Introduction

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Telomeres are specialized heterochromatic structures at the ends of eukaryotic chromosomes, and are characterized by tandemly repeated short DNA with the sequence T TAGGG (1). Telomerases, which synthesize T TAGGG telomeric DNA repeats onto chromosomal ends de novo, are important for the stability, replication, and function of chromosomes (4). In normal cells, shortening of telomeric repeats progress with senescence (8), whereas in immortal cells, such as most cancer cells, the length of telomeric repeats has been stabilized, probably as a result of telomerase activation. Its activity is presumed to be associated with the unlimited proliferative capacity of the tumor cells (9, 10).

Recent evidence suggests that telomerase activity is detectable in certain noncancerous cells, especially less differentiated stem or germ-line cells, and cells from renewal tissues, such as the intestinal epithelium (11, 12). When expressed in certain less differentiated or undifferentiated cells, their expression is suppressed upon cellular differentiation (13, 14). Further, induction of telomerase activity is not restricted to cancer cells. Activation of both B and T lymphocytes can induce telomerase activity in these cells (15). Ultraviolet irradiation is known to induce this activity in certain cells (16), consistent with the observation that sun-damaged skin expresses higher levels of telomerase activity than does nondamaged skin (17). Partial hepatectomy followed by liver regeneration is accompanied by telomerase induction (18). Inflamed skin with psoriasis, and liver with hepatitis or cirrhosis, are associated with expression of telomerase activity (19). Thus, there is abundant evidence that expression or induction of telomerase activity is not restricted to cancerous or transformed cells. The association of telomerase expression or induction with tissue injury and fibrosis (18, 19) suggests a potential role for this enzyme in the pathogenesis of fibrotic lesions.

Despite this compelling evidence for the inducibility of telomerase activity in noncancerous cells and its association with injured and inflamed tissues (18, 19), its role in noncancerous pathologic processes, characterized by increased proliferation or survival of cells, is unknown. Pulmonary fibrosis is one such process, which is characterized by increased proliferation of fibroblasts. In bleomycin-induced lung injury and fibrosis, the de novo appearance of myofibroblasts with their distinct -smooth-muscle actin (-SMA)- expressing phenotype heralds the onset of the period of heightened interstitial collagen gene expression (20). Peribronchial and perivascular adventitial fibroblasts appear to be the source of these myofibroblasts, which are also key sources of cytokines with inflammatory and fibrogenic properties (21). In addition to cell proliferation, this differentiation event, namely from fibroblast to myofibroblast, appears to be an important component of the overall process of pulmonary fibrosis. Previous studies have found that fibroblasts isolated from lungs with active fibrosis have increased proliferative capacity and elevated collagen and cytokine gene expression. They also have a significantly higher content of myofibroblasts (22). Increased numbers and persistence of such cells would be expected to favor the progression of fibrosis to end-stage lung disease. In view of the association of telomerase activity with cell proliferation and survival, as well as cellular differentiation, this study is designed to determine whether telomerase activity is expressed during the fibroblast proliferative phase in a rat model of bleomycin-induced pulmonary fibrosis.

	Materials and Methods

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Animals and Induction of Pulmonary Fibrosis

Specific pathogen-free male Fisher 344 rats weighing from 170 to 200 g were obtained from Charles River Breeding Laboratories (Wilmington, MA). The rats were provided with food and water ad libitum and kept on a regular light-dark cycle in a temperature- and humidity-controlled environment. Rats were randomly chosen for the control or bleomycin-treated groups. They were treated by endotracheal injection on Day 0 with 7.5 units/kg body weight of bleomycin (Blenoxane; Nippon Kayaku Co. Ltd., Tokyo, Japan) diluted in sterile phosphate-buffered saline (PBS). Control animals received endotracheal injections of sterile PBS only. On Days 1, 3, 7, 14, 21, and 28 after bleomycin treatment, the animals were killed and the lungs promptly removed for determination of telomerase activity and isolation of fibroblasts.

Tissues and Cells

The lungs were rapidly dissected free of extraneous tissues, immediately frozen in liquid nitrogen, and stored at 80°C until analysis for telomerase activity. Other lung samples were used for isolation of lung fibroblasts by trypsinization of lung mince, as described previously (21). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and passaged by trypsinization. Only cells earlier than the third passage after primary culture were used for these studies.

Telomerase Assay

Telomerase activity in cells or tissue samples was measured using the Telomerase PCR ELISA kit (Boehringer Mannheim GmbH, Mannheim, Germany) in accordance with the manufacturer's instructions. Cells or tissue samples were homogenized with a motorized pestle in ice-cold lysis buffer. After centrifugation, the supernates were analyzed for telomerase activity using a modified telomeric repeat amplification protocol (TRAP) assay. Telomeric repeats were added to a biotin-labeled primer during the first reaction at 25°C for 20 min. After the telomerase inactivation at 94°C for 5 min, the elongation products were amplified by polymerase chain reaction (PCR) using a PTC-200 DNA Engine thermal cycler (MJ Research, Inc., Waltham, MA). Each of 30 cycles included denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and polymerization at 72°C for 1.5 min, followed by a single 10-min extension at 72°C.

An aliquot of the PCR product was denatured and hybridized to a digoxygenin (DIG)-labeled telomeric repeat-specific detection probe, followed by immobilization of the resulting product via the biotin-labeled primer to a streptavidin-coated microtiter plate. Finally, the immobilized PCR product was detected with a peroxidase-conjugated antibody to DIG using the substrate tetramethyl benzidine, and then quantified using an enzyme-linked immunosorbent assay (ELISA) reader.

Telomerase activity was also assayed using conventional detection of the DNA-laddering pattern typical for the TRAP assay. The procedure followed that for the ELISA method, but instead of measuring the PCR products generated by ELISA, they were separated using a 10% nondenaturing polyacrylamide gel and then blotted onto positively charged nylon membranes. Blotted TRAP products were subsequently visualized using the Biotin Luminescent Detection kit (Boehringer Mannheim) by binding of streptavidin-alkaline phosphatase (AP) to the 5' biotin-labeled amplicons, followed by detection of the AP label using nitroblue tetrazolium (NBT)/X-phosphate.

Telomerase Messenger RNA Expression

Detection of telomerase messenger RNA (mRNA) expression was undertaken using in situ hybridization. This was carried out using the In Situ Hybridization and Detection System (GIBCO BRL, Gaithersburg, MD) with minor modifications. The complementary DNA (cDNA) probe for telomerase mRNA detection was synthesized using reverse transcription-PCR. This was carried out using the Superscript One-Step PR-PCR System (GIBCO BRL) with the following primers: upstream, 5'-GCCTGAAGTGAACCAGAAGC-3'; downstream, 5'-GAGACAAGCTTTGCTGACCC-3'.

These primers were chosen on the basis of the published sequence for rat telomerase mRNA (23) and generated a 239-base pair product, which was confirmed by sequencing. This probe was separated and isolated using agarose gel electrophoresis, followed by biotinylation using the Bioprime DNA Labeling System (GIBCO BRL).

Fibroblasts from control and bleomycin-treated rat lungs were cultured on poly-L-lysine-coated coverslips (BIOCOAT; Becton Dickinson Labware, Franklin Lakes, NJ) in DMEM supplemented with 10% FBS. After approximately 5 d, they were fixed with CELL-FIXX (Shandon, Inc., Pittsburg, PA) and washed with PBS several times before hybridization. After 12 h of hybridization, coverslips were washed in NaCl-Na citrate followed by blocking buffer. Detection of the biotin-labeled hybridized probe was accomplished using streptavidin-AP conjugate and visualized using NBT/4-bromo-5-chloro-3-indiolyphosphate.

These same coverslips were then subjected to immunostaining with a murine monoclonal antibody to -SMA (Boehringer Mannheim). The Vectastain ABC KIT and AEC Substrate Kit for Peroxidase (Vector Laboratories, Inc., Burlingame, CA) were used to visualize positive staining. Detection of the biotin-labeled second antibody was accomplished using streptavidin-horseradish peroxidase conjugate and the substrate 3-amino-9-ethyl-carbazole. Coverslips were washed in water and embedded in Immu-mount (Shandon).

Immunofluorescence Analysis for Telomerase Reverse Transcriptase

To further analyze the cellular localization of telomerase activity vis-à-vis myofibroblast phenotype, double immunofluorescence analysis for telomerase reverse transcriptase (TRT) and -SMA was undertaken. The method is essentially as described earlier for assessment of telomerase mRNA expression. Briefly, Day 14 rat-lung fibroblasts were cultured on coverslips. After 48 h of culture, the cells were fixed in 4% paraformaldehyde and the coverslips stained with Cy3-conjugated monoclonal mouse anti--SMA antibody (Sigma Chemical Co., St. Louis, MO) at a working dilution of 1:300 for 1 h at room temperature. After washing with PBS, the same coverslips were immunostained with rabbit polyclonal anti-TRT antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at a concentration of 2 µg/ml overnight at 4°C. This was followed by incubation with 5 µg/ml of Alexa Fluor 488-conjugated antirabbit immunoglobulin G antibody (Molecular Probes, Eugene, OR) for 30 min at room temperature. The coverslips were then embedded with Mowiol mounting medium and examined with a Zeiss Axiophot 2 fluorescence microscope. A minimum of 10 randomly selected high-power fields were examined per coverslip. Cells positive for TRT (green fluorescence) or -SMA (red fluorescence) or both were counted and expressed as a percentage of total cells counted for each coverslip. A total of five coverslips were examined.

	Results

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Telomerase Activity in Lung Tissues and Fibroblasts

Because no information is available as to the inducibility of lung telomerase activity, baseline data are needed regarding expression of this enzyme in normal, injured, and inflamed lungs. This was undertaken using a rat model of lung injury and fibrosis induced by endotracheal injection of bleomycin. Using the telomerase ELISA assay, no detectable activity was found in control uninjured lungs (PBS in Figure 1A). Consistent with this finding was the lack of detectable activity in fibroblasts isolated from such control lungs (PBS in Figure 1B). When bleomycin-treated lungs were examined, telomerase activity remained undetectable on Days 1 and 3 after injury. However, significant activity was detected in lung tissue extracts beginning on Day 7, reaching a peak increase on Day 14 and a gradual decline on Day 21 after induction of injury (Figure 1A). By Day 28 after injury, the lung telomerase activity again became undetectable.

View larger version (26K): [in this window] [in a new window]   View larger version (21K): [in this window] [in a new window]   Figure 1.   Lung tissue (A) and fibroblast (B) telomerase activity by ELISA. Lungs from control and bleomycin-treated rats were harvested and either homogenized or used as sources of fibroblast cultures. Extracts of lung tissue (A) or fibroblast cultures (B) were analyzed for telomerase activity using the TRAP procedure combined with product analysis by ELISA. Control rat-lung samples for all time points were negative and the Day 14 controls are shown. Mean values ± standard deviation of three independent experimental results are shown. Telomerase activity was undetectable after heat treatment (85°C, 10 min) of all the samples, and the result from a heat-inactivated Day 14 sample is shown as a negative control []. For a positive control [+], the commercial assay kit supplied a cell extract prepared from immortalized telomerase-expressing human kidney cells (293 cells).

When fibroblasts isolated from these injured lungs were analyzed for telomerase activity, the results paralleled those for lung tissue. Thus cells from control and Days 1, 3, and 28 bleomycin-injured lungs did not express detectable activity (Figure 1B); whereas those from Days 7, 14, and 21 bleomycin-injured lungs expressed significant activity, with maximal expression found in Day 14 cells. The telomerase activity found in the positive lung and fibroblast extracts was inactivated by heat pretreatment (85°C for 10 min) of the samples, and the result from a heat-inactivated Day 14 sample is shown as a negative control in Figure 1.

To confirm the results using the ELISA assay, the products of PCR amplification were also analyzed by polyacrylamide gel electrophoresis. Telomerase activity using this method showed up as a laddering pattern consisting of bands of increasing size (Figure 2). As with the ELISA data, only lung tissues from Days 7, 14, and 21 showed detectable telomerase activity (Figure 2A), with maximal induction again occurring on Day 14 after bleomycin treatment. When the lung fibroblast extracts were analyzed, virtually identical results were observed; namely, that only cells from Days 7, 14, and 21 bleomycin-treated lungs expressed telomerase activity, with maximal activity detected in cells from Day 14 bleomycin-treated lungs (Figure 2B). As with the ELISA assay, heat treatment also eliminated activity in the positive samples.

View larger version (96K): [in this window] [in a new window]   View larger version (105K): [in this window] [in a new window]   Figure 2.   Lung tissue (A) and fibroblast (B) telomerase activity by electrophoretic analysis. Extracts of lung tissue (A) and fibroblast cultures (B) were prepared as described in the legend to Figure 1, and assayed for telomerase activity using the same TRAP procedure. However, the products were analyzed by polyacrylamide gel electrophoresis instead of by ELISA. Control rat-lung samples for all time points were negative and the Day 14 controls are shown. The results of a representative experiment, out of a total of three independent experiments, are shown. Telomerase activity was undetectable after heat treatment (85°C, 10 min) of all the samples, and the result from a heat-inactivated Day 14 sample is shown as a negative control (neg). The positive control (pos) is identical to that used in Figure 1.

Telomerase mRNA Expression

The telomerase mRNA component can serve as a template for TRT. Exploiting this property of the telomerase enzyme enables the detection of telomerase by in situ hybridization. Inasmuch as rat lung fibroblasts are known to be heterogeneous with respect to -SMA expression (22, 24), measurements of bulk cell extracts for telomerase activity do not provide information with regard to the distribution of enzyme expression by fibroblasts vis-à-vis myofibroblasts. To assess possible heterogeneity with respect to telomerase expression, cells from control and bleomycin-treated lungs were subjected to simultaneous analysis for telomerase mRNA expression by in situ hybridization, and for -SMA expression by immunohistochemical staining.

The results from this technique confirmed the finding of telomerase induction using the telomerase assays described earlier (Figure 3). Thus, only cells from lungs of Days 7, 14, and 21 bleomycin-treated rats showed detectable levels of telomerase mRNA. Expression was much reduced or undetectable at other time points on Days 1, 3, and 28. Telomerase mRNA was not uniformly expressed by all cells in the positive samples from Days 7, 14, and 21 bleomycin-injured lungs. This heterogeneity of telomerase mRNA expression may be related to the heterogeneity of lung fibroblasts.

View larger version (157K): [in this window] [in a new window]   Figure 3.   (left) Fibroblast telomerase mRNA expression by in situ hybridization. Rat lung fibroblasts from control and bleomycin-treated rats were analyzed for heterogeneity with respect to telomerase RNA expression and -SMA expression. In situ hybridization for detection of telomerase mRNA (blue) was carried out with the biotin-labeled telomerase cDNA probe as described in MATERIALS AND METHODS. The same slides were then immunostained for detection of -SMA expression ( purple). Telomerase mRNA was undetectable in cells isolated from control (not shown) and Days 1 (A) and 3 (B) bleomycin-treated rats. Cells from lungs of Days 7 (C), 14 (D), and 21 (E) bleomycin-treated animals showed detectable levels of telomerase mRNA (blue granular staining). Telomerase mRNA expression was detected mostly in cells that did not express -SMA, as shown in Days 14 (D), 21 (E), and 28 (F) cells. By Day 28 the cells predominantly consisted of -SMA positive cells that did not express telomerase mRNA.

Previous studies have shown that myofibroblasts appear de novo beginning on Day 4 after bleomycin treatment, peaking between Days 7 and 14, and subsequently gradually disappear after Day 21 (20). Cells isolated from animals at these various time points reflect these changes in myofibroblast composition (22), and essentially confirmed in the present study. When these cells were also stained for -SMA expression to assess distribution of telomerase mRNA expression in myofibroblasts versus fibroblasts, only a small minority of the actin-positive cells were found to express telomerase mRNA (Figures 3D and 3E). Conversely, only a few of the telomerase mRNA-expressing fibroblasts expressed -SMA (Figures 3C-3E). A third group of cells expressed neither telomerase mRNA nor -SMA. These latter cells predominate in cultures isolated from control and Days 1 and 3 bleomycin-treated rat lungs, wherein fibrosis was nonexistent or at its earliest stages, respectively.

TRT Expression

Because TRT expression correlates with telomerase activity, further confirmation of predominant telomerase expression in nonmyofibroblasts was sought using dual immunofluorescence for TRT and -SMA. Further, immunofluorescence for TRT exhibited a lower background and thus yielded a more specific and clear-cut signal as compared with the in situ hybridization analysis for telomerase mRNA (Figure 3). In this experiment, Day 14 bleomycin-treated rat-lung fibroblasts were examined for localization of these two antigens. Sequential staining for TRT and -SMA confirmed the previous telomerase mRNA results. A representative photomicrograph of the double immunofluorescence is shown in Figure 4, wherein TRT expression, as with telomerase mRNA expression (Figure 3), occurred predominantly in cells that are negative for -SMA. There were, however, a few cells that appeared positive for both antigens, suggesting that a minority of the myofibroblasts do express some level of TRT. To confirm this visual inspection, the cells that were positive for TRT or -SMA or both were counted for each coverslip. The results of this counting are shown in Figure 5. Consistent with the observations of telomerase mRNA localization, TRT expression was found primarily in cells that did not express -SMA. Only less than 20% of myofibroblasts (-SMA-positive cells) expressed TRT, while only about a third of all TRT-expressing cells were myofibroblasts. Thus, predominantly nonmyofibroblasts expressed TRT.

View larger version (93K): [in this window] [in a new window]   Figure 4.   Cellular localization of telomerase and -SMA expression. Rat fibroblasts from lungs of Day 14 bleomycin-treated animals were cultured on coverslips and immunostained sequentially for -SMA (red fluorescence) and TRT (green fluorescence) as described in MATERIALS AND METHODS. The slides were viewed and the images digitally captured separately for green and red fluorescence (original magnification, ×400). The separate green and red fluorescence images of the same field were then superimposed and printed using computer software (Adobe Photoshop).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Quantitation of cells expressing telomerase or -SMA or both. The coverslips of rat lung fibroblasts similar to the one shown in Figure 4 were counted for the numbers of cells positive for antigens as indicated in the x-axis labels. Cells positive for the indicated antigen or antigens (as indicated on the x-axis) were counted and expressed as a percentage of total cells counted. Results are expressed as means ± standard error from a total of five separate coverslips.

	Discussion

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Induction of telomerase activity has previously been reported in activated lymphocytes, injured and inflamed skin, and regenerating or inflamed and fibrotic liver (15- 19). Its precise role or roles in these situations are uncertain, although on the basis of its known activity it would be logical to hypothesize its participation in extending survival or life span of cells expressing this enzyme. Cellular localization of the telomerase expression in fibrotic lesions is unknown. Because expansion of the fibroblast population is a key characteristic of active fibrotic lesions (21), the possible role of telomerase in this process was investigated in this study.

Expression of telomerase activity in normal lung is variable and strain-dependent in the mouse (25). In the present study, normal Fisher 344 rat lungs showed no detectable telomerase activity. Whether it can be induced under pathologic conditions is unknown. Bleomycin-induced lung injury causes inflammation followed rapidly by fibrosis (21). Using this model of pulmonary fibrosis in the rat, the results in the present study showed for the first time that telomerase activity, mRNA, and RT were induced in fibroblasts derived from injured lungs in a time-dependent manner. Cells isolated from control uninjured lungs and from lungs exhibiting the early or late stages of fibrosis contained no or very low levels of enzyme activity. The peak of fibrosis based on the maximal increase in procollagen gene expression, for instance, brackets the time period in which maximal telomerase activity was observed, namely at approximately Day 14 after bleomycin treatment. These findings in isolated lung fibroblasts are reflected as well in the lung tissues themselves, thus confirming the in vivo relevance of the in vitro cell-culture findings. The kinetics of induction and its coincidence with the period of active fibrosis with expansion of the fibroblast population suggest the possible role of telomerase in the expansion of the fibroblast population, either by prolonging their survival or life span, and/or by enhancing the proliferative capacity of these cells.

When the cells were assessed for telomerase mRNA expression, this was found to parallel that for telomerase activity. Interestingly, however, the expression was found to be heterogeneous in those positive samples with detectable expression. Concomitant immunostaining for -SMA expression revealed that telomerase mRNA expression was primarily localized to cells without appreciable actin expression or nonmyofibroblasts. This finding was confirmed by double immunofluorescence analysis for TRT and -SMA expression, wherein only about a third of TRT-expressing cells were myofibroblasts. Because these immunologic approaches are not quantitative with respect to the staining intensity, the degree or level of TRT expression in nonmyofibroblasts vis-à-vis myofibroblasts could not be precisely determined. It is unclear at this time whether this predominant expression of telomerase in nonmyofibroblasts is due to loss of telomerase expression upon differentiation of fibroblasts to myofibroblasts, similar to what has been shown in the differentiation of neuronal and HL-60 cells (26). Alternatively, only telomerase-negative cells can differentiate to myofibroblasts. More studies need to be undertaken to distinguish between these various possibilities.

In contrast to human tissues and somatic cells, previous reports show that telomerase activity is expressed in several murine normal tissues and cultured somatic cells. These earlier authors speculate that stringent control of cell proliferative capacity may not occur in such relatively short-lived animals as mice, thus explaining persistence of telomerase activity in tissues of adult animals (25, 27). The data from the study by Blasco and associates cannot rule out the possibility that enhanced activity in cultured tissues could be due to selective outgrowth of telomerase-positive cells (27). In the present study telomerase activity was not detectable in control lung tissue and fibroblast samples, thus suggesting that the telomerase activity induction in injured lung samples was associated with the inflammation induced by the injury. The mechanism of this telomerase induction in the bleomycin model is unclear. Some reports reveal that telomerase activity is sometimes inducible by environmental factors. For example, it is induced in human lymphocytes by in vitro mitogenic or antibody stimulation, and in human hematopoietic progenitor cells upon their proliferation and differentiation (15). Certain cytokines may be involved in this regulation of telomerase expression, and may also participate in the induction of lung telomerase in this model.

Human and mouse telomerases are reported to differ in both their functional properties and their regulation (7, 9). The precise mechanism for regulating its activity at the level of transcription, molecular assembly, or enzymatic activity, remains to be determined. However, if telomerase plays a significant role in prolonging the survival and increasing the proliferative capacity of fibroblasts, this may be decisive in whether lung injury resolves or progresses to fibrosis and end-stage lung disease. Hence, it may represent a new target for the design of future therapeutic strategies in management of pulmonary fibrosis.