行动。这些研究不仅应该为 维生素D作用的核受体假说，但应该 有助于我们深入了解临床维生素D的基本机制 1，25(OH)2D3的抗药性及其潜在的抗癌作用。

This renewal application proposes to continue the investigation of 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3) hormone action in cell culture. We plan to expand on published observations made during the first three years of this grant in mouse fibroblasts (3T6), rat osteosarcoma (ROS 17/2.8) osteoblast-like cells, porcine (LLC- PK1) and monkey (LLC-MK2) kidney cells, human leukemia (HL- 60) cells and human fibroblasts from patients with vitamin D- dependent rickets type II. Biological responses that will be monitored include 1,25(OH)2D3 receptor upregulation and modification via phosphorylation, calbindin D28k (CaBP) induction, 25(OH)vitamin D3-240Hase catabolic enzyme activity, and cell growth and differentiation. The hypothesis to be tested is that the nuclear receptor for 1,25(OH)2D3, in its occupied and phosphorylated form, binds enhancer sequences in DNA and this association triggers the transcription of vitamin D induced genes which ultimately code for proteins affecting the myriad of bioresponses. We have recently screened an expression vector library with our receptor monoclonal antibody to obtain a cDNA to the avian 1,25(OH)2D3 receptor and propose to exploit this to select for mammalian 1,25(OH)2D3 receptor cDNAs via nucleic acid hybridization screening of appropriate libraries. If successful, we propose to use mammalian 1,25(OH)2D3 receptor cDNA to screen mouse, rat and human genomic libraries for the respective natural receptor genes. These reagents, if obtained even in part, would facilitate the following novel experiments in cultured cells. i) Characterization of 1,25(OH)2D3 receptor autoregulation by identifying enhancer regions in the natural receptor gene. ii) Elucidation of the role of receptor phosphorylation through transfection with receptor cDNAs possessing various deletions. iii) Transfection of CaBP negative but receptor-rich 3T6 cells with CaBP-promoter constructs. iv) Transfection of 1,25(OH)2D3 receptor DNA into receptor deficient LLC-MK2 kidney cells and fibroblasts from patients with resistance to 1,25(OH)2D3 to determine if bioresponsiveness is restored. v) Probe the mechanism of HL-60 cell differentiation by transfecting resistant HL-60 blast cells with the human 1,25(OH)2D3 receptor. Finally, antisense mRNA to the receptor will be incorporated to determine its effect on 1,25(OH)2D3 action. These studies should not only provide the final test for the nuclear receptor hypothesis of vitamin D action, but should add to our insight into the basic mechanisms of clinical vitamin D resistance and the potential anticancer effect of 1,25(OH)2D3.