Department of Physiology & Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.
Department of Medicine, Boston University School of Medicine, Boston, Massachusetts.
Am J Physiol Lung Cell Mol Physiol. 2020 May 1;318(5):L852-L863. doi: 10.1152/ajplung.00324.2019. Epub 2020 Mar 11.
Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.
特发性肺纤维化（IPF）导致肺组织过度细胞外基质（ECM）产生而发生瘢痕形成。固有成纤维细胞是参与 ECM 沉积的主要细胞类型。导致病理性成纤维细胞激活和异常 ECM 沉积的生化途径尚未完全阐明。Tank 结合蛋白激酶-1（TBK1）是一种激酶，可调节多种信号通路，最近在大规模小干扰 RNA（siRNA）筛选中被鉴定为成纤维细胞激活的候选调节剂。为了确定 TBK1 对成纤维细胞激活的影响，我们在正常和 IPF 人肺成纤维细胞中通过药理学（MRT-68601）和遗传学（siRNA）抑制 TBK1。降低 TBK1 的活性或表达可使 TGF-β刺激的正常和 IPF 成纤维细胞中α-平滑肌肌动蛋白应激纤维水平降低 40-60%，胶原 I 和纤维连接蛋白等 ECM 成分的沉积降低 50%。YAP 和 TAZ 是同源的机械调节性促纤维化转录共激活因子，已知可调节成纤维细胞激活。TBK1 敲低或抑制可降低 YAP/TAZ 的总蛋白和核蛋白水平。此外，低细胞-细胞接触和增加 ECM 底物硬度可增强 TBK1 的磷酸化和激活，这与调节 YAP/TAZ 的信号一致。TBK1 对 YAP/TAZ 激活的作用不依赖于 LATS1/2 和经典的 TBK1 下游信号转导介质 IRF3，但依赖于细胞的蛋白酶体机制。本研究鉴定 TBK1 为人类肺成纤维细胞的促纤维化激活剂，表明 TBK1 可能是肺纤维化的一个新的治疗靶点。
