Department of Medicine, Cedars-Sinai Medical Center, Women's Guild Lung Institute, 127 S San Vicente Blvd., AHSP A9315, Los Angeles, CA, 90048, USA.
Laboratory of Dynamic Imaging, Mechanical and Aerospace Engineering, Monash University, Clayton, VIC, 3800, Australia.
BMC Pulm Med. 2019 Aug 29;19(1):165. doi: 10.1186/s12890-019-0922-7.
Recent studies have highlighted the contribution of senescent mesenchymal and epithelial cells in Idiopathic Pulmonary Fibrosis (IPF), but little is known regarding the molecular mechanisms that regulate the accumulation of senescent cells in this disease. Therefore, we addressed the hypothesis that the loss of DNA repair mechanisms mediated by DNA protein kinase catalytic subunit (DNA-PKcs) in IPF, promoted the accumulation of mesenchymal progenitors and progeny, and the expression of senescent markers by these cell types.
Surgical lung biopsy samples and lung fibroblasts were obtained from patients exhibiting slowly, rapidly or unknown progressing IPF and lung samples lacking any evidence of fibrotic disease (i.e. normal; NL). The expression of DNA-Pkcs in lung tissue was assessed by quantitative immunohistochemical analysis. Chronic inhibition of DNA-PKcs kinase activity was mimicked using a highly specific small molecule inhibitor, Nu7441. Proteins involved in DNA repair (stage-specific embryonic antigen (SSEA)-4 cells) were determined by quantitative Ingenuity Pathway Analysis of transcriptomic datasets (GSE103488). Lastly, the loss of DNA-PKc was modeled in a humanized model of pulmonary fibrosis in NSG SCID mice genetically deficient in PRKDC (the transcript for DNA-PKcs) and treated with Nu7441.
DNA-PKcs expression was significantly reduced in IPF lung tissues. Chronic inhibition of DNA-PKcs by Nu7441 promoted the proliferation of SSEA4 mesenchymal progenitor cells and a significant increase in the expression of senescence-associated markers in cultured lung fibroblasts. Importantly, mesenchymal progenitor cells and their fibroblast progeny derived from IPF patients showed a loss of transcripts encoding for DNA damage response and DNA repair components. Further, there was a significant reduction in transcripts encoding for PRKDC (the transcript for DNA-PKcs) in SSEA4 mesenchymal progenitor cells from IPF patients compared with normal lung donors. In SCID mice lacking DNA-PKcs activity receiving IPF lung explant cells, treatment with Nu7441 promoted the expansion of progenitor cells, which was observed as a mass of SSEA4 CgA expressing cells.
Together, our results show that the loss of DNA-PKcs promotes the expansion of SSEA4 mesenchymal progenitors, and the senescence of their mesenchymal progeny.
最近的研究强调了衰老的间充质和上皮细胞在特发性肺纤维化（IPF）中的贡献，但对于调节这种疾病中衰老细胞积累的分子机制知之甚少。因此，我们提出了这样一个假设，即在 IPF 中，DNA 蛋白激酶催化亚基（DNA-PKcs）介导的 DNA 修复机制的丧失，促进了间充质祖细胞和后代的积累，并导致这些细胞类型表达衰老标志物。
从表现为缓慢、快速或未知进展的 IPF 的患者的手术肺活检样本和肺成纤维细胞以及缺乏任何纤维化疾病证据的肺样本（即正常；NL）中获得肺组织。通过定量免疫组织化学分析评估肺组织中 DNA-Pkcs 的表达。使用高度特异性的小分子抑制剂 Nu7441 模拟慢性抑制 DNA-PKcs 激酶活性。通过定量分析转录组数据集（GSE103488）中的 Ingenuity 通路分析，确定参与 DNA 修复的蛋白质（阶段特异性胚胎抗原（SSEA）-4 细胞）。最后，在 PRKDC（DNA-PKcs 的转录物）基因缺失的 NSG SCID 小鼠的人源化肺纤维化模型中，对 DNA-Pk 的缺失进行建模，并使用 Nu7441 进行治疗。
DNA-PKcs 的表达在 IPF 肺组织中显著降低。慢性抑制 DNA-PKcs 可促进 SSEA4 间充质祖细胞的增殖，并显著增加培养的肺成纤维细胞中衰老相关标志物的表达。重要的是，来自 IPF 患者的间充质祖细胞及其成纤维细胞后代显示出编码 DNA 损伤反应和 DNA 修复成分的转录本的缺失。此外，与正常肺供体相比，来自 IPF 患者的 SSEA4 间充质祖细胞中编码 PRKDC（DNA-PKcs 的转录物）的转录本显著减少。在缺乏 DNA-PKcs 活性并接受 IPF 肺外植体细胞的 SCID 小鼠中，Nu7441 的治疗促进了祖细胞的扩增，这表现为大量表达 SSEA4 CgA 的细胞团。
综上所述，我们的研究结果表明，DNA-PKcs 的缺失促进了 SSEA4 间充质祖细胞的扩增及其间充质后代的衰老。
