Telomerase Dysfunction Induces Lung Epithelial Aging

Along with the increase of aging population, the morbidity and mortality of lung disease was rising rapidly in the past twenty years. Elderly people experience more environmental exposures, including tobacco smoke, air pollution, respiratory infections and occupational dusts. They also get impaired organ function, resulting in increased susceptibility to injury and development of lung diseases. Among the lung diseases, Idiopathic pulmonary fibrosis(IPF) is a progressive lung disease with a usual interstitial pneumonia pattern and has poor prognosis. IPF patients live on average 3 years after diagnosis, and no effective treatments present up-to-date. Lungs from patients with idiopathic pulmonary fibrosis are characterized by heterogeneous distribution of normal or mildly affected regions, alternating with regions of alveolar epithelial cell injury and hyperplasia, Fibrotic areas contain honeycombing, and fibroblastic foci. Though the etiology of IPF is not folly understood, the histological characteristics lead to the notion that the epithelial micro injuries trigger abnormal repair and hyperplasia. Lung alveolar epithelial type II cells(AECII) in alveolar space have been suggested as region-specific epithelial stem/progenitor cell populations in the adult lung of mice and humans, the impairment and senescence of AECII is the vital factor of lung aging, and highly associated with onset and development of lung diseases with age. The molecular mechanism of AECII senescence is still a challenge in lung aging research.Part I:Knockout of Smad7 mediated telomerase down-regulation induces lung epithelial agingDuring the initial injury phase, activated alveolar epithelial cells and recruited inflammatory cells release profibrogenic growth factors like TGFβ, TNF-a and PDFG to perpetuate the cycle of injury, repair failure and fibrosis, especially the strongest profibrotic factor TGFβ. TGFβ pathway is tightly associated to IPF. Whereas most research has focused on the different phenotypes resulted from activation of TGFβ pathway and the relieving effects induced by its inhibition, less is known about the molecular mechanism of the pathogenesis of IPF. Here we take advantage of Smad7 knockout in AECII mice as a model to unveil the molecular mechanism of this TGFP pathway mediated lung aging.1. First we investigated the expression pattern of Smad7 in lungs of normal human and IPF patients, we found Smad7 deceased in IPF patient lungs. And Smad7 down regulated with age in mice lungs. Smad7 is an inhibitor of TGFP signaling pathway. Less Smad7 in elderly lungs may indicate high activity of TGFβ and the predisposition for IPF since TGFβ1 is likely the strongest profibrogenic cytokine.2. Second, we found the number of AECII in KO mice lung tissue is reduced compared with wild type. In contrast to wild type, KO mice exhibited more aging markers (like p16, p21 and HPlγ) expression in AECII as well as increasedβ gal staining and a-SMA expression. These results showed knockout of Smad7 can induce lung aging. As known, AECII cells proliferate, differentiate into AECI cells, and contribute to epithelial repair, they also help in the synthesis and secretion of surface-active material. Thus AECII is the key factor to lung epithelial aging and has a close relationship with the initial and development of aging associated lung diseases.3. Later, we did the micro-array assay in AECII and among these affected gene we saw the decreased mTert level in AECII from KO mice. The transcriptional levels of telomere binding proteins like shelterin and telomerase were detected by Q-PCR in Smad7 knockdown A549 cells, and we noted a strong inhibitory effect of TERT in this population.4. Since down regulation Smad7 leads to mTert repression, we tracked lung epithelial aging phenotype of Smad7 and mTert knockout mice in 2m,3m and 8m age mice, we found the p21 positive ratio in AECII between Smad7 and mTert knockout mice lungs got a similar result,5. Further we want to know whether Smad7 knockout can shorten telomere length, hence we used tissue FISH and Q-FISH to detect AECII telomere length in lung tissue and that of isolated AECII respectively, both of the results demonstrated that AECII got short telomere length and increased TIFs in KO mice. This led us to conclude deletion of Smad7 mimics an activated TGFPsignaling and inhibited the maintenance of telomere length, and further leads to the recognition of chromosome ends as DNA damage sites and recruitment of DNA damage responsers.6. Treating A549 cells with TGFβ1 cytokine can induced TIFs, while overexpression TERT can rescue and inhibit TIFs formation, this indicates that TGFβ/Smad signaling pathway may inhibit TERT and lead to dysfunction of adding de novo telomere repeats by telomerase, thus shorten telomere length and cause senescence ultimately. In AECII double knockout (Smad7 KO and mTert KI) mice, increased β-gal positive staining phenotype or β gal activity in AECII were rescued in contrast to AECII Smad7 KO mice, also the AECII counts in DK mice elevated compared with that of KO mice. It indicate the phenotype occurred in Smad7 KO mice may mediated by mTert inhibition.In conclusion, the data presented here show that knockout Smad7 mimics TGFP signaling activation, nuclear localization of phosphorylated Smad3 inhibit Tert expression, and consequently impair telomerase function and result in telomere shortening and increased TIFs formation. Furthermore smad7 knockout induces cell cycle arrest and makes cell enter senescence state as well as reducing AECII number. Excitingly, In AECII DK (Smad7 KO and mTert KI) mice, increased p-gal positive staining phenotype can be rescued in contrast to AECII Smad7 KO mice. For first time, we reported Smad7 conditional knockout in AECII resulted TGFβ signaling activation, impaired maintenance of telomere length and AECII senescence. This could be a novel molecular mechanism of IPF initiation and thus offers us a further understanding about aging associated lung diseases and its prevention.Part Ⅱ:Telomerase deficiency causes alveolar stem cell senescence-associated low-grade inflammation in lungsTGFβ pathway is tightly associated to IPF while other gene mutations found in IPF patients. 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. Whereas most research has focused on the different phenotypes resulted from activation of TGFβ pathway and the relieving effects induced by its inhibition, less is known about the molecular mechanism of the pathogenesis of IPF. In part Ⅰ, we found Smad7 knockout mice got a repression of mTert expression in lungs and resulted in a lung epithelial aging phenotype. Here we take advantage of telomerase deficiency mice as a model to unveil the molecular mechanism of AECⅡ senescence mediated lung aging. In this report, we show that knockout (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 (α-SMA) and collagen 1α1 (Col1α1) suggesting myofibroblast differentiation, TERC deficiency also leads to marked cellular infiltrations of a mononuclear cell population positive for the leukocyte common antigen CD45 (LCA), 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.