The vitamin D receptor gene start codon polymorphism:: A functional analysis of FokI variants
The vitamin D receptor gene start codon polymorphism:: A functional analysis of FokI variants
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DOI：
10.1359/jbmr.1998.13.11.1691
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发表时间：
1998-11-01
影响因子：
6.2
通讯作者：
Feldman, D
中科院分区：
文献类型：
--
作者：
Gross, C;Krishnan, AV;Feldman, D
维生素D受体(VDR)基因含有起始密码子多态性(SCP)，它是位于第二起始位点(ATG)上游的三个密码子。SCP基因可以用限制性内切酶FokI确定，其中“f”表示存在限制位点和第一个ATG，而“F”表示它不存在。最近的证据表明，在某些人群中，ff基因与较低的骨密度(BMD)相关。SCP导致结构不同的交替VDR，F变异体(F-VDR)比f变异体(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。蛋白质在Western blotts上通过不同的迁移率相互区分，证实了较大的f-VDR配体结合研究表明两种VDR形式对[H-3]-1，25(OH)(2)D-3([H-3]-1，25(OH)(2)D-3)的亲和力没有显著差异(K-d=131+/-78 pm，f-VDR；K-d=237+/-190 pm，F-VDR；p=0.24)；但是，这种方法不能区分亲和力的2倍差异。通过电泳迁移率改变分析，两种受体形式与DNA结合的能力没有差异。使用三种不同的维生素D反应荧光素酶报告结构：24-羟基酶、骨钙素和骨桥蛋白，研究了两种VDR形式反式激活靶基因的能力。在这些激活实验中，1，25(OH)(2)D-3剂量-反应曲线(0.1-10 nm)显示，两种VDR形式的激活ED50值是不可区分的。此外，具有Ff、Ff和ff基因的培养的人成纤维细胞对1，25(OH)(2)D-3在诱导24-羟基酶mRNA方面具有类似的敏感性。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结合或反式激活活性方面的显著差异。然而，我们必须强调，所用方法的敏感性限制了我们检测VDR亲和力和功能的微小差异的能力。总而言之，我们不能定义VDR中SCP可能导致人群BMD差异的机制。
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 [H-3]-1,25-dihydroxyvitamin D-3 ([H-3]-1,25(OH)(2)D-3) (K-d = 131 +/- 78 pM, f-VDR; K-d = 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)(2)D-3 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)(2)D-3 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.