Introduction

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Pulmonary fibrosis is a progressive disorder characterized by the loss of alveolar architecture through the apoptosis of epithelial and endothelial cells, proliferation of myofibroblasts (fibroblasts expressing -smooth muscle actin), and extensive deposition of extracellular matrix proteins, especially collagens type I and III (1, 2). Despite the identification of many of the changes that occur during pulmonary fibrosis, the mechanism of this pathology is not completely understood.

Growing evidence suggests that the protease angiotensin converting enzyme (ACE) and one of its proteolytic products, angiotensin II (Ang II), may be dysregulated in fibrosis. The normal physiologic role of ACE is maintenance of blood pressure homeostasis by converting Ang I to Ang II, a potent vasoconstrictor, and proteolytically inactivating bradykinin, a vasodilator. ACE controls the key regulatory step in Ang II formation, and the critical site for this formation appears to be locally at the endothelial cell surface, rather than in the blood, despite the occurrence of a circulating form of ACE (3). Although the normal activity of Ang II is vasoconstriction, recent studies suggest that in some cases Ang II directly induces the growth of fibroblasts and smooth muscle cells, and causes apoptosis in epithelial and endothelial cells (4). Ang II also regulates cytokines and growth factors, potentially further affecting the balance of fibroblast/smooth muscle cell versus epithelial/endothelial cell growth. Ang II additionally upregulates the expression of the cytokine transforming growth factor (TGF)-1, which exerts mitogenic signaling and phenotypic alterations in fibroblasts (6). Both Ang II and TGF-1 downregulate factors known to sustain endothelial/epithelial cell growth, such as hepatocyte and keratinocyte growth factors (11). Together, these changes in the expression of growth factors and cytokines may contribute to the development and progression of fibrosis.

ACE activity has been shown to be increased in fibrotic heart tissue and in the bronchoalveolar lavage (BAL) fluid of animals with bleomycin-induced pulmonary fibrosis (17, 18). Studies of fibrotic tissue in culture show that ACE inhibitors slow myofibroblast growth, possibly via the reduction of Ang II production (18, 19). ACE inhibitors, such as captopril and lisinopril, attenuate fibrosis in the heart, kidney, and lung, both by reducing fibroblast proliferation and by preventing apoptosis of epithelial and endothelial cells (7, 8, 11, 19, 20).

ACE expression is regulated by a number of biologic and pharmacologic agents. Endothelial cells express ACE in a confluence-dependent manner; preconfluent cells express low ACE, but expression increases after confluence, reaching a maximal level at 4 to 6 d postconfluence (21). This expression is independent of serum (21). ACE in confluent cells can be further upregulated by basic fibroblast growth factor, atrial natriuretic peptide, phorbol 12-myristate 13-acetate (PMA), losartan (an Ang II receptor antagonist), and by several ionophores (22). Steroids, including dexamethasone, hydrocortisone, aldosterone, and cortisosterone-21- acetate also stimulate ACE production (27). ACE mRNA and protein levels are reduced by estrogen and the RU38486 steroid receptor antagonist, and also by Ang II via its receptor in a negative feedback loop (25, 27, 28). With the exception of PMA, the mechanisms by which these agents regulate ACE are unknown. Phorbol ester-induced gene expression of ACE may be regulated by the activation of the early growth response 1 (Egr-1) transcription factor downstream of protein kinase C (24). Neither the extracellular signal nor the specific mechanism that increases ACE expression in fibrotic tissue is known.

The chemotherapeutic agent bleomycin induces pulmonary fibrosis in both humans and animals (29, 30). Bleomycin has been used extensively to identify and study alterations in cellular composition and protein expression (including ACE) that occur during pulmonary fibrosis in animals (2, 18, 29). In the present study, we show that bleomycin directly increases both ACE enzymatic activity and mRNA in primary bovine pulmonary artery endothelial cells (BPAEC). Bleomycin increases expression of ACE mRNA by causing the nuclear translocation and increased DNA binding activity of the Egr-1 transcription factor. Bleomycin also activates the p42/p44 mitogen activated protein kinase (MAPK). Activation of Egr-1 by bleomycin is blocked by the MAPK kinase1/2 (MEK1/2) inhibitor U0126, suggesting that Egr-1 activation is dependent on the MAPK pathway.

	Materials and Methods

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Reagents

Bleomycin, Blenoxane, was from Mead Johnson, Princeton, NJ. The MEK1/2 inhibitor U0126 was purchased from New England Biolabs, Beverly, MA. Other reagents are described below.

Cell Culture and Bleomycin Treatment

BPAECs were obtained from freshly slaughtered calves as described previously (27). Passage 3-8 cells were used for all experiments. BPAECs were cultured in RPMI 1640 with antibiotics (penicillin and streptomycin), fungisone, and 10% fetal bovine serum (FBS); cells were grown in 5% CO₂ at 37°C in a humidified atmosphere. For treatment with bleomycin, postconfluent BPAEC were switched to RPMI containing 0.2% FBS. Bleomycin, freshly dissolved in 0.9% NaCl, was added to the culture medium at final concentrations of 0.1-1.0 µg/ml.

ACE Hippuryl-L-Histidyl-L-Leucine Assay

BPAEC were washed twice in phosphate-buffered saline (PBS) to remove dead cells, and then scraped into PBS and sonicated (3 times for 10 s on ice) to extract cellular protein. ACE catalytic activity was determined by a fluorimetric assay with the synthetic substrate hippuryl-L-histidyl-L-leucine (HHL) as described by Friedland and Silverstein (32). Specificity of substrate hydrolysis was confirmed by complete inhibition of ACE activity by lisinopril, a specific inhibitor of ACE. Protein concentrations were determined according to Lowry and coworkers (33).

Quantitative Polymerase Chain Reaction

Total cell RNA was obtained from cultured cells using RNAzol-B (Tel-Test Inc., Friendswood, TX). RNA concentrations were determined spectrophotometrically at 260 nm. One microgram of RNA from each sample was reverse transcribed for 1 h at 37°C, using 200 U Moloney murine leukemia virus reverse transcriptase (MMLV-RT; Life Technologies, Grand Island, NY) in 10 µL of reaction buffer containing 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 6 mM MgCl₂, 1 mM dithiothreitol (DTT), 660 pmol oligo-(dT)_12-18, 0.5 mM each deoxynucleoside triphosphate (dNTP), and 1 U RNase inhibitor. Samples were heated to 95°C for 5 min to inactivate the MMLV-RT and stored at 40°C. Competitive quantitative polymerase chain reaction (PCR) for ACE mRNA was performed as described previously (34, 35). Briefly, cDNA was used in a PCR reaction buffer (120 mM Tris-HCl, 400 mM KCl, 12 mM MgCl₂, 0.08% gelatin, pH 8.5) with 0.4 µM each forward and reverse primer, 200 µM each dNTP, and 1 U/50 µL Taq DNA polymerase. PCR was performed for 25-35 cycles with 30 s, 94°C denaturation; 1 min, 60°C annealing; and 1 min, 72°C extension. The last cycle extension was for 10 min. A deletion mutant internal standard was used to normalize for differences in PCR efficiency. ACE PCR results were normalized to PCR results of tubulin, also determined using a mutant internal standard (34, 35).

Cell Transfection

BPAECs grown in 12-well plates to 60-80% confluence were transfected with promoter-reporter constructs by Lipofectamine (Life Technologies). Cells in serum-free RPMI were transfected with 500 ng construct DNA and Lipofectamine for 5 h. Transfection mixtures also contained 83 ng of the renilla luciferase vector, pRL-TK (Promega, Madison, WI), to normalize transfection efficiency. After 16 h, medium was switched to RPMI with 0.2% FBS and cells were treated with bleomycin for up to 96 h.

Plasmids

The 5'-flanking region of the ACE gene was obtained by PCR amplification of human genomic DNA and cloned into the pGL3 luciferase reporter vector (Promega). PCR was performed in a buffer (60 mM Tris-sulfate, 19 mM NH₄SO₄, 2 mM MgSO₄, pH 8.9) containing 200 µM each dNTP, 1 M betaine, 10 µM each primer, 0.04 U/µl Platinum Taq DNA polymerase High Fidelity (GIBCO BRL, Grand Island, NY), and a 150-fold dilution of human genomic DNA (cat #p1600, lot #cos108; Oncor, Gaithersburg, MD). The primers were 5'-cccgggaggtaccgaCAACCCACCGT GTTCTTTGACATC-3' (forward) and 5'-tgacgtaagcttCAGCAG CAACAGCAGCGGCAG-3' (reverse), with upper case bases corresponding to ACE gene sequences and lower case bases indicating nonhomologous 5' extensions with restriction enzyme sites (bold) for KpnI and HindIII, respectively, to allow for directional cloning. These primers amplified a 5576-bp amplicon encompassing a region from -5,461 to +88 of exon-1 of the ACE gene (the pGL3 vector containing this amplicon is referred to as pACE5461GLKa). After PCR, the amplicon was sequenced on both strands by dideoxy cycle sequencing and the sequence was submitted to the GenBank Database (GenBank Accession No. AF229986). Samples were cycled as follows: 95°C, 1 min; then 40 cycles with 95°C, 0.5 min denaturation, 68°C, 5 min combined annealing/extension; with a final extension at 68°C for 8 min. Three shorter ACE promoter constructs were also prepared from pACE5461GLKa: (1) pACE4742GLKa, extending from -4742 to +88 of the ACE gene; (2) pACE2066GLKa, extending from -2066 to +88; and (3) pACE97GLKa, extending from -97 to +88. These additional constructs were prepared using the reverse primer above with the following forward primers: (1) 5'-cccgggaggtac cgaAAAGAAGAGGCTGGGAGCGGT-3'; (2) 5'-cccgggaggtac cgaAGTGGAGGCGGAGGCTGTTTA-3'; and (3) 5'-cccgggag gtaccgaGACTTTGGAGCGGAGGAGGAA-3'. The reaction buffers and cycles were the same as above.

Dual Luciferase Assay

Transfected BPAECs were washed twice with PBS, lysed with passive lysis buffer (Promega), and assayed for firefly and renilla luciferase activities by the Dual Luciferase Assay (Promega) according to the manufacturer's instructions, in a Turner TD-20/20 luminometer.

Western Blot Analysis

Western blots were performed as previously described (36). To prepare lysates, cells were washed in PBS and solubilized with 50 mM Hepes solution (pH 7.4) containing 1% (v/v) Triton X-100, 4 mM ethylenediaminetetraacetic acid (EDTA), 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, 2 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, and 10 µg/ml aprotinin. Lysate was cleared by centrifugation at 4°C for 15 min. Nuclear extracts for Western blots were prepared according to Slomiany and colleagues (37). Protein concentrations in the supernatant were determined as above (33).

Cell lysates or nuclear extracts (10 µg protein) were electrophoresed through reducing (5% -mercaptoethanol) SDS polyacrylamide:bis gels (10%) and electroblotted onto nitrocellulose membranes. After the transfer, membranes were blocked in 5% BSA in PBS and blotted with the Tween + Tris buffered saline (TBST, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween 20) with 0.5% BSA and antibodies at concentrations as recommended by the manufacturers. Antibodies were: phospho-specific p44/42 MAPK (New England Biolabs); p44/42 MAPK, and Egr-1 (Santa Cruz Biotechnology, Santa Cruz, CA). Levels of proteins and phosphoproteins were detected with horseradish peroxidase-linked secondary antibodies and ECL System (Amersham Life Science, Arlington Heights, IL). All Western blots were repeated at least three times.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA) was performed as described by Garner and Revzin (38). To prepare nuclear extracts, cells were washed in PBS and incubated in 10 mM Hepes (pH 7.8), 10 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 0.1 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM NaF, 0.1 mM sodium orthovanadate, and 1 mM tetrasodium pyrophosphate for 15 min at 4°C. IGEPAL CA-630 (Sigma) was then added at a final concentration of 0.6% (v/v). Samples were vortexed and centrifuged. Pelleted nuclei were resuspended in extraction buffer (50 mM Hepes [pH 7.8], 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 0.1 mM PMSF, 5 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM tetrasodium pyrophosphate, and 1% [v/v] glycerol), then mixed vigorously for 20 min and centrifuged for 5 min. The supernatants were harvested, and protein concentrations determined (33). To perform EMSA, binding reaction mixtures containing 2 µg protein of nuclear extract, 1 µg poly(dI-dC) poly(dI-dC) and ³²P-labeled double stranded oligonucleotide probe containing consensus Egr-1 sequence (Santa Cruz Biotechnology) in 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% (v/v) glycerol, and 20 mM Tris-HCl (pH 7.5) were incubated for 20 min at 25°C. Electrophoresis of samples through a native 6% polyacrylamide gel (acrylamide:bis, 29:1) was followed by autoradiography. Supershift experiments were performed by incubating 2 µg Egr-1 antibody (Santa Cruz Biotechnology) in the binding reaction mixture for 1 h at 4°C before the addition of the ³²P-labeled oligonucleotide probe to start the binding reaction. All experiments were repeated at least three times.

Statistics

Statistical comparisons were performed using a Student's t test for unpaired samples and a two-way ANOVA for multiple comparisons. Statistical significance was determined P < 0.05.

	Results

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Bleomycin Regulates ACE Expression Through the ACE Promoter

Treatment of BPAECs with bleomycin resulted in the upregulation of cell-associated ACE activity in a dose- and time-dependent manner (Figure 1). Increased ACE activity could be consistently detected with the addition of 0.1 µg/ml bleomycin; the increase in ACE expression was statistically significant with the addition of 1.0 µg/ml bleomycin for 72 h. These concentrations of bleomycin were selected to minimize the level of apoptosis induced in postconfluent BPAECs at low serum concentrations (RPMI, 0.2% FBS). Apoptosis in postconfluent BPAEC treated with 1.0 µg/ml bleomycin was 15-25% after 24-72 h, determined by propidium iodide staining and fluorescent cell sorting analysis (data not shown). The increase in ACE activity induced by bleomycin was not restricted to pulmonary endothelial cells; experiments in human umbilical vein endothelial cells (HUVECs) also showed a dose- and time-dependent induction in response to bleomycin (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 1.   Bleomycin upregulation of ACE enzyme activity in BPAECs. Two days postconfluent BPAECs in RPMI medium with 0.2% FBS were treated with 0 (control), 0.1, or 1.0 µg/ml bleomycin for the indicated times. Cell lysates were assayed for ACE activity. Results were normalized to ACE levels in untreated cells at the 24 h time point. Because basal levels of ACE increase in endothelial cells as a function of time after confluence, each time point of cells treated with bleomycin must be compared with untreated cells from the same time point. Values represent mean fold increases ± SEM, n = 5. * Indicates significantly different from control at 72 h, P < 0.05.

The upregulation of ACE activity in BPAEC was preceded by a time-dependent increase in ACE mRNA, at 2 and 4 h, as measured by competitive quantitative RT-PCR. Figure 2 shows the time course of increased ACE mRNA with 1.0 µg/ml bleomycin treatment in a representative experiment with an internal standard for ACE PCR. Increased ACE mRNA was maintained for at least 24 h (data not shown). The delay between the observed upregulation of the mRNA and increased ACE activity is possibly due to the requirement for ACE export to the cell surface and subsequent activation, which may be limiting.

View larger version (36K): [in this window] [in a new window]   Figure 2.   Bleomycin upregulates ACE mRNA expression in BPAECs. Two days postconfluent BPAEC in RPMI, 0.2% FBS were treated with 1.0 µg/ml bleomycin for the indicated times, followed by RNA isolation. Equal RNA amounts from each sample were used for competitive, quantitative RT-PCR for both ACE and tubulin. (A) Agarose gel of bleomycin-induced of native ACE mRNA relative to an internal standard for ACE. (B) Densitometry of ACE mRNA ratio to the internal standard, normalized to the ratio of tubulin mRNA to its internal standard. Values represent means ± SEM (n = 5). * Indicates significantly different from the control value at t = 0, P < 0.05.

To determine whether the upregulation of ACE mRNA was mediated through the ACE promoter, we examined the effect of bleomycin on a luciferase reporter gene downstream of three different lengths of the ACE promoter: 4742 base pair (bp), 2066 bp, and 97 bp (Figure 3A). Preconfluent BPAEC were transfected for 16 h and the medium was then changed to RPMI, 0.2% FBS before the addition of bleomycin. The preconfluent transfected cells were more susceptible to apoptosis induced by long exposures to bleomycin, so for these experiments, we used a lower dose of bleomycin (0.1 µg/ml instead of 1.0 µg/ml). We found that treatment of cells with bleomycin for 96 h increased the level of luciferase 4- to 10-fold in all three promoter constructs (Figure 3B). The 97-bp core promoter showed the highest increase in response to bleomycin treatment, suggesting that a cis element(s) within this region is/are able to bind transcription factor(s) activated by bleomycin. A shorter time course with the core 97-bp promoter showed that bleomycin induced a twofold increase in luciferase reporter within 6 h of treatment (Figure 3C). These results indicate both immediate and sustained increases of ACE are induced through the ACE promoter.

View larger version (28K): [in this window] [in a new window]   Figure 3.   Bleomycin upregulates expression from the luciferase reporter gene downstream of the ACE promoter. (A) Three constructs of the luciferase reporter gene were made using ACE promoter segments from 4742 bp, 2066 bp, or 97 bp to +88 bp. (B) 60-80% preconfluent BPAEC were transfected with the ACE-luciferase constructs; the renilla reporter gene was cotransfected to normalize for transfection efficiency. 16 h later, cells were placed in RPMI, 0.2% FBS and were treated with ± 0.1 µg/ml bleomycin for 96 h. Values represent means ± SD (n = 6). (C) BPAECs were transfected with the pACE97 luciferase construct and treated with 0.1 µg/ ml bleomycin for 6 h. Values represent means ± SD (n = 6). * Indicates significantly different from the control value, P < 0.05.

Bleomycin Activates the Egr-1 Transcription Factor

Although a number of studies have shown that bleomycin is involved in the regulation of gene expression, the mechanism(s) of the regulation is unknown (2, 18, 29). The core 97-bp ACE promoter contains a number of binding sites for transcription factors, including consensus sequences for AP2, ets-1, Sp1 and Egr-1 sites (Figure 4A). Only Egr-1, a zinc finger transcription factor, has been shown to positively regulate ACE expression in response to PMA (23). We wished to determine whether this pathway was also regulated by bleomycin. BPAECs were placed in serum- free medium for 48 h to reduce background transcription factor activity, and cells were then treated with 1.0 µg/ml bleomycin. Nuclear extracts were purified and analyzed by immunoblotting. Western blots showed that untreated control nuclei contained a low amount of Egr-1, which was increased by bleomycin (Figure 4B). Nuclear Egr-1 levels were increased within 30 min and maintained for at least 6 h (data not shown).

View larger version (37K): [in this window] [in a new window]   Figure 4.   Bleomycin upregulates the nuclear translocation and DNA binding activity of the Egr-1 transcription factor. (A) ACE core promoter sequence, indicating the positions and directionality of the predicted binding sites for known transcription factors. (B) BPAECs were serum-starved for 48 h before treatment for the indicated times with 1.0 µg/ml bleomycin. Nuclear extracts were purified and equal amounts of protein were used in Western blot analysis for Egr-1 protein. (C) Confluent BPAEC were serum-starved for 48 h before treated with 1.0 µg/ml bleomycin for 24 h. Nuclear extracts were purified for EMSA with an oligonucleotide containing the Egr-1 consensus sequence. The position of Egr-1 (indicated) was confirmed by a supershift assay with an antibody specific for Egr-1. Experiments were performed three times.

EMSA experiments on BPAEC nuclear extracts from cells treated ± bleomycin for 24 h showed that bleomycin induced DNA binding activity toward the oligonucleotide containing an Egr-1 consensus binding site (Figure 4C). A supershift experiment with an Egr-1-specific antibody was used to confirm the identity of the factor as Egr-1. These results indicate that bleomycin induced the nuclear import and DNA binding activity of Egr-1.

Bleomycin Activation of the Egr-1 Transcription Factor Is Dependent upon Activation of the MAPK Pathway

The Egr-1 transcription factor is activated by a variety of growth factors via the MAPK pathway (39). We therefore examined the ability of bleomycin to activate MAPK in primary endothelial cells. Previous studies have shown a correlation between activation of MAPK and the level of MAPK phosphorylation, as detected by specific anti-phospho-MAPK antibodies (36). BPAEC were placed in serum-free RPMI medium for 48 h to reduce endogenous levels of MAPK activity, and cells were treated with 1.0 µg/ml bleomycin. Western blot analysis of total cell lysates showed that bleomycin treatment led to the phosphorylation of MAPK p44 and p42 within 10 min, which was sustained for at least 3 h (Figure 5A). Blots of total MAPK protein showed equal levels in all samples (Figure 5B). MAPK phosphorylation was also induced by 0.1 µg/ml bleomycin within 10 min (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 5.   Bleomycin induces MAPK activity in BPAECs. BPAECs were serum-starved for 48 h before treatment for the indicated times with 1.0 µg/ml bleomycin. Hepatocyte growth factor (HGF) activates MAPK in endothelial cells and was used as a positive control. Whole-cell lysates were normalized for protein concentration and used in Western blots for phospho-MAPK (A) or total MAPK (B). Data show representative blots of three sets of experiments.

We next determined whether bleomycin-induced nuclear translocation and activation of Egr-1 was dependent upon MAPK. BPAEC were placed in serum-free medium for 48 h; cells were pretreated with the MEK1/2 inhibitor U0126 (30 µM) for 60 min to block upstream activation of MAPK before treatment with 1.0 µg/ml bleomycin. Western blotting for phosphorylated MAPK in cell lysates showed that this concentration of U0126 completely blocked bleomycin mediated phosphorylation of MAPK (Figure 6A); the endogenous level of phosphorylated MAPK was also reduced. A blot of total MAPK showed that levels of MAPK protein are unchanged (data not shown). A blot for nuclear levels of Egr-1 showed that bleomycin was unable to induce nuclear translocation in the presence of the MEK1/2 inhibitor (Figure 6B). Band shift analysis using nuclear extracts of cells pretreated with U0126 showed that bleomycin-induced Egr-1 DNA-binding activity was also blocked (Figure 6C). Together, these data show that Egr-1 activation by bleomycin is dependent upon activation of the MAPK pathway.

View larger version (40K): [in this window] [in a new window]   Figure 6.   Bleomycin activation and upregulation of Egr-1 is dependent upon MAPK activation. BPAEC were placed in serum-free medium for 48 h. Cells were pretreated ± 30 µM U0126 (MEK1/2 inhibitor) for 1 h and then treated with 1.0 µg/ml bleomycin for the indicated times. (A) Equal amounts of protein from whole-cell lysates were blotted for phospho-MAPK. (B) Nuclear extracts were purified from cells and normalized for protein concentrations; equal amounts of protein were Western blotted for Egr-1 protein. (C) Nuclear extracts from treated cells were used in EMSA with an oligonucleotide containing the Egr-1 consensus sequence. Arrow indicates the position of Egr-1.

	Discussion

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The bleomycin model for pulmonary fibrosis has been used extensively to study cellular remodeling as well as changes in protein expression which occur during the progression of the fibrosis. TGF-1, one of the proteins believed to have a causative function in pulmonary fibrosis, has altered expression both in animals and cultured cells treated with bleomycin (2, 30, 31). Although a number of proteins, including TGF-1, have been shown to change expression levels in response to bleomycin (2, 18, 20, 29), the mechanism for the induced changes of these genes has not been determined. We show in this study that bleomycin induces the expression of ACE in primary culture of pulmonary artery endothelial cells, and that the upregulation of ACE expression by bleomycin occurs via the activation of the Egr-1 transcription factor and the MAPK pathway.

By examining the effect of 500 µg/ml metal-free bleomycin on ACE enzymatic activity, a previous study showed that bleomycin inhibited ACE activity of pulmonary artery endothelial cells in culture (40). This effect was ascribed to the chelation of zinc divalent cations by the metal-free bleomycin from the ACE catalytic site, resulting in the inactivation of the ACE (40). In contrast, our study, which shows increased ACE activity, examined the regulation of ACE gene expression by 0.1-1.0 µg/ml metal-bound bleomycin, the form normally used therapeutically.

Although much research has focused on the function of TGF-1 in pulmonary fibrosis, increasing evidence suggests that tissue ACE, through the formation of Ang II, also participates in the progression of fibrosis. Ang II is a potent growth factor for fibroblasts and smooth muscle cells, and induces apoptosis in epithelial and endothelial cells (4). Ang II increases TGF-1 expression (6), and both Ang II and TGF-1 downregulate factors known to sustain endothelial/epithelial cell growth, such as hepatocyte and keratinocyte growth factors (11). ACE activity is increased in fibrotic heart tissue and in the BAL fluid of animals with experimental bleomycin-induced pulmonary fibrosis (17, 18). Furthermore, ACE inhibitors, such as captopril and lisinopril, attenuate fibrosis in the heart, kidney, and lung, both by reducing fibroblast proliferation and by preventing apoptosis of epithelial and endothelial cells (7, 8, 11, 19, 20).

ACE expression is known to be regulated by a number of growth factors and pharmacologic agents (22), but the mechanism of ACE regulation is not well understood. The ACE promoter contains a number of consensus transcription factor binding sites, including Egr-1, Sp1, ets-1, and AP2. Our results show that increased ACE expression in response to bleomycin correlates with the nuclear translocation and activation of Egr-1. Our results are in agreement with previous work which showed that the regulation of ACE by PMA occurs through the Egr-1 transcription factor (24). At this time, the actions of the SP1, ets-1, and AP2 transcription factors on ACE expression are unknown.

In summary, our results demonstrate that treatment of BPAEC with clinically relevant concentrations of bleomycin stimulates gene expression of ACE, an important mediator of lung fibrosis. A putative regulator of ACE gene transcription, Egr-1, was found to be activated by bleomycin. Furthermore, we showed that bleomycin signaling is dependent on the MEK1/2-MAPK pathway. We propose that this signal transduction cascade (bleomycin  MEK1/2  MAPK  Egr-1, leading to ACE gene expression) plays an essential role in the pathogenesis of pulmonary fibrosis.