MECHANISM OF CULTURED CELL RESPONSES TO VITAMIN D3

1，25-二羟基维生素D3（1，25（OH2D3））被认为是功能性的 维生素D3的代谢产物，刺激 通过基因组作用表达特定基因。 最近，这种甾醇被 在调节蛋白质等多种生物现象中， 合成和分泌、酶活性和细胞形态变化 和差异化。 我们建议利用几种哺乳动物培养细胞 已经确定了对维生素D3的适当生物反应的品系， 包括人肠细胞（407）、人白血病细胞（HL-60）、猪 肾细胞（LLC-PK1）、大鼠骨肉瘤细胞（ROS 17/2.8）和小鼠骨肉瘤细胞（ROS 17/2.8） 成纤维细胞（3T6），以阐明甾醇的作用机制。 我们将首先研究 1，25（OH）2D3和完整细胞，其与特异性细胞质 受体及其对转录参数的影响。 调控 将在4个水平检查1，25（OH）2D3的生物活性：i） 分子（CaBP、胶原蛋白和肌动蛋白合成），ii）酶（维生素 D3 - 24-OHase），iii）质膜（Ca ++转运），和iv）细胞 对照（形态学和分化）。 我们会调查 维生素D3代谢产物对培养细胞反应的影响，并试图 将反应程度与给予的维生素D3相关联 代谢物浓度、受体存在和受体 占用率 抑制剂研究（放线菌素D、丁酸盐、放线菌酮）将 用于进一步确定培养细胞响应的水平 至1，25（OH）2D3。 最后，DNA结合位点特异性抗受体抗体 将在显微注射研究中用作选择性阻断剂， 受体介导的激素的核作用，从而允许评估 受体需求和非基因组活性的鉴定 1，25（OH）2D3。 这一做法有望进一步明确 维生素D3和新发现的细胞生物反应之间的关系， 特别是促进这些活动的机制。 我们觉得 提出的实验可能会揭示重要的线索， 调节细胞分化过程，一种现象 对我们理解人类癌症至关重要。

1,25-Dihydroxyvitamin D3 (1,25(OH2D3) is considered to be the functional metabolite of vitamin D3 which stimulates the expression of hormone-specific genes via genomic action. Recently, this sterol has been identified in the regulation of such diverse biologic phenomena as protein synthesis and secretion, enzyme activity, and changes in cell morphology and differentiation. We propose to utilize several mammalian cultured cell lines in which appropriate bioresponses to vitamin D3 have been identified, including human intestine cells (407), human leukemic cells (HL-60), pig kidney cells (LLC-PK1), rat osteosarcoma cells (ROS 17/2.8), and mouse fibroblasts (3T6), to elucidate the mechanism(s) of the sterol's action. We will initially examine the characteristics of interaction between 1,25(OH)2D3 and the intact cell, its binding to specific cytoplasmic receptors, and its effects on transcriptional parameters. Regulation of biologic activity by 1,25(OH)2D3 will be examined at 4 levels: i) molecular (CaBP, collagen, and actin synthesis), ii) enzymatic (vitamin D3-24-OHase), iii) plasma membrane (Ca++ transport), and iv) cellular control (morphology and differentiation). We will investigate the effects of vitamin D3 metabolites on cultured cell responses, and attempt to correlate the extent of the response with both the administered vitamin D3 metabolite concentration, receptor presence, and the degree of receptor occupancy. Inhibitor studies (actinomycin D, butyrate, cycloheximide) will be utilized to further define the level at which the cultured cell responds to 1,25(OH)2D3. Finally, DNA binding site-specific antireceptor antibodies will be utilized in microinjection studies as selective blockers of the receptor-mediated nuclear action of the hormone, thus permitting evaluation of receptor requirements and identification of non-genomic ativities of 1,25(OH)2D3. This approach is expected to further define the relationship between vitamin D3 and newly identified cellular bioresponses, and in particular, the mechanism by which these activities are promoted. We feel that the proposed experiments may reveal important clues as to the regulation of processes involved in cell differentiation, a phenomenon crucially important to our understanding of human cancers.