Telomerase Deficiency Causes Alveolar Stem Cell Senescence-associated Low-grade Inflammation in Lungs

Ruping Chen,Kexiong Zhang,Hao Chen,Xiaoyin Zhao,Jianqiu Wang,Li Li,Yusheng Cong,Zhenyu Ju,Dakang Xu,Bryan R.G Williams,Jihui Jia,Jun-Ping Liu

doi:10.1074/jbc.m115.681619

Abstract

Mutations of human telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT) are associated with a subset of lung aging diseases, but the mechanisms by which TERC and TERT participate in lung diseases remain unclear. In this report, we show that knock-out (KO) of the mouse gene Terc or Tert causes pulmonary alveolar stem cell replicative senescence, epithelial impairment, formation of alveolar sacs, and characteristic inflammatory phenotype. Deficiency in TERC or TERT causes a remarkable elevation in various proinflammatory cytokines, including IL-1, IL-6, CXCL15 (human IL-8 homolog), IL-10, TNF-α, and monocyte chemotactic protein 1 (chemokine ligand 2 (CCL2)); decrease in TGF-β1 and TGFβRI receptor in the lungs; and spillover of IL-6 and CXCL15 into the bronchoalveolar lavage fluids. In addition to increased gene expressions of α-smooth muscle actin and collagen 1α1, suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45, low-affinity Fc receptor CD16/CD32, and pattern recognition receptor CD11b in the lungs. Our data demonstrate for the first time that telomerase deficiency triggers alveolar stem cell replicative senescence-associated low-grade inflammation, thereby driving pulmonary premature aging, alveolar sac formation, and fibrotic lesion.

Highlights

Telomerase is a ribonucleoprotein complex that operates to maintain telomeres, counteracting cell division-associated telomere shortening in the stem cell compartment and cancer [1, 2]
Decreased alveolar numbers (Fig. 1C), detection of alveolar fusion and formation of alveolar sacs (Fig. 1D), and increased total alveolar surface areas (Fig. 1, A, B, and E), indicative of pulmonary epithelial aging and atrophy. ␤-Gal staining for cellular senescence was markedly increased in G3 telomerase RNA component (TERC)-null and G3 telomerase reverse transcriptase (TERT)-null lung sections compared with control (Fig. 1, A and B)
Upon examination of the alveolar stem cell AECII population that express surfactant protein-C (SPC) and telomerase activity [25, 32, 37], we found that senescent ␤-gal-positive cells were significantly increased among the AECII population from G2 TERC-null and G3 TERT-null animals by flow-␤-gal analysis (Fig. 1, F–H)

Summary

Experimental Procedures

Animals and AECII Isolation—All of the mice used were on the C57BL/6J genetic background. Homozygous TERT-null mice [31], and homozygous TERC-null mice [18] were kept in the Animal Experimental Center of Hangzhou Normal University. Telomere Fluorescence in Situ Hybridization (FISH)—The sorted AECII were attached to microscope glass slides using a cytospin machine (Thermo); fixed at Ϫ20 °C in 100% cold methanol for 10 min; dehydrated in ethanol with 70, 95, and 100% EtOH for 5 min consecutively; and air-dried for ϳ2 min. Cells were fixed with 2% (w/v) paraformaldehyde for 5 min at room temperature; washed in PBS twice; dehydrated in ethanol with 70, 95, and 100% EtOH for 5 min consecutively; and dried in air. The incubation was stopped with prechilled PBS, which was followed by incubating the cell suspension with AECII markers at 4 °C for 40 min.

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