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. 2007 Dec;117(12):3800-9.
doi: 10.1172/JCI32369.

Telomerase activity is required for bleomycin-induced pulmonary fibrosis in mice

Tianju Liu  1 , Myoung Ja Chung, Matthew Ullenbruch, Hongfeng Yu, Hong Jin, Biao Hu, Yoon Young Choi, Fuyuki Ishikawa, Sem H Phan
Affiliations

Telomerase activity is required for bleomycin-induced pulmonary fibrosis in mice

Tianju Liu et al. J Clin Invest. 2007 Dec.

Abstract

In addition to its well-known expression in the germline and in cells of certain cancers, telomerase activity is induced in lung fibrosis, although its role in this process is unknown. To identify the pathogenetic importance of telomerase in lung fibrosis, we examined the effects of telomerase reverse transcriptase (TERT) deficiency in a murine model of pulmonary injury. TERT-deficient mice showed significantly reduced lung fibrosis following bleomycin (BLM) insult. This was accompanied by a significant reduction in expression of lung alpha-SMA, a marker of myofibroblast differentiation. Furthermore, lung fibroblasts isolated from BLM-treated TERT-deficient mice showed significantly decreased proliferation and increased apoptosis rates compared with cells isolated from control mice. Transplantation of WT BM into TERT-deficient mice restored BLM-induced lung telomerase activity and fibrosis to WT levels. Conversely, transplantation of BM from TERT-deficient mice into WT recipients resulted in reduced telomerase activity and fibrosis. These findings suggest that induction of telomerase in injured lungs may be caused by BM-derived cells, which appear to play an important role in pulmonary fibrosis. Moreover, TERT induction is associated with increased survival of lung fibroblasts, which favors the development of fibrosis instead of injury resolution.

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Figures

Figure 1
Figure 1. Effects of BLM treatment on lung HYP content.
Whole-lung homog­enates from the indicated strains were collected at day 21 after BLM or saline administration as indicated. Results (mean ± SEM) are expressed as μg per lung. n = 5 per group.
Figure 2
Figure 2. Histopathological changes of the lung tissues at day 21.
Representative H&E-stained lung tissue sections from WT and TERT–/– mice treated by BLM or saline are shown, with higher-magnification views in insets. (A) Saline-treated TERT–/–. (B) BLM-treated TERT–/–. (C) Saline-treated WT. (D) BLM-treated WT. Original magnification, ×100; ×400 (insets).
Figure 3
Figure 3. Effects of BLM on lung α-SMA expression.
Lung total RNA and protein were isolated from the indicated mice at days 7 and 21 after BLM or saline treatment, respectively. (A) α-SMA mRNA was detected by real-time PCR, and results were expressed as 2–ΔΔCT, with GAPDH used as the endogenous control and the level in saline-treated WT mice as reference. Data are mean ± SEM. n = 3 per group. (B) α-SMA protein was detected by Western blotting and expressed as relative integration units (RIU) after measurement of net intensity of scanned bands. The same amount (5 μg) of tissue protein was loaded on the 12% SDS-polyacrylamide gel and blotted with anti–α-SMA antibody. A typical blot from 3 independent experiments and the quantitative plot are shown.
Figure 4
Figure 4. Assessment of BLM genotoxicity.
DNA damage was visualized by SYBR green I staining of lung cellular DNA after single-cell electrophoresis. Damaged DNA fragments exhibited comet-like tails in cells exposed to H2O2, as shown in positive controls. Representative lung cells from whole-lung homogenates of WT or TERT–/– mice treated with BLM or saline are shown. Original magnification, ×400.
Figure 5
Figure 5. Effects of FITC on lung collagen.
WT and TERT–/– mice were treated as indicated, and lung tissue homogenates were prepared for analysis of type I collagen by Western blotting. Equal amounts of protein (20 μg) were loaded per lane, and the resulting blot was scanned and digitized for quantitative analysis of band intensity. Results are mean ± SEM. n = 5 per group.
Figure 6
Figure 6. Effects of BLM on fibroblast proliferation.
BLF and NLF were isolated from the indicated mice after BLM or saline treatment (cultured in DMEM supplemented with 10% PDS, 10 ng/ml EGF, and 5 ng/ml PDGF), respectively. Cell proliferation was determined using the WST-1 assay; results (mean ± SEM) are expressed as the difference between the absorbance at 450 nm and that at 690 nm. n = 5 per group.
Figure 7
Figure 7. Effects of BLM on fibroblast apoptosis.
Apoptosis of lung fibroblasts were detected by TUNEL staining (A) and flow cytometry after annexin V–FITC staining (B–I). (A) Lung fibroblasts from the indicated groups were cultured in 4-well chamber slides and treated with buffer only or TNF-α plus CHX. Apoptosis was analyzed by TUNEL staining after 4 hours of treatment: 500 cells in randomly selected high-power fields (original magnification, ×400) were counted, and apoptotic cells were expressed as a percentage of the total cells counted. Data are mean ± SEM. n = 3 per group. (B–I) For annexin V analysis, the cells were treated similar to that described above in 60-mm dishes and trypsinized after TNF-α and CHX treatment for annexin V–FITC and PI staining. The x axis (FL1) reflects annexin V–FITC fluorescence; the y axis (FL3) reflects PI fluorescence. Early apoptotic (annexin V–FITC–positive) cells appear in the lower right quadrant of the dot plot. A typical result from 3 independent experiments is shown.
Figure 8
Figure 8. Effects of BM transplantation on lung telomerase and fibrosis.
BM chimera mice were generated as described in Methods by transplanting WT BM into TERT–/– and TERT–/– BM into WT mice (WT-KO and KO-WT, respectively). BM transplantation from TERT–/– to TERT–/– or WT to WT mice were used as sham controls. Whole-lung lysates were collected at day 21 after BLM or saline treatments. (A) Lung telomerase activity was measured by TRAP-based ELISA, and results were expressed as the difference between absorbance at 450 nm and that at 690 nm. (B) Lung collagen content was analyzed with HYP assay. (C) α-SMA expression was detected by Western blotting with equal amounts (5 μg) of protein loaded per lane. A typical blot and the quantitative plot are shown. Data are mean ± SEM. n = 5 (BLM); 3 (saline).
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