VITAMIN D HORMONE MECHANISM OF ACTION IN CULTURED CELLS

本更新申请建议继续调查 1，25-二羟维生素D_3（1，25（OH）_2D_3）激素在细胞中的作用 文化 我们计划扩大发表的意见， 这项资助的前三年在小鼠成纤维细胞（3 T6）、大鼠 骨肉瘤（ROS 17/2.8）成骨细胞样细胞，猪（LLC- PK 1）和猴（LLC-MK2）肾细胞、人白血病（HL- 60)细胞和人成纤维细胞， 依赖性佝偻病II型 生物反应， 监测包括1，25（OH）2D 3受体上调， 钙结合蛋白D28 k（CaBP） 诱导，25（OH）维生素D3- 240水解酶分解代谢酶活性， 以及细胞生长和分化。 待测试的假设 是1，25（OH）2D 3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 最终编码的蛋白质会影响 生物反应 我们最近筛选了一个表达载体 文库与我们的受体单克隆抗体，以获得cDNA 鸟类1，25（OH）2D 3受体，并建议利用这一点， 通过核酸筛选哺乳动物1，25（OH）2D 3受体cDNA 酸杂交筛选合适的文库。 如果 成功后，我们建议使用哺乳动物1，25（OH）2D 3受体 cDNA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 即使部分，将促进以下新的实验， 培养细胞 i）1，25（OH）2D 3受体的表征 通过识别天然增强子区域的自我调节 受体基因 （二）阐明受体的作用 通过用受体cDNA转染的磷酸化 具有各种缺失的。 iii）CaBP阴性的转染 但富含CaBP启动子的3 T6细胞。 iv） 将1，25（OH）2D 3受体DNA转染到受体中 来自患者的LLC-MK2缺陷型肾细胞和成纤维细胞 对1，25（OH）2D 3具有抗性，以确定生物反应性 恢复了 v）探讨HL-60细胞分化的机制 通过将耐药HL-60母细胞与人 1，25（OH）2D 3受体。 最后，受体的反义mRNA 将被纳入，以确定其对1，25（OH）2D 3的影响 行动上 这些研究不仅应提供最终的测试， 维生素D作用的核受体假说，但应 增加我们对临床维生素D的基本机制的了解， 耐药性和1，25（OH）2D 3的潜在抗癌作用。

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.