OXIDANT/ANTIOXIDANT EFFECTS OF TGF BETA1 ON LUNG CELLS

TGF β是一个多功能多肽家族， 与肺重塑过程和肺部疾病有关。 我们有 发现TGF β 1对内皮细胞产生“促氧化”作用 和成纤维细胞，表现为细胞凋亡减少， 谷胱甘肽，细胞氧化还原状态的主要决定因素，和 刺激细胞外H2 O2的释放，这与 NADH升高：细胞表面的黄素氧化还原酶活性。 抗氧化剂预防TGF β 1诱导的内皮细胞生长抑制 细胞，而活性氧，降低细胞谷胱甘肽 与此相反，当生长停滞的成纤维细胞 显示对单独的TGF β 1没有增殖反应，生长刺激因子 某些生长因子的作用被TGF β 1增强。 从这些 研究中，我们假设TGF β 1对细胞氧化还原的作用 地位可能是一个重要的和以前未被认识到的调节器， 内皮细胞和成纤维细胞的生长过程。在 我们计划进一步验证这一假设， 具体将：1）进一步表征细胞表面相关的 NADH氧化酶被TGF β 1激活，并确定其与 细胞内H2 O2和氧化还原状态; 2）检查 细胞的氧化还原状态对TGF β 1的自分泌产生， TGF β 1作用的延续者，如通过定量PCR测量的， TGF β 1 mRNA、TGF β 1蛋白的ELISA和人肝癌细胞株的分析 TGF β 1基因启动子使用瞬时转染的细胞; 3）确定 刺激细胞H2 O2产生的中间过程 通过评估TGF β 1和细胞氧化还原的影响 转录因子AP-1和细胞激酶的状态;以及4） 评估H2 O2产生和细胞氧化还原状态的可能作用 在成纤维细胞产生的成纤维细胞增殖的增强中， 在TGF β 1存在下的生长因子。 我们预计 复合研究将有助于建立细胞氧化还原状态， TGF β 1作用的重要中介， 到疾病状态下肺的重塑

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.