Bilezikjian L M, Corrigan A Z, Vaughan J M, Vale W M
Clayton Foundation Laboratories for Peptide Biology, Salk Institute, La Jolla, California 92037.
Endocrinology. 1993 Dec;133(6):2554-60. doi: 10.1210/endo.133.6.8243277.
The activin-binding protein, follistatin (FS), was immunoprecipitated from metabolically labeled rat anterior pituitary cells or their media using a specific antiserum to purified porcine FS (anti-FS). Several immunoreactive proteins, including one that had a mobility in the range of 42-44 kilodaltons (kDa), were detected in the cell lysates. When immunoprecipitates of the culture medium were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, a broad 35- to 46-kDa or 39- to 53-kDa band was visualized under unreducing or reducing conditions, respectively. Upon deglycosylation by treatment with N-glycosidase-F, the secreted product migrated as a sharp protein band with an apparent size of 35 kDa. The identity or the relatedness of the immunoprecipitated proteins to FS was verified by the ability of the C-terminally truncated form of recombinant human FS (rhFS288) to compete for binding to anti-FS. When the cultured rat anterior pituitary cells were treated with either forskolin or 12-O-tetradecanoylphorbol acetate, the accumulation of FS in the culture medium was stimulated by approximately 2.5-fold. These observations suggest that the activation of either the protein kinase A or the protein kinase C signaling pathway has a stimulatory effect on anterior pituitary FS production. A more dramatic stimulation of FS secretion (up to 7-fold) was observed when the rat anterior pituitary cells were treated with activin-A. The concentration dependence for this effect was within the same range that has been reported for most of the actions of activin-A. Inhibin-A suppressed basal FS secretion and blocked its stimulation by activin-A. To determine if locally produced FS exerts an influence on the response of gonadotropes to activins, the effects of anti-FS on FSH secretion were monitored. The ability of this FS antiserum to immunoneutralize the activity of FS was initially confirmed; anti-FS attenuated the inhibitory action of exogenous follistatin on FSH secretion. Treatment of cells with the antiserum increased the apparent sensitivity of gonadotropes to submaximal concentrations of activin-A. Moreover, the presence of the antiserum lowered the concentration of activin-A that was required to produce the maximum amount of FSH secretion, without changing the magnitude of the response. These results suggested that locally produced FS interferes with the secretory response of gonadotropes to activins. Changes in locally secreted FS may, therefore, represent a mechanism by which the response of rat anterior pituitary cells to incoming stimuli are tightly regulated.
使用针对纯化猪卵泡抑素（抗FS）的特异性抗血清，从经代谢标记的大鼠垂体前叶细胞或其培养基中免疫沉淀激活素结合蛋白卵泡抑素（FS）。在细胞裂解物中检测到几种免疫反应性蛋白，包括一种迁移率在42 - 44千道尔顿（kDa）范围内的蛋白。当对培养基的免疫沉淀物进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳时，在非还原或还原条件下，分别可见一条宽的35至46 kDa或39至53 kDa的条带。用N - 糖苷酶 - F处理进行去糖基化后，分泌产物迁移为一条明显大小为35 kDa的清晰蛋白条带。重组人FS（rhFS288）的C末端截短形式竞争结合抗FS的能力验证了免疫沉淀蛋白与FS的同一性或相关性。当用福斯可林或12 - O - 十四烷酰佛波醇乙酸酯处理培养的大鼠垂体前叶细胞时，培养基中FS的积累受到约2.5倍的刺激。这些观察结果表明，蛋白激酶A或蛋白激酶C信号通路的激活对垂体前叶FS的产生具有刺激作用。当用激活素 - A处理大鼠垂体前叶细胞时，观察到FS分泌有更显著的刺激（高达7倍）。这种效应的浓度依赖性在已报道的激活素 - A大多数作用的相同范围内。抑制素 - A抑制基础FS分泌并阻断其被激活素 - A的刺激。为了确定局部产生 的FS是否对促性腺激素细胞对激活素的反应有影响，监测了抗FS对促卵泡激素（FSH）分泌的作用。最初证实了这种FS抗血清免疫中和FS活性的能力；抗FS减弱了外源性卵泡抑素对FSH分泌的抑制作用。用抗血清处理细胞增加了促性腺激素细胞对亚最大浓度激活素 - A的明显敏感性。此外，抗血清的存在降低了产生最大量FSH分泌所需的激活素 - A浓度，而不改变反应的幅度。这些结果表明局部产生的FS干扰促性腺激素细胞对激活素的分泌反应。因此，局部分泌的FS的变化可能代表一种机制，通过该机制大鼠垂体前叶细胞对传入刺激的反应受到严格调节。
