Wang Y Z, Christakos S
Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, UMD-New Jersey School and Graduate School of Biomedical Sciences, Newark 07103-2714, USA.
Mol Endocrinol. 1995 Nov;9(11):1510-21. doi: 10.1210/mend.9.11.8584029.
It is a well established fact that the calcium-binding protein, calbindin-D28k, is influenced by vitamin D in intestine and kidney. However, very little is known concerning the regulation of calbindin-D28k in brain. Although few genes that are regulated by retinoic acid (RA) have been identified in the nervous system, we now report that the human medulloblastoma cell line D283 (which is derived from cerebellum and has a distinctly neuronal phenotype) contains calbindin-D28k endogenously and that calbindin protein and mRNA can be induced 10- to 15-fold in these cells by 10(-7) M RA. These findings are the first evidence of RA-mediated regulation of calbindin. The time course of response, as determined by Northern blot analysis, indicated that the first significant increase in calbindin-D28k mRNA is at 12 h with a plateau of calbindin mRNA induction at 72 h after RA treatment. The induction of calbindin mRNA by RA was preceded by an induction of retinoic acid receptor-alpha mRNA and was accompanied by an induction of retinoid X receptor-alpha mRNA. Calbindin-D28k mRNA levels in D283 medulloblastoma cells as well as the induction of calbindin mRNA by RA were not significantly affected by 1,25-dihydroxyvitamin D3 treatment. Deletion mutant analysis of the native calbindin-D28k promoter and cotransfection of CV-1 or D283 medulloblastoma cells in the presence of retinoic acid receptor-alpha and/or retinoid X receptor-alpha expression vectors as well as results of nuclear transcription assays did not indicate transcriptional regulation of calbindin-D28k by RA. Studies of calbindin-D28k mRNA in control and RA-pretreated D283 medulloblastoma cells at various times (3-24 h) after treatment with 4 micrograms/ml actinomycin D indicated that the half-life of calbindin-D28k mRNA was significantly increased in the presence of RA, suggesting regulation of calbindin-D28k mRNA stability by RA. Thus, calbindin-D28k is one of the few known targets of RA action in cells that express a neuronal phenotype. In addition, our findings present further evidence of an interrelationship between the actions of 1,25-dihydroxyvitamin D3 and the active metabolites of vitamin A.
钙结合蛋白calbindin-D28k受维生素D影响，这在小肠和肾脏中已是公认的事实。然而，关于calbindin-D28k在大脑中的调节机制却知之甚少。尽管在神经系统中已鉴定出少数受视黄酸（RA）调节的基因，但我们现在报告，人髓母细胞瘤细胞系D283（源自小脑，具有明显的神经元表型）内源性地含有calbindin-D28k，并且在这些细胞中，10(-7) M的RA可将calbindin蛋白和mRNA诱导10至15倍。这些发现是RA介导的calbindin调节的首个证据。通过Northern印迹分析确定的反应时间进程表明，calbindin-D28k mRNA的首次显著增加在12小时出现，RA处理后72小时calbindin mRNA诱导达到平台期。RA诱导calbindin mRNA之前先诱导了视黄酸受体α mRNA，并伴随着类视黄醇X受体α mRNA的诱导。1,25 - 二羟基维生素D3处理对D283髓母细胞瘤细胞中calbindin-D28k mRNA水平以及RA诱导calbindin mRNA的作用没有显著影响。对天然calbindin-D28k启动子的缺失突变分析以及在视黄酸受体α和/或类视黄醇X受体α表达载体存在的情况下对CV-1或D283髓母细胞瘤细胞进行共转染，以及核转录分析结果均未表明RA对calbindin-D28k有转录调节作用。在用4微克/毫升放线菌素D处理后的不同时间（3 - 24小时）对对照和RA预处理的D283髓母细胞瘤细胞中calbindin-D28k mRNA的研究表明，在RA存在的情况下，calbindin-D28k mRNA的半衰期显著延长，提示RA对calbindin-D28k mRNA稳定性有调节作用。因此，calbindin-D28k是在表达神经元表型的细胞中已知的少数RA作用靶点之一。此外，我们的发现进一步证明了1,25 - 二羟基维生素D3和维生素A的活性代谢产物之间存在相互关系。
