Harendza Sigrid, Lovett David H, Panzer Ulf, Lukacs Zoltan, Kuhnl Peter, Stahl Rolf A K
Department of Medicine, Division of Nephrology, University of Hamburg, Hamburg D-20246, Germany.
J Biol Chem. 2003 Jun 6;278(23):20490-9. doi: 10.1074/jbc.M211536200. Epub 2003 Mar 25.
Gelatinase A (matrix metalloproteinase-2) plays a prominent role in multiple biologic processes. Prior studies have established critical roles for gelatinase A transcriptional regulation by defined enhancer elements. To determine possible functional single nucleotide polymorphisms within these elements, we determined the single nucleotide polymorphism distribution within 1,665 bp of the gelatinase A 5'-flanking region, using a healthy homogeneous Caucasian study group of 463 individuals. Among the polymorphisms detected, a G --> A transition at bp -1575 was located immediately 5' to a half-palindromic potential estrogen receptor binding site. In estrogen receptor-positive MCF-7 cells the -1575G allele functioned as an enhancer, whereas the -1575A allele reduced transcription activity significantly. Gel shift assays confirmed that the differences in allelic expression affected binding of the estrogen receptor-alpha to this region. Cotransfection experiments with an estrogen receptor-alpha expression vector in MDA-MB-231 cells, which do not constitutively express an estrogen receptor, revealed that estrogen receptor is absolutely required for enhancing activity. Allelic distribution analysis indicated that a previously reported C --> T transition within an Sp1 binding site at -1306 was in linkage disequilibrium with the -1575G --> A transition. Luciferase reporter studies of the linked variant -1575A -1306T allele versus the wild type -1575G -1306C allele demonstrated an additive reduction in estrogen-dependent reporter activity. The frequency of the -1575G --> A transition deviated significantly from the expected Hardy-Weinberg distribution in two independently assembled study populations consisting of healthy adult blood donors and newborns of Caucasian origin, both with a calculated 21% reduction in genetic fitness. Gelatinase A is a known estrogen-responsive gene and the demonstration of a loss of function polymorphism within an operational estrogen receptor binding site associated with a decrease in genetic fitness underscores the biologic significance of promoter polymorphism analyses.
明胶酶A（基质金属蛋白酶-2）在多种生物学过程中发挥着重要作用。先前的研究已经确定了特定增强子元件对明胶酶A转录调控的关键作用。为了确定这些元件内可能的功能性单核苷酸多态性，我们使用了一个由463名健康的同质白种人组成的研究组，确定了明胶酶A 5'侧翼区域1665 bp内的单核苷酸多态性分布。在检测到的多态性中，-1575 bp处的G→A转换紧邻一个半回文潜在雌激素受体结合位点的5'端。在雌激素受体阳性的MCF-7细胞中，-1575G等位基因起增强子作用，而-1575A等位基因则显著降低转录活性。凝胶迁移试验证实等位基因表达的差异影响雌激素受体α与该区域的结合。在不组成性表达雌激素受体的MDA-MB-231细胞中，用雌激素受体α表达载体进行共转染实验表明，增强活性绝对需要雌激素受体。等位基因分布分析表明，先前报道的-1306处Sp1结合位点内的C→T转换与-1575G→A转换处于连锁不平衡状态。对连锁变体-1575A -1306T等位基因与野生型-1575G -1306C等位基因进行荧光素酶报告基因研究，结果显示雌激素依赖性报告基因活性呈累加性降低。在两个独立组建的研究群体中，即由健康成年献血者和白种人新生儿组成的群体中，-1575G→A转换的频率显著偏离预期的哈迪-温伯格分布，两者的遗传适应性均计算降低了21%。明胶酶A是一个已知的雌激素反应基因，在一个与遗传适应性降低相关的功能性雌激素受体结合位点内证明存在功能丧失多态性，突出了启动子多态性分析的生物学意义。
