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

BLM-induced pulmonary fibrosis was reduced in TERT^–/– mice.

TERT^–/– mice were generated as previously described (26). To evaluate the amplitude of BLM-induced lung fibrosis in TERT^–/– mice, lung collagen content was compared in TERT^–/– and WT mice by hydroxyproline (HYP) analysis at day 21 after BLM administration. The results showed that pulmonary fibrosis was significantly reduced in TERT^–/– mice compared with WT mice (Figure 1). This marked reduction in biochemically assessed fibrosis was confirmed by morphological analysis of lung tissue sections. The results showed that while the lungs from BLM-treated WT mice exhibited typical severe pulmonary fibrosis, characterized by increased cellularity, loss of normal alveolar architecture, and thickening of alveolar septa, the lungs from BLM-treated TERT^–/– mice had substantially less fibrosis, with more patchily distributed lesions that were much smaller in size and extension (Figure 2). Saline-treated control lungs showed no morphological abnormalities in both WT and TERT^–/– mice, indicating that TERT deficiency did not impair normal lung development. Thus both biochemical and morphological assessment indicated that TERT deficiency resulted in significantly reduced BLM-induced pulmonary fibrosis.

TERT deficiency reduced genesis of myofibroblasts in pulmonary fibrosis.

Previously, alterations in telomerase expression were found to affect myofibroblast differentiation (11). To investigate whether TERT deficiency also affects the genesis of myofibroblasts in pulmonary fibrosis in vivo, lung α-SMA expression, a marker of myofibroblast differentiation, was analyzed by real-time PCR at day 7 after BLM-induced lung injury. As expected, BLM treatment in WT mice caused a significant increase (>2-fold over saline-treated controls; P < 0.001) in lung α-SMA mRNA levels; conversely in TERT^–/– mice, a slight, nonsignificant reduction was noted in the BLM-treated group relative to the saline-treated control group (Figure 3A). Consistent with these differences in lung tissue mRNA levels, α-SMA protein expression analyzed by Western blotting showed similar differences between the various groups (Figure 3B). Protein levels in WT mice were significantly higher (almost 2-fold increase; P < 0.001) in the BLM-treated compared with the saline-treated group, but in TERT^–/– mice were slightly decreased in the BLM-treated group and not significantly different from saline-treated controls. This pattern of differences in α-SMA expression was also reflected in fibroblasts isolated from these lungs (data not shown). These findings indicated that myofibroblast differentiation as determined by α-SMA expression was significantly reduced in TERT^–/– compared with WT mice.

Because BLM is also known to cause DNA strand scission and damage (27–29), the protective effect of TERT deficiency in the BLM model may be specific only for genotoxic injury–induced pulmonary fibrosis. To examine this possibility, DNA damage was evaluated in the BLM model using the Comet assay. The results showed that lung cell samples from both WT and TERT^–/– mice at day 1 after BLM treatment did not exhibit comet tails (Figure 4). In contrast, positive control samples from H₂O₂-treated cells induced marked comet tails, as expected. Lung cells from mice at day 7 after BLM treatment and primary cultured isolated fibroblasts from mice at days 1 and 7 after BLM treatment also showed no evidence of DNA damage (data not shown). Thus, BLM treatment in vivo to induce pulmonary fibrosis, at the indicated dose (1.5 U/kg body weight), showed no evidence of genotoxicity using this assay. This made it unlikely that TERT deficiency protects only fibrosis resulting from a genotoxic insult.

To confirm this finding, the effect of TERT deficiency was studied in another model that is not known to be induced by a genotoxic insult, namely, one induced by FITC. While this model did not cause as severe a fibrosis as did BLM, the results did show a significant increase (~68%; P < 0.05) in lung type I collagen in WT FITC-treated mice (Figure 5). In contrast, lung type I collagen content was not significantly altered by FITC-treatment in TERT^–/– mice, reminiscent of the protection against BLM-induced fibrosis in these mice. These findings suggested that TERT deficiency also has a protective effect against FITC-induced pulmonary fibrosis, and thus the protective effect of TERT deficiency is not specific only to the BLM model.

TERT deficiency affected lung fibroblast proliferation and apoptosis.

To explore the possible mechanisms by which fibrosis was reduced in TERT^–/– mice, the effects of TERT deficiency on lung fibroblast proliferation and apoptosis were analyzed to determine whether reduction in fibroblast numbers could be a factor. The cell proliferative responses of lung fibroblasts isolated from BLM- or saline-treated mice (BLF and NLF, respectively) at day 14 were determined as described in Methods. The results showed that WT BLF proliferated at a significantly higher rate (>2-fold increase) than did WT NLF, whereas KO BLF proliferation was not significantly different from KO NLF (Figure 6). Both the KO NLF and the KO BLF proliferation rates were not significantly different from the WT NLF rate, but were all significantly lower than the WT BLF rate. Thus TERT deficiency did not significantly affect proliferation in normal lung fibroblasts, but appeared to impair the emergence of an activated fibroblast population with higher proliferative rate in response to lung injury and remodeling.

The effect of TERT deficiency on fibroblast survival was also analyzed by assessing the response to a known apoptotic stimulus (5 ng/ml TNF-α with 500 ng/ml cycloheximide [CHX]). The apoptotic response to TNF-α was evaluated by TUNEL staining in the same lung fibroblast populations described above. The results showed that in the absence of apoptotic stimuli, both WT NLF and WT BLF exhibited low levels of apoptotic cells (<4%), which were significantly lower than the levels in KO NLF (7.6%) or KO BLM (20.3%; Figure 7A). Treatment with TNF-α caused an increase in the apoptotic rate in all cells, but the increase was significantly higher in both the KO NLF (21.5%; P < 0.001) and the KO BLF cells (47.8%; P < 0.001) compared with their respective untreated groups and with both treated WT groups (P < 0.05). Analysis of apoptosis using flow cytometric assessment of annexin V–FITC–propidium iodide (annexin V–FITC–PI) staining confirmed the markedly higher apoptotic potential of lung fibroblasts from control and BLM-treated TERT^–/– mice (Figure 7, B–I). Consistent with the data shown by TUNEL staining, the apoptotic cells in the absence of apoptotic stimuli were 1.86% and 2.96% in WT NLF and WT BLF, respectively, whereas the corresponding values in KO NLF and KO BLF were more than 2-fold higher at 7.76% and 7.74%, respectively. Treatment with TNF-α caused increases in apoptosis in all cell groups, which again were almost 3-fold higher in the KO compared with the WT groups. The results of these apoptosis experiments indicated that, relative to cells from WT mice, lung fibroblasts from TERT^–/– mice are substantially more susceptible to apoptotic stimuli, especially in cells from injured lungs undergoing fibrosis. Thus, in addition to reduced proliferative capacity, the increased susceptibility to apoptosis may affect the survival of these cells and result in reduced cell numbers in TERT^–/– mice.

Contribution of BM-derived cells to lung telomerase induction and fibrosis.

Previously, GFP BM chimera mice have been used to determine the presence of BM-derived fibroblast-like cells in BLM-injured lung as well as which are TERT positive (30). To investigate the possibility that BLM-induced lung telomerase expression may be caused by recruitment of these BM-derived cells, and to assess their role in fibrosis, BM chimera WT and TERT^–/– mice, transplanted with corresponding TERT^–/– and WT BM, were created and analyzed for effects on BLM-induced lung telomerase and fibrosis. Sham controls consisted of TERT^–/– and WT mice receiving TERT^–/– and WT BM, respectively. The results showed that transplantation of WT BM into TERT^–/– mice reconstituted BLM-induced lung telomerase activity to a level indistinguishable from that in WT mice (Figure 8A). Interestingly, transplantation of BM from TERT^–/– mice to WT mice caused a marked reduction in BLM-induced lung telomerase induction. These findings showed that BM-derived cells were the main source of induced lung telomerase activity in BLM-induced pulmonary fibrosis.

In view of the ability of BM-derived cells to reconstitute the induction of lung telomerase activity by BLM treatment in TERT^–/– mice, the same BM chimera mice were analyzed for responses to BLM-induced pulmonary fibrosis. Analysis of lung HYP content on day 21 after BLM treatment showed the expected increase in sham-transplanted WT mice relative to saline-treated controls, which was significantly higher than that in sham-transplanted TERT^–/– mice (Figure 8B). However, as in the case of telomerase induction (Figure 7A), transplantation of WT BM to TERT^–/– mice reconstituted the WT response to BLM-induced fibrosis, while transplantation of BM from TERT^–/– mice to WT mice reduced the response relative to that in sham-transplanted WT mice.

Because myofibroblast differentiation correlated with the fibrotic response to BLM, these groups of BM chimera mice were also analyzed for BLM-induced alterations in lung α-SMA protein levels by Western blotting. Consistent with the whole-lung HYP changes, the lung α-SMA expression was similarly altered in the various groups of mice (Figure 8C). Thus transplantation of WT BM to TERT^–/– mice caused α-SMA induction by BLM to be at a level comparable to that in WT mice, while the converse transplantation of BM from TERT^–/– mice to WT mice ablated this response. Taken together, these findings suggested that lung telomerase induction from BM derived cells in response to BLM treatment was important for the pathogenesis of pulmonary fibrosis.

Animals and induction of pulmonary fibrosis.

C57BL/6 mice were purchased from The Jackson Laboratory. TERT^–/– mice on C57BL/6 background were produced as previously described (26). Briefly, a 4-kb region containing exons γ, δ, and ε was replaced by neomycin resistance gene neo. Exon γ codes for part of motif A of the mTERT protein (amino acids 701–753 of mTERT). The first-generation mTERT^–/– mice were fertile and did not show any noticeable gross or microscopic abnormality during early generations (G1 and G2). mTR expression levels were essentially same in WT and mTERT^–/– mice. All tissue cells derived from mTERT^–/– mice lacked telomerase activity, which suggests that mTERT is the only gene encoding the telomerase catalytic subunit. Telomeric DNA in mTERT^–/– mice has G-strand 5′-overhangs, indicating that these telomeric 5′-overhangs are produced by telomerase-independent mechanisms. These mice were propagated at the University of Michigan for these studies. G1–G3 mTERT^–/– mice were used for these studies.

For induction of BLM-induced pulmonary fibrosis, BLM (Blenoxane) was dissolved in sterile PBS and instilled endotracheally at a dose of 1.5 U/kg body weight on day 0 as previously described (54). Control groups received the same volume of sterile PBS only. Mice (n = 3–5) were randomly assigned to each of the indicated treatment groups. At the indicated time points after BLM treatment, the mice were sacrificed, and their lungs were harvested rapidly. Where indicated, the lung tissue samples were immediately placed in TRIzol reagent (Invitrogen) for total RNA isolation or in lysis buffer (50 mM Tris.Cl, pH 7.5, and 1% Nonidet P-40) for protein analysis. For histopathological analysis, 21 days after BLM injection the lungs were formalin-fixed and stained with routine H&E, as previously described. FITC-induced pulmonary fibrosis was performed as previously described (55). Briefly, 20 mg FITC (Sigma-Aldrich) was dissolved in 10 ml sterile PBS, vortexed extensively, and followed by 30 seconds of sonication. This slurry was injected endotracheally into mice (5 mg/kg) using a 23-gauge needle. Twenty-one days after treatment, whole lung was homogenized in PBS supplemented with proteinase inhibitor (Roche Diagnostics) for analysis of type I collagen content by Western blotting. All animal studies were reviewed and approved by the University Committee on Use and Care of Animals at the University of Michigan.

Isolation of lung fibroblasts.

Fibroblasts were isolated from isolated lung tissue by trypsinization as described previously (54). The cells were cultured in DMEM supplemented with 10% plasma-derived fetal bovine serum (PDS), 10 ng/ml EGF, and 5 ng/ml PDGF (R&D Systems Inc.). Cells were passaged by trypsinization and used at the third to fifth passage after primary culture. After plating as indicated, the cells were allowed to grow until they were 85% confluent before being used in the indicated experiments.

mRNA analysis by quantitative RT-PCR.

For quantitative mRNA analysis, total RNA was isolated from lung tissue or fibroblasts. Primer Express 2.0 software (Applied Biosystems) was used to design TaqMan primers and MGB probes (6-FAM conjugated) for α-SMA, which were then purchased from Applied Biosystems. The primer and probe sequences for α-SMA were as follows: forward, 5′-CAACAGGATGAAGACTGCAACCT-3′; reverse, 5′-GGGACCATCAGCTAAAGAAG-3′; probe, 5′-6FAM-CCCTTCTCATCTGCGTCT-3′. Primers and probe for GAPDH were purchased from Applied Biosystems. For each assay, 100 ng total RNA was used as template. GAPDH mRNA was used as internal control to normalize the amount of input RNA. One-step real-time RT-PCR (48°C for 30 minutes, 95°C for 10 seconds, followed by 45 cycles of 95°C for 10 seconds and 60°C for 1 minutes) was performed with TaqMan One Step RT-PCR Master Mix (Applied Biosystems) using a GeneAmp 7500 Sequence Detection System (Applied Biosystems). Results were expressed as 2^–ΔΔCT as previously described (56).

Western blotting analysis.

Western blotting to detect α-SMA and type I collagen protein expressions were performed as previously described (57). Briefly, 5 μg (α-SMA) and 50 μg (type I collagen) of cell extract proteins were loaded and separated by 12% and 8% SDS-PAGE, respectively. Mouse anti–α-SMA (Sigma-Aldrich) and rabbit anti–type I collagen (Biodesign International) antibodies were used as primary antibodies, and HRP-labeled anti-mouse and anti-rabbit IgG (Amersham Biosciences) were used as secondary antibodies. The immunostained bands were visualized using a chemiluminescent substrate (Cell Signaling Technology) followed by exposure to ECL Hyperfilm (Amersham Biosciences). The films were then scanned and quantitated using 1D Image Analysis software (Kodak).

Telomerase activity assay (TRAP-based ELISA).

Telomerase activity was assayed using a telomerase PCR ELISA kit (Roche) in accordance with the manufacturer’s protocols. Briefly, cell extracts were prepared by lysing the cultured fibroblasts with ice-cold lysis reagent. The protein concentrations were determined by the method of Bradford reagent (Bio-Rad Laboratories) and stored at –80°C until assayed. For each sample, 0.5 μg total cell protein was added to 25 μl reaction mixture containing telomerase substrate, including the biotin-labeled P1-TS and P2 primers, as well as nucleotides, Taq polymerase, and sterile water to a final volume of 50 μl. These mixtures were transferred to a PTC-200 DNA Engine thermal cycler (MJ research Inc.) for primer elongation at 25°C for 25 minutes, telomerase inactivation at 94°C for 5 minutes, and 30 cycles of amplification (94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 90 seconds). Heat treatment of the cell extracts at 80°C for 10 minutes prior to the TRAP reaction was used as negative control. The positive control (human kidney 293 cells) was supplied by the assay kit. The PCR product (5 μl) was denatured and hybridized to a digoxigenin-labeled telomeric repeat–specific probe on the precoated microtiter plates at 37°C for 2 hours with shaking (300 rpm). Finally, anti-digoxigenin peroxidase conjugate and TMB substrate were used for ELISA assay, and the absorbance at 450 nm (with a reference wave length of 630 nm) was measured using an ELx 800 UV universal microplate reader (Bio-Tek Instruments Inc.). Based on the negative controls, samples with absorbance values less than 0.25 were considered negative.

HYP assay.

Lung collagen deposition was estimated by measuring the HYP content of whole-lung homogenates as previously described (58). Briefly, the lungs were excised, homogenized in 0.5 M acetic acid, and hydrolyzed in 6N HCl overnight at 110°C. HYP was assessed by colorimetric assay, and the results were expressed as μg HYP per lung.

Cell proliferation assay.

Isolated fibroblasts from BLM- and saline-treated TERT^–/– and WT mice (cultured in DMEM supplemented with 10% PDS, 10 ng/ml EGF, and 5 ng/ml PDGF) were passaged once and then plated into 96-well plate at a density of 1 × 10⁴ cells/well in a final volume of 100 μl medium. When the cells reached approximately 85% confluency on the next day, 10 μl WST-1 reagent (1:10 final dilution, WST-1 Cell Proliferation Kit II; Roche) was added to each well. After an additional 4 hours of incubation, cell proliferation was determined by measuring the absorbance at 450 nm with reference wavelength of 690 nm using an ELx 800 UV Universal microplate reader. Only absorbance readings in the linear range (0.4–1.5) were used. The cells treated with 10 ng/ml PDGF were used as positive controls.

Induction and detection of cell apoptosis.

The fibroblasts were plated into 4-well chamber slides (Nalge Nunc International), with a cell density of 5 × 10³ cells/well, and allowed to reach subconfluence. Serum deprivation was achieved by maintenance in DMEM with 0.5% PDS for 24 hours. Apoptosis was induced by treating cells with 5 ng/ml TNF-α (R&D Systems) and 500 ng/ml CHX (Sigma-Aldrich) for 4 hours. Apoptosis was evaluated using TUNEL assay for in situ detection of apoptotic cells using the ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (Chemicon). After induction of apoptosis, cells were washed with PBS, fixed with 1% paraformaldehyde for 10 minutes at room temperature, and labeled by TUNEL in accordance with the manufacturer’s protocol. Briefly, after fixation, cells were labeled for 60 minutes at 37°C using terminal deoxynucleotidyl transferase. The reaction was terminated in stop/wash buffer for 10 minutes at room temperature. The cells were then incubated with an anti-digoxigenin conjugate for 30 minutes at room temperature. Nuclei were labeled with PI. The cells were observed under a fluorescence microscope. For each slide, the number of positive cells were counted in randomly chosen, noncontiguous, high-power fields (original magnification, ×400) until a minimum of 500 total cells was counted.

Additionally, annexin V–FITC–PI staining (TACS annexin V–FITC; R&D Systems) was performed for early apoptosis detection. Briefly, the mouse lung fibroblasts were plated in 60-mm dishes with a cell density of 5 × 10⁵ cells/dish. The cell were treated as described above with TNF-α and CHX for 4 hours and then collected after trypsinization, washed with ice-cold PBS, resuspended in 100 μl annexin V incubation reagent (10 μl 10× binding buffer, 10 μl PI, 1 μl annexin V–FITC, and 79 μl dH₂O) at a concentration of 3–5 × 10⁵ cells/100 μl, and incubated for 15 minutes at room temperature in the dark. Samples were washed with binding buffer and analyzed by FACSCalibur (BD). Apoptotic cells were identified as an annexin V–FITC–positive/PI-negative population.

Single-cell gel electrophoresis assay.

Single and double DNA breaks were evaluated using the Comet assay (R&D Systems) in accordance with the manufacturer’s manual. Briefly, fresh lung tissue was minced into small pieces in 1 ml ice-cold PBS (Ca⁺⁺ and Mg⁺⁺ free) and 20 mM EDTA and let stand for 5 minutes. Cell suspension was recovered and pelleted by centrifugation and resuspended at 1 × 10⁵ cells/ml in ice-cold PBS. For analysis of primary cultured lung fibroblasts, the cells were scraped using a rubber policeman and then resuspended in ice-cold PBS at 1 × 10⁵ cell/ml after centrifugation. The cells were combined with 1% molten low–melting point agarose at a ratio of 1:10 (v/v) and immediately pipetted onto a CometSlide. After further incubation in the dark to lyse the cells and unwind DNA, the slides were subjected to electrophoresis and then treated with SYBR green I for viewing the DNA under a fluorescence microscope (Nikon Eclipse E600; Diagnostic Instruments Inc.). The cells treated with 400 μM H₂O₂ for 30 minutes on ice were used as a positive control.

Generation of BM chimera mice.

BM chimeras were prepared as previously described (30), with minor modifications. Donor BM cells were collected from femurs and tibias of WT and TERT^–/– mice by aspiration and flushing. Recipient WT or TERT^–/– mice were exposed to 2 doses of 5 Gy given 3 hours apart using a ¹³⁷Cs irradiator and then maintained on acidified water and autoclaved feed ad libitum. After irradiation, 4 × 10⁶ BM cells from donor mice in a volume of 200 μl sterile PBS were injected into recipient mice retro-orbitally under anesthesia. Six weeks after durable BM engraftment had been established, pulmonary fibrosis was induced by endotracheal BLM injection as described above.

Statistics.

All data were expressed as mean ± SEM unless otherwise indicated. Differences between means of various treatment and control groups were assessed for statistical significance by ANOVA followed by post-hoc analysis using Scheffé’s test for comparison between any 2 groups. A P value less than 0.05 was considered significant.