Enomoto H, Hendy G N, Andrews G K, Clemens T L
Bone Center, Cedars-Sinai Medical Center/University of California, Los Angeles 90048.
Endocrinology. 1992 Jun;130(6):3467-74. doi: 10.1210/endo.130.6.1375904.
Vitamin D-dependent calcium-binding protein (calbindin-D28K), is regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], and several other factors in a tissue-specific manner, but the controlling mechanisms are still poorly understood. In this study we examined the relative contributions of transcriptional and posttranscriptional mechanisms in the 1,25-(OH)2D3 control of calbindin-D28K mRNA expression in primary chick kidney cells and investigated the effect of extracellular Ca2+ on calbindin-D28K gene expression in the presence and absence of hormone. 1,25-(OH)2D3 treatment (10(-8) M) of cells grown in serum-free medium resulted in a marked 20- to 30-fold increase in calbindin-D28K mRNA peaking at 12-18 h, which then rapidly declined to basal levels by 24 h. The abrupt decline in mRNA appeared to be associated with a reduction in size of the calbindin-D28K transcripts. Nuclear run-off assays showed only a slight (1.5-fold) increase in calbindin-D28K gene transcription 2 h after 1,25-(OH)2D3, whereas parallel assays clearly demonstrated a marked (7-fold) induction in the rate of metallothionein gene transcription 2 h after treatment of chick kidney cells with 10 microM zinc. The induction of calbindin-D mRNA by 1,25-(OH)2D3 required ongoing protein synthesis, since it was blocked by cycloheximide. Calbindin-D28K mRNA was stable for 12 h in the presence of actinomycin-D in both vitamin D-deficient and 1,25-(OH)2D3-treated cells. Both basal and 1,25-(OH)2D3-induced calbindin-D28K mRNA were modulated by the extracellular Ca2+, with maximum expression occurring at 1-2 mM. We conclude that 1,25-(OH)2D3 induces kidney calbindin-D28K mRNA by producing a small increase in its transcriptional rate, which is accompanied by pronounced posttranscriptional effects(s). The striking modulation of calbindin-D28K expression by extracellular Ca2+ is consistent with a putative role for this protein in the regulation of this ion in the kidney cell.
维生素D依赖性钙结合蛋白（钙结合蛋白-D28K）受1,25-二羟基维生素D3 [1,25-(OH)2D3]及其他多种因素以组织特异性方式调控，但其调控机制仍知之甚少。在本研究中，我们检测了转录和转录后机制在1,25-(OH)2D3对原代鸡肾细胞中钙结合蛋白-D28K mRNA表达调控中的相对作用，并研究了在有无激素存在的情况下细胞外Ca2+对钙结合蛋白-D28K基因表达的影响。在无血清培养基中生长的细胞用1,25-(OH)2D3（10(-8) M）处理后，钙结合蛋白-D28K mRNA显著增加20至30倍，在12至18小时达到峰值，然后在24小时迅速降至基础水平。mRNA的突然下降似乎与钙结合蛋白-D28K转录本大小的减少有关。核转录分析显示，1,25-(OH)2D3处理2小时后，钙结合蛋白-D28K基因转录仅略有增加（1.5倍），而平行分析清楚地表明，用10 microM锌处理鸡肾细胞2小时后，金属硫蛋白基因转录速率显著增加（7倍）。1,25-(OH)2D3对钙结合蛋白-D mRNA的诱导需要持续的蛋白质合成，因为它被放线菌酮阻断。在维生素D缺乏和1,25-(OH)2D3处理的细胞中，钙结合蛋白-D28K mRNA在放线菌素-D存在下均稳定12小时。基础和1,25-(OH)2D3诱导的钙结合蛋白-D28K mRNA均受细胞外Ca2+调节，最大表达出现在1至2 mM。我们得出结论，1,25-(OH)2D3通过使其转录速率略有增加来诱导肾钙结合蛋白-D28K mRNA，同时伴有明显的转录后效应。细胞外Ca2+对钙结合蛋白-D28K表达的显著调节与该蛋白在肾细胞中对该离子的调节作用一致。
