Transcriptional activation of transforming growth factor-β1 in mesangial cell culture by high glucose concentration
Transcriptional activation of transforming growth factor-β1 in mesangial cell culture by high glucose concentration
复制标题
DOI：
10.1046/j.1523-1755.1998.00119.x
复制
发表时间：
1998-10-01
影响因子：
19.6
通讯作者：
Ziyadeh, FN
中科院分区：
文献类型：
--
作者：
Hoffman, BB;Sharma, K;Ziyadeh, FN
背景。转化生长因子-β (TGF-β) 是糖尿病肾病发病机制中重要的肥厚性和促硬化细胞因子。肾细胞中高环境葡萄糖调节 TGF-β 系统的机制尚不完全明确。本研究探讨了高葡萄糖对培养的小鼠系膜细胞 (MMC) 中 TGF-β1 表达的调节机制。方法。 MMC 在正常 (100 mg/dl) 或高 (450 mg/dl) D-葡萄糖浓度下培养。测定总TGF-β1蛋白分泌及生物活性、mRNA表达及稳定性、基因转录率；进行启动子报告基因氯霉素乙酰转移酶 (CAT) 测定和电泳迁移率变动测定 (EMSA) 以研究假定的葡萄糖反应元件的存在。结果。提高环境 D-葡萄糖浓度 72 小时可使细胞培养基中的 TGF-PI 生物活性增加 47%，总 TGF-β1 分泌增加约 90%。 Northern 分析表明，在高葡萄糖条件下生长 48 小时后，稳态 TGF-β 1 mRNA 水平增加了近两倍。这种增加并不是由于稳定性增加，因为在正常和高葡萄糖条件下，消息的半升时间约为五个小时。在高葡萄糖条件下生长 24 小时的细胞中，TGF-β 1 基因的转录活性（核连载测定）增加了 73%。用含有不同长度的鼠TGF-PI启动子的CAT构建体瞬时转染MMC，证明高葡萄糖选择性地增加仅其中一种构建体pA835的表达。序列检查揭示了该构建体中存在假定的葡萄糖反应元件 CACGTG。当使用 EMSA 进行分析时，MMC 培养物中的高葡萄糖持续 24 小时会增加核蛋白与含有该元素的探针的结合。结论。高葡萄糖刺激总 TGF-β1 蛋白的产生和生物活性以及 TGF-β1 mRNA 的稳态水平。后一种效应主要是由于基因转录率的刺激而不是信息稳定性。高葡萄糖引起的转录激活可能涉及 TGF-β1 启动子中含有推定葡萄糖反应元件的区域。
Background. Transforming growth factor-beta (TGF-beta) is an important hypertrophic and prosclerotic cytokine in the pathogenesis of diabetic nephropathy. The mechanisms of regulation of the TGF-beta system by high ambient glucose in kidney cells are incompletely defined. This study examined the mechanisms of regulation of TGF-beta 1 expression by high glucose in murine mesangial cells (MMCs) in culture.Methods. MMCs were cultured in either normal (100 mg/dl) or high (450 mg/dl) D-glucose concentration. Total TGF-beta 1 protein secretion and bioactivity, mRNA expression and stability, and gene transcription rate were measured; promoter-reporter chloramphenicol acetyltransferase (CAT) assays and electrophoretic mobility shift assay (EMSA) were performed to investigate the presence of putative glucose-response elements.Results. Raising the ambient D-glucose concentration for 72 hours increased TGF-PI bioactivity in cell culture medium by 47% and total TGF-beta 1 secretion by approximately 90%. Northern analysis demonstrated that the steady-state TGF-beta 1 mRNA level was increased nearly twofold after 48 hours of growth in high glucose. This increase was not due to increased stability, as the half-lift: of the message was approximately five hours in both normal and high glucose conditions. Transcriptional activity of the TGF-beta 1 gene (nuclear run-on assay) was increased by 73% in cells grown in high glucose for 24 hours. Transiently transfected MMCs with CAT constructs containing varying lengths of the murine TGF-PI promoter demonstrated that high glucose selectively increased the expression of only one of the constructs, pA835. Sequence inspection revealed the presence of a putative glucose responsive element, CACGTG, within this construct. High glucose in MMC culture for 24 hours increased nuclear protein binding to a probe containing this element when analyzed using EMSA.Conclusions. High glucose stimulates total TGF-beta 1 protein production and bioactivity as well as the steady-state level of TGF-beta 1 mRNA. The latter effect is due primarily to stimulation of gene transcription rate rather than message stability. Transcriptional activation by high glucose may involve a region in the TGF-beta 1 promoter containing a putative glucose-response element.