Nahman N S, Rothe K L, Falkenhain M E, Frazer K M, Dacio L E, Madia J D, Leonhart K L, Kronenberger J C, Stauch D A
Division of Nephrology, Department of Internal Medicine, Ohio State University, Columbus 43210, USA.
J Lab Clin Med. 1996 Jun;127(6):599-611. doi: 10.1016/s0022-2143(96)90151-1.
We assessed the effect of angiotensin II on fibronectin biosynthesis a nd transcription factor activation in adult human mesangial cells in culture. We found that 10(-5) mol/L angiotensin II tended to increase fibronectin mRNA expression within 1 hour (1.2-fold +/- 0.3-fold of that in controls), with a significant increase after 4 hours (0.3-fold +/- 0.1-fold of that in controls, p < 0.05) and 24 hours (1.9-fold +/- 0.3-fold of that in controls, p < 0.02). In conjunction with increased fibronectin mRNA levels, angiotensin II exposure resulted in a significant elevation in immunoreactive fibronectin concentrations and the incorporation of (35S)-labeled methionine into fibronectin after 2 hours (224% +/- 23% of controls, p < 0.05). Angiotensin II also induced mesangial cell activation of the cyclic adenosine monophosphate response element binding protein (CREB) transcription factor, a DNA binding protein known to recognize specific regulatory elements present on the fibronectin gene promoter. Using the electrophoretic mobility shift assay, we showed that angiotensin II increased mesangial cell expression of the activated form of CREB after 4 hours (1.2-fold +/- 0.04-fold of that in controls, p < 0.05). To determine the importance of the CREB regulatory elements in mediating angiotensin II induction of fibronectin gene transcription, JEG-3 cells were transfected with plasmids containing fibronectin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs with (FN510) or without (FN122) the CREB regulatory motifs. Angiotensin II resulted in a significant increase in CAT activity in FN510 transfectants (1.6-fold +/- 0.2-fold of that in controls, p < 0.05), but there was no effect of angiotensin II on FN122 transfected cells. These data demonstrate that angiotensin II stimulates fibronectin biosynthesis in adult human mesangial cells and suggest that the process may be regulated at the transcriptional level.
我们评估了血管紧张素II对培养的成人肾小球系膜细胞中纤连蛋白生物合成及转录因子激活的影响。我们发现，10(-5) mol/L血管紧张素II在1小时内倾向于增加纤连蛋白mRNA表达（为对照的1.2倍±0.3倍），4小时后显著增加（为对照的0.3倍±0.1倍，p < 0.05），24小时后增加（为对照的1.9倍±0.3倍，p < 0.02）。伴随着纤连蛋白mRNA水平的升高，血管紧张素II作用2小时后，免疫反应性纤连蛋白浓度显著升高，且(35S)标记的甲硫氨酸掺入纤连蛋白的量增加（为对照的224%±23%，p < 0.05）。血管紧张素II还诱导肾小球系膜细胞中环状腺苷单磷酸反应元件结合蛋白（CREB）转录因子的激活，CREB是一种已知能识别纤连蛋白基因启动子上特定调控元件的DNA结合蛋白。使用电泳迁移率变动分析，我们发现血管紧张素II作用4小时后增加了系膜细胞中活化形式的CREB的表达（为对照的1.2倍±0.04倍，p < 0.05）。为了确定CREB调控元件在介导血管紧张素II诱导纤连蛋白基因转录中的重要性，用含有纤连蛋白启动子-氯霉素乙酰转移酶（CAT）报告基因构建体（有（FN510）或无（FN122）CREB调控基序）的质粒转染JEG-3细胞。血管紧张素II导致FN510转染细胞中CAT活性显著增加（为对照的1.6倍±0.2倍，p < 0.05），但血管紧张素II对FN122转染细胞无影响。这些数据表明，血管紧张素II刺激成人肾小球系膜细胞中纤连蛋白的生物合成，并提示该过程可能在转录水平受到调控。
