Varghese S, Lee S, Huang Y C, Christakos S
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.
J Biol Chem. 1988 Jul 15;263(20):9776-84.
We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.
我们报道了使用克隆的哺乳动物钙结合蛋白-D28k（28 kDa维生素D依赖性钙结合蛋白）的cDNA来研究大鼠钙结合蛋白基因的表达。利用Northern印迹分析进行的组织分布研究表明，在大鼠肾脏和脑中可检测到钙结合蛋白-D28k mRNA，但在大鼠肠道、睾丸、骨骼、胰腺、肝脏、肺或骨骼肌中未检测到。大鼠肾脏和脑均含有三种RNA种类（1.9、2.8和3.2千碱基对）。通过Northern印迹分析和狭缝印迹分析对该基因的调控进行了表征。研究了激素调控、钙结合蛋白-D28k mRNA的发育表达以及饮食改变的影响。在肾脏中，所有三种mRNA的诱导均依赖于1,25-二羟基维生素D3（1,25-(OH)2D3）的存在。肾脏钙结合蛋白-D28k mRNA诱导的时间进程表明，单次注射1,25-(OH)2D3（200 ng/100 g体重）后2小时即可检测到钙结合蛋白-D mRNA显著增加，在12小时达到最大值。与肾脏不同，在维生素D缺乏大鼠的脑中观察到高水平的钙结合蛋白-D28k mRNA。给予1,25-(OH)2D3后，脑中钙结合蛋白-D28k mRNA的浓度未发生变化。发育研究表明，大鼠肾脏和脑中的钙结合蛋白-D mRNA在出生前就已存在，但以组织特异性方式受到发育调控。肾脏钙结合蛋白-D28k mRNA丰度最显著的变化发生在出生至1周龄之间。与肾脏不同，脑钙结合蛋白-D28k mRNA在较晚时间（1至2周龄之间，即主要突触形成期）大幅增加。在饮食改变研究中，Northern印迹分析结果表明，低磷饮食会导致肾脏中钙结合蛋白-D mRNA增加，但脑中不会。这些研究代表了对大鼠钙结合蛋白-D28k基因及其体内调控的首次分析。我们的研究结果表明，在大鼠肾脏和脑中，钙结合蛋白-D28k基因的表达及其受1,25-(OH)2D3调控均存在显著差异。
