Introduction

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Iron is an essential element for all forms of life. A wide variety of metabolic processes are dependent upon this metal. Iron and iron metabolism are also inextricably linked to a variety of pathophysiologic events, including inflammation and bacterial infections (1).

Intracellular iron levels modulate gene expression by transcriptional and post-transcriptional mechanisms. This has been elegantly demonstrated in the regulation of proteins directly involved in iron homeostasis, transferrin receptor and ferritin. Under iron-limited conditions, cytoplasmic aconitase is converted to an RNA-binding protein (iron regulatory protein 1) that binds to specific RNA sequences called iron-responsive elements (IREs). Binding of this protein to IREs in the 3' untranslated region of the transferrin receptor messenger RNA (mRNA) stabilizes the transcript, resulting in increased expression of the receptor; whereas binding of the protein to an IRE in the 5' untranslated region of ferritin mRNA suppresses translation of this intracellular iron storage protein (2). The net effect of these changes in the expression of transferrin receptor and ferritin is to increase intracellular iron availability.

More recently, it has become clear that iron metabolism is intimately linked with other important metabolic pathways. For example, nitric oxide (NO), as well as other reactive oxygen species, triggers the conversion of aconitase from the cytoplasmic enzyme form to the RNA-binding form of the molecule (3, 4). Of note: hyperoxia inactivates aconitase activity in A549 human pulmonary epithelial cells and in rat lungs. Feedback regulatory mechanisms exist between iron metabolism and the NO/NO synthase pathway; i.e., intracellular iron levels modulate transcription and activity of inducible NO synthase (5). Potential interactions with other metabolic pathways have not been fully explored.

The plasmin/plasminogen activator (PA) system is a key component in tissue repair processes, including the extensive remodeling process that follows acute lung injury (6). Alveolar epithelial cells play a central role in this process (7, 8). We and others have previously demonstrated that rat alveolar epithelial cells (9) and A549 human carcinoma-derived pulmonary epithelial cells express urokinase-type PA (u-PA), u-PA receptor (u-PAR), and PA inhibitor-type 1 (PAI-1) (12, 13) and therefore may participate directly in the repair process. Further, interleukin (IL)-1, an important inflammatory mediator in acute lung injury, upregulates u-PA and u-PAR expression, resulting in increased cell-surface plasmin generation (12, 13).

Reports of acute lung injury in a subset of iron-overloaded individuals treated with the iron chelator deferoxamine (14, 15) spurred us to explore the relationship between intracellular iron levels and expression of the PA/ plasmin system. Herein we report that decreased intracellular iron availability markedly suppresses cell-surface plasmin generation by A549 human carcinoma-derived pulmonary epithelial cells. Further, this effect is due to transcriptional and post-transcriptional effects on u-PA and PAI-1 expression.

	Materials and Methods

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Materials

Tissue-culture plasticware was obtained from Costar (Cambridge, MA); fetal calf serum (FCS) from Life Technologies, Inc. (Grand Island, NY); actinomycin D, antifoam A, bovine serum albumin, cycloheximide, deferoxamine mesylate (DEF), dextran sulfate, dithiothreitol (DTT), L-glutamine, ethylenediaminetetraacetic acid (EDTA), Ficoll, Ham's F-12 medium, glycerol, N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes), N-lauroyl sarcosine, 3-(N-morpholino)propanesulfonic acid, Nonidet P-40 (NP-40), penicillin-streptomycin-amphotericin B, phorbol myristate acetate (PMA), plasmin salmon sperm DNA, sodium acetate, tranexamic acid, and Triton X-100 from Sigma Chemical Co. (St. Louis, MO); H-D-Val-Leu-Lys-p-nitroanilide (S2251) and plasminogen from Pharmacia Upjohn (Franklin, OH); n-acetyl cysteine from Dey Laboratories (Napa, CA); agarose, mercaptoethanol, and sodium dodecyl sulfate (SDS) from Bio-Rad Laboratories (Richmond, CA); Sep-Pak C-18 column from Waters Division of Millipore (Milford, MA); IL-1 from Genzyme Corp. (Cambridge, MA); formamide, guanidine thiocyanate, glycogen, nucleotides, phenol, poly(dI-dC), proteinase K, Sephadex columns, ribonuclease (RNase)-free deoxyribonuclease (DNase), random-primed DNA labeling kit, adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), T4 polynucleotide kinase, and torula transfer RNA (tRNA) from Boehringer-Mannheim Biochemicals (Indianapolis, IN); chloroform, ferric chloride (Fe³⁺), and formaldehyde from Mallinckrodt Specialty Chemicals Co. (Paris, KY); [-³²P]deoxycytidine triphosphate (dCTP) (3,000 Ci/mmol), [-³²P]deoxyuridine triphosphate (3,000 Ci/mmol), and Reflections X-ray film from Dupont NEN (Boston, MA); low molecular-weight urokinase and RNase T1 from Calbiochem (La Jolla, CA); sodium heparin from Elkins-Sinn (Cherry Hill, NJ); RNase inhibitor from Molecular Biology Resources (Milwaukee, WI); Nytran 0.2-µm nylon membranes and BA 85 nitrocellulose membranes from Schleicher and Schuell, Inc. (Keene, NH); PAI-1 and u-PA enzyme-linked immunosorbent assay (ELISA) kits from American Diagnostica, Inc. (Greenwich, CT); and A549 human pulmonary epithelial cell line and the 1.5-kb Pst 1 fragment of the human u-PA complementary DNA (cDNA) from American Type Culture Collection (Rockville, MD). The 2-kb EcoR1 fragment of the human PAI-1 cDNA was kindly provided by Dr. M. Ginsburg (University of Michigan, Ann Arbor, MI); and the 0.74-kb CHO-B cDNA fragment was kindly provided by Dr. W. T. Garvey (VA Medical Center, Indianapolis, IN).

Tissue Culture

A549 human carcinoma-derived pulmonary epithelial cells were cultured in Ham's F-12 medium supplemented with 10% FCS, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and amphotericin B (250 ng/ ml). Cells were grown to confluence in complete media, then washed with phosphate-buffered saline (PBS) and changed to serum-free medium. Fresh serum-free medium was added 12 h before the experimental period unless otherwise indicated.

Cell-Surface Plasmin Assay

Confluent cells in 12-well plates were pretreated with DEF (100 µm), DEF (100 µm) + Fe³⁺ (100 µm), or no additive for 16 h, and then were treated with IL-1 (10 U/ml) or no additive for 24 h. The medium was discarded and the cells were then washed in PBS and incubated in serum-free Ham's F-12 containing plasminogen (20 µg/ml) for 3 h. The supernatants were collected and cells were again washed with PBS. Plasmin was then dissociated from the cell membrane by incubation of the cells in 250 µl of 1 mM tranexamic acid in PBS, pH 7.4, for 15 min (16). Aliquots of the samples (50 µl) were assayed in duplicate in a total assay volume of 125 µl of 0.1 M Tris, pH 8.0, containing 1.0 mM final concentration of the chromagenic substrate S-2251. The amount of p-nitroaniline released after 2 h at 37°C was measured at 405 nm with a Molecular Devices UV MAX plate reader and referenced to a plasmin (Catalog #P4895; Sigma) standard curve run in parallel with the samples. One unit of plasmin activity was defined as a change in absorbance of 2.5 per minute in the stated assay conditions. Data was expressed as milliunits (10³ units) per well. Comparisons were made with a Student's t test.

RNA Band-Shift Assay

Cytoplasmic extracts were prepared by minor modifications of the method of Leibold and Munro (17). Confluent monolayers of cells were chilled on ice immediately after the treatment period. Cells were scraped into 25 ml ice-cold PBS containing 2 mM DTT and 1 mM phenylmethylsulfonyl fluoride (PMSF) and pelleted by centrifugation. The cell pellets were disrupted by pipetting up and down over 5 min in 1 ml of ice-cold lysis buffer containing 20 mM Hepes (pH 7.6), 25 mM KCl, 50% (vol/vol) glycerol, 0.5% NP-40, 2 mM DTT, 1 mM PMSF, 10 mM  glycerophosphate, 2.5 mM benzamidine, 1 mM NaF, 1 mM NaVO₄, and 10 mg/ml leupeptin. The lysates were centrifuged at 14,000 × g for 10 min and the supernatants were collected and frozen in aliquots in liquid nitrogen. Protein content of the cytoplasmic extracts was determined by the Micro-BCA assay (Pierce Biochemical, Rockford, IL). [³²P]-labeled RNA was synthesized from the Sma1-digested pGL-66, which results in a 118-base pair (bp) transcript containing the first 65 bp of the rat liver ferritin L-subunit mRNA encompassing the IRE (18). The labeled RNA was gel-purified before the binding studies. The cytoplasmic extracts (30 µg) were incubated with labeled RNA probe (8-10,000 cpm) at room temperature for 30 min in 2 mM Hepes (pH 7.6), 3 mM MgCl₂, 40 mM KCl, 5% (vol/vol) glycerol, and 1 mM DTT in a final volume of 20 µl. RNase T1 (1 U) was then added, followed in 10 min by the addition of 10 U of sodium heparin. After another 10 min, the samples were loaded on a 4% nondenaturing polyacrylamide gel (acrylamide/bisacrylamide ratio 60:1) that had been pre-electrophoresed for 30 to 60 min at 13 V/cm. Electrophoresis was carried out for 90 min at the same voltage, followed by drying of the gel and autoradiography at 70°C with an intensifier screen.

Measurement of u-PA Activity

Aliquots of samples were assayed in duplicate in a total assay volume of 125 µl of 1% Triton X-100 in PBS containing 0.15 µm plasminogen and 1.5 mM S2251 in 96-well plates at 37°C for 8 h. The amount of p-nitroaniline released was measured at 405 nm with a Molecular Devices plate reader and referenced to a standard urokinase preparation (Catalog #6172101; Calbiochem) run in parallel with the samples. In the stated assay conditions, 1 U of activity was defined as 0.086 change in absorbance per minute at 37°C. Comparisons were made with a Student's t test.

Measurement of Immunoreactive PAI-1 and u-PA

Confluent cells in 12-well plates were pretreated with DEF (100 µM), DEF (100 µM) + Fe³⁺ (100 µM), or no additive for 16 h and then were incubated with serum-free medium alone or the same medium containing IL-1 (10 U/ml) for 24 h. Supernatants were then collected and cell lysates obtained by the addition of 1% Triton X-100 in PBS on ice for 1 h. Samples were assayed for immunoreactive PAI-1 and u-PA by ELISA. Data were expressed as nanograms of antigen per well. Comparisons were made with a Student's t test.

Northern Blot Analyses

Total RNA was prepared from the cells by acid guanidine thiocyanate-phenol-chloroform extraction and quantitated by measuring absorbance at 260 nm. RNA (10 to 20 µg) was size-fractionated on 1% agarose/2.2 M formaldehyde gels and transferred to nylon membranes by capillary action. RNA was crosslinked to the nylon membrane by exposure to ultraviolet light and prehybridized in 5× saline sodium citrate (SSC), 50 mM Na₂HPO₄, 10× Denhardt's solution, 2.5% dextran sulfate, 50% formamide, 0.75% SDS, and 10 µg/ml salmon sperm DNA. The cDNA probes were labeled with [³²P]dCTP by the random-primer method and hybridized to the immobilized RNA overnight in 5× SSC, 20 mM Na₂HPO₄, 1× Denhardt's solution, 10% dextran sulfate, 50% formamide, 0.5% SDS, and 10 µg/ml salmon sperm DNA at 42°C. The membranes were washed at high stringency and then exposed to X-ray film at 70°C with an intensifier screen. The CHO-B signal (a constitutively expressed gene) served to verify that approximately equal amounts of RNA had been loaded and transferred to the nylon membrane.

Nuclear Runoff Transcription Assays

Nuclei were prepared according to the method described by Greenberg and Bender (19). In brief, the cells were washed and then resuspended in 1% NP-40 lysis buffer containing 2 mM MgCl₂, 10 mM Tris (pH 7.5), and 3 mM CaCl₂, and incubated on ice for 15 min. This lysis step was repeated; then, after centrifugation, the pellets were suspended in 125 µl of 40% glycerol storage buffer containing 10 mM Tris (pH 7.5), 5 mM MgCl₂, 80 mM KCl, and 0.5 mM DTT, and stored frozen in liquid nitrogen until labeling. Nuclei were thawed on ice and 2 µl of RNase inhibitor and 2 µl of 3% antifoam A were added to each sample. Nascent RNA transcripts were elongated 20 to 30 min at 30°C with 4 mM each of ATP, CTP, and GTP, and 200 µCi of [-³²P]uridine triphosphate. The reaction was continued 5 min after the addition of 20 mM CaCl₂ and 5 µl of 20 mg/ ml RNase-free DNase, and then terminated by incubation for 30 min at 30°C after the addition of 25 µl of 10× SET solution containing 5% SDS, 50 mM EDTA, 100 mM Tris (pH 7.4), 2 µl of 10 mg/ml proteinase K, and 5 µl of 10 mg/ ml torula tRNA. RNA extraction was performed by acid guanidine thiocyanate-phenol-chloroform extraction. The dried RNA pellets were dissolved in 200 µl of 10 mm (TES) (pH 7.5) and 10 mM EDTA with 2 µl RNase inhibitor, and the counts normalized to 10⁶ cpm per sample (mean ± standard error [SE] of total cpm was 1.79 ± 0.33 × 10⁶). Reaction volumes were adjusted to 650 µl with the same buffer and the samples were denatured at 65°C for 15 to 20 min, followed by storage on ice before hybridization. The plasmids containing cDNA sequences of CHO-B, PAI-1, and u-PA genes were linearized by restriction enzyme digestion, purified, denatured, and applied to BA 85 nitrocellulose membranes according to the manufacturer's recommendations at a concentration of 5 µg per slot. After baking for 2 h at 80°C, the membranes were prehybridized for 4 h at 65°C in glass scintillation vials in buffer containing 10 mM N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid (TES) (pH 7.5), 5 mM EDTA, 0.15 M NaCl, 1× Denhardt's solution, and 0.2% SDS. To each of the 650-µl RNA solutions we added 100 µl of 10 mg/ml torula tRNA and 750 µl of 2× TES hybridization solution containing 10 mM TES, 10 mM EDTA, 0.3 M NaCl, 2× Denhardt's solution, and 0.4% SDS. The mixtures were then applied to the prehybridized membranes. Hybridization was carried out for 36 h at 65°C within the glass scintillation vials in a Hybaid Minioven. Membranes were then washed twice for 15 min in 2× SSC and 0.1% SDS at room temperature with vigorous agitation, and then exposed to Reflections X-ray film for 14 d at 70°C with an intensifier screen.

	Results

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Effect of Decreased Intracellular Iron Availability on the Induction of Cell-Surface Plasmin Generation

We have previously reported that IL-1 is a potent upregulator of cell-surface plasmin generation (13). Pretreatment of cells with DEF (100 µM) for 16 h suppressed cell-surface plasmin to undetectable levels (not shown) and markedly suppressed the IL-1-induced increase in plasmin generation (Figure 1). This effect was not observed when cells were incubated with equimolar amounts (100 µM) of DEF and Fe³⁺ (Figure 1). Further experiments focused on the effects of DEF on IL-1-stimulated cells.

View larger version (41K): [in this window] [in a new window]   Figure 1.   Effect of DEF on IL-1-induced cell-surface plasmin generation. Confluent cells were preincubated for 16 h with no additive (Ctl and IL-1 groups), with DEF (100 µM), or with DEF (100 µM) + Fe3+ (100 µM). After preincubation, the cells were incubated for an additional 24 h without (Ctl) or with 10 U/ml of IL-1 and then assayed for cell-surface plasmin generation as described in MATERIALS AND METHODS. Plasmin activity is expressed as µU/well (mean ± SEM, n = 4 for each group). *P < 0.01 for IL-1 + DEF versus IL-1; #P < 0.01 for IL-1 + DEF + Fe3+ versus IL-1 + DEF.

To confirm that DEF decreased available intracellular iron, we performed RNA band-shift assays using the IRE as the target RNA. We found, as expected, that DEF treatment of cells markedly increased protein binding to the IRE (Figure 2).

View larger version (24K): [in this window] [in a new window]   Figure 2.   Effect of DEF on protein binding to IRE. Confluent cells were incubated for 19 h with no additive (Ctl) or with DEF (100 µM). Cell lysates were prepared and RNA band-shift assays performed as described in MATERIALS AND METHODS using labeled IRE as the RNA target. The RNA-protein complex can be seen at the top of the gel and the unbound, free probe at the bottom.

Effect of Decreased Intracellular Iron on u-PA Activity and u-PA and PAI-1 Antigen Levels

To define the factors involved in the DEF effect on cell-surface plasmin generation, we first examined its effects on u-PA antigen and activity levels. We found that DEF blocked the IL-1 induction of u-PA antigen in both the supernatant and lysate fractions (Table 1). The increase in u-PA activity due to IL-1 was also blocked by DEF (Table 1). These effects were not observed when cells were incubated with equimolar amounts of DEF and Fe³⁺. Notably, DEF reduced u-PA activity below the limits of detection despite the presence of u-PA antigen, suggesting that it might have a concomitant effect on PAI-1 expression. Indeed, we found that DEF increased PAI-1 antigen levels in the IL-1-treated cells in both supernatant and lysate fractions. Figure 3 shows total PAI-1 antigen levels.

View larger version (43K): [in this window] [in a new window]   Figure 3.   Effect of DEF on PAI-1 antigen levels. Confluent cells in 12-well plates were preincubated for 16 h with no additive (Ctl and IL-1 groups), with DEF (100 µM), or with DEF (100 µM) + Fe3+ (100 µM). After preincubation, the cells were incubated for an additional 24 h without (Ctl) or with 10 U/ml of IL-1. Supernatants and lysates were assayed for immunoreactive PAI-1 by ELISA, and the total is shown in the graph in ng/well (mean ± SEM, n = 4 for each group). *P < 0.01 for IL-1 + DEF versus IL-1; #P < 0.01 for IL-1 + DEF + Fe3+ versus IL-1 + DEF.

Effect of Decreased Intracellular Iron Availability on Steady-State u-PA and PAI-1 mRNA Levels

To further define the effect of DEF on u-PA and PAI-1 expression, we performed Northern blot analyses. We have previously reported that IL-1 causes a relatively rapid accumulation of u-PA mRNA that peaks at 3 h after stimulation and returns to near baseline by 24 h (12). Pretreatment of cells with DEF attenuated the IL-1-induced increase in steady-state u-PA mRNA levels observed at the 3-h time point (Figure 4). In contrast, steady-state levels of PAI-1 mRNA were markedly increased by DEF pretreatment at both 3- and 24-h time points (Figure 4). Analogous to our findings at the antigen level, these effects on mRNA levels were not observed when cells were incubated with equimolar concentrations of DEF and Fe³⁺. The effects of DEF were not unique to the IL-1 stimulus. The same pattern of response was observed when DEF-pretreated cells were stimulated with PMA (not shown).

View larger version (51K): [in this window] [in a new window]   Figure 4.   Effect of DEF on the induction of PAI-1 mRNA and u-PA mRNA by IL-1. Confluent cells were preincubated in serum-free medium with no additive (Ctl and IL-1 groups), with DEF (100 µM), or with DEF (100 µM) + Fe3+ (100 µM). After a 16-h preincubation, IL-1 (10 U/ml) was added to selected flasks and the cells were incubated for an additional 3 or 24 h. Total RNA was then prepared for Northern analysis. The upper panel shows PAI-1 mRNA, the middle panel shows u-PA mRNA, and the lower panel shows the constitutively expressed CHO-B mRNA. This result is representative of three separate experiments.

Effect of Decreased Intracellular Iron Availability on Transcriptional and Post-transcriptional Events

We used nuclear runoff transcription assays and half-life experiments using actinomycin to explore whether the effect of DEF on steady-state u-PA and PAI-1 mRNA levels was mediated by transcriptional or post-transcriptional mechanisms. Nuclear runoff assays showed that DEF pretreatment modulates the transcription rate of u-PA and PAI-1 genes (Figure 5). The effect of DEF on gene transcription rates paralleled the changes in steady-state u-PA and PAI-1 mRNA levels shown in Figure 4. The magnitude of the effect of DEF on the transcription rate of the u-PA gene roughly corresponded to its effect on steady-state mRNA levels, but the marked increase in steady-state PAI-1 mRNA level seemed out of proportion to the modest increase in transcription rate of the PAI-1 gene. We therefore investigated the effect of DEF on PAI-1 mRNA stability. Actinomycin was added to cell cultures to block gene transcription 3 h after the addition of IL-1. One set of cell cultures had been pretreated with DEF and the other received no pretreatment. Total RNA was prepared 8 and 20 h after the addition of actinomycin, and steady-state PAI-1 mRNA levels were determined by Northern analysis. We found that DEF clearly prolongs the half-life of the PAI-1 mRNA transcript (Figure 6). Thus, the increased steady-state PAI-1 mRNA level caused by DEF is due to stabilization of the mRNA transcript as well as transcriptional activation of the gene.

View larger version (51K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Figure 5.   Effect of DEF on PAI-1 and u-PA gene transcription rate. Confluent cells were incubated in serum-free medium with no additive (Ctl and IL-1 groups), with DEF (100 µM), or with DEF (100 µM) + Fe3+ (100 µM). After a 16-h preincubation, IL-1 (10 U/ml) was added to selected flasks and the cells were incubated for an additional 30 min. Nuclei were then prepared and runoff transcription assays performed as previously described. In A, the lower row shows u-PA, the middle row shows PAI-1, and the upper row shows the constitutively expressed CHO-B. In B, the bar graph shows transcription rates for the u-PA (hatched columns) and PAI-1 (solid columns) genes normalized to the CHO-B gene transcription rate as determined by densitometric analysis. This result is representative of two separate experiments.

View larger version (50K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Figure 6.   Effect of DEF on PAI-1 mRNA half-life. Confluent cells were incubated in serum-free medium with no additive or with DEF (100 µM). After a 16-h preincubation, IL-1 (10 U/ml) was added to the flasks, followed by the addition of actinomycin 3 h later. Total RNA was then prepared for Northern analysis at 0, 8, and 20 h time points. A shows PAI-1 mRNA and the constitutively expressed CHO-B mRNA. The line graph in B shows results of densitometric analysis, normalizing the 3.2-kb PAI-1 transcript to the corresponding CHO-B band. The PAI-1/CHO-B ratio at the 0 h time point was arbitrarily considered 100%, and results at the other time points referenced to that baseline. This result is representative of three separate experiments.

	Discussion

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Acute fibrin deposition and subsequent resorption occurs in a variety of acute inflammatory processes, including acute lung injury. The plasmin/PA system likely plays a key role in the extensive repair process that follows lung injury, and pulmonary epithelial cells are important contributors to the fibrinolytic balance in the bronchoalveolar space. Increasing evidence that iron may play a critical role in lung injury and repair led us to explore its influence on epithelial-cell plasmin generation. We found that decreased intracellular iron availability markedly suppresses the IL-1-mediated induction of cell-surface plasmin by downregulation of u-PA expression and upregulation of PAI-1 expression. Similar changes were observed in PMA-stimulated cells, thus the effects of decreased iron on u-PA/ PAI-1 expression are not due to modifications in IL-1 signal transduction pathways. Although the specific mechanisms have not been fully elucidated, we have shown that regulation occurs at transcriptional and post-transcriptional levels.

Decreased intracellular iron availability suppresses transcriptional activation of the u-PA gene by IL-1 and has the opposite effect on the PAI-1 gene. The most prominent effects of changes in intracellular iron availability on transferrin receptor and ferritin expression are mediated by post-transcriptional mechanisms, but modest changes in gene transcription rate have also been reported (20, 21). In addition, other genes such as NO synthase (5), protein kinase C- (22), and tartrate-resistant acid phosphatase (23) are also modulated at a transcriptional level by changes in intracellular iron. To date, the specific mechanisms for these transcriptional effects have not been fully elucidated. Thus, our findings are compatible with previous reports; and indeed, a detailed promoter analysis of the u-PA and PAI-1 genes may provide insight into the mechanisms of iron's effects on gene transcription.

We found that steady-state u-PA mRNA levels parallel changes in gene transcription; however, the marked increase in PAI-1 mRNA level is also partly due to stabilization of the mRNA transcript. Such post-transcriptional regulation of steady-state PAI-1 mRNA levels has been demonstrated previously (24, 25). The effect of DEF on PAI-1 stability is analogous to its effect on transferrin receptor expression; i.e., decreased intracellular iron availability leads to an increased steady-state mRNA level, predominantly due to stabilization of the mRNA transcript (26). Conformational changes in aconitase/iron-binding protein result in loss of aconitase activity and binding to IREs in the 3' untranslated region of the transferrin receptor mRNA transcript, protecting it from degradation and thereby prolonging its half-life. No consensus IREs exist in the PAI-1 mRNA transcript, but we speculate that other RNA-protein interactions are determinants of PAI-1 mRNA stability.

Another possible explanation for the effects of DEF on u-PA/PAI-1 expression is disruption of the oxygen-sensing system by chelation of a critical iron-containing protein. Several genes that are upregulated by hypoxia are similarly regulated by deferoxamine, including erythropoietin (27), vascular endothelial growth factor (28, 29), several glycolytic enzymes (30), and the glucose transporter GLUT-1 (31). Of note: the urokinase receptor gene has recently been added to this list (32). Both transcriptional and post-transcriptional mechanisms have been reported in the regulation of these other genes. We are currently exploring the effects of hypoxia on expression of the PA/ plasmin system.

One other potential regulatory mechanism should be pointed out with respect to u-PA expression. DEF attenuates the increase in steady-state u-PA mRNA levels induced by IL-1; however, the levels remain significantly higher than those of control cells. In contrast, u-PA antigen levels in DEF-treated cells fall far below those of control cells. This may be due to an increased turnover rate of u-PA antigen or an inhibition of u-PA translation. We have no direct evidence for either of these possibilities, but have found that increased intracellular iron stimulates translation of u-PA mRNA (T. Hasegawa and B. C. Marshall, unpublished observation). Further investigation of the potential role of intracellular iron levels on translation of u-PA mRNA is continuing in our laboratory.

A significant body of information indicates an important role for iron in lung injury and repair (33). There is increased extracellular iron in the setting of acute (34, 35) and chronic (36) lung injury, and this is potentially harmful via the generation of reactive oxygen species by a Fenton mechanism (37). However, we know very little about intracellular iron availability in the setting of lung injury, particularly within different cellular compartments of the lung. Of course, systemic conditions may also affect intracellular iron availability. For example, iron deficiency is a common clinical condition that often results in anemia, but may also have more subtle effects in other organ systems. Some commonly used drugs such as ibuprofen, with its iron-chelating properties (38), may also decrease intracellular iron availability. In any event, our data suggest that intracellular iron stores have a marked effect on expression of the PA/plasmin system and this may be relevant to lung injury and repair.

Although data does exist showing that iron affects the coagulation system (39), to our knowledge this is the first report linking iron metabolism and cell-surface plasmin generation. Moreover, because of the central role that the PA/plasmin system plays in a variety of physiologic and pathophysiologic conditions, our findings may have wide-ranging implications.