Gross C, Krishnan A V, Malloy P J, Eccleshall T R, Zhao X Y, Feldman D
Department of Medicine, Stanford University School of Medicine, California 94305-5103, USA.
J Bone Miner Res. 1998 Nov;13(11):1691-9. doi: 10.1359/jbmr.1998.13.11.1691.
The vitamin D receptor (VDR) gene contains a start codon polymorphism (SCP) which is three codons upstream of a second start site (ATG). The SCP genotype can be determined with the restriction enzyme FokI, where "f" indicates the presence of the restriction site and the first ATG, while "F" indicates its absence. Recent evidence suggests that the ff genotype is correlated with lower bone mineral density (BMD) in some populations. The SCP results in alternate VDRs that differ structurally, with the F variant (F-VDR) being three amino acids shorter than the f variant (f-VDR). To determine whether there are functional differences between the f-VDR and the F-VDR, we studied the two VDR forms expressed in COS-7 cells. The proteins were distinguishable from one another on Western blots by their different mobilities, confirming the larger size of f-VDR. Ligand binding studies showed no significant differences between the affinities of the two VDR forms for [3H]-1,25-dihydroxyvitamin D3 ([3H]-1,25(OH)2D3) (Kd = 131+/-78 pM, f-VDR; Kd = 237+/-190 pM, F-VDR; p = 0.24); however, a 2-fold difference in affinity can not be discriminated by this method. There were no differences in the abilities of the two receptor forms to bind DNA as determined by electrophoretic mobility shift assays. The ability of the two VDR forms to transactivate target genes was investigated using three different vitamin D responsive luciferase reporter constructs: 24-hydroxylase, osteocalcin, and osteopontin. In these transactivation experiments, 1,25(OH)2D3 dose-response (0.1-10 nM) curves revealed that the ED50 values for transactivation were indistinguishable between the two VDR forms. Additionally, cultured human fibroblasts with FF, Ff, and ff genotypes had similar sensitivity to 1,25(OH)2D3 with respect to the induction of 24-hydroxylase mRNA. In summary, we were unable to detect significant differences in ligand affinity, DNA binding, or transactivation activity between f-VDR and F-VDR forms. We must emphasize, however, that the sensitivity of the methods used limits our ability to detect minor differences in VDR affinity and function. In conclusion, we cannot define a mechanism whereby the SCP in the VDR might contribute to population differences in BMD.
维生素D受体（VDR）基因包含一个起始密码子多态性（SCP），它位于第二个起始位点（ATG）上游三个密码子处。SCP基因型可通过限制性内切酶FokI来确定，其中“f”表示存在限制性位点和第一个ATG，而“F”表示其不存在。最近的证据表明，在某些人群中，ff基因型与较低的骨矿物质密度（BMD）相关。SCP导致结构不同的交替VDR，F变体（F-VDR）比f变体（f-VDR）短三个氨基酸。为了确定f-VDR和F-VDR之间是否存在功能差异，我们研究了在COS-7细胞中表达的两种VDR形式。通过蛋白质印迹法，这两种蛋白质因其不同的迁移率而可区分，证实了f-VDR的尺寸更大。配体结合研究表明，两种VDR形式对[3H]-1,25-二羟基维生素D3（[3H]-1,25(OH)2D3）的亲和力没有显著差异（解离常数Kd = 131±78 pM，f-VDR；Kd = 237±190 pM，F-VDR；p = 0.24）；然而，这种方法无法区分2倍的亲和力差异。通过电泳迁移率变动分析确定，两种受体形式结合DNA的能力没有差异。使用三种不同的维生素D反应性荧光素酶报告构建体（24-羟化酶、骨钙素和骨桥蛋白）研究了两种VDR形式激活靶基因的能力。在这些激活实验中，1,25(OH)2D3剂量反应（0.1 - 10 nM）曲线显示，两种VDR形式激活的半数有效剂量（ED50）值无法区分。此外，具有FF、Ff和ff基因型的培养人成纤维细胞在诱导24-羟化酶mRNA方面对1,25(OH)2D3具有相似的敏感性。总之，我们无法检测到f-VDR和F-VDR形式之间在配体亲和力、DNA结合或激活活性方面的显著差异。然而，我们必须强调，所用方法的灵敏度限制了我们检测VDR亲和力和功能微小差异的能力。总之，我们无法确定VDR中的SCP可能导致人群BMD差异的机制。
