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. 2019 May 31;294(22):8861-8871.
doi: 10.1074/jbc.RA118.006615. Epub 2019 Apr 18.

Telomerase reverse transcriptase ameliorates lung fibrosis by protecting alveolar epithelial cells against senescence

Tianju Liu  1 , Francina Gonzalez De Los Santos  2 , Yuyue Zhao  2 , Zhe Wu  2 , Andrew E Rinke  2 , Kevin K Kim  3 , Sem H Phan  4
Affiliations

Telomerase reverse transcriptase ameliorates lung fibrosis by protecting alveolar epithelial cells against senescence

Tianju Liu et al. J Biol Chem. .

Abstract

Mutations in the genes encoding telomerase reverse transcriptase (TERT) and telomerase's RNA components as well as shortened telomeres are risk factors for idiopathic pulmonary fibrosis, where repetitive injury to the alveolar epithelium is considered a key factor in pathogenesis. Given the importance of TERT in stem cells, we hypothesized that TERT plays an important role in epithelial repair and that its deficiency results in exacerbation of fibrosis by impairing this repair/regenerative process. To evaluate the role of TERT in epithelial cells, we generated type II alveolar epithelial cell (AECII)-specific TERT conditional knockout (SPC-Tert cKO) mice by crossing floxed Tert mice with inducible SPC-driven Cre mice. SPC-Tert cKO mice did not develop pulmonary fibrosis spontaneously up to 9 months of TERT deficiency. However, upon bleomycin treatment, they exhibited enhanced lung injury, inflammation, and fibrosis compared with control mice, accompanied by increased pro-fibrogenic cytokine expression but without a significant effect on AECII telomere length. Moreover, selective TERT deficiency in AECII diminished their proliferation and induced cellular senescence. These findings suggest that AECII-specific TERT deficiency enhances pulmonary fibrosis by heightening susceptibility to bleomycin-induced epithelial injury and diminishing epithelial regenerative capacity because of increased cellular senescence. We confirmed evidence for increased AECII senescence in idiopathic pulmonary fibrosis lungs, suggesting potential clinical relevance of the findings from our animal model. Our results suggest that TERT has a protective role in AECII, unlike its pro-fibrotic activity, observed previously in fibroblasts, indicating that TERT's role in pulmonary fibrosis is cell type-specific.

Keywords: epithelial cell; fibrosis; idiopathic pulmonary fibrosis (IPF); inflammation; lung disease; pulmonary dysfunction; senescence; telomerase; telomerase reverse transcriptase (TERT).

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Figures

Figure 1.
Figure 1.
Generation of doxycycline-induced SPC-Tert cKO mice. A, the strategy for generation of SPC-Tert cKO. Double-transgenic mice bearing the SPC-rtTA and (tetO) 7-CMV-Cre transgenes were bred to mice bearing the floxed Tert to generate triple-transgenic progeny. In the presence of doxycycline (DOX), the activated rtTA bound to (tetO) 7-CMV led to activation of Cre recombinase, which then excised the floxed Tert sequence (part of the promoter and exons 1 and 2) in AECII. B, PCR genotyping using mouse tail DNA. The PCR fragments for Tertfl/fl (343 bp) using primers P1 and P2, Spc (200 bp), Cre (500 bp), and Cre-excised SPC-Tert cKO (215 bp) using primers P1 and P5 are shown. C, Tert ablation in AECII and lung tissue. AECII and lung tissue total RNA were isolated from WT or SPC-Tert cKO mice (cKO) and analyzed for TERT (Tert) mRNA expression by qPCR. TERT mRNA levels were calculated as 2−ΔΔCT and shown as -fold change over WT controls. D, cell lysates were collected from AECII isolated from mice 7 days after doxycycline treatment and analyzed for telomerase activity by telomerase PCR ELISA and are shown as -fold change over WT controls. E, Tert levels in the indicated cell types were analyzed as described in C. *, p < 0.05 between the two indicated groups, with n = 3 for AECII, fibroblasts, T and B cells, and macrophages and n = 5 mice for lung tissue analyses.
Figure 2.
Figure 2.
TERT deficiency enhanced alveolar epithelial damage and pulmonary fibrosis in SPC-Tert cKO mice. A, WT and SPC-Tert cKO mouse lungs were lavaged on day 7 after PBS or BLM treatment. Total protein was measured using a BCA protein assay kit. BALF, bronchoalevolar lavage fluid. B, total BAL cells were counted in BAL fluids, and the numbers of F4/80+, CD3+, B220+, and Gr1+ BAL cells were determined using flow cytometry. C, representative H&E-stained lung tissue sections are shown. Original magnification, ×20. Scale bars = 500 μm. D, lung tissues from WT or SPC-Tert cKO mice were homogenized, and the lung collagen content was measured by HYP assay in whole-lung homogenates. E, lung tissue RNA was analyzed for fibrotic marker type I collagen (Col1a2) and α-SMA (Acta2) expression at the mRNA level by qPCR analysis. F and G, lung tissue RNA was also analyzed for the profibrotic cytokines FIZZ1 (Retnla), FIZZ2 (Retnlb), TGFβ1 (Tgfb1), and amphiregulin (Areg) (F) and the inflammatory cytokines MCP1 (Ccl2) and TNFα (Tnfa) (G). *, p < 0.05 between the two indicated groups; n = 3 in A and B and n = 5 mice in C and E–G; in D, n = 11 for WT-PBS, n = 13 for WT-BLM, n = 12 for cKO-PBS, and n = 13 for cKO-BLM.
Figure 3.
Figure 3.
TERT deficiency suppressed AECII proliferation in BLM-induced pulmonary fibrosis. A and B, whole-lung single-cell suspensions were analyzed for SPC- and Ki67-positive cells by flow cytometry. The total SPC-positive population was further analyzed to show the percentage of Ki67-positive cells in the SPC-positive population, which is shown in B. A representative analysis from three separate flow cytometry runs showing similar results is shown in A. Analysis from the combination of all three runs showing the average percentage of proliferated AECII that were SPC+ and Ki67+ in total SPC+ AECII is shown as a bar graph in B. C, SPC and Ki67 double immunofluorescence staining was performed on paraffin-embedded lung tissue sections. SPC signals are shown in green, Ki67 in red, and nuclei in purple-blue (DAPI). Original magnification, ×400. Scale bars = 20 μm. D, results of direct cell counting from the relevant micrographs are expressed as percentages of all lung cells (top panel) or SPC+ cells (bottom panel), with the latter indicating the percentage of proliferating AECII. *, p < 0.05 between the two indicated groups; n = 5 for WT-PBS, WT-BLM, and cKO-BLM and n = 6 for the cKO-PBS group. E, telomere length assay in AECII. Tert cKO and their control mice received doxycycline for 30 days, and AECII were isolated from mice without BLM treatment and then embedded with agarose gel. Telomere length was performed using TRF-Southern blotting. F, the proportion of the shortest telomere signals is shown as the intensity percentage of shortest telomere (<15 kb) of the total telomere signal. n = 3 mice/group.
Figure 4.
Figure 4.
TERT deficiency enhanced BLM-induced senescence in lung cells. A, AECII were isolated from WT or SPC-Tert cKO lungs on day 3 and day 7 after PBS or BLM treatment and analyzed for senescence marker p16 (Ink4a) mRNA expression by qPCR. B, the p21 (Cdkn1a) mRNA levels in isolated AECII and MLF on day7 after PBS or BLM treatment were analyzed. C, AECII and lung tissue RNA samples were analyzed for IL-6 (Il6) mRNA. *, p < 0.05 between the two indicated groups; n = 3 for PBS and n = 4 for the BLM group in A–C. D, cellular senescence in IPF. SPC and p16 double immunofluorescence microscopy was performed on paraffin-embedded human lung tissue sections from control or IPF patients. SPC signals are shown in green, p16 in red, and nuclei in purple-blue with DAPI. Representative single and merged images from control and IPF lung sections are shown. Original magnification, ×400. Scale bars = 20 μm.
Figure 5.
Figure 5.
Schematic of the cell type–specific role of TERT in pulmonary fibrosis. TERT is shown as a suppressor of senescence, which, in turn, interrupts the cell cycle in AECII and fibroblasts by derepression of p53/p21/p16. This, in turn, will suppress epithelial cell repair/regeneration, a critical factor in limiting development and progression of pulmonary fibrosis. Thus, the effect of TERT deficiency in AECII is to enhance fibrosis, as demonstrated in this study. In contrast, suppression of fibroproliferation in the case of senescence in fibroblasts should limit fibrosis, as noted previously (57).
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References

    1. Mitchell J. R., Wood E., and Collins K. (1999) A telomerase component is defective in the human disease dyskeratosis congenita. Nature 402, 551–555 10.1038/990141 - DOI - PubMed
    1. Blasco M. A., Lee H. W., Hande M. P., Samper E., Lansdorp P. M., DePinho R. A., and Greider C. W. (1997) Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell 91, 25–34 10.1016/S0092-8674(01)80006-4 - DOI - PubMed
    1. Alder J. K., Chen J. J., Lancaster L., Danoff S., Su S. C., Cogan J. D., Vulto I., Xie M., Qi X., Tuder R. M., Phillips J. A. 3rd, Lansdorp P. M., Loyd J. E., and Armanios M. Y. (2008) Short telomeres are a risk factor for idiopathic pulmonary fibrosis. Proc. Natl. Acad. Sci. U.S.A. 105, 13051–13056 10.1073/pnas.0804280105 - DOI - PMC - PubMed
    1. Armanios M. Y., Chen J. J., Cogan J. D., Alder J. K., Ingersoll R. G., Markin C., Lawson W. E., Xie M., Vulto I., Phillips J. A. 3rd, Lansdorp P. M., Greider C. W., and Loyd J. E. (2007) Telomerase mutations in families with idiopathic pulmonary fibrosis. N. Engl. J. Med. 356, 1317–1326 10.1056/NEJMoa066157 - DOI - PubMed
    1. Chibbar R., Gjevre J. A., Shih F., Neufeld H., Lemire E. G., Fladeland D. A., and Cockcroft D. W. (2010) Familial interstitial pulmonary fibrosis: a large family with atypical clinical features. Can. Respir. J. 17, 269–274 10.1155/2010/591523 - DOI - PMC - PubMed

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