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Clinical Trial
. 2013 Aug;49(2):260-8.
doi: 10.1165/rcmb.2012-0514OC.

Telomerase and telomere length in pulmonary fibrosis

Tianju Liu  1 , Matthew Ullenbruch, Yoon Young Choi, Hongfeng Yu, Lin Ding, Antoni Xaubet, Javier Pereda, Carol A Feghali-Bostwick, Peter B Bitterman, Craig A Henke, Annie Pardo, Moises Selman, Sem H Phan
Affiliations
Clinical Trial

Telomerase and telomere length in pulmonary fibrosis

Tianju Liu et al. Am J Respir Cell Mol Biol. 2013 Aug.

Abstract

In addition to its expression in stem cells and many cancers, telomerase activity is transiently induced in murine bleomycin (BLM)-induced pulmonary fibrosis with increased levels of telomerase transcriptase (TERT) expression, which is essential for fibrosis. To extend these observations to human chronic fibrotic lung disease, we investigated the expression of telomerase activity in lung fibroblasts from patients with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). The results showed that telomerase activity was induced in more than 66% of IPF lung fibroblast samples, in comparison with less than 29% from control samples, some of which were obtained from lung cancer resections. Less than 4% of the human IPF lung fibroblast samples exhibited shortened telomeres, whereas less than 6% of peripheral blood leukocyte samples from patients with IPF or hypersensitivity pneumonitis demonstrated shortened telomeres. Moreover, shortened telomeres in late-generation telomerase RNA component knockout mice did not exert a significant effect on BLM-induced pulmonary fibrosis. In contrast, TERT knockout mice exhibited deficient fibrosis that was independent of telomere length. Finally, TERT expression was up-regulated by a histone deacetylase inhibitor, while the induction of TERT in lung fibroblasts was associated with the binding of acetylated histone H3K9 to the TERT promoter region. These findings indicate that significant telomerase induction was evident in fibroblasts from fibrotic murine lungs and a majority of IPF lung samples, whereas telomere shortening was not a common finding in the human blood and lung fibroblast samples. Notably, the animal studies indicated that the pathogenesis of pulmonary fibrosis was independent of telomere length.

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Figures

<i>Figure 1.</i>
Figure 1.
Telomerase activity and telomere length analysis in lung fibroblasts from patients with interstitial lung disease (ILD). Human lung fibroblasts were isolated from control subjects or patients with the indicated diagnoses. Fibroblast lysates were harvested from passage 6–10 cells. (A) Five micrograms of cell lysates were used for a telomerase activity assay with telomerase repeat amplification protocol (TRAP)-ELISA. The samples with absorbance at greater than or equal to 0.25, after subtracting the absorbance reading of the negative control, were considered positive for telomerase activity. The results are shown as percentages of samples that were telomerase-positive in each disease category, with the total number of samples indicated. (B) Genomic DNA from these fibroblasts was analyzed for telomere lengths by terminal restriction fragment (TRF) analysis and Southern blotting. The results are plotted as mean telomere length. The median is indicated with a solid line inside the shaded box, whereas the mean is indicated with a dashed line. The lower error bar indicates the 10th percentile, whereas the upper error bar indicates the 90th percentile. The bottom boundary of the shaded box indicates the 25th percentile, and the upper boundary indicates the 75th percentile. Each data point represents results from cells from a single donor. HP, hypersensitivity pneumonitis; IPF, idiopathic pulmonary fibrosis; NSIP, nonspecific interstitial pneumonia; UIP, usual interstitial pneumonia; SSc, systemic sclerosis.
<i>Figure 2.</i>
Figure 2.
Measurement of telomere length in peripheral blood leukocytes. Genomic DNA (1 μg) was extracted from peripheral blood leukocytes and used for telomere length assessment. Average telomere length was measured by performing mean TRF analysis and Southern blotting. (A) Representative blots for IPF and control samples are shown, with the asterisk indicating a sample exhibiting a shortened telomere. “Hi” Lad, DNA ladder control with high telomere length; “Lo” Lad, DNA ladder control with low telomere length. (B) Telomere length was plotted versus age. The control samples (open circles) were used to generate a regression line, with the predicted 10th and 90th percentile indicated by the lower and upper dashed curves, respectively. The total number of samples per group (upper right) is indicated, as well as the percentage of samples in each group that exhibited telomere length lower than the 10th percentile (upper left). (C) The proportions of the shortest telomere signals in the three groups are shown. Mean values were not significantly different between all three groups. The average TRF is indicated for each group.
<i>Figure 3.</i>
Figure 3.
Effects of telomerase RNA component (TR) or telomerase catalytic reverse transcriptase (TERT) deficiency on telomere length, TERT expression, and pulmonary fibrosis. (A) Genomic DNA from lung fibroblasts were isolated from bleomycin (BLM)–treated or saline (SAL)–treated wild-type (WT) or TR knockout (KO) mice, and analyzed for telomere length by TRF assay, followed by Southern blotting. The quantitative analysis is shown at left (n = 3, *P < 0.05), and a representative blot is shown at right. G, generation. The effect of TR deficiency on fibrosis is shown as lung hydroxyproline content in whole-lung homogenates on Day 21 after BLM or SAL administration (B) and as procollagen I mRNA concentrations (C). Data are presented as means ± SEs, with n = 5 animals per group. Asterisks indicate a statistically significant difference (P < 0.05) from the corresponding SAL control groups. (D) The effect of TR deficiency on the BLM induction of TERT was analyzed. Lung-tissue lysates from WT and TR KO mice on Day 21 after BLM or SAL treatment were analyzed for TERT protein expression by Western blotting. Representative blots are shown. The lane on the right shows a negative control for the TERT antibody, using TERT KO lung lysate. (E) The effects of prolonged TERT deficiency on pulmonary fibrosis were analyzed by hydroxyproline assay in SAL-treated or BLM-treated G4 TERT KO mice. Data are expressed as percentages of respective SAL-treated groups (n = 5 mice per group). An asterisk indicates a statistically significant difference (P = 0.012) from the corresponding SAL-treated control group.
<i>Figure 4.</i>
Figure 4.
Human lung fibroblast TERT expression and histone acetylation. (A) Lung fibroblasts isolated from patients with IPF and control subjects (at passages 6–10) were analyzed for TERT protein expression by Western blotting. Glyceraldehyde 3–phosphate dehydrogenase (GAPDH) signals were used as a loading control. Data are expressed as relative integration units, and are normalized as percentages of GAPDH signals (n = 7 per group). Each data point represented result from cells from a single donor, and the significance of the bars and lines in the box plot is as described in the legend to Figure 1B. The global histone acetylation at H3K9 were detected by acetylated H3K9 antibody using Western blotting, as already described, and the correlation between the global acetylated H3K9 and TERT concentrations in lung fibroblasts from patients with IPF (B, n = 9) or control subjects (C, n = 7) were analyzed by linear regression and the results shown in the graph. Representative blots are shown in Figure E5 of the online supplement.
<i>Figure 5.</i>
Figure 5.
Trichostatin A (TSA) induction of human TERT (hTERT) in human lung fibroblasts. (A) Lung fibroblasts from a patient with IPF were treated with the indicated doses of TSA for the indicated times, and then analyzed for TERT mRNA by quantitative PCR. Untreated cells at 4 hours were used as the calibrator for the calculation of 2−ΔΔCT. Asterisks indicate statistically a significant difference (P < 0.01) from the corresponding untreated control samples. Representative results are shown from at least four separate experiments with IPF cells from individual donors. Cells were also analyzed by Western blotting for TERT and acetylated H3K9 (H3K9Ac) protein concentrations after 24-hour treatment with the indicated doses of TSA. (B) Representative blots.
<i>Figure 6.</i>
Figure 6.
Chromatin immunoprecipitation (ChIP) assay of H3K9 acetylation status at the human TERT promoter. (A) The ChIP quantitative PCR analysis was performed in cells from human control and IPF samples, and the results are shown as means ± SE (n = 3). The asterisk indicates a statistically significant difference (P = 0.005) from control cells. (B) The cells as already described were treated with the indicated doses of TSA for 12 hours, and then subjected to ChIP assay. The cell DNA immunoprecipitated by acetylated H3K9 antibody was amplified by quantitative PCR. One tenth of the supernatant before immunoprecipitation was used for the DNA input control. Data are expressed as fold changes over untreated cells. The representative result is shown from at least three separate experiments. The inset depicts a typical gel image of quantitative PCR products. bp, base pairs. Asterisks indicate statistically significant differences (P < 0.02) from untreated cells.
<i>Figure 7.</i>
Figure 7.
Mouse TERT (mTERT) regulation by histone H3K9 acetylation. Mouse lung fibroblasts were isolated 14 days after BLM or SAL treatment and analyzed for inductions of TERT protein at 24 hours (A) and mRNA at 8 hours (B) after TSA treatment by Western blotting and quantitative PCR, respectively. Asterisks indicate statistically significant differences (P < 0.01) from the corresponding untreated cells. (C) Acetylated H3K9 protein induction was detected in Day 14 BLM-treated lung tissue by Western blotting.
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