Transcriptional and Proteomic Characterization of Telomere-Induced Senescence in a Human Alveolar Epithelial Cell Line

Cellular senescence due to telomere dysfunction has been hypothesized to play a role in age-associated diseases including idiopathic pulmonary fibrosis (IPF). It has been postulated that paracrine mediators originating from senescent alveolar epithelia signal to surrounding mesenchymal cells and contribute to disease pathogenesis. However, murine models of telomere-induced alveolar epithelial senescence fail to display the canonical senescence-associated secretory phenotype (SASP) that is observed in senescent human cells. In an effort to understand human-specific responses to telomere dysfunction, we modeled telomere dysfunction-induced senescence in a human alveolar epithelial cell line. We hypothesized that this system would enable us to probe for differences in transcriptional and proteomic senescence pathways in vitro and to identify novel secreted protein (secretome) changes that potentially contribute to the pathogenesis of IPF. Following induction of telomere dysfunction, a robust senescence phenotype was observed. RNA-seq analysis of the senescent cells revealed the SASP and comparisons to previous murine data highlighted differences in response to telomere dysfunction. We conducted a proteomic analysis of the senescent cells using a novel biotin ligase capable of labeling secreted proteins. Candidate biomarkers selected from our transcriptional and secretome data were then evaluated in IPF and control patient plasma. Four novel proteins were found to be differentially expressed between the patient groups: stanniocalcin-1, contactin-1, tenascin C, and total inhibin. Our data show that human telomere-induced, alveolar epithelial senescence results in a transcriptional SASP that is distinct from that seen in analogous murine cells. Our findings suggest that studies in animal models should be carefully validated given the possibility of species-specific responses to telomere dysfunction. We also describe a pragmatic approach for the study of the consequences of telomere-induced alveolar epithelial cell senescence in humans.

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell

Purpose. Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. Methods. FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. Results. FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. Conclusion. AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.