OXIDANT/ANTIOXIDANT EFFECTS OF TGF BETA1 ON LUNG CELLS

TGFbetas是一个多功能多肽家族，一直以来都具有很强的 与肺重塑过程和肺部疾病有关。我们有 发现TGFbeta1对内皮细胞有“促氧化”作用 和成纤维细胞在培养中表现为细胞减少 谷胱甘肽，细胞氧化还原状态的主要决定因素，以及 刺激细胞外过氧化氢释放与血管紧张素转换酶 NADH的升高：细胞表面的黄素氧化还原酶活性。 抗氧化剂拮抗TGFbeta1诱导的内皮细胞生长抑制 细胞内活性氧产生时，细胞内谷胱甘肽含量降低 而Fe3+则增强了这一作用。相比之下，虽然生长受阻的成纤维细胞 对单独的生长刺激因子TGFbeta1没有增殖反应 某些生长因子的作用被TGFbeta1增强。从这些 研究表明，我们假设TGFbeta1对细胞氧化还原的作用 地位可能是一个重要的、以前未被承认的调节器 内皮细胞和成纤维细胞的生长过程。在 目前的建议，我们计划进一步检验这一假设，并 具体将：1)进一步表征与细胞表面相关的 TGFbeta1激活的NADH氧化酶及其与NADH的关系 细胞内过氧化氢和氧化还原状态；2)检测 TGFbeta1在自分泌过程中细胞的氧化还原状态 通过定量聚合酶链式反应检测的转化生长因子β1作用的永久化 人TGFbeta1mRNA、TGFbeta1蛋白的酶联免疫吸附试验及分析 利用瞬时转基因细胞检测TGFbeta1基因启动子 细胞内过氧化氢刺激产生的中间过程 通过评估TGFbeta1和细胞氧化还原的影响而产生 转录因子AP-1和细胞激酶的研究现状；4) 评估过氧化氢产生和细胞氧化还原状态的可能作用 在促进成纤维细胞增殖中的作用 生长因子在TGFbeta1上的存在。我们预计， 综合研究将有助于建立细胞氧化还原状态作为 在TGFbeta1的行动中的重要中介可能有助于 到疾病状态下的肺重塑。

TGFbetas, a family of multifunctional polypeptides, have been strongly implicated in pulmonary remodeling processes and lung disease. We have found that TGFbeta1 produces a "pro-oxidant" effect on endothelial cells and fibroblasts in culture as manifested by a reduction in cellular glutathione, a major determinant of cellular redox status, and stimulation of extracellular H2O2 release that is associated with an elevation in NADH: flavin oxidoreductase activity at the cell surface. Anti-oxidants prevent TGFbeta1-induced growth inhibition of endothelial cells, while reactive oxygen species, lowering of cellular glutathione and Fe3+ potentiate it. In contrast, while growth-arrested fibroblasts show no proliferative response to TGFbeta1 alone, the growth stimulatory effect of certain growth factors is enhanced by TGFbeta1. From these studies, we hypothesize that the action of TGFbeta1 on cellular redox status may be an important and previously unrecognized regulator of growth processes of both endothelial cells and fibroblasts. In the present proposal we plan to further test this hypothesis and specifically will: 1) Further characterize the cell surface-associated NADH oxidase activated by TGFbeta1 and determine its relationship to intracellular H2O2 and redox status; 2) Examine the influence of the redox status of the cell on autocrine production of TGFbeta1, a likely perpetuator of TGFbeta1 action, as measured by a quantitative PCR for TGFbeta1 mRNA, ELISA for TGFbeta1 protein and analysis of the human TGFbeta1 gene promoter using transiently transfected cells; 3) Determine intermediary processes resulting from stimulation of cellular H2O2 production by assessing the influence of TGFbeta1 and cellular redox status on the transcription factor AP-1 and cellular kinases; and 4) Evaluate the possible role of H2O2 production and cellular redox status in the augmentation of fibroblast proliferation produced by fibroblast growth factor in the presence on TGFbeta1. We anticipate that the composite studies will help to establish cellular redox status as an important intermediary in the actions of TGFbeta1 that may contribute to remodeling of the lung in disease states.