Gong Y, Blok L J, Perry J E, Lindzey J K, Tindall D J
Department of Urology, Mayo Foundation, Rochester, Minnesota 55905, USA.
Endocrinology. 1995 May;136(5):2172-8. doi: 10.1210/endo.136.5.7720667.
Elevation of intracellular calcium levels in the presence of normal androgen levels has been implicated in apoptotic prostate cell death. Since the androgen receptor (AR) plays a critical role in the regulation of growth and differentiation of the prostate, it was of interest to determine whether Ca2+ would affect the expression of androgen receptor messenger RNA (mRNA) and protein, thus affecting the ability of androgens to control prostate function. AR-positive human prostate cancer cells, LNCaP, were incubated with either the calcium ionophore A23187 or the intracellular endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin. Subsequently, AR mRNA and protein levels were assessed by Northern and Western blot analysis. Both A23187 and thapsigargin were found to down-regulate steady state AR mRNA levels in a time- and dose-dependent manner. AR mRNA began to decrease after 6-8 h of incubation with 10(-6) M A23187 or 10(-7) M thapsigargin, reaching a nadir at 16 and 10 h of incubation, respectively. In contrast, control mRNA (glyceraldehyde 3-phosphate dehydrogenase) did not change significantly during the treatments with either A23187 or thapsigargin. AR protein levels were found to be decreased after 12 h of incubation with either 10(-6) M A23187 or 10(-7) M thapsigargin. The decrease in AR mRNA and protein seemed to precede apoptosis, since neither A23187 (24 h) nor thapsigargin (30 h) was found to alter cell morphology within the treatment time. Cycloheximide and actinomycin D were unable to change the calcium-mediated decrease in AR mRNA, ruling out the necessity for de novo protein synthesis or a change in mRNA stability. Moreover, the decrease in AR mRNA induced by calcium does not seem to involve protein kinase C- or calmodulin-dependent pathways, since inhibitors of these cellular components had no effect. Nuclear run-on assays demonstrated little or no effects of either A23187 or thapsigargin treatment on AR gene transcription (8 h and 10 h). In conclusion, these studies show that intracellular calcium seems to be a potent regulator of AR gene expression in LNCaP cells.
在雄激素水平正常的情况下，细胞内钙水平的升高与前列腺细胞凋亡性死亡有关。由于雄激素受体（AR）在前列腺生长和分化的调节中起关键作用，因此确定Ca2+是否会影响雄激素受体信使核糖核酸（mRNA）和蛋白质的表达，从而影响雄激素控制前列腺功能的能力，这一点很有意义。将雄激素受体阳性的人前列腺癌细胞LNCaP与钙离子载体A23187或细胞内内质网Ca(2+)-ATP酶抑制剂毒胡萝卜素一起孵育。随后，通过Northern印迹和Western印迹分析评估AR mRNA和蛋白质水平。发现A23187和毒胡萝卜素均以时间和剂量依赖性方式下调AR mRNA的稳态水平。用10(-6) M A23187或10(-7) M毒胡萝卜素孵育6-8小时后，AR mRNA开始下降，分别在孵育16小时和10小时时达到最低点。相比之下，对照mRNA（甘油醛-3-磷酸脱氢酶）在用A23187或毒胡萝卜素处理期间没有明显变化。在用10(-6) M A23187或10(-7) M毒胡萝卜素孵育12小时后，发现AR蛋白质水平下降。AR mRNA和蛋白质的下降似乎先于细胞凋亡，因为在处理时间内未发现A23187（24小时）或毒胡萝卜素（30小时）改变细胞形态。环己酰亚胺和放线菌素D无法改变钙介导的AR mRNA下降，排除了从头合成蛋白质或mRNA稳定性改变的必要性。此外，钙诱导的AR mRNA下降似乎不涉及蛋白激酶C或钙调蛋白依赖性途径，因为这些细胞成分的抑制剂没有作用。核转录分析表明，A23187或毒胡萝卜素处理对AR基因转录几乎没有影响（8小时和10小时）。总之，这些研究表明细胞内钙似乎是LNCaP细胞中AR基因表达的有效调节因子。
