Vallée M, Perron S, Tremblay Y, Bélanger A
MRC Group in Molecular Endocrinology, CHUL Research Center, Quebec, Canada.
J Steroid Biochem Mol Biol. 1995 Jul;54(1-2):31-8. doi: 10.1016/0960-0760(95)00059-9.
The role of ACTH, forskolin and 8Br-cAMP on the regulation of mRNA abundance, protein levels and enzymatic activity of cytochrome P450 21-hydroxylase (P450c21, CYP21) were investigated in guinea pig adrenal cell cultures. In untreated cells, 21-hydroxylase activity was diminished throughout a 48 h period of incubation. Although incubation with forskolin and 8Br-cAMP restored 21-hydroxylase activity to normal levels, the addition of ACTH did not prevent the decrease of 21-hydroxylase activity. Treatment of cells with RU486 for 24 h inhibited 21-hydroxylase activity by 93%; however, after removal of the drug a slight increase of enzyme activity was observed; this rise was enhanced by the addition of ACTH. Forskolin and 8Br-cAMP increased the levels of 21-hydroxylase activity to the same range as seen in untreated cells. In cells that were not pretreated with RU486, incubation with cycloheximide for 1 h had no effect on 21-hydroxylase activity and could not prevent the modest increase of 21-hydroxylase activity induced by forskolin or 8Br-cAMP after 48 h of incubation. In RU486-treated cells, cycloheximide blocks the stimulation of enzyme activity induced by ACTH, forskolin and 8Br-cAMP. Our findings indicate that 21-hydroxylase activity can be stimulated by ACTH, forskolin or 8Br-cAMP solely in the presence of reduced enzymatic activity. Western immunoblot analysis of P450c21 protein levels in untreated or RU486-treated adrenal cells indicate that P450c21 protein levels were in the same range and further incubation with ACTH caused a similar elevation of P450c21 protein levels in both the untreated and RU486-treated cells. Northern blot analysis on RNA isolated from adrenal cells showed that RU486 did not alter the basal steady state levels of P450c21 mRNA. As well, incubation with ACTH or 8Br-cAMP increased the levels of P450c21 transcript to the same extent in both untreated and RU486-treated cells. These results taken together provide additional evidence for the presence of an adrenal specific protein factor(s) modulating 21-hydroxylase activity.
在豚鼠肾上腺细胞培养物中研究了促肾上腺皮质激素（ACTH）、福斯高林和8-溴环磷腺苷（8Br-cAMP）对细胞色素P450 21-羟化酶（P450c21，CYP21）的mRNA丰度、蛋白水平和酶活性调节的作用。在未处理的细胞中，整个48小时的孵育期间21-羟化酶活性均降低。虽然用福斯高林和8Br-cAMP孵育可使21-羟化酶活性恢复到正常水平，但添加ACTH并不能阻止21-羟化酶活性的降低。用RU486处理细胞24小时可使21-羟化酶活性抑制93%；然而，去除药物后观察到酶活性略有增加；添加ACTH可增强这种升高。福斯高林和8Br-cAMP使21-羟化酶活性水平增加到与未处理细胞中所见相同的范围。在未用RU486预处理的细胞中，用放线菌酮孵育1小时对21-羟化酶活性没有影响，并且不能阻止孵育48小时后福斯高林或8Br-cAMP诱导的21-羟化酶活性适度增加。在RU486处理的细胞中，放线菌酮可阻断ACTH、福斯高林和8Br-cAMP诱导的酶活性刺激。我们的研究结果表明，仅在酶活性降低的情况下，ACTH、福斯高林或8Br-cAMP可刺激21-羟化酶活性。对未处理或RU486处理的肾上腺细胞中P450c21蛋白水平进行的蛋白质免疫印迹分析表明，P450c21蛋白水平在相同范围内，并且用ACTH进一步孵育导致未处理和RU486处理的细胞中P450c21蛋白水平有相似的升高。对从肾上腺细胞分离的RNA进行的Northern印迹分析表明，RU486不会改变P450c21 mRNA的基础稳态水平。同样，用ACTH或8Br-cAMP孵育在未处理和RU486处理的细胞中均使P450c21转录本水平增加到相同程度。综合这些结果为存在调节21-羟化酶活性的肾上腺特异性蛋白因子提供了额外证据。
