Introduction

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Telomerase is a specific multi-subunit ribonucleoprotein that synthesizes TTAGGG telomere DNA onto chromosomal ends by using its intrinsic RNA component as a template, thereby compensating for telomere loss that normally occurs with each cell division (1, 2). Telomerase has been shown to be essential for unlimited cell proliferation and has been linked to immortality (3). Evidence of telomerase activation in transformed cells and tumors suggests its possible importance in carcinogenesis. Additionally however, recent evidence indicates that telomerase is also present in some injured and inflamed tissues or cells, as well as in selected normal tissues and cells including spleen, thymus, and testis (4). In those tissues and cells that do express telomerase activity, it is usually growth-regulated. Mouse mammary tissue and skin samples exhibit low levels of telomerase activity that become greatly elevated when cells are isolated and grown in tissue culture (7). More recent data show that telomerase could be induced in silica-induced lung injury (8) and in cultured synoviocytes from patients with rheumatoid arthritis (9). The latter study also indicates that the telomerase in these cells can be upregulated by basic fibroblast growth factor (bFGF). Bleomycin-induced lung injury and fibrosis is also known to induce telomerase activity in the affected lung tissue and isolated lung fibroblasts (10). The activity, however, is expressed only during the period of active fibrosis, and appears to be localized primarily in fibroblasts vis-à-vis myofibroblasts. The activity also declines with increased passaging in vitro, which is associated with increased differentiation to myofibroblasts. These studies suggest that induction of telomerase in fibroblasts may be important in the fibroproliferative response in chronic inflammation and fibrosis. The factors responsible for regulating telomerase activity in these tissues and cells are unknown for the most part.

Telomerase contains two components, an RNA component and a catalytic subunit referred to as the telomerase reverse transcriptase (TERT). There is mounting evidence to indicate that it is the TERT but not the RNA component that is highly correlated with the presence of telomerase activity (11, 12). The proposed telomerase catalytic subunits are phylogenetically conserved (12). The human gene encodes a 1,132-amino acid polypeptide with a predicted molecular weight greater than 100 kD. Sequence analysis shows that the TERT promoter is GC-rich, lacks TATA and CAAT boxes, but contains binding sites for several transcription factors that may be involved in its regulation. The abundance of these sites suggested that TERT expression may be subject to multiple levels of control and regulated by different factors in different cellular context (13).

Bleomycin-induced lung injury causes increased fibroblast numbers in the lung associated with fibrosis. The fibroblasts isolated from such lungs undergoing fibrosis show increased intrinsic proliferative capacity and are able to differentiate into -smooth muscle actin (-SMA) expressing myofibroblasts, which are also a key source of cytokines with inflammatory and fibrogenic properties (14, 15). Induction of telomerase activity is associated with increased fibroblast numbers in bleomycin-induced lung fibrosis, and the -SMA and TERT mRNA expression pattern indicate that telomerase expression localizes primarily to fibroblasts, presumably before their differentiation to myofibroblasts or in cells that do not undergo myofibroblast differentiation. These findings suggest that the injured lung fibroblast population may contain telomerase expressing cells with extended life span, which could contribute to the observed increased numbers of lung fibroblasts (10). However, the mechanism of induction and regulation of telomerase expression in the context of lung injury and fibrosis is still unknown. The objective of this study is to identify potential mediators capable of regulating telomerase expression in lung fibroblasts cultivated from primary cultures of normal and bleomycin-treated rat lung fibroblasts.

	Materials and Methods

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Induction of Animal Models for Pulmonary Fibrosis

Male specific pathogen-free Fisher 344 rats (7-8 wk of age) were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Pulmonary fibrosis was induced on Day 0 by the endotracheal injection of 7.5 U/kg body weight of bleomycin (Blenoxane; Nippon Kayaku Co. Ltd., Tokyo, Japan) in sterile phosphate-buffered saline (PBS). The control group received the same volume of sterile PBS only. On Days 7, 14, 21, and 28 after bleomycin treatment, the rats were killed and the lungs were removed, and either immediately frozen in liquid nitrogen for mRNA analysis or used for isolation of fibroblasts as described previously (14). Rat lung fibroblasts from control (NRF) and bleomycin-treated (BRF) rats were maintained in culture and passaged as previously described (14). Only cells between passages 3 and 5 after primary culture were used. Preliminary studies with passage 1 cells exhibited similar behavior in terms of telomerase activity expression (data not shown), but were not used in subsequent studies to ensure purity of the cells.

Treatment of Fibroblasts

To avoid the effects of transforming growth factor (TGF)- in in vitro culture, serum was not used in cultivating these cells. Instead, cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% plasma-derived fetal bovine serum (PDS), 10 ng/µl epidermal growth factor (EGF), and 5 ng/µl platelet-derived growth factor (PDGF) (R&D Systems, Inc., Minneapolis, MN). For each experiment, the cells were plated in the desired tissue culture wells and allow to grow to ~ 75% confluence, and then made quiescent by culturing in DMEM containing 0.5% PDS for 48 h. Recombinant human bFGF (R&D Systems) or recombinant rat IL-4 (rrIL-4, R&D Systems) was then added at the indicated concentrations and further incubated for 24, 48, and 72 h before harvest for telomerase activity assay and Western blotting. Alternatively, the treated fibroblasts were incubated for 4, 6, and 12 h for total mRNA analysis.

Telomerase Activity Assays

Telomerase activity was assayed by a telomerase polymerase chain reaction (PCR) enzyme-linked immunosorbent assay (ELISA) kit (Roche Molecular Biochemicals, Indianapolis, IN) using a modified telomeric repeat amplification protocol (TRAP) in accordance with the manufacturer's instructions as previously described (10). Briefly, cell extracts prepared by lysing the cultured fibroblasts were used immediately or stored frozen at 80°C until used. Samples (0.5 µg protein) from each extract were added to a reaction mixture containing telomerase substrate, primers, nucleotides, Taq polymerase, and sterile water. These mixtures were transferred to a PTC-200 DNA Engine thermal cycler (MJ Research, Inc. Waltham, MA) for amplification (10). The PCR products were then denatured and hybridized to a digoxygenin (DIG)-labeled telomeric repeat specific probe in microtiter plates. Finally, peroxidase-conjugated anti-DIG antibody was used in an ELISA to measure the PCR products. Based on the manufacturer's recommendations, an absorbance value of less than 0.25 was considered as negative. Also, given the observed variances in readings, only absorbance values  0.2 units above their respective negative control were considered positive for telomerase activity. Heat-treated (80°C for 10 min before the TRAP reaction) cell extracts were used as negative controls. The positive control (human kidney 293 cell extract) was supplied by the manufacturer of the assay kit. All assays were undertaken with 0.5 µg of total protein, thus all reported activities were normalized to this value.

Additionally, in selected experiments, telomerase activity was also determined by analyzing the DNA-laddering pattern obtained upon electrophoresis in a 12.5% nondenaturing polyacrylamide gel (PAG) of samples generated by the TRAP reaction. Upon completion of the run, each gel was transferred to a positive charged nylon membrane (Amersham Pharmacia Biotech Ltd. Buckinghamshire, UK) using a TE 22 Mini Tank Transphor Unit (Amersham) at constant current of 350 mA for approximately 1 h. Bands on the membrane were visualized by a biotin luminescent detection kit (Roche Molecular Biochemicals). Telomerase activity was indicated by the presence of the entire 6-bp telomere ladders consisting of bands of increasing size.

Western Blotting Analysis

Western blotting was used to quantify TERT and -SMA protein expression. Fibroblast extracts were subjected to electrophoresis on 10% SDS-polyacrylamide gels, followed by transfer to a Hybond-P membrane (Amersham). Equal amounts of protein (5 µg for -SMA, and 15 µg for TERT analysis) were loaded per lane. After blocking with 5% nonfat dry milk, the membrane was incubated with rabbit anti-TERT polyclonal antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse anti--SMA monoclonal antibody (1:1,000, Roche). This was followed by incubation with horseradish peroxidase-labeled anti-rabbit or anti-mouse IgG (Amersham), and then developed for 1 min with Lumi Glo reagent (New England Bio Lab Inc., Beverly, MA). The membrane was visualized immediately by exposing to ECL Hyperfilm (Amersham) for 3-10 min.

mRNA Analysis by Reverse Transcriptase/Polymerase Chain Reaction

For analysis of TERT and bFGF mRNA expression, total RNA was isolated from lung tissue or fibroblasts for reverse transcriptase/polymerase chain reaction (RT-PCR) analysis, essentially as previously described (10). RT-PCR was undertaken with the SuperScript one-step RT-PCR system (Gibco BRL, Gaithersburg, MD) and using the following protocol: 1 cycle each of 50°C for 25 min, and 94°C for 2 min, followed by 26 cycles of 94°C for 15 s, 56°C for 30 s, 72°C for 1 min, and finally by 1 cycle of 72°C for 10 min. To normalize the amounts of input RNA, amplification of the glyceraldehydes-3-phosphate dehydrogenase (GAPDH) mRNA signal was used as internal control. The following primers were used: TERT, upstream 5'-GACATGGAGAACAAGCTGT TTGC-3', downstream 5'-ACAGGGAAGTTCACCACTGTC-3'; bFGF, upstream 5'-TATGAAGGAAGATGGACGGC-3', downstream, 5'-CCGTTTTGGATCCGAGTTTA-3'; GAPDH, upstream 5'-GTCTTCTGAGTGGCAGTGATG-3', downstream, 5'-TCCAG TATGACTCTACCCACG-3'.

Immunofluorescence

Rat lung fibroblasts were cultured and treated with either bFGF or IL-4 as described above, except the cells were plated on 22 × 22 mm coverslips at a density of 2 × 10⁴ cells/coverslip. The cells were then fixed in 4% of paraformaldehyde and stained with rabbit anti-TERT polyclonal antibody (1:150, Santa Cruz) followed by incubation with Alexa Fluor 488-labeled anti-rabbit IgG (Molecular Probe, Eugene, OR). The coverslips were then embedded with Mowiol mount medium (Calbiochem, San Diego, CA) and examined with a Zeiss Axiophot 2 fluorescence microscope (Carl Zeiss, Thornwood, NY). As a negative control, nonspecific rabbit IgG was used instead of the anti-TERT antibody. A minimum of ten randomly selected high-power fields was examined per coverslip to obtain at least a minimum total cell count of 500. The TERT-positive cells (green fluorescence) were counted and expressed as a percentage of total cells counted. A total of five separate coverslips were examined.

Statistical Analysis

All data were expressed as means ± SE 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 two groups. A P value < 0.05 was considered to indicate statistical significance.

	Results

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Effects of Cytokines on Expression of Telomerase Activity by Fibroblasts

Bleomycin-induced lung injury in rats is known to induce lung telomerase activity, which appears to be localized preferentially to fibroblasts relative to myofibroblasts (10). To clarify the mechanism for the induction of telomerase activity, a number of potential agonists were examined for their ability to regulate expression of this activity in isolated rat lung fibroblasts from control saline-treated (NRF) and bleomycin-treated (BRF) animals. As noted previously (10), NRF exhibited very low or negligible levels of telomerase activity using the TRAP-based ELISA assay (Figure 1A). Treatment with IFN, PDGF, and IL-7 did not have significant effects on cellular telomerase activity (data not shown). However, upon treatment with bFGF for 48 h, there was a slight, but dose-dependent, increase in telomerase activity (Figure 1A). In contrast, IL-4 caused a small dose-dependent reduction in the already low level of telomerase activity in NRF. Day 21 BRF exhibited high levels of telomerase activity (Figure 1B). Similar dose-dependent stimulatory and inhibitory effects on telomerase activity by bFGF and IL-4, respectively, were seen in BRF, except the magnitude of the effects was significantly larger (Figure 1B). The stimulation by bFGF was still increasing at a dose of 10 ng/ml. The results of the TRAP based ELISA assay were confirmed by analysis of the TRAP PCR products by nondenaturing polyacrylamide gel electrophoresis, which revealed laddering of telomeric TTAGGG DNA repeats consistent with the ELISA results (data not shown). The kinetics of the effects of bFGF and IL-4 on telomerase activity is shown in Figures 2A and 2B for NRF and BRF, respectively. As previously noted (10), telomerase activity in isolated lung fibroblasts (NRF and BRF) gradually declined as a function of time in culture (Figure 2). Stimulation by bFGF in both NRF and BRF was noted by 24 h of treatment, which increased further at 48 and 72 h relative to the untreated controls. Although treatment with bFGF did not completely reverse the gradual decline in activity in both NRF and BRF as a function of time in culture, it did reduce the magnitude of the reduction seen in untreated control cells. As expected, IL-4 inhibited expression of telomerase activity in both NRF and BRF relative to that in untreated control cells, by increasing the magnitude of the spontaneous decrease in culture.

View larger version (11K): [in this window] [in a new window]   Figure 1.   Dose-response curves for bFGF (solid line) and IL-4 (dashed line) effects on lung fibroblast telomerase activity. Lung fibroblasts from control (NRF) and bleomycin-treated (BRF) rats were treated with the indicated doses of cytokine for 48 h. Extracts of NRF (A) and BRF (B) were analyzed for telomerase activity using the ELISA procedure as described in MATERIALS AND METHODS. Telomerase activity was expressed as the absorbance at 450 nm after subtracting the background absorbance at 630 nm, and normalized to 0.5 µg of protein. Because the entire experiment was performed at the same time, the untreated controls ("0 ng/ml") apply to both the bFGF- and IL-4-treated samples. Mean values ± SD of triplicate samples are shown. Telomerase activity was undetectable after heat-treatment (85°C, 10 min) of all the samples (not shown).

View larger version (29K): [in this window] [in a new window]   Figure 2.   Kinetics of the effects of bFGF and IL-4 on lung fibroblast telomerase activity. NRF (A) and BRF (B) were prepared as described in the legend to Figure 1, and treated without (Control, open bars), or with bFGF (2 ng/ml, striped bars) or IL-4 (5 ng/ml, hatched bars) for the indicated times. Cell extracts were then prepared for assay of telomerase activity by the ELISA method, and expressed as described in the legend to Figure 1. Means ± SD of triplicate samples are shown.

Although the bFGF stimulatory effect on telomerase activity appears not to be shared by other growth factors (e.g., PDGF), the uniqueness of the inhibitory effect of IL-4 is unknown. In view of the latter's ability to induce myofibroblast differentiation, the effect of TGF-, which has similar effects on fibroblasts (16), was examined. The results showed similar dose-dependent inhibitory effects of TGF on BRF telomerase activity (Figure 3A). However, the kinetics of inhibition was slower compared with that for IL-4 at a comparable dose, such that maximum relative inhibition was observed at 48 h of treatment (Figure 3B). Thus the inhibitory effect of IL-4 was also seen with TGF-, consistent with their similar activities on myofibroblast differentiation.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Effects of TGF- on fibroblast telomerase activity. Rat lung fibroblasts from bleomycin-treated rats were treated with the indicated doses of TGF- (A) or for the indicated times (B) without (Control, open bars) or with 2 ng/ml TGF- (striped bars). Cell extracts were then prepared for assay of telomerase activity by the ELISA method, and expressed as described in the legend to Figure 1. Means ± SD of triplicate samples are shown.

Effects of bFGF and IL-4 on Fibroblast TERT Expression

To explore further the effects of bFGF and IL-4 on fibroblast telomerase activity, their effects on NRF and BRF TERT protein expression were examined. Cell extracts from NRF (from rats on Day 0 or Day 21 after saline treatment) and BRF (from rats on Days 7, 14, and 21 after bleomycin treatment) were prepared and analyzed by Western blotting using TERT antibody. The TERT-specific bands (> 100 kD) were barely detectable in NRF from both Day 0 and Day 21 saline-injected rats, but were clearly visible in BRF from all three time points (Figure 4A). Peak increase in TERT protein expression in BRF occurred after Day 7. These differences in TERT protein expression between NRF and BRF were consistent with the telomerase activity differences in these cells.

View larger version (36K): [in this window] [in a new window]   Figure 4.   Effects of bFGF on TERT expression. Lung fibroblasts were isolated from rats at the indicated time points after bleomycin treatment at time 0. Cell extracts were then prepared for Western blotting to analyze for TERT protein expression (A). Extracts from saline-treated control rats on Days 0 and 21 were also shown as controls. The effects of bFGF on NRF (from Day 21 saline-treated controls) and BRF (from lungs of Day 21 bleomycin-treated animals) TERT protein expression were similarly examined by Western blotting. The dose dependence (B) and kinetics (C) of the bFGF effect are shown. Equal amounts of protein were loaded for each data point. The kinetics of the bFGF effect on TERT mRNA levels was determined using RT-PCR (D). The inset shows a representative electropherogram of PCR products, and the graph shows the means ± SD (n = 3) after quantitation of the bands by scanning densitometry and normalization to the respective GAPDH mRNA signals. Open bars, control; striped bars, +bFGF.

Treatment of both NRF and BRF with bFGF also caused a dose-dependent increase in TERT protein expression, albeit the levels of expression in BRF were substantially higher than those seen in NRF at all doses of bFGF examined (Figure 4B). Of all doses tested, the greatest effect was seen at the 2 ng/ml dose. Relative stimulation in TERT protein expression by bFGF was observed at all time points examined from 24 to 72 h of treatment, although as with telomerase activity, the gradual decline in TERT protein expression in culture was not completely prevented by this growth factor (Figure 4C). Inhibitory effects of IL-4 on TERT protein expression were also comparable to those seen with telomerase activity in terms of both dose-dependence and kinetics (data not shown).

The effects of bFGF on TERT protein expression were also reflected in the TERT mRNA levels in BRF (Figure 4D). The stimulatory effect of bFGF on TERT mRNA was rapid, being noticeable beginning at 4 h after treatment, with maximal relative stimulation at 6 h, and disappearing at 12 h. Consistent with the telomerase activity and TERT protein results, IL-4 caused a decline in TERT mRNA levels in BRF (data not shown).

Immunofluorescence Analysis of TERT Protein Expression

The observed stimulatory effect of bFGF on fibroblast TERT expression may be a result of uniform upregulation in all cells or in a select subgroup of cells. Furthermore, the intracellular localization of TERT expression is undetermined. To address these issues, NRF and BRF cultured on coverslips were treated without or with bFGF for 24 h, followed by immunofluorescence analysis for TERT protein expression. The results showed that < 8% of NRF were weakly positive for TERT protein, whereas > 15% of BRF showed strong staining for TERT (data not shown). When stimulated with bFGF, the number of cells positive for TERT increased to 14% and 29% for NRF and BRF, respectively, consistent with the stimulation of telomerase activity and TERT protein expression. Thus the observed increases in the latter two parameters appear to be due to a greater percentage of cells expressing TERT protein, rather than a uniform stimulation in all cells. Consistent with previous results (10), the TERT-positive cells did not stain with anti- -smooth muscle actin antibody (data not shown), indicating that bFGF-induced induction of TERT occurred exclusively in fibroblasts, whereas myofibroblasts appear to be unresponsive to this growth factor.

Examination of the intracellular localization for TERT expression in bFGF-treated BRF showed that the fluorescent signal could be found in both cytoplasmic and nuclear compartments. Only a minority (6%) of cells showed nuclear staining exclusively, whereas most of the positive cells exhibited either cytoplasmic (34%) or both cytoplasmic and nuclear (59%) staining. This distribution of cells according to their TERT staining pattern did not differ significantly between BRF treated without or with bFGF (data not shown), thus indicating that stimulation of telomerase activity in BRF was not associated with a change in the localization of TERT.

TERT and bFGF mRNA Levels in Bleomycin-Injured Lung Tissue

To examine the potential in vivo relevance of the in vitro effects of bFGF on TERT expression, the levels of both bFGF and TERT mRNA were examined in lung tissue of rats treated with bleomycin. The results revealed evidence of a gradual increase in lung TERT mRNA in bleomycin-injured lung tissue beginning as early as Day 7 after induction of injury, and approaching a maximal increase after Day 14 (Figure 5). Bleomycin treatment also caused a comparable increase in bFGF mRNA with similar kinetics. Thus, there is good correlation between an upregulation of lung bFGF and TERT mRNAs in bleomycin-induced lung injury and fibrosis. Interestingly, BRF constitutively expressed higher levels of bFGF mRNA than NRF (data not shown), which could account for the constitutively expressed telomerase activity in BRF through autocrine and/or juxtacrine regulation.

View larger version (32K): [in this window] [in a new window]   Figure 5.   Kinetics of lung tissue TERT and bFGF mRNA expression in bleomycin-induced lung fibrosis. Lung tissues from bleomycin-treated rats at the indicated time points after treatment at time 0, were analyzed for TERT (open bars) and bFGF (striped bars) mRNAs by RT-PCR. A representative electropherogram of the PCR products is shown in the inset. The graph shows the means ± SD (n = 5) after quantitation of the bands by scanning densitometry and normalization to the respective GAPDH mRNA signals.

	Discussion

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Telomerase activity has been detected in over 85% of human cancers (17). Recent evidence, however, shows that telomerase is expressed in some normal rodent organs having a self-renewal potential such as liver, spleen, thymus, and testis, even in adult animals (18, 19). This activity has also been found in activated lymphocytes, injured and inflamed skin, as well as in fibrotic liver (20). Directly relevant to the study reported herein, recent findings have demonstrated that telomerase activity was transiently induced in bleomycin-injured rat lung tissue and fibroblasts (10). Although the upregulation of telomerase in immortal tumor cells have been described in detail (23, 24), the regulation of telomerase expression and its role in noncancerous or nontransformed tissues and cells remain unclear. Bleomycin injection causes lung inflammation followed rapidly by fibrosis characterized by expansion of the lung fibroblast population and emergence of myofibroblasts. The latter cells represent a key contributor to the increasing matrix synthesis and deposition characteristic of fibrosis (25). The fact that telomerase is selectively induced in fibroblasts isolated from bleomycin-injured lungs relative to myofibroblasts (10), suggests that telomerase may participate in the pathogenesis of fibrosis at a stage before the emergence of the myofibroblast, or the differentiation of the fibroblast to myofibroblast.

To identify potential regulators of telomerase activity in lung injury and fibrosis, lung fibroblasts were isolated from normal and bleomycin-injured lungs. A number of potential candidate agonists were tested to see if they could induce telomerase activity in these cells, based on their known expression in lung injury and fibrosis. Of the growth factors or cytokines tested, only three have consistent and significant effects on telomerase expression, namely bFGF, IL-4, and TGF. The growth factor, bFGF, is a potent mitogenic and chemotactic factor for most cells, including fibroblasts, with autocrine and paracrine stimulatory effects on cell proliferation (26). In this study, bFGF was found to induce and/or stimulate telomerase expression in vitro in fibroblasts isolated from both control and bleomycin-injured rat lungs. This stimulation was observed both in terms of enzymatic activity and TERT protein expression. Because TERT mRNA levels were also elevated, transcriptional regulation may be an important mechanism. Stimulation of telomerase expression by bFGF in cells from bleomycin-injured lungs was several fold higher than that seen in cells from control uninjured lungs, suggesting that a fibroblast phenotypic or functional alteration in injured lungs caused an amplified response to subsequent bFGF treatment. The increased expression of bFGF in BRF versus NRF could play such a role in an autocrine or juxtacrine manner. Additionally this may imply a role perhaps for a costimulatory signal present in injured lung to optimize the response to bFGF. The increase in telomerase activity seen in bFGF-treated cells was not associated with a significant alteration in the intracellular localization or compartmentalization of the TERT protein, but was apparently due mainly to an increase in the percentage of cells expressing this protein. Despite the growth promoting activities of other growth factors such as PDGF and EGF, only bFGF was found to upregulate telomerase expression, suggesting some specificity for the bFGF effect and that cell proliferation per se was not sufficient to produce such an effect on telomerase expression. The in vivo or pathophysiologic relevance of these in vitro effects of bFGF was supported by evidence of its induction in bleomycin-injured lung tissue, which correlated very closely with TERT expression in terms of their kinetics. This association suggests a possible role for bFGF in the induction of telomerase expression in injured lungs undergoing fibrosis. Increased bFGF expression in vivo has also been reported in renal fibrosis (26).

In contrast to bFGF, the induced telomerase expression in fibroblasts from bleomycin-injured lungs was suppressed by IL-4 as well as TGF. Since TERT mRNA was also decreased by IL-4 treatment, transcriptional regulation may also be an important mechanism. These findings are consistent with previous studies showing that IL-4 inhibits the telomerase activity in hepatoblastoma and acute myelogenous leukemia cells (27, 28). IL-4, a Th2 type cytokine, is an inducer of -smooth muscle actin expression and myofibroblast differentiation, similar to the effects of the pro-fibrogenic cytokine TGF on fibroblasts in vitro (16). Thus the inhibition of telomerase expression by these two cytokines may be related, or due, to the differentiation to myofibroblasts promoted by these mediators. This conclusion would be consistent with the previous observation that telomerase expression is preferentially localized in fibroblasts relative to myofibroblasts (10). Interestingly, myofibroblast differentiation induced by IL-4 or TGF is inhibited by bFGF (16, 29). It appears then that differentiation to myofibroblasts is associated with the loss of telomerase expression, and/or resistance to bFGF induction of telomerase expression. Cell differentiation leading to loss of telomerase expression has been previously reported as well in HL-60 cells (30).

Telomerase activity has been reported to be mainly modulated at the TERT transcriptional level in cells of various origins, whereas the RNA component and other telomerase associated proteins are constitutively expressed in both normal and tumor cells (12, 31, 32). The results of the current study would be consistent with these observations since the effects of bFGF and IL-4 on telomerase activity paralleled their effects on TERT protein and mRNA levels, albeit with dissimilar kinetics. Other studies however indicate that bFGF induced telomerase activity does not correlate with increasing TERT mRNA levels, although activity and mRNA determinations are done at the same time point, namely at 24 h of stimulation (18, 33). This discrepancy may be due to the fact that bFGF induced elevation in TERT mRNA levels was shown in this current study to be a rapid and transient event (maximal at 6 h and undetectable by 12 h) compared with its protein and telomerase activity (> 24 h). Nevertheless, the possibility of both transcriptional and post-transcriptional regulation of telomerase expression, such as by phosphorylation (34), cannot be ruled out by the available evidence. There is evidence for the presence of TERT mRNA in some cells that do not express telomerase activity, raising the possibility of a noncatalytic function for the telomerase complex. Finally, enzyme activity in telomerase-negative human cell lines can be restored by the ectopic expression of TERT (35).

When taken together, the results of the current study suggest that induction of telomerase expression, perhaps by bFGF, in bleomycin-induced lung injury and fibrosis may represent an intermediate step in the activation of fibroblasts toward their differentiation to myofibroblasts. The expression of telomerase activity appears to be associated with the period of expansion of the lung fibroblast population, but that subsequent differentiation to myofibroblasts, perhaps under the influence of IL-4 and/or TGF-, results in a decline in telomerase activity. The precise role, if any, of this transiently induced telomerase activity is currently unknown, although it may be related to the increased proliferation of lung fibroblasts. Future studies to examine the effect of abrogation of this induction of telomerase activity on lung fibrosis are necessary to examine this potential role.