Mechanism of tumor necrosis factor gene expression in the development of heart failure and cardiac hypertrophy

临床观察表明，肿瘤坏死因子（TNF）的浓度升高，在晚期心力衰竭或肥厚型心肌病患者。然而，在这些疾病的背景下TNF产生的机制知之甚少。我们最近发现，血管紧张素II（ANGII）以及机械拉伸刺激心脏成纤维细胞中TNF的产生。为了扩展这些观察结果，我们研究了ANGII上调TNFα基因表达的分子机制。使用新生大鼠心脏成纤维细胞。北方印迹分析显示，血管紧张素Ⅱ、内皮素-1（ET-1）或脂多糖（LPS）均可诱导TNFαmRNA表达。TNFαmRNA水平的增加在转录水平上受到TNFα启动子基因荧光素酶活性的调节。对TNFα启动子基因进行性缺失分析表明，ANGII反应区位于转录起始位点的-100 ~-70bp之间。另一方面，LPS反应区位于-200至-100 bp之间。进一步的突变分析表明ANGII反应元件可以与Bs和NF-κ B相互作用，而LPS反应元件可以与Sp1相互作用。进行了额外的研究，以评价与LPS诱导的TNFα基因表达相关的细胞内信号传导途径。PKG特异性抑制剂KT 5823可抑制TNFαmRNA表达的增加，而MAP激酶、PKA或PKC抑制剂对TNFαmRNA表达的增加无影响。此外，8溴环GMP诱导TNFαmRNA表达。结论：ANG Ⅱ、ET 1和LPS可转录激活心肌成纤维细胞TNFα基因的表达。然而，这种反应是由ANGII和LPS通过不同的转录因子介导的。因此，心力衰竭或心脏肥大中TNF产生的机制应该与感染性疾病中的机制不同。此外，PKG依赖性信号通路可能对这种反应很重要。

Clinical observations indicate that the concentration of tumor necrosis factor (TNF) is elevated in patients with advanced heart failure or hypertrophic cardiomyopathy. However, the mechanisms for TNF production in the setting of these diseases are poorly understood. We recently showed that angiotensin II (ANGII) as well as mechanical stretch stimulate the production of TNF in cardiac fibroblasts. To extend these observations, we have examined the moleculer mechanisms by which ANGII up regulates TNFα gene expression. Using neonatal rat cardiac fibroblasts. Northern blot analysis showed that treatment with ANGII, endothelin-1 (ET1) or lipopolysaccharide (LPS) induced TNFαmRNA expression. These increase in TNFαmRNA level were regulated at transcriptional level as determined by luciferase activity of TNFα promoter gene. Progressive deletion analysis of TNFα promoter gene showed that the ANGII-responsive region lies between -100 to -70bp from the transcription start site. On the other hand, LPS-responsive region lies between -200 to -100bp. Further, mutation analysis indicated that the ANGII-responsive element could interact with Bs and NF-_KB, but the LPS-responsive element could interact with Sp1 . Additional studies were performed to evaluate the intracelluler signaling pathway associated LPS-induced TNFα gene expression. The increase of TNFαmRNA expression was attenuated by KT5823 which is a PKG specific inhibitor, whereas MAP kinase, PKA or PKC inhibitors had no effect on this response. Moreover, 8 bromo-cyclic GMP induced TNFαmRNA expression. We conclude that TNFα gene expression is transcriptionaly activated by ANGII, ET1 and LPS in cardiac fibroblasts. However, this response is mediated through different transcriptional factors by ANGII and LPS. Thus, the mechanisms for TNF production in heart failure or cardiac hypertrophy should differ from those in infectious disease. Further, PKGdependent signaling pathway may be important for this response.