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Published ahead of print on June 19, 2003, doi:10.1165/rcmb.2002-0305OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 69-75, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2002-0305OC

Urokinase Receptor mRNA Stability Involves Tyrosine Phosphorylation in Lung Epithelial Cells

Sreerama Shetty and Steven Idell

Department of Specialty Care Services, The University of Texas Health Center at Tyler, Tyler, Texas

Address correspondence to: Sreerama Shetty, Ph.D., Associate Professor of Medicine, University of Texas Health Center at Tyler, 11937 US HWY 271, Lab C-6, Tyler, TX, 75708. E-mail: sreerama.shetty{at}uthct.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) localizes cellular proteolysis and promotes cellular proliferation and migration, effects that may contribute to the pathogenesis of lung inflammation and neoplasia. Enhanced uPAR expression as well as stabilization of uPAR mRNA by transforming growth factor-ß and phorbol myristate acetate (PMA) shares a common mechanism involving phosphorylation and dephosphorylation of a uPAR mRNA-binding protein (uPAR mRNABp). PMA-induced tyrosine phosphorylation of the uPAR mRNABp inhibited the uPAR mRNA–uPAR mRNABp interaction, stabilized uPAR mRNA and enhanced uPAR protein expression. Downregulation of the uPAR mRNA and uPAR mRNABp interaction by PMA and transforming growth factor-ß can be reversed by pretreatment of cells with herbimycin which in turn inhibits expression of uPAR protein via a decrease in uPAR mRNA stability. Our experiments indicate that post-transcriptional regulation of uPAR expression requires activation of tyrosine kinases. Cytokines can regulate uPAR expression of lung-derived epithelial cells at the post-transcriptional level by tyrosine phosphorylation of the uPAR mRNA binding protein and may thereby influence tissue remodeling in lung injury or neoplasia.

Abbreviations: bronchial epithelial cells, Beas2B • bovine serum albumin, BSA • cycloheximide, cycD • glycosyl phospatidyl inositol, GPI • lipopolysaccharide, LPS • plasminogen activator inhibitor-1, PAI-1 • plasminogen activator inhibitor-2, PAI-2 • phosphate-buffered saline, PBS • phorbol myristate acetate, PMA • sodium dodecyl sulfate, SDS • SDS-polyacrylamide gel electrophoresis, SDS-PAGE • saline sodium citrate, SSC • transforming growth factor-ß, TGF-ß • tumor necrosis factor-{alpha}, TNF-{alpha} • tissue-type plasminogen activator, tPA • urokinase-type plasminogen activator, uPA • urokinase-type plasminogen activator receptor, uPAR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Degradation of extracellular matrix by serine proteinases and metalloproteinases has been implicated in the pathogenesis of lung injury and lung neoplasia (1–3). These enzymes influence inflammatory cell traffic or cancer cell invasiveness via the dissolution of basement membranes and extracellular matrix (4–7). Plasmin, a serine protease, is involved in the breakdown of extracellular matrix and basement membrane during tissue degradation. This protease is generated via the action of plasminogen activators such as urokinase (uPA) or tissue plasminogen activator (tPA), and can influence tissue remodeling either directly or through activation of latent collagenases. uPA is mainly involved in extravascular proteolysis, and is implicated in stromal remodeling in acute and chronic lung injury (8–10) and in metastatic neoplasia (7). Interaction of uPA with its cellular receptor has several unique properties that promote plasminogen activation. During the last decade, evidence for the involvement of the uPA system in lung injury and repair or lung neoplasia (1) has steadily increased, and it now seems beyond reasonable doubt that uPA-dependent plasminogen activation is central to these processes. Because many biological activities of uPA depend on association with its receptor, uPAR plays a central role in localized uPA-mediated plasminogen activation. Increased expression of uPA or uPAR has, for example, been inversely correlated with prognosis in lung cancer (11, 12). Better understanding of the specific pathways that regulate uPAR expression is therefore germane to the pathogenesis of lung injury or the spread of lung neoplasms.

The steady-state of any mRNA reflects its stability as well as synthesis. Moreover, stability of many mRNAs are major determinants of their abundance. Among the various mechanisms by which different cell types influence mRNA stability, regulation of mRNA decay is a potentially important process determining the level of gene expression. Synthesis of uPAR is regulated by a variety of hormones, growth factors, and cytokines either at the transcriptional or post-transcriptional level (13–20). Both uPA and uPAR, as well as plasminogen activator inhibitor (PAI)-1 and -2, are expressed by lung epithelial cells (21–23), and it is now clear that posttranscriptional regulation contributes to the regulation of uPA, uPAR, and PAI-1 by these cells (16, 22–26).

Expression of uPA and uPAR controls several cellular functions, including epithelial cell adhesion, signaling, and mitogenesis, and most of the biological activities of uPA are dependent on its association with the uPAR (4, 13, 27–33). The expression of these components by the lung epithelium is tightly regulated during normal physiologic processes and is disordered in lung injury or lung cancer. It is noteworthy that the signaling pathways activated by proinflammatory cytokines induce uPAR expression (30). We therefore postulated that cooperativity between cytokine-mediated signaling and post-transcriptional regulation of uPAR mRNA stability was plausible, albeit not previously described. This possibility was investigated using cultured bronchial epithelial (Beas2B) cells as a model system. In these studies, we describe a novel regulatory pathway by which phorbol myristol acetate (PMA) and transforming growth factor (TGF)-ß induce uPAR expression at the post-transcriptional level.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Material
Culture media, penicillin, streptomycin, and fetal calf serum were purchased from Gibco BRL laboratories (Grand Island, NY); tissue culture plastics were from Becton Dickinson Labware (Lincoln Park, NJ). Herbimycin from Calbiochem (La Jolla, CA), bovine serum albumin (BSA), ovalbumin, Tris-base, aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, ammonium persulfate, and PMA were from Sigma Chemical Co. (St. Louis, MO). Acrylamide, bisacrylamide, and nitrocellulose were from BioRad laboratories (Richmond, CA). Anti-uPAR antibody was obtained from American Diagnostics (Greenwich, CT). XAR X-ray film was purchased from Eastman Kodak (Rochester, NY).

Cell Cultures
Human Beas2B cells were maintained in LHC-9, or RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 1% glutamine, and 1% antibiotics as previously described (30).

Total Cellular Membrane Extraction and Western Blotting
Beas2B cells grown to confluence were serum-starved overnight with RPMI-glutamine media containing 0.5% BSA. The cells were treated with or without various agents for indicated times and were washed with phosphate-buffered saline (PBS). Receptor-bound uPA was removed by glycine-HCl treatment as described earlier (29–30). We used sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and Western blotting to measure functional uPAR at the cell surface. Membrane proteins isolated as described earlier (29–30) from Beas2B cells were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1% BSA in wash buffer for 1 h at room temperature followed by overnight hybridization with uPAR monoclonal antibody in the same buffer at 4°C, washed and uPAR proteins were detected by enhanced chemiluminescence.

Plasmid Construction
Plasmid uPAR/pBluescript was obtained from the ATCC. The human uPAR mRNA template containing a complete sequence of uPAR cDNA (nucleotides –16 to 1144) from uPAR pBluescript was subcloned to Hind III and Xba I sites of pRC/CMV (Invitrogen, Carlsbad, CA) and the sequences of the clones were confirmed by sequencing. The uPAR insert was released by Hind III or Xba I, purified on 1% agarose gels, extracted with phenol/chloroform, and used as cDNA probe for Northern blotting.

In Vitro Transcription
Linearized plasmids containing the human uPAR mRNA transcriptional template of complete uPAR cDNA were transcribed in vitro with T7 or Sp6 polymerase (Ambion, Austin, TX). The uPAR mRNA transcripts were synthesized according to the supplier's protocol except that 50 µCi of [32-P]UTP was substituted for unlabeled UTP in the reaction mixture. Passage through a Sephadex G-25 column removed unincorporated radioactivity. The specific activities of the product were 4.9 x 108.

Random Priming of uPAR cDNA
The full-length template of uPAR was released with Hind III or Xba I, purified on 1% agarose gels, and labeled with 32P-dCTP using a rediPrime labeling kit (Amersham, Arlington Heights, IL). Passage through a Sephadex G-25 column removed unincorporated radioactivity. The specific activity of the product was 6 x 108 cpm/µg.

Northern Blotting of uPAR mRNA
A Northern blotting assay was used to assess the level of uPAR mRNA. Beas2B cells grown to confluence were serum-starved overnight in RPMI-BSA media, then treated with PMA, TGF-ß, and cycloheximide for varying times (0–24 h) in the same media. Total RNA was isolated using TRI reagent. RNA (20 µg) was isolated on agarose/formaldehyde gels. After electrophoresis, the RNA was transferred to Hybond N+ according to the instructions of the manufacturer. Prehybridization and hybridization was done at 65°C in NaCl (1 M)/SDS (1%) and 100 µg/ml salmon sperm DNA. Hybridization was performed with a uPAR cDNA probe (1 ng/ml) labeled to ~ 6 x 108 cpm/µg of DNA overnight. After hybridization, the filters were washed twice for 15 min at 65°C, with: 2 x saline sodium citrate (SSC), 1% SDS; 1 x SSC, 1% SDS, and 0.1% SSC, 1% SDS, respectively. The membranes were next exposed to X-ray film at –70°C overnight. The intensity of the bands was measured by densitometry and normalized against that of ß-actin.

We also measured the stability of uPAR mRNA in the presence of PMA by transcription chase experiments as described earlier (16).

Preparation of Cytosolic Extracts
Beas2B cells treated with or without PMA or TGF-ß were detached from the culture flasks. The cells were washed in PBS, and cytosolic extracts were prepared by suspending the cell pellet in a buffer containing 25 mM Tris-HCl (pH 7.9), 0.5 mM EDTA, and 0.1 mM phenylmethylsulfonyl fluoride. The cells lysates were prepared as described earlier (16).

Gel Mobility Shift Assay
Cytoplasmic protein (20 µg) was incubated with 2 x 104 cpm of 32P-labeled transcript in a mixture containing 15 mM KCl, 5 mM MgCl2, 0.25 mM dithiothreitol, 12 mM Hepes (pH 7.9), 10% glycerol, and Escherichia coli tRNA (200 ng/µl) in a total volume of 20 µl at 30°C for 30 min. The reaction mixture was treated with 50 U of RNase T1 or A and incubated at 37°C for 30 min. To avoid nonspecific binding, 5 mg/ml of heparin was added, and the mixture was incubated at room temperature for an additional 10 min. Samples were separated by electrophoresis on a 5% native polyacrylamide gel with 0.25x tris-borate-EDTA running buffer. The gels were dried and developed by autoradiography at –70°C.

Ultraviolet Cross-Linking
We used ultraviolet cross-linking assay to confirm the molecular weight of uPAR mRNABp, and we performed uPAR mRNA–protein interaction reaction essentially same as described above. After heparin digestion, the reaction mixture was kept on ice and ultraviolet-irradiated for 30 min at 2,500 µJ. The uPAR mRNA-protein complex was separated on 8% SDS-PAGE; radiolabeled uPAR mRNA–protein complex was visualized by autoradiography.

Northwestern Assay
Alternatively, we confirmed the molecular weight and uPAR mRNA-uPAR mRNABp interaction by Northwestern assay. Cytosolic proteins from Beas2B cells separated on 8% SDS-PAGE, blotted to nitrocellulose membrane. The membrane was blocked with gel shift buffer containing 1% BSA and 20 µg ribosomal RNA for 1 h. The membrane was replaced with fresh buffer containing 32P-labeled uPAR mRNA (200,000 cpm/ml) and incubated for an additional 1 h at RT. The membrane was later washed thrice with 50 ml of gelshift buffer for 10 min each, air dried and exposed to X-ray film. The membrane was later stripped and developed by Western blot using a ß-actin monoclonal antibody as described above for equal loading.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of uPAR in Lung Epithelial Cells
Because we previously found that lung epithelial cells, fibroblasts, and pleural mesothelial/mesothelioma cells express uPAR in vitro (24–26, 29–30), we initially wanted to determine if uPAR expression is induced by proinflammatory agents in cultured nonmalignant Beas2B cells. Western blotting assays were used to determine the level of uPAR expression at the surface of Beas2B cells treated with PBS, PMA, TGF-ß, tumor necrosis factor (TNF)-{alpha}, lipopolysaccharide (LPS), and cycloheximide. All these agents, except cycloheximide, induced uPAR expression. However, expression of uPAR was greatest in PMA- and TGF-ß–stimulated cells (Figure 1A). We also found that both PMA and TGF-ß induced maximum amounts of uPAR expression between 12 and 24 h after treatment (Figure 1B).



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  		Figure 1. Expression of uPAR in bronchial epithelial cells. (A) Beas2B cells grown to confluence were treated with PBS, PMA (150 nM), LPS (10 µg/ml), TGF-ß (2 ng/ml), TNF-{alpha} (10 ng/ml), and cycloheximide (CycD, 10 µg/ml) for 24 h. The membrane proteins were isolated from Beas2B cells and treated with various agents were separated on 8% SDS-PAGE and electroblotted to nitrocellulose membranes. The membranes were subjected to Western blotting using a urokinase receptor monoclonal antibody. (B) Beas2B cells treated with PMA or TGF-ß for 0–24 h. uPAR expression was measured by Western blot as described in A.

 
We next analyzed the expression of uPAR mRNA by Northern blot, and found that PMA, TGF-ß, and cycloheximide induced uPAR mRNA expression (Figure 2A). PMA enhanced uPAR mRNA expression by increasing the stability of the message (Figure 2B).



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  		Figure 2. Urokinase receptor mRNA expression in Beas2B cells. (A) Confluent Beas2B cells were treated with PBS, PMA, TGF-ß, and cycD for 12 h at 37°C in basal medium containing 0.5% BSA. Total RNA was isolated and uPAR mRNA was measured by Northern blot using 32P-labeled uPAR cDNA. The data illustrated is representative of two independent experiments. (B) Beas2B cells treated with PBS or PMA for 12 h, after which ongoing transcription was inhibited by treating the cells with DRB for 0–24 h in the same medium. Total RNA was isolated and uPAR was analyzed by Northern blot.

 
PMA Downregulates the uPAR mRNA–uPAR mRNABp Interaction
Strong and rapid induction of uPAR and uPAR mRNA stability by PMA in Beas2B cells prompted us to next evaluate the role of the uPAR mRNA–uPAR mRNABp interaction in the process. This interaction has previously been demonstrated to regulate uPAR mRNA stability (16). We confirmed that PMA and TGF-ß reduced the uPAR mRNA–uPAR mRNABp interaction by ~ 50% using gel mobility shift (Figure 3). Similar results were also found by independent Northwestern and ultraviolet cross-linking assays (data not shown). We also confirmed that the PMA-mediated decrement in the uPAR mRNA–uPAR mRNABp complex was apparent by 3 h after the treatment, and that the inhibitory effect is sustained over 24 h (Figure 4).



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  		Figure 3. Downregulation of uPAR mRNA and uPAR mRNABp interaction by PMA. (A) Beas2B cells grown to confluence were serum-starved overnight and treated with PBS, PMA, or TGF-ß for 24 h. The cytosolic extracts were subjected to gel mobility shift assay using uPAR mRNA coding region transcripts. Fp, free probe. (B) The bar graph illustrates the mean density of individual bands from five independent experiments.

 


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  		Figure 4. Time-dependent inhibition of uPAR mRNA and uPAR mRNABp interaction by PMA. Beas2B cells were treated with PMA for 0–24 h at 37°C. The cytosolic extracts were subjected to gel mobility shift assay as described in Figure 3A.

 
Activation of uPAR mRNABp Involves Phosphorylation of the uPAR mRNABp
We next wanted to determine whether PMA-mediated induction of uPAR (Figure 1) involved regulation through the interaction of uPAR mRNA with the uPAR mRNABp. We therefore treated Beas2B cells with PMA for varying time periods and prepared cell lysates. The cell lysates were immunoprecipitated with antibody developed against purified rabbit uPAR mRNABp (26). The immune complexes were separated on SDS-PAGE under denaturing condition and were then blotted to nitrocellulose membranes. The membrane was later developed to detect tyrosine phosphorylation activation, using an antiphosphotyrosine antibody. The results indicate that PMA induces tyrosine phosphorylation of the uPAR mRNABp (Figure 5A). When the same samples were subjected to Western blotting using an anti-uPAR mRNABp antibody, we found no significant quantitative variation of the uPAR mRNABp expression after PMA treatment (Figure 5B). The results show that PMA induced tyrosine phosphorylation of the uPAR mRNABp in a time-dependent manner and that the protein was detectable in phosphorylated form from 6–24 h.



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  		Figure 5. Time-dependent tyrosine phosphorylation of the uPAR mRNABp by PMA. (A) Beas2B cells were treated with PMA for 0–24 h, and the uPAR mRNABp was immunoprecipitated using an anti-uPAR mRNABp antibody. The membrane was developed by antiphosphotyrosine antibody. M, molecular weight markers. (B) The cytosolic extracts of PMA treated Beas2B cells were subjected to Western blot using anti-uPAR mRNABp polyclonal antibody. The same membrane was stripped and developed with ß-actin monoclonal antibody.

 
Inhibition of Tyrosine Phosphorylation Inhibits PMA as Well as TGF-ß-Mediated uPAR Expression
Because PMA induced tyrosine phosphorylation of the uPAR mRNABp, we next explored the possibility that post-transcriptional regulation of uPAR expression in epithelial cells involves activation of tyrosine kinases. We treated these cells with PBS, PMA, or TGF-ß alone or with the tyrosine kinase inhibitor, herbimycin A, for 24 h. uPAR expression at the cell surface was determined by Western blotting using an anti-uPAR antibody as described in Figure 1. Results of these experiments (Figure 6A) demonstrate that PMA and TGF-ß induce uPAR, and that pretreatment of these cells with that herbimycin reversed its induction by these agents.




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  		Figure 6. Inhibition of PMA- and TGF-ß–mediated urokinase receptor expression by herbimycin. (A) Beas2B cells grown to confluence were treated with PBS, PMA, or TGF-ß in the presence or absence of herbimycin for 24 h. The membrane proteins were separated and uPAR was analyzed by Western blot using anti-uPAR antibody. (B) Inhibition of PMA- and TGF-ß–mediated uPAR mRNA expression by herbimycin. Beas2B cells treated with or without PMA or TGF-ß in the presence or absence of herbimycin for 12 h. Total RNA was isolated and uPAR mRNA expression was measured by Northern blot using uPAR cDNA. (C) Herbimycin decreases PMA-mediated uPAR mRNA stability. Beas2B cells were treated with PBS or PMA in the presence or absence of herbimycin for 12 h. Ongoing transcription was then inhibited by treating the cells with DRB for varying time periods and uPAR mRNA was analyzed by Northern blot.

 
Inhibition of Tyrosine Phosphorylation Inhibits PMA and TGF-ß-Mediated uPAR mRNA Expression as Well as mRNA Stability
We also confirmed that herbimycin reversed PMA- and TGF-ß–mediated uPAR expression by inhibiting uPAR mRNA expression (Figure 6B). It is well known that PMA and TGF-ß enhance uPAR mRNA stability as well as transcription (14–16). We therefore analyzed uPAR mRNA stability in the presence of herbimycin and found that PMA enhanced uPAR mRNA stability, and that herbimycin reversed PMA-mediated stabilization of uPAR mRNA (Figure 6C).

Inhibition of the uPAR mRNA-uPAR mRNABp Interaction by Herbimycin
We next studied the effect of tyrosine phosphorylation on the PMA and TGF-ß-mediated uPAR mRNA-uPAR mRNABp interaction by gel mobility shift assay. Results of these experiments indicate that PMA and TGF-ß downregulate the uPAR mRNA–uPAR mRNABp interaction (Figure 7A). However, inactivation of tyrosine kinases by pretreatment of the cells with herbimycin A reversed the inhibitory effect of PMA and TGF-ß. Similar reversal of the uPAR mRNA and uPAR mRNABp interaction was also confirmed by Northwestern assay (Figure 7B). We also found that herbimycin reversed PMA- and TGF-ß–mediated tyrosine phosphorylation of the uPAR mRNABp by Western blotting (Figure 7C). In a separate experiment, we inhibited PMA-mediated tyrosine phosphorylation of uPAR mRNABp by pretreating the cells with herbimycin and the uPAR mRNABp was immunoprecipitated using an anti-uPAR mRNABp antibody. The immune complexes were separated and the tyrosine phosphorylation of uPAR mRNABp was analyzed by Western blotting using an anti-phosphotyrosine antibody. As shown in Figure 7D, pretreatment of Beas2B cells with herbimycin A inhibited PMA-mediated tyrosine phosphorylation of uPAR mRNABp (Figures 5A and 7B).





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  		Figure 7. (A) Effect of herbimycin on the uPAR mRNA and uPAR mRNABp interaction in Beas2B cells. Beas2B cells treated with PBS, PMA, or TGF-ß in the presence or absence of herbimycin for 24 h. Total cytosolic extracts were subjected to gel mobility shift assay using uPAR mRNA coding region transcripts. Fp, free probe. (B) Cytosolic proteins were subjected to Northwestern assay using a uPAR coding region mRNA transcript. (C) The same membrane as in B was stripped and reprobed with anti-phosphotyrosine antibody. (D) Inhibition of PMA-mediated tyrosine phosphorylation of uPAR mRNABp by herbimycin A. The cells were pretreated with PMA for 0–24 h in the presence of herbimycin A and the uPAR mRNABp was immunoprecipitated using an anti-uPAR mRNABp antibody. The membrane was developed using an antiphosphotyrosine antibody. M, molecular weight marker.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Synthesis of uPAR is controlled both at transcriptional and post-transcriptional levels by a variety of hormones and cytokines (14–16, 18). In previous studies, we and other investigative teams observed that PMA, TGF-ß, and cycloheximide increased uPAR mRNA stability in MS-1 cells (16) and other cell lines (14–15, 18). We have previously examined various aspects of post-transcriptional regulation in lung epithelial cells (22–24). The choice to examine PMA and TGF-ß effects in Beas2B cells was influenced by the fact that lung epithelial cell responses are germane to the pathogenesis of acute lung injury and repair. We therefore now extend these observations and demonstrate a newly recognized underlying mechanism by which these same stimuli can induce uPAR expression at the post-transcriptional level in Beas2B cells. We also confirm that these responses involve a post-transcriptional regulatory component.

Expression of uPAR at the cell surface is germane to a variety of cellular responses involved in the pathogenesis of cancer and inflammation. The interaction between uPA and uPAR at the cancer cell surface appears to influence neoplastic growth and metastasis by mediating tissue remodeling, tumor cell invasion, adhesion, and proliferation (1, 28–33). Epithelial-derived tumor cell invasion is also facilitated by saturation of uPAR with either exogenously supplemented uPA or overexpressed endogenous uPA (34–39). In addition, the binding of uPA to uPAR mediates cellular proteolysis and cell proliferation in several cell types, including nonmalignant and malignant epithelial cells and mesothelioma cells (21, 26, 29–30, 32). Post-transcriptional regulation of uPAR could therefore influence a wide range of pathophysiologic responses. We chose Beas2B cells as an in vitro lung epithelial cell model system to further elucidate mechanisms that regulate uPAR expression.

It has previously been reported that uPAR expression as well as the specific capacity to bind uPA is increased by PMA, EGF, LPS, TGF-ß, and TNF-{alpha} in various cell lines (14–15, 24, 30). These agents strongly and rapidly induce cell surface uPAR expression, an effect that can be traced back to a rapid, antecedent increase in the cellular level of uPAR mRNA. PMA increases the stability of uPAR mRNA in Beas2B cells, a phenomenon consistent with that we previously reported in MS-1 cells (16). We found that cytokine-induced uPAR was regulated at the post-transcriptional level in these cells (14, 16). The increased uPAR mRNA stability in lung-derived epithelial and mesothelioma cells correlates with increased uPAR mRNA and cell surface expression of uPAR. There are precedents for this mode of regulation. Lymphocyte engagement, for example, also stabilizes uPAR mRNA, a process that involves AU-rich sequences present in the uPAR 3'untranslated region (3'UTR) (18).

Our data now demonstrate that post-transcriptional regulation of uPAR expression further involves activation of tyrosine kinases. The sustained effect of proinflammatory agents on both uPAR protein and mRNA levels suggested the likelihood that tyrosine kinase signaling prolongs the half-life of uPAR mRNA. Supporting this inference, we found that pretreatment of cells with herbimycin reverses the PMA- or TGF-ß-mediated increase of uPAR expression. Similarly, we found that genestein, a tyrosine kinase inhibitor, also decreased uPAR expression, whereas the tyrosine phosphatase inhibitor sodium orthovanadate potentiated uPAR expression in resting Beas2B lung epithelial cells and also has an additive effect with uPA (40). Even though our present experiments do not directly demonstrate the rate of dephosphorylation of the uPAR mRNABp in Beas2B cells, orthovanadate-potentiated expression of uPAR in resting and uPA-stimulated Beas2B cells clearly suggests that a balance between ongoing dephosphorylation and phosphorylation of the uPAR mRNABp contributes to maintenance of the level of uPAR expression. We also found that herbimycin pretreatment decreased uPAR expression by reducing the stability of uPAR mRNA. This effect was achieved through increased interaction between uPAR mRNA and the uPAR mRNABp. These observations extend our previous reports and show that the post-transcriptional regulation of uPAR by assembly of this regulatory complex is controlled by tyrosine phosphorylation of the uPAR mRNABp. Translational inhibition analyses in Beas2B cells indicated that the uPAR mRNABp is very stable (S. Shetty, unpublished results), suggesting that functional alteration rather than degradation of the uPAR mRNABp probably plays the principal role in post-transcriptional regulation of uPAR expression. Proinflammatory cytokines implicated in the pathogenesis of inflammatory lung disease and lung neoplasia can increase uPAR expression of Beas2B cells by inducing phosphorylation of the uPAR mRNABp, thereby disrupting the uPAR mRNA–uPAR mRNABp complex. These responses in turn lead to increased expression of uPAR at the cell surface.

Previous reports confirm that inactivation of MAP kinases with the specific inhibitor SB203580 also inhibited cellular expression of uPA and uPAR (41–42), PMA-induced matrix metalloproteinase-9 (43), or TNF-{alpha} (44), indicating the involvement of these signaling intermediates in expression of genes implicated in tissue remodeling. These studies suggest the possibility that mitogen-activated protein kinase signal transduction is involved in 3'UTR AU-rich element–mediated mRNA stability. Given that uPAR expression may involve transcriptional or translational control as well as cycling of the receptor from the cell surface (1), our present observations suggest the possibility that there could be mechanistic overlap involving signaling at these levels as well. The uPAR mRNABp interacts with a 5' coding sequence to stabilize uPAR mRNA and increase uPAR expression at the cell surface (24). Although it is now clear that tyrosine phosphorylation of the uPAR mRNABp blocks its ability to interact with the uPAR mRNA binding sequence, it is unclear if this pathway is operative in all forms of cytokine-induced expression of uPAR in Beas2B cells. The precise intermediates involved in the signaling mechanism that controls the phosphorylation (activation) state of the uPAR mRNABp remain to be elucidated.

In summary, we confirmed that proinflammatory agents regulate uPAR mRNA expression at the post-transcriptional level in cultured Beas2B lung epithelial cells. This pathway involves tyrosine phosphorylation of uPAR mRNABp by the proinflammatory agents. This newly recognized mechanism represents yet another pathway by which uPAR-dependent responses of the lung epithelium may be controlled in the context of lung injury and repair, neoplastic transformation or in the growth and spread of lung neoplasms.


   Acknowledgments
 
This work was supported by grants from the NHLBI Grants R01-HL-62453, R01-HL71147, and R01-HL45018. The authors are grateful to Kathy Johnson, Brad Low, and M. B. Harish for technical assistance.

Received in original form December 17, 2002

Received in final form June 17, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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