Brehier A, Thomasset M
INSERM U120, Alliée CNRS, Le Vésinet, France.
Endocrinology. 1990 Aug;127(2):580-7. doi: 10.1210/endo-127-2-580.
We have used organ cultures of 18-day fetal rat duodena to study the regulation of calbindin-D9K (CaBP9K) gene expression by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) and calcium. The epithelium of the fetal rat duodena maintained in culture for 10 days in a defined serum-free medium with no exogenous 1,25(OH)2D3 contains differentiated enterocytes. Treatment with 10(-8) M 1,25(OH)2D3 resulted in a 2-fold increase in CaBP9K messenger RNA (mRNA) after 1 h; CaBP9K content was increased 2.5-fold in 24 h. Simultaneous treatment of cultures with 1,25(OH)2D3 and 1 microM actinomycin D selectively blocked 1,25(OH)2D3-stimulated CaBP9K mRNA and protein synthesis. Fifty micromolars of cycloheximide significantly decreased CaBP9K protein accumulation but not mRNA. These findings indicate that 1,25(OH)2D3 controls CaBP9K gene transcription. Enterocytes exhibit some CaBP9K gene expression after 10 days under basal culture conditions, suggesting that CaBP9K synthesis is not dependent on 1,25(OH)2D3 alone. This basal expression drops after 24 h in a calcium free medium with 1 mM EGTA, suggesting that extracellular calcium is involved in the control of this gene. Increasing the calcium concentration to 1.2 mM resulted in a 6-fold increase in CaBP9K mRNA after 3 h and a 10-fold increase in CaBP9K protein content after 24 h. Calcium may increase CaBP9K gene transcription, since simultaneous addition of 1 microM actinomycin D and calcium blocked the calcium effect on CaBP9K mRNA. Therefore, this in vitro study demonstrates that both calcium and 1,25(OH)2D3 stimulate CaBP9K gene expression in the rat duodenum.
我们利用18日龄胎鼠十二指肠的器官培养物，研究1,25 - 二羟胆钙化醇（1,25(OH)2D3）和钙对钙结合蛋白-D9K（CaBP9K）基因表达的调控。在不含外源性1,25(OH)2D3的特定无血清培养基中培养10天的胎鼠十二指肠上皮含有分化的肠上皮细胞。用10(-8)M 1,25(OH)2D3处理1小时后，CaBP9K信使核糖核酸（mRNA）增加了2倍；24小时内CaBP9K含量增加了2.5倍。用1,25(OH)2D3和1微摩尔放线菌素D同时处理培养物，选择性地阻断了1,25(OH)2D3刺激的CaBP9K mRNA和蛋白质合成。50微摩尔的环己酰亚胺显著降低了CaBP9K蛋白质的积累，但对mRNA没有影响。这些发现表明，1,25(OH)2D3控制CaBP9K基因转录。在基础培养条件下10天后，肠上皮细胞表现出一些CaBP9K基因表达，这表明CaBP9K的合成并非仅依赖于1,25(OH)2D3。在含有1毫摩尔乙二醇双四乙酸（EGTA）的无钙培养基中培养24小时后，这种基础表达下降，这表明细胞外钙参与了该基因的调控。将钙浓度提高到1.2毫摩尔，3小时后CaBP9K mRNA增加了6倍，24小时后CaBP9K蛋白质含量增加了10倍。钙可能增加CaBP9K基因转录，因为同时添加1微摩尔放线菌素D和钙可阻断钙对CaBP9K mRNA的作用。因此，这项体外研究表明，钙和1,25(OH)2D3均可刺激大鼠十二指肠中CaBP9K基因的表达。
