Department of Human Anatomy and Cell Science, University of Manitoba, Manitoba, Canada.
Biology of Breathing Group, Children's Hospital Research Institute of Manitoba, Manitoba, Canada.
Am J Physiol Lung Cell Mol Physiol. 2018 Mar 1;314(3):L493-L504. doi: 10.1152/ajplung.00372.2017. Epub 2017 Oct 26.
Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β (TGF-β) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.
特发性肺纤维化（IPF）是一种致命的成人肺纤维化疾病，治疗选择有限。自噬和未折叠蛋白反应（UPR）是细胞应激诱导的基本过程，在 IPF 患者的肺成纤维细胞和上皮细胞中失调。用转化生长因子-β（TGF-β）刺激来自非 IPF 和 IPF 供体的人原代培养的肺实质和气道成纤维细胞，并用自噬或 UPR（IRE1 抑制剂）抑制剂处理。通过免疫印迹，我们监测了自噬（LC3βII 和 Atg5-12）、UPR（BIP、IRE1α 和 cleaved XBP1）和纤维化（胶原 1α2 和纤连蛋白）蛋白标志物的时间变化。使用荧光免疫组织化学，我们对非 IPF 和 IPF 肺组织中的自噬（LC3βII）和 UPR（BIP 和 XBP1）标志物进行了分析。与非 IPF 供体的肺和气道成纤维细胞相比，IPF 肺成纤维细胞中 TGF-β 诱导的胶原 1α2 和纤连蛋白蛋白产生显著更高。TGF-β 诱导 LC3βII 的积累与胶原 1α2 和纤连蛋白平行，但 IPF 患者的肺成纤维细胞中的自噬标志物含量明显较低。在非 IPF 和 IPF 供体的成纤维细胞中，抑制自噬流可显著降低 TGF-β 诱导的胶原和纤连蛋白生物合成。相反，仅在 IPF 供体的肺成纤维细胞中，TGF-β 诱导 UPR 标志物。IRE1 抑制剂处理可降低 IPF 肺成纤维细胞中 TGF-β1 诱导的胶原 1α2 和纤连蛋白生物合成，但不能降低非 IPF 供体的成纤维细胞中 TGF-β1 诱导的胶原 1α2 和纤连蛋白生物合成。IRE1 途径的 UPR 反应仅在 IPF 供体的肺成纤维细胞中被 TGF-β 诱导，并且是这些细胞中胶原和纤连蛋白过度生物合成所必需的。
