CHARACTERIZATION AND THE REGULATION OF INHIBIN ACTIVIN SUBUNIT PROTEINS OF CULTURED RAT ANTERIOR-PITUITARY-CELLS
CHARACTERIZATION AND THE REGULATION OF INHIBIN ACTIVIN SUBUNIT PROTEINS OF CULTURED RAT ANTERIOR-PITUITARY-CELLS
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DOI：
10.1210/en.133.6.2545
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发表时间：
1993-12-01
期刊：
影响因子：
4.8
通讯作者：
VALE, WW
中科院分区：
文献类型：
--
作者：
BILEZIKJIAN, LM;VAUGHAN, JM;VALE, WW
用抑制素/激活素α、β(A)和β(B)亚单位蛋白(抗-α、抗-β(A)和抗-β(B))抗血清检测培养的大鼠垂体前叶细胞产生抑制素/激活素的能力。从代谢性标记细胞中免疫沉淀被抗血清识别的细胞或分泌的蛋白质，然后用变性聚丙烯酰胺凝胶电泳法进行分析。抑制素/激活素β(B)蛋白在细胞裂解物和培养上清液中均可见。抗-β(B)实验证实激活素-B(beta(B)beta(B))是培养的大鼠垂体前叶细胞的局部分泌产物。分泌的β(B)免疫反应蛋白条带的表观大小分别为24-25千道尔顿(KDa)或14-15 kDa，分别与未还原的β(B)二聚体或还原单体的大小一致。细胞裂解产物含有两种由抗-β(B)特异性免疫沉淀的蛋白质。其中一个具有大于95 kDa(未还原)或55-60 kDa(还原)的迁移率，可能分别代表β(B)前体的二聚体或单体。第二条14-15 kDa(还原和未还原)免疫活性β(B)蛋白条带被证实为成熟的β(B)单体。抗-α或抗-β(B)抗体均未检测到成熟的异二聚体抑制素-B(Alphabeta(B))。然而，通过细胞裂解产物与抗α抗体孵育，观察到多种蛋白质物种被特异性地免疫沉淀。在所有样品中均未检测到成熟的β(A)单体。通过评估脉冲标记细胞中β(B)的合成速度来监测细胞β(B)产生的调节。Forskolin或12-O-十四酰佛波醇醋酸酯处理增加了[S-35]半胱氨酸掺入细胞14~15 kDaβ(B)单体的速率，表明蛋白激酶A或蛋白激酶C的激活调节了半胱氨酸的合成。激活素-A、抑制素-A和卵泡抑素也可调节细胞内β(B)的积累速度；激活素-A对细胞的抑制作用为30%，而抑制素-A或卵泡抑素的刺激作用为70%。等摩尔浓度的激活素-A和卵泡抑素阻止了单独由这两个因素产生的净影响。在类似的脉冲标记条件下，均未检测到免疫反应阳性的α-亚型，标记至平衡后(至48h)，其水平无明显变化。因此，观察到的β(B)积累的变化可能反映了垂体激活素-B的调节产生。综上所述，这些结果表明，局部产生的激活素-B或性腺激活素对垂体激活素-B的产生起抑制作用，这种负反馈控制反过来又受到抑制素和叶利他汀类药物的调节。垂体和性腺激活素、抑制素和叶立他汀类在拟议的调控环路中的相对重要性仍有待确定。
The production of inhibin/activin by Cultured rat anterior pituitary cells was evaluated using specific antisera to inhibin/activin alpha, beta(A), and beta(B) subunit proteins (anti-alpha, anti-beta(A), and anti-beta(B)). Cellular or secreted Proteins recognized by the antisera were immunoprecipitated from metabolically labeled cells then analyzed by denaturing polyacrylamide gel electrophoresis. Immunoreactive inhibin/activin beta(B) proteins were visualized in both cell lysates and the media. Experiments with anti-beta(B) confirmed that activin-B (beta(B)beta(B)) is a local secretory product of cultured rat anterior pituitary cells. The secreted beta(B)-immunoreactive protein band had an apparent size of 24-25 kilodaltons (kDa) or 14-15 kDa, consistent with the size of unreduced beta(B) dimer or reduced monomer, respectively. Cell lysates contained two proteins that were specifically immunoprecipitated by anti-beta(B). One of these had a mobility of greater than 95 kDa (unreduced) or 55-60 kDa (reduced), probably representing dimers or monomers of the beta(B) precursor, respectively. The second 14- to 15-kDa (reduced and unreduced) immunoreactive beta(B) protein band was verified to be the mature beta(B) monomer. Mature heterodimeric inhibin-B (alphabeta(B)) was not detected by either anti-alpha or anti-beta(B). Multiple protein species, however, were observed to be specifically immunoprecipitated by incubation of cell lysates with anti-alpha. Mature beta(A) monomer was not detected in any of the samples. The regulation of cellular beta(B) production was monitored by evaluating its rate of synthesis in pulse-labeled cells. Treatment with either forskolin or 12-O-tetradecanoylphorbol acetate enhanced the rate of [S-35]cysteine incorporation into the cellular 14- to 15-kDa beta(B) monomer, indicating that the activation of either protein kinase A or protein kinase C regulates its production. The rate of cellular beta(B) accumulation was also regulated by activin-A, inhibin-A, and follistatin; activin-A caused a 30% inhibition in contrast to the 70% stimulation by treatment with either inhibin-A or follistatin. Equimolar concentrations of activin-A and follistatin prevented the net effect produced by either factor alone. None of the immunoreactive a-forms was detectable under similar pulse-labeling conditions, and there was no apparent change in their level after labeling to equilibrium (up to 48 h). The observed changes in beta(B) accumulation may, therefore, reflect the regulated production of pituitary activin-B. Taken together, these results suggest that locally produced activin-B or gonadal activins exert an inhibitory tone on the production of pituitary activin-B and that this negative-feedback control is in turn modulated by inhibins and follistatins. The relative importance of pituitary and gonadal activins, inhibins, and follistatins in the proposed regulatory loop remains to be established.