Investigation of p21 in the molecular mechanisms of lung injury and pulmonary fibrosis

1.转化生长因子-β1促进fas介导的肺上皮细胞凋亡转化生长因子-β1（TGF-β1）在肺纤维化中起重要作用，并在多种类型的细胞中具有诱导凋亡的潜力。我们之前证明了Fas结扎诱导的肺上皮细胞凋亡可能参与肺纤维化的发展。本研究表明，TGF-β1通过激活caspase-3和下调周期蛋白依赖性激酶抑制剂p21诱导原代培养的细支气管上皮细胞凋亡。单独不足以诱导细胞凋亡的TGF-β1浓度可促进培养细支气管上皮细胞的抗Fas抗体或重组Fas配体介导的凋亡。特发性肺纤维化（IPF）患者支气管肺泡灌洗液（BALF）中的可溶性Fas配体也能诱导培养的细支气管上皮细胞凋亡，而抗tgf -β抗…此外，来自过敏性肺炎（HP）患者的BALF不能诱导细支气管上皮细胞凋亡，尽管其可溶性Fas配体的数量相当。IPF患者BALF中TGF-β1的浓度明显高于HP患者或对照组。此外，低浓度TGF-β1与HP BALF共孵育可产生与IPF BALF相当的促凋亡作用。在体内，TGF-β1可通过激活caspase-3促进fas介导的小鼠上皮细胞凋亡和肺损伤。我们的研究结果证明了TGF-β1作为fas介导的肺上皮细胞凋亡的增强剂在肺纤维化病理生理中的新作用。MCP-1突变体的体内基因转移减弱肺纤维化已知肺泡上皮细胞存在于肺纤维化肺损伤的原发部位。细胞凋亡与上皮细胞损伤和肺纤维化有关。由于细胞周期蛋白依赖性激酶抑制剂p21可诱导G1阻滞和DNA修复，并且它也可阻止某些细胞的凋亡，我们假设p21基因转移可减轻博莱霉素诱导的小鼠肺纤维化，其发病机制可能涉及上皮细胞凋亡。人p21基因在体外培养1 ~ 7天时在小鼠肺泡上皮细胞中表达，在腺病毒转染后1 ~ 7天时在肺上皮细胞中主要检测到p21蛋白。模型第7天炎症细胞浸润和纤维化已开始。气管内注射博来霉素后7天腺病毒转染人p21基因，14天凋亡细胞数量减少，肺部炎症和纤维化减少。因此，p21的强制表达具有抗凋亡和抗纤维化的双重作用，有助于达到治疗肺纤维化的最终目的。少

1.Transforming Growth Factor-β1 as an Enhancer of Fas-mediated Apoptosis of Lung Epithelial CellsTransforming growth factor-β1(TGF-β1) has important roles in lung fibrosis and the potential to induce apoptosis in several types of cells. We previously demonstrated that apoptosis of lung epithelial cells induced by Fas ligation may be involved in the development of pulmonary fibrosis. Here we show that TGF-β1 induces apoptosis of primary cultured bronchiolar epithelial cells via caspase-3 activation and downregulation of cyclin-dependent kinase inhibitor p21. Concentrations of TGF-β1 that were not sufficient to induce apoptosis alone could enhance agonistic anti-Fas antibody or recombinant Fas ligand-mediated apoptosis of cultured bronchiolar epithelial cells. Soluble Fas ligand in the bronchoalveolar lavage fluid(BALF) from patients with idiopathic pulmonary fibrosis(IPF) also induced apoptosis of cultured bronchiolar epithelial cells that was significantly attenuated by anti-TGF-β anti … More body. Otherwise, BALF from patients with hypersensitivity pneumonitis(HP) could not induce apoptosis on bronchiolar epithelial cells despite its comparable amounts of soluble Fas ligand. The concentrations of TGF-β1 in BALF from patients with IPF were significantly higher compared with those in BALF from patients with HP or controls. Furthermore, co-incubation with the low concentration of TGF-β1 and HP BALF created pro-apoptotic effects comparable to the IPF BALF. In vivo, the administration of TGF-β1 could enhance Fas-mediated epithelial cell apoptosis and lung injury via caspase-3 activation in mice. Our results demonstrate a novel role of TGF-β1 in the pathophysiology of pulmonary fibrosis as an enhancer of Fas-mediated apoptosis of lung epithelial cells.2.In vivo gene transfer of mutant MCP-1 attenuates pulmonary fibrosisAlveolar epithelial cells are known to be present at the primary site of lung damage in pulmonary fibrosis. Apoptosis has been implicated as being involved in epithelial cell damage and pulmonary fibrosis. Since the cyclin-dependent kinase inhibitor p21 induces G1 arrest and DNA repair, and since it also prevents apoptosis in some cells, we hypothesized that p21 gene transfer may attenuate bleomycin-induced pulmonary fibrosis in mice, the pathogenesis of which likely involves epithelial cell apoptosis. Human p21 protein was expressed in mouse alveolar epithelial cells at 1 to 7 days in vitro and was detected predominantly in lung epithelial cells at 1 to 7 days in vivo after adenoviral transfer of the human p21 gene. Inflammatory cell infiltration and fibrosis had already begun at 7 days in this model. Adenoviral transfer of the human p21 gene at 7days after intratracheal instillation of bleomycin led to a decrease in the number of apoptotic cells, lung inflammation and fibrosis at 14 days. Therefore, the forced expression of p21 exerted both anti-apoptotic and anti-fibrotic effects, which would facilitate the ultimate goal of treatment for pulmonary fibrosis. Less