Michel U, Farnworth P
Prince Henry's Institute of Medical Research, Monash Medical Centre, Clayton, Victoria, Australia.
Endocrinology. 1992 Jun;130(6):3684-93. doi: 10.1210/endo.130.6.1375907.
The regulation of steady state levels of follistatin (FS) messenger RNA (mRNA) was examined in a rat renal mesangial cell line in tissue culture. A specific 32P-radiolabeled antisense probe was used which corresponds to the 3' end of exon 5 together with the 5' end of exon 6 of the rat FS gene, and which distinguishes between the two different forms of FS mRNA. In addition, a specific 35S-radiolabeled probe for the ubiquitous protein cyclophilin was developed and used as an internal standard. Total RNA was harvested from confluent cell cultures to yield four independent samples per treatment/time point, and equal amounts of RNA from every sample in a given experiment were subjected to S1-nuclease analysis for the estimation of specific mRNA levels. Treatment of the cultured cells with epidermal growth factor (10 nM) caused an 8- to 9-fold increase in the FS mRNA level after 4 h, but no consistent change was observed after treatment with basic fibroblast growth factor (0.28 or 0.56 nM), somatostatin (3.7-73 nM), angiotensin II (0.1-2500 nM), or FS itself (0.29 nM) for between 4 and 48 h. Neither activin (0.5 or 1.2 nM) nor inhibin (0.64 nM) changed the FS mRNA level in the mesangial cell line during a 24-h treatment. FS mRNA levels in the cells also were not affected by a 48-h treatment with the steroids dihydrotestosterone (1-1000 nM), estradiol (1 and 100 nM), and the antiprogesterone RU 486 (1000 nM), whereas 100 nM RU 28362 (a synthetic glucocorticoid) caused a 5- to 6-fold increase and 1000 nM progesterone increased the FS mRNA level up to 3.5-fold above control. Retinoic acid, a vitamin A derivative, significantly increased the FS steady state mRNA level at 3 nM, and at 1000 nM stimulated FS mRNA up to 5-fold within 4 h, whereas incubation of the cells with 30 microM prostaglandin E2 for 4 h caused a 10-fold increase. The FS mRNA level increased 3- and 4-fold within 4 h during incubation of the cells with 100 nM phorbol 12-myristate, 13-acetate, and 25 microM forskolin, respectively, whereas the calcium ionophore A23187 (1-100 microM) caused no change within this timespan. None of the tested hormones had an obvious effect on the ratio of the two different forms of FS mRNA (FS 344:FS 317).(ABSTRACT TRUNCATED AT 400 WORDS)
在组织培养的大鼠肾系膜细胞系中研究了卵泡抑素（FS）信使核糖核酸（mRNA）稳态水平的调节。使用了一种特异性的32P放射性标记反义探针，它对应于大鼠FS基因外显子5的3'端以及外显子6的5'端，并且能够区分FS mRNA的两种不同形式。此外，还制备了一种针对普遍存在的亲环蛋白的特异性35S放射性标记探针，并用作内参。从汇合的细胞培养物中收获总RNA，每个处理/时间点产生四个独立样本，并且在给定实验中，将每个样本等量的RNA进行S1核酸酶分析以估计特异性mRNA水平。用表皮生长因子（10 nM）处理培养细胞4小时后，FS mRNA水平增加了8至9倍，但用碱性成纤维细胞生长因子（0.28或0.56 nM）、生长抑素（3.7 - 73 nM）、血管紧张素II（0.1 - 2500 nM）或FS本身（0.29 nM）处理4至48小时后，未观察到一致的变化。在24小时处理期间，激活素（0.5或1.2 nM）和抑制素（0.64 nM）均未改变系膜细胞系中的FS mRNA水平。细胞中的FS mRNA水平也不受甾体类药物双氢睾酮（1 - 1000 nM）、雌二醇（1和100 nM）和抗孕激素RU 486（1000 nM）48小时处理的影响，而100 nM RU 28362（一种合成糖皮质激素）导致增加5至6倍，1000 nM孕酮使FS mRNA水平比对照增加高达3.5倍。视黄酸，一种维生素A衍生物，在3 nM时显著增加FS稳态mRNA水平，在1000 nM时在4小时内刺激FS mRNA增加高达5倍，而用30 microM前列腺素E2孵育细胞4小时导致增加10倍。在用100 nM佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯和25 microM福斯高林孵育细胞期间，FS mRNA水平分别在4小时内增加了3倍和4倍，而钙离子载体A23187（1 - 100 microM）在此时间段内未引起变化。所测试的激素均未对FS mRNA的两种不同形式（FS 344:FS 317）的比例产生明显影响。（摘要截断于400字）
