Radiation pneumonitis is a major complication of radiation therapy. However, the detailed cellular mechanisms have not been clearly defined. Based on the recognition that basement membrane disruption occurs in acute lung injury and that matrix metalloproteinase (MMP)-2 can degrade type IV collagen, one of the major components of the basement membrane, we hypothesized that ionizing radiation would modulate MMP-2 production in human lung epithelial cells. To evaluate this, the modulation of MMP-2 with irradiation was investigated in normal human bronchial epithelial cells as well as in A549 cells. We measured the activity of MMP-2 in the conditioned medium with zymography and the MMP-2 mRNA level with RT-PCR. Both of these cells constitutively expressed 72-kDa gelatinolytic activity, corresponding to MMP-2, and exposure to radiation increased this activity. Consistent with the data of zymography, ionizing radiation increased the level of MMP-2 mRNA. This radiation-induced increase in MMP-2 expression was mediated via p53 because the p53 antisense oligonucleotide abolished the increase in MMP-2 activity as well as the accumulation of p53 after irradiation in A549 cells. These results indicate that MMP-2 expression by human lung epithelial cells is involved in radiation-induced lung injury.
- radiation pneumonitis
radiation therapy is now commonly used as one of the conventional modalities for the treatment of malignant neoplasms in the thorax. The pulmonary parenchyma is relatively radiosensitive, which makes the lung the dose-limiting organ in therapeutic radiation. The percentage of symptomatic radiation pneumonitis was reported to be ∼7% (29). However, the detailed cellular mechanisms of radiation-induced lung injury have not been established.
The histopathological changes after pulmonary radiation can be divided into early, intermediate, and late stages. In the early stage, vascular congestion and increased capillary permeability are typical findings. The alveolar walls are generally thickened, and the alveolar epithelium desquamates into the alveolar spaces. It has been reported that the mRNA level of type IV collagen is elevated in irradiated rat lungs after 1 wk and that the elevation is sustained over 26 wk (37). Type IV collagen is one of major components of the basement membrane, and this elevation may reflect alteration of the basement membrane. And this finding could imply that disruption of the basement membrane may contribute to increased capillary permeability.
The basement membrane plays a crucial role in maintaining the integrity of the lung epithelium. It has been suggested that early disruption of the basement membrane may be implicated in the pathogenesis of lung fibrosis, enhancing the migration of fibroblasts and deposition of interstitial collagen into the alveolar spaces. In addition, disruption of the basement membrane may also result in failure to replace damaged alveolar type I epithelial cells after severe injury, which appears to be an important condition contributing to the progression of radiation pneumonitis followed by fibrosis.
Matrix metalloproteinases (MMPs) are a large family of related proteolytic enzymes that includes collagenases, gelatinases, stromelysins, elastases, and membrane-type (MT) MMPs (40). MMPs are known to degrade the extracellular matrix, and these enzymes have been reported to play a critical role in the pathogenesis of acute and chronic lung diseases. Because the gelatinases MMP-2 and MMP-9 are capable of degrading several components of the basement membrane, including type IV collagen, both enzymes are thought to be major players in basement membrane disruption in various pathological conditions.
Indeed, Hayashi and colleagues (16, 17) have demonstrated, in a series of pathological studies in various pulmonary disorders, that type IV collagen and MMP-2 showed focal colocalization in disrupted epithelial basement membrane. It has been reported that the increase in MMP-2 activity was recognized in epithelial lining fluid (ELF) obtained from patients with adult respiratory distress syndrome (ARDS) (9, 45). Pardo et al. (31) have also demonstrated the increased expression of gelatinases and collagenase in rat lungs exposed to subacute hyperoxia.
Previous studies (7, 36, 37) have shown that ionizing radiation is associated with increased expression of cytokines such as tumor necrosis factor-α and transforming growth factor-β in a variety of cell types. These cytokines may contribute to the acute-phase inflammation and late-phase fibrosis of the lung.
Ionizing radiation is known as a representative of DNA-damaging agents. The DNA cleavage by irradiation leads to the accumulation of p53 and its translocation into the nucleus (41). Then the activated p53 binds to DNA in a sequence-specific manner and modulates a set of genes (1). A recent study (5) has demonstrated that the promoter region of the MMP-2 gene has a putative p53 binding site and that p53 transcriptionally upregulates the expression of MMP-2 mRNA. Indeed, it has been reported that ionizing radiation increased MMP-2 activity in rat astrocytes (39). However, in lung cells, the modulation of MMPs by ionizing radiation and the involvement of p53 in the signaling pathways have not been clearly elucidated.
In the present study, we analyzed the effect of ionizing radiation on the expression of MMP-2 in normal human bronchial epithelial (NHBE) cells as well as in A549 human type II-like pneumocytes. We found that ionizing radiation enhanced the expression of MMP-2 but had no effect on tissue inhibitor of metalloproteinase-2 (TIMP-2) expression. The effect of ionizing radiation on the expression of MMP-2 appears to be mediated via a p53 transcription factor. Finally, glucocorticoid dramatically inhibited the increased expression of MMP-2 induced by ionizing radiation.
MATERIALS AND METHODS
Polyacrylamide, gelatin, cycloheximide (CHX), actinomycin D (Act D), dexamethasone (Dex), and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (Tokyo, Japan). Trypsin-EDTA and LIPOFECTIN were purchased from GIBCO BRL (Life Technologies, Rockville, MD).
Cryopreserved primary NHBE cells were purchased from Clonetics (San Diego, CA) and grown in 60-mm tissue culture dishes in bronchial epithelial cell growth medium supplied by Clonetics. The cultures were incubated at 37°C in a humidified, 5% CO2-95% air atmosphere. When the cells were grown to confluence, the culture medium was changed to hydrocortisone-free defined medium. NHBE cells were used within the first five passages. A549 cells, a tumor cell line from a human lung carcinoma with properties of type II alveolar epithelial cells, were purchased from American Type Culture Collection (Manassas, VA). This cell line has been used as a model of human type II alveolar epithelial cells in the literature (10, 32). The cells were grown in 60-mm tissue culture dishes in a humidified, 5% CO2-95% air atmosphere. The culture medium was RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) containing 10% heat-inactivated fetal calf serum (GIBCO BRL), 2 mM l-glutamine, 50 IU/ml of penicillin, and 50 μg/ml of streptomycin. When the cells reached confluence, the culture medium was changed to the serum-free defined medium of the above-mentioned composition except for heat-inactivated fetal calf serum. After incubation for 1 h, the cells were irradiated at room temperature with an X-ray source (dose rate of 2.54717 Gy/min; MBR-1505R, Hitachi Medical, Hitachi, Japan). The conditioned medium was collected after cell culture for an additional 24 h.
Aliquots (10 μl) of the conditioned medium collected from the culture of NHBE cells were directly analyzed for gelatinolytic activity. For A549 cells, aliquots (500 μl) of the conditioned medium were concentrated 50-fold by centrifugation through an Amicon membrane (Millipore), with a cutoff of 10 kDa. These conditioned media were loaded onto 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels containing 1.5 mg/ml of gelatin. After electrophoresis, the gels were washed at room temperature for 1 h in 2.5% Triton X-100 to remove SDS. The gels were then incubated overnight at 37°C in incubation buffer (50 mM Tris · HCl, pH 7.5, 0.05% NaN3, 5 mM CaCl2, and 1 μM ZnCl2). To check the inhibition of gelatinolytic activity, 20 mM EDTA or 2 mM PMSF was added to the incubation buffer. The gel was stained with 0.1% Coomassie brilliant blue in 10% acetic acid and 10% isopropanol and subsequently destained for 1 h. Gelatinolytic activities were identified as clear zones of lysis against a blue background. The intensity of the bands was quantified by densitometric analysis with National Institutes of Health Image 1.59.
Measurement of Cell Number and Viability
Cells were grown to confluence in 60-mm tissue culture dishes and then irradiated. The cells were collected with trypsin-EDTA after culture for an additional 24 h after irradiation. Cell numbers and viability were determined with a conventional hemacytometer by trypan blue exclusion.
Detection of Apoptosis
Flow cytometric analysis.
Apoptosis was assessed by fluorescence-activated cell-sorting analysis carried out on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) with the CellQuest software system. The irradiated cells were harvested and immediately immobilized by 70% ice-cold ethanol overnight. Low molecular weight fragmented DNA was washed away with 40 mM citrate buffer. Then the cells were incubated with 100 μg/ml of RNase in PBS-Tween 80 for 30 min and stained with 30 μl of a propidium iodide solution (1 mg/ml) for 30 min in the dark. The quantity of cells with hypodiploid DNA was measured on a FACScan at the FL2 channel. Ten thousand cells were examined for each determination.
4′,6-Diamidino-2-phenylindole dihydrochloride staining.
We performed nuclear staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Harvested cells were stained with DAPI-methanol (10 μg/ml) for 30 min in the dark. Then the treated cells were washed twice with distilled water, seeded on a glass slide, and air-dried. The glass slides were viewed and photographed with a fluorescence microscope (Nikon, Tokyo, Japan).
Total cellular RNA was extracted from the cells with the acid guanidinium thiocyanate-phenol-chloroform extraction method with ISOGEN (Nippon Gene, Tokyo, Japan). The amount of RNA was quantified by absorbance at 260 nm.
All reagents for RT-PCR were obtained from Takara Shuzo (Kyoto, Japan). Two micrograms of total RNA were reverse transcribed to cDNA as previously described (48). One-fourth of the cDNA product was used in the PCR. Amplification of a specific PCR product was carried out separately in a different tube. The primers used were MMP-2 sense primer, 5′-ACCTGGATGCCGTCGTGGAC-3′; MMP-2 antisense primer, 5′-TGTGGCAGCACCAGGGCAGC-3′; MMP-9 sense primer, 5′-GGTCCCCCCACTGCTGGCCCTTCTACGGCC-3′; MMP-9 antisense primer, 5′-GTCCTCAGGGCACTGGAGGATGTCATAGGT-3′; TIMP-2 sense primer, 5′-TGCAGCTGCTCCCCGGTGCAC-3′; TIMP-2 antisense primer, 5′-TTATGGGTCCTCGATGTCGAG-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer, 5′-CAAAAGGGTCATCATCTCTG-3′; and GAPDH antisense primer, 5′-CCTGCTTCACCACCTTCTTG-3′. These primer sets yielded PCR products of 447, 590, 530, and 446 bp for MMP-2, MMP-9, TIMP-2, and GAPDH, respectively. Reaction mixtures were incubated in a Perkin-Elmer Cetus DNA thermal cycler (Perkin-Elmer, Norwalk, CT). Aliquots of the PCR products were subjected to agarose gel electrophoresis in Tris-acetate-EDTA buffer and visualized by ethidium bromide staining.
Our data were obtained before the amplification products reached the plateau phase. For this purpose, optimal PCR conditions were chosen after amplification kinetics were studied by collecting samples up to 50 cycles. Amplified products were obtained in the exponential phase for each set of primers at 30–35 cycles. Examples of the relationship between the number of amplification cycles and the amplified products are shown in Fig.1 A for MMP-2 and Fig.1 B for GAPDH.
We used phosphorothioate antisense (5′-CCCTGCTCCCCCCTGGCTCC-3′) and sense (5′-GGAGCCAGGGGGGAGCAGGG-3′) oligonucleotides of p53 with a length of 20 bases as previously described (18). These oligonucleotides were purchased from Greiner Japan (Tokyo, Japan).
Oligonucleotide Treatment of the Cells
Cells were treated with oligonucleotide at a final concentration of 2 μM for 24 h in serum-free defined medium containing LIPOFECTIN (10 μg/ml). After treatment, the medium was replaced with oligonucleotide-free defined medium and irradiated at room temperature. After 24 h, the conditioned medium was collected for gelatin zymography and the cell lysates were analyzed by RT-PCR.
Western Blot Analysis
Immunoblot analysis was used to confirm the inhibitory effect of p53 antisense oligonucleotide on p53 protein induction by ionizing radiation. After stimulation with ionizing radiation, the cells were washed twice with ice-cold PBS and lysed with Triton X-based lysis buffer. The concentration of total cellular protein was measured with a Bio-Rad (Richmond, CA) protein assay. Thirty micrograms of each protein preparation were separated by electrophoresis on 10% gradient SDS-polyacrylamide gels and electrically transferred to nitrocellulose membranes. After blockade of nonspecific binding, the blots were incubated overnight at 4°C with mouse antibodies against p53 (Transduction Laboratories, Lexington, KY). Goat anti-mouse IgG-horseradish peroxidase was used for chemiluminescence detection.
We repeated each type of experiment at least three times and confirmed that similar data were obtained. All values are means ± SD. Comparisons were made with one-way ANOVA with Fisher's post hoc test. Differences between means were evaluated with Student'st-test. A P value of <0.05 was considered significant.
Ionizing Radiation Enhances Production of MMP-2 in Lung Epithelial Cells
Both NHBE and A549 cells constitutively secreted MMP-2 into the culture medium as previously reported (32). Ionizing radiation enhanced the production of MMP-2 in a dose-dependent manner, and the maximal production of MMP-2 was observed at doses of 4 and 8 Gy in NHBE and A549 cells, respectively (Fig.2). The molecular mass of MMP-2 was 72 kDa, corresponding to the pro form. The active form was not detected. MMP-9 was found as a 92-kDa faint band corresponding to the pro form, and this band was little affected by ionizing radiation. Gelatinolytic bands completely disappeared with the addition of EDTA but not of PMSF to the incubation buffer, indicating that MMP(s) is responsible for this activity (data not shown). Cell viability of both NHBE and A549 cells as assessed by trypan blue dye was >95%. Cell number had a tendency to decrease with irradiation (Tables1 and2), but no significance was observed. It has been reported that ionizing radiation induces apoptosis in a variety of cells (15). Hence we evaluated the effect of ionizing radiation on apoptosis in NHBE and A549 cells by measuring the percentage of the cells with hypodiploid DNA with propidium iodide staining followed by flow cytometric analysis. In the whole range of doses we utilized, we were not able to detect an apoptosis-inducing effect of ionizing radiation on A549 cells (Fig.3) and NHBE cells (data not shown). We further confirmed this finding by counting cells with nuclear condensation and fragmentation with DAPI staining followed by fluorescence microscopy (data not shown).
Ionizing Radiation Induces Expression of MMP-2 mRNA in Lung Epithelial Cells
The effect of ionizing radiation on MMP-2 mRNA expression in NHBE and A549 cells is shown in Fig. 4. The level of MMP-2 mRNA was evaluated with RT-PCR, and the results were normalized to the levels of the housekeeping gene GAPDH. After ionizing radiation, there was a dose-dependent increase in MMP-2 mRNA levels at 24 h, and the maximal expression was observed at doses of 2 Gy in NHBE cells and 8 Gy in A549 cells. The discrepancy between the data from gelatin zymography and RT-PCR in NHBE cells is presumably affected by the difference in the batch utilized for each experiment. The levels of MMP-2 mRNA peaked 12 h after ionizing radiation in A549 cells (data not shown). In contrast, the expression of the MMP-9 gene in NHBE cells decreased in response to irradiation (Fig. 4 A), and the transcript of the MMP-9 gene in A549 cells was not detected by RT-PCR. Ionizing radiation had no effect on TIMP-2 mRNA expression in NHBE cells and A549 cells (Fig. 4).
Effect of Various Synthesis Inhibitors on MMP-2 Production in Ionizing Radiation-Stimulated A549 Cells
It has been reported that MMP-2 associates with the integrin αvβ3 on the cell surface (6), and MMP-2, TIMP-2, and MT-MMP become a trimolecular complex on the cell surface (43). To rule out the possibility that the increase in MMP-2 in the conditioned medium after irradiation might be influenced by the shedding of MMP-2 from the cell surface of A549 cells, CHX and Act D, known as protein and RNA synthesis inhibitors, respectively, were added to serum-free defined medium before ionizing radiation. Both CHX and Act D abolished the increase in MMP-2 protein in the conditioned medium, indicating that the increase in MMP-2 after ionizing radiation needs the de novo synthesis of its mRNA and protein (Fig. 5).
p53 Mediates the Increase in MMP-2 Expression by Ionizing Radiation in A549 Cells
A previous study (5) has demonstrated that the promoter region of the MMP-2 gene has a putative p53 binding site and that p53 upregulates the expression of MMP-2 mRNA. Ionizing radiation generates DNA damage, and, subsequently, p53 accumulates in the nucleus and acts as a transcription factor (41). To examine whether MMP-2 production was mediated via p53, we used a p53 antisense oligonucleotide. Ionizing radiation (8 Gy) resulted in a significant increase in the level of p53 in A549 cells (Fig.6 A). The kinetics of accumulation of p53 was biphasic after irradiation, with the first peak at ∼3 h and the second peak between 12 and 24 h. Similar biphasic accumulation of p53 after exposure to ionizing radiation was reported in human embryo cells (12). In contrast to treatment with p53 sense oligonucleotide or with LIPOFECTIN alone, treatment with p53 antisense oligonucleotide almost perfectly abrogated the irradiation-induced accumulation of p53 protein in A549 cells 3 h after radiation exposure (Fig. 6 B).
We therefore analyzed the effect of the p53 antisense oligonucleotide on the expression of MMP-2 after ionizing radiation. The activity of MMP-2 was markedly diminished in p53 antisense oligonucleotide-treated cells (Fig. 7 A). Consistent with the data from gelatin zymography, the level of MMP-2 mRNA transcript was dramatically diminished in p53 antisense oligonucleotide-treated cells (Fig. 7 B). These results suggest that the expression of MMP-2 induced by ionizing radiation may be largely mediated via a p53 transcription activator.
Dex Suppresses the Increase in MMP-2 Expression Induced by Ionizing Radiation in A549 Cells
Because corticosteroid is the mainstay of the treatment of radiation pneumonitis (29), we examined the effect of Dex on the production of MMP-2 after ionizing radiation. Dex inhibited the increase in MMP-2 production on exposure to radiation (Figs. 5 and8). This inhibition was clearly observed at a dose of 10−7 M Dex. However, dose-dependent inhibition was not observed within the range of 10−7 to 10−5 M (data not shown). Even in the nonirradiated condition, Dex suppressed the basal production of MMP-2 (Fig.8 A).
The present study has demonstrated that ionizing radiation enhances the production of MMP-2 but not of MMP-9 in human lung epithelial cells and that this enhancement is mainly mediated by a p53 transcription factor. Corticosteroid markedly inhibited MMP-2 expression.
MMPs are a family of extracellular matrix-degrading enzymes associated with numerous physiological and pathological events such as malignant tumor cell invasion and inflammatory processes (21). Particularly, gelatinases such as MMP-2 and MMP-9 play a crucial role in remodeling of the basement membrane in various lung diseases because these enzymes are capable of degrading type IV collagen, which is one of the major constituents of the basement membrane (19,20). And alteration of the basement membrane has various pathological effects on the progression of lung diseases. A recent study (34) with bovine pulmonary microvascular endothelial cells has shown that MMP-9 induced by tumor necrosis factor-α contributes to the increase in lung permeability through a modification of extracellular matrix components. Delclaux et al. (9) have shown that MMP-2 in ELF is a more sensitive and specific index of ARDS or at least of alveolar injury than MMP-9 in ELF. In an experiment of bleomycin-induced lung injury in rats, Bakowska and Adamson (4) have demonstrated that an intense band at 72 kDa, consistent with MMP-2, was observed in bronchoalveolar lavage fluids over a 6-wk period after bleomycin treatment as determined by gelatin zymography, whereas a faint band at 92 kDa, consistent with MMP-9 in bronchoalveolar lavage fluids, was observed only 1 wk after treatment. And recently, Lemjabbar et al. (25) have reported that patients with idiopathic pulmonary fibrosis had a higher activity of MMP-2 in ELF compared with that of MMP-9. These results indicate that MMP-2 rather than MMP-9 is a major contributor to the alteration of alveolar structure in lung injury due to various insults.
In the lung, two gelatinases, MMP-2 and MMP-9, are known to be produced by a variety of cells in vitro. MMP-2 is preferentially secreted from fibroblasts and various epithelial cells including airway epithelial cells, and MMP-9 is preferentially expressed by inflammatory cells (13). We have shown that NHBE and A549 cells have obvious gelatinolytic activity corresponding to MMP-2 in contrast to very little activity corresponding to MMP-9. Pardo et al. (33) have reported that the MMP-2 mRNA transcript is highly expressed in type II alveolar epithelial cells as well as in alveolar macrophages in rat lungs exposed to hyperoxia. Hayashi et al. (17) have localized by confocal microscopy the presence of both gelatinases and also of TIMP-1 and TIMP-2 in the lungs from patients with diffuse alveolar damage and idiopathic pulmonary fibrosis. Immunoreactive sites were found particularly in alveolar epithelial cells and were colocalized with a disrupted basement membrane, indicating that gelatinases play an important role in the derangement of alveolar structure in these diseases. It has also been reported in an immunohistochemical study of the lung tissues of patients with bronchiolitis obliterans organizing pneumonia (11) that MMP-2 was detected in the regenerated epithelial cells covering intra-alveolar fibrosis. These studies indicate that type II pneumocytes are one of the major players in the pathophysiology of basement membrane disruption in various pulmonary disorders.
In the present investigation, we studied the acute effect of ionizing radiation on the expression of MMP-2 in human airway epithelial cells. After ionizing radiation, the levels of MMP-2 mRNA peaked at 12 h, whereas the expression of MMP-9 did not change (data not shown). In radiation-induced injury, after the acute phase of shedding of epithelial cells, the remaining viable epithelial cells would dedifferentiate, spread, and migrate over the denuded basement membrane to cover the deepithelialized zone. Using an in vitro model of respiratory epithelium wound repair, Legrand et al. (24) have demonstrated that MMP-9 is actively expressed by migrating human bronchial epithelial cells during wound repair and that the wound repair process depends on the activity of this MMP. In an in vivo porcine wound healing model, it has been reported that MMP-9 activity is linked to the reepithelialization process (28). Our findings therefore suggest that MMP-2 generated by airway epithelial cells may be, at least in part, involved in the early phase after irradiation.
One of the primary responses of the lung to ionizing radiation is the increase in pulmonary epithelial permeability. Potential mechanisms for the increase in permeability might include desquamation of the epithelial cells, cytolytic injury of the cells, increases in transcellular transport, and increases in paracellular transport. In this study, it is unlikely that the increased permeability was the result of the cytolytic action of ionizing radiation because there is no evidence of cell death, including apoptosis, as measured by trypan blue dye exclusion and cell cycle DNA analysis. Although it is probable that ionizing radiation leads to increases in transcellular and paracellular permeability of the epithelial cell layer and desquamation of the epithelial cells, degradation of the basement membrane by gelatinases may also contribute to the increase in pulmonary epithelial permeability. It has been reported that alveolar instillation of TIMP-2, a potent inhibitor of MMP-2, has significantly reduced lung permeability in a rat model of acute lung injury (30). Delclaux et al. (9) demonstrated a correlation between ELF albumin and the sum of activated gelatinases in the ELF from patients with ARDS. These findings suggest that MMP-2 may be, at least in part, involved in lung permeability after irradiation.
MMP-2 is secreted as a 72-kDa latent proenzyme, and this protein, in turn, is proteolytically processed to the active 64- and 62-kDa forms by MT1-MMP, the action of which is tightly regulated by the level of TIMP-2 (14, 23). Although treatment of human airway epithelial cells with ionizing radiation resulted in enhancement of 72-kDa proMMP-2 secretion, the active forms of MMP-2 were not detected in our study (Fig. 2). We were not able to detect transcripts of the MT1-MMP gene after irradiation in A549 cells with RT-PCR (data not shown). In line with our results, D′Ortho et al. have shown that A549 cells under various conditions expressed only 72-kDa proMMP-2 (10). It therefore seems likely that airway epithelial cells may have very little activity with which to process the latent form of MMP-2 to the active form, presumably due to very little expression of MT1-MMP. Because fibroblasts have been reported to express MT1-MMP (38), it is probable that the latent form of MMP-2 secreted from airway epithelial cells may be activated by neighboring stromal cells such as fibroblasts in vivo.
Our results indicate that ionizing radiation induces the enhancement of MMP-2 production in NHBE and A549 cells. It has been reported that calcium influx, vitronectin, cAMP, and type I insulin-like growth factor modulate the expression of MMP-2 (3, 22, 26, 44). Although Sawaya et al. (39) have recently demonstrated that ionizing radiation enhanced MMP-2 expression in rat astrocytes, this report is the first investigation to clearly show that ionizing radiation enhances the expression of MMP-2 in lung epithelial cells. Ionizing radiation damages genomic DNA, and the DNA strand breaks induce accumulation of p53 protein (8, 41). Recently, it has been reported that the 5′-flanking region of the MMP-2 gene contains a perfect p53 consensus binding sequence and that the binding of p53 to that site upregulates the expression of the MMP-2 gene (5). Considering the above results, in this study, ionizing radiation appears to enhance the production of MMP-2 via increased transcription of the MMP-2 gene through the binding of the p53 transcription factor to the promoter.
Furthermore, we examined the effect of the p53 antisense oligonucleotide on the production of MMP-2 in A549 cells. The p53 antisense oligonucleotide dramatically abrogated the enhanced production of MMP-2 induced by ionizing radiation. This result also supports the notion that ionizing radiation enhances the production of MMP-2 through the accumulation of p53 protein.
In contrast to MMP-2, the transcript levels of TIMP-2 mRNA in human lung epithelial cells did not change on exposure to ionizing radiation. Irradiation is therefore likely to shift the MMP-2 protease-TIMP-2 protease inhibitor balance in favor of the protease. Together with the fact that various polarized cells preferentially secrete MMP-2 toward the basal pole of the cells (10, 47), MMP-2 secreted from lung epithelial cells seems to be increased in the immediate vicinity of its basement membrane substrates in the lungs of patients with radiation pneumonitis.
Dex markedly suppressed the production of MMP-2 in A549 cells. It has been reported that, in addition to the consensus binding sequence for p53, the 5′-flanking region of the human MMP-2 gene contains putative binding sites for a variety of transcription factors including activator protein-2, cAMP response element binding protein (CREB), and Ets-1 (5). Glucocorticoids are known to be capable of modulating the production of some transcription factors such as Ets-1 (46). And ligand-bound glucocorticoid receptors are able to control the transcriptional activity of several transcription factors such as c-Jun through protein-protein interaction (42). As for p53, recently, it was reported (2, 27,35) that activated glucocorticoid receptors suppressed the transactivation function of p53 through direct interaction or by means of p300/CREB binding protein. These results raise the possibility that Dex may inhibit the expression of the MMP-2 gene at the transcriptional level.
In conclusion, we demonstrated in this study that ionizing radiation enhances the expression of MMP-2 in human lung epithelial cells. In addition, our data indicate that Dex is potent in inhibiting MMP-2 activity of the cells, suggesting that glucocorticoids would be beneficial for the treatment of radiation pneumonitis. Taken together, the results of the present investigation support the hypothesis that MMP-2 produced by lung epithelial cells may be deeply involved in acute lung injury due to radiation exposure.
Address for reprint requests and other correspondence: M. Maruyama, The First Dept. of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical Univ., 2630 Sugitani, Toyama 930-0194, Japan (E-mail:).
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