Activation of the transcription factors nuclear factor-kappaB and activator protein-1 in bladder smooth muscle exposed to outlet obstruction and mechanical stretching.
Activation of the transcription factors nuclear factor-kappaB and activator protein-1 in bladder smooth muscle exposed to outlet obstruction and mechanical stretching.
复制标题
暴露于出口阻塞和机械拉伸的膀胱平滑肌中转录因子核因子-kappaB 和激活蛋白-1 的激活。
作者：
K. Persson;T. Dean;W. Steers;J. Tuttle
目的
膀胱出口梗阻，生长因子和机械力的基因转录控制知之甚少。我们分析了膀胱梗阻，机械拉伸和血小板衍生生长因子对主要生长控制转录因子核因子-κ B和激活蛋白-1的激活的影响。
材料和方法
结扎雌性大鼠近端尿道造成出口完全梗阻，分别于3、6和24小时后收集膀胱。膀胱细胞在培养物中生长，并用10 ng/ml刺激。ml.血小板衍生生长因子或10个循环/分钟机械拉伸0.5至4小时。高盐提取核蛋白，并与含有激活蛋白-1或核因子-kappaB的共有结合序列的32个磷双链寡核苷酸一起孵育。通过电泳迁移率变动测定分析所得DNA蛋白复合物。
结果
从阻塞膀胱中分离的核提取物在阻塞后3、6和24小时显示出强烈的激活蛋白-1结合活性，并且在6和24小时后显示出核因子-kappaB结合活性增加。在假手术大鼠中不存在结合活性或结合活性极小。与未拉伸的细胞相比，暴露于机械拉伸2和4小时的培养细胞显示出增加的激活蛋白-1和核因子-κ B DNA结合。同样地，用血小板衍生生长因子刺激引起激活蛋白-1和核因子-κ B结合活性的一致增加。核蛋白的结合被取消了40倍过量的未标记的特定寡核苷酸，但不是过量的无关寡核苷酸。因此，试验对所涉及的因子具有特异性。
结论
膀胱梗阻和机械拉伸导致激活蛋白-1和核因子-kappaB DNA复合物的形成，这与这些转录因子在控制肥大相关基因激活中的作用一致。
PURPOSE
Transcriptional control of bladder genes in response to outlet obstruction, growth factors and mechanical force is poorly understood. We analyzed the effects of bladder obstruction, mechanical stretching and platelet derived growth factor on the activation of the major growth controlling transcription factors nuclear factor-kappaB and activator protein-1.
MATERIALS AND METHODS
Complete outlet obstruction was created in female rats by proximal urethral ligation and bladders were harvested 3, 6 and 24 hours later, respectively. Bladder cells were grown in culture and stimulated with 10 ng./ml. platelet derived growth factor or 10 cycles per minute of mechanical stretching for 0.5 to 4 hours. Nuclear proteins were high salt extracted and incubated with 32phosphorus double strand oligonucleotides containing a consensus binding sequence for activator protein-1 or nuclear factor-kappaB. The resulting DNA protein complexes were analyzed by electrophoretic mobility shift assay.
RESULTS
Nuclear extract isolated from obstructed bladders showed intense activator protein-1 binding activity 3, 6 and 24 hours after obstruction as well as increased nuclear factor-kappaB binding activity after 6 and 24 hours. Binding activity was absent or minimal in sham operated rats. Cultured cells exposed to mechanical stretching for 2 and 4 hours showed increased activator protein-1 and nuclear factor-kappaB DNA binding compared with unstretched cells. Likewise stimulation with platelet derived growth factor caused a consistent increase in activator protein-1 and nuclear factor-kappaB binding activity. The binding of nuclear proteins was abolished by a 40-fold excess of an unlabeled specific oligonucleotide but not by excess irrelevant oligonucleotide. Thus, the assays were specific for the factors involved.
CONCLUSIONS
Bladder obstruction and mechanical stretching cause the formation of activator protein-1 and nuclear factor-kappaB DNA complexes, consistent with a role of these transcription factors in the control of hypertrophy associated gene activation.
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