Nimmer D, Bergtrom G, Hirano H, Amrani D L
J Biol Chem. 1987 Jul 25;262(21):10369-75.
Plasma fibronectin is an acute-phase reactant synthesized by hepatocytes. Glucocorticoids are one of the major factors implicated in controlling the hepatic acute-phase response. To study the regulatory effects of glucocorticoids on plasma fibronectin biosynthesis, a model chick hepatocyte culture system under serum- and hormone-free conditions was used. In the presence of either dexamethasone or corticosterone, secreted plasma fibronectin increased maximally to 2.8-fold above basal levels. The stimulatory effect of the hormones was maintained only in their continuous presence, since plasma fibronectin production dropped to near basal levels within 16 h of glucocorticoid withdrawal. Pulse-chase studies indicated that pretreatment of cells with dexamethasone stimulated the level of secreted plasma fibronectin but had no effect on its rate of secretion. The increase in plasma fibronectin production by dexamethasone was abolished in a dose-dependent manner by the addition of progestin, an antagonist of dexamethasone known to compete specifically for the liver glucocorticoid receptor. Actinomycin D and alpha-amanitin, which are inhibitors of transcription, also blocked the early dexamethasone effect on plasma fibronectin synthesis. Slot blot hybridization of total RNA samples from dexamethasone-treated cultures revealed a 6-fold stimulatory rise in fibronectin mRNA during the first 6 h of treatment, which later declined and was no longer evident at 48 and 72 h. However, fibronectin mRNA levels were elevated to about the same extent in control and dexamethasone-treated cells at the later time points. During the same time period (0 to 72 h), plasma fibronectin protein levels rose and remained elevated. Evaluation of pulse-chase experiments following pretreatment with hormone for 48 h demonstrated that equal amounts of plasma fibronectin were translated by dexamethasone-treated and control cells, but only 42% of labeled plasma fibronectin was secreted by control cells compared with 93% for dexamethasone-treated cells. These findings suggest that the early phase of glucocorticoid regulation of plasma fibronectin biosynthesis occurs at the transcriptional level and is mediated through the specific action of the glucocorticoid receptor. A later phase of glucocorticoid-stimulated plasma fibronectin biosynthesis results from modulation of post-translational processing events leading to secretion of an increased amount of newly translated plasma fibronectin polypeptides.
血浆纤连蛋白是一种由肝细胞合成的急性期反应物。糖皮质激素是参与控制肝脏急性期反应的主要因素之一。为了研究糖皮质激素对血浆纤连蛋白生物合成的调节作用，使用了一种在无血清和无激素条件下的雏鸡肝细胞培养系统。在地塞米松或皮质酮存在的情况下，分泌的血浆纤连蛋白最大增加至基础水平的2.8倍。激素的刺激作用仅在其持续存在时维持，因为在糖皮质激素撤除后16小时内血浆纤连蛋白产量降至接近基础水平。脉冲追踪研究表明，用地塞米松预处理细胞可刺激分泌的血浆纤连蛋白水平，但对其分泌速率没有影响。通过添加孕激素（一种已知可特异性竞争肝脏糖皮质激素受体的地塞米松拮抗剂），地塞米松引起的血浆纤连蛋白产量增加以剂量依赖性方式被消除。放线菌素D和α-鹅膏蕈碱（转录抑制剂）也阻断了地塞米松对血浆纤连蛋白合成的早期作用。来自地塞米松处理培养物的总RNA样品的狭缝印迹杂交显示，在处理的前6小时内纤连蛋白mRNA有6倍的刺激升高，随后下降，在48和72小时时不再明显。然而，在后期时间点，对照细胞和地塞米松处理细胞中的纤连蛋白mRNA水平升高到大致相同的程度。在同一时间段（0至72小时）内，血浆纤连蛋白蛋白水平上升并保持升高。在用激素预处理48小时后的脉冲追踪实验评估表明，地塞米松处理的细胞和对照细胞翻译的血浆纤连蛋白量相等，但对照细胞中只有42%的标记血浆纤连蛋白被分泌，而地塞米松处理的细胞为93%。这些发现表明，糖皮质激素调节血浆纤连蛋白生物合成的早期阶段发生在转录水平，并通过糖皮质激素受体的特异性作用介导。糖皮质激素刺激血浆纤连蛋白生物合成的后期阶段是由于翻译后加工事件的调节，导致分泌增加量的新翻译血浆纤连蛋白多肽。
