Regulation of urokinase receptor expression by phosphoglycerate kinase is independent of its catalytic activity
Sreerama Shetty,
Malathesha Ganachari,
Ming-Cheh Liu,
Ali Azghani,
Harish Muniyappa, and
Steven Idell
Department of Medicine, University of Texas Health Center at Tyler, Tyler, Texas
Submitted 25 August 2004
; accepted in final form 30 May 2005
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ABSTRACT
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Posttranscriptional regulation of urokinase-type plasminogen activator receptor (uPAR) mRNA involves the interaction of a uPAR mRNA coding region sequence with phosphoglycerate kinase (PGK), a 50-kDa uPAR mRNA binding protein. PGK catalyzes a reversible transfer of a phosphoryl group from 1,3-biphosphoglycerate to ADP in the glycolytic pathway. Our previous studies showed that overexpression of PGK in uPAR-overproducing H157 lung carcinoma cells results in decreased cytoplasmic uPAR mRNA and cell surface uPAR protein expression through destabilization of the mRNA. In order to determine the role of PGK enzymatic activity on uPAR mRNA stability we mutated PGK by changing amino acid P204H and amino acid D219A. The mutant proteins were expressed in Epicurian coli BL21 cells, and the purified proteins were analyzed for PGK activity. We found that mutation of amino acid P204H and D219A reduced PGK activity by 99 and 83%, respectively. By gel mobility shift and Northwestern assay, we found that the mutant proteins were able to bind to uPAR mRNA as effectively as wild-type PGK. Overexpression of mutant, inactive PGK in H157 cells reduced cell surface uPAR protein as well as uPAR mRNA expression. Run-on transcription analysis indicated that overexpression of mutant PGKs fails to alter the rate of synthesis of uPAR mRNA, whereas transcription chase experiments demonstrated that both mutants and wild-type PGK reduce the stability of the uPAR mRNA transcripts to a similar extent. Overexpression of mutant PGK also inhibited the rate of DNA synthesis and the invasion-migration ratio. These results demonstrate that uPAR mRNA binding activity as well as PGK-mediated regulation of uPAR mRNA are independent of PGK enzymatic activity.
messenger ribonucleic acid stability; messenger ribonucleic acid binding proteins
SUPPORT FOR THE INVOLVEMENT of urokinase-type plasminogen activator (uPA)-dependent plasminogen activation in lung injury and repair or lung neoplasia has been steadily increasing (19). Because many biological activities of uPA depend on association with urokinase-type plasminogen activator receptor (uPAR), this receptor plays a central role in localized uPA-mediated plasminogen activation at the cell surface. Increased expression of uPA or uPAR has, for example, been inversely correlated with the pathogenesis of lung injury and lung neoplasia (8, 10, 11, 16, 22, 44). Interaction of uPA with its receptor also promotes cellular signaling, which can thereby influence the course of lung inflammation or cancer. Better understanding of the specific pathways that regulate uPAR expression is therefore germane to the pathogenesis of lung injury or the growth of lung neoplasms.
The expression of a number of proteins is regulated by specific and rapid decay of their transcripts. The steady state of any mRNA in turn reflects a balance between its synthesis and degradation. Among the various mechanisms that regulate mRNA stability, control of mRNA decay is a potentially important determinant of the level of gene expression in eukaryotic cells. Synthesis of uPAR is regulated by a variety of hormones, growth factors, and cytokines at either the transcriptional or posttrancriptional level (3, 17, 18, 30, 39, 42, 46, 47). The responsible mechanisms involve cis-trans interactions that involve the 3'-untranslated region (UTR) or the coding region of the specific mRNA.
uPA regulates diverse functions, such as cell adhesion, signaling, and mitogenesis, and most of the biological activities of uPA are dependent on its association with the uPAR (24, 13, 36, 40, 44, 45, 48). It is noteworthy that squamous cell lung carcinoma is characterized by elevated uPAR expression and inhibition of uPAR reduces proliferation as well as migration in H157 cells (37, 38). Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate. This protein contains a common nucleotide binding domain with unique catalytic domains. We previously reported that PGK specifically binds to the coding region (CDR) of uPAR mRNA and consequently inhibits cell surface uPAR expression (41). In this study, we observed that purified PGK mutant proteins lacking catalytic activity specifically bound to the uPAR mRNA CDR. We also found that overexpression of PGK mutant proteins in H157 lung squamous carcinoma cells reduced cell surface uPAR expression and inhibited uPAR-mediated cellular functions. These effects were mediated at the posttranscriptional level. The inhibitory effects of the mutant proteins were comparable to those of wild-type PGK, as we previously described (41).
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EXPERIMENTAL PROCEDURES
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Materials.
Culture media, penicillin, streptomycin, and fetal calf serum (FCS) were purchased from GIBCO-BRL Laboratories (Grand Island, NY); Tissue culture plastics were from Becton Dickinson Labware (Lincoln Park, NJ). Bovine serum albumin (BSA), ovalbumin, Tris-base, aprotinin, dithiothreitol (DTT), phenylmethylsulfonyl fluoride, and ammonium persulfate were from Sigma Chemical (St. Louis, MO). QuikChange site-directed mutagenesis kit and XL1-Blue Epicurian coli-competent cells were purchased from Stratagene (La Jolla, CA). Oligonucleotide primers were synthesized by MWG Biotech (High Point, NC). pGEX-2TK glutathione S-transferase (GST) gene fusion vector, E. coli BL21 host cells, and glutathione-Sepharose were products of Amersham Biosciences (Piscataway, NJ). Acrylamide, bisacrylamide, and nitrocellulose were from Bio-Rad Laboratories (Richmond, CA). Isopropyl
-D-1-thiogalactoside (IPTG) was purchased from Gold Biotechnology (St. Louis, MO). Anti-uPAR antibody was obtained from American Diagnostics (Greenwich, CT). XAR X-ray film was purchased from Eastman Kodak (Rochester, NY).
Cell culture.
Human lung squamous cell carcinoma (H157) cells were maintained in RPMI-1640 containing 10% heat-inactivated FCS, 1% glutamine, and 1% antibiotics as previously described (40).
Plasmid construction.
Plasmid uPAR/pBluescript was obtained from ATCC. The human uPAR mRNA template containing a complete sequence of uPAR cDNA (nucleotides 16 to 1144) from uPAR pBluescript was subcloned to HindIII and XbaI sites of pRC/CMV (Invitrogen), and the sequences of the clones were confirmed by sequencing.
In vitro transcription.
Linearized plasmids containing the human uPAR mRNA transcriptional templates of uPAR cDNA were transcribed in vitro with T7 or Sp6 polymerase (Ambion, TX). The uPAR mRNA transcripts were synthesized according to the supplier's protocol except that 50 µCi of [32P]UTP were 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 cpm/µg.
Molecular cloning, expression, and purification of the wild-type PGK.
The coding sequence of wild-type PGK was PCR-amplified using a previously cloned full-length cDNA packaged in pcDNA 3.1 plasmid, in conjunction with sense (CGC GGA TCCATG TCG CTT TCT AAC AAG CTG) and antisense (CGC GGA TCC CTA AAT ATT GCT GAG AGC ATC CA) oligonucleotide primers designed based on 5'- and 3'-region of the open reading frame. The 1272-base pair PCR product, purified upon agarose electrophoresis, was restricted with the BamHI enzyme and subcloned into the BamHI site of pGEX-2TK prokaryotic expression vector and transformed into E. coli BL21. The cDNA insert was then subjected to nucleotide sequencing to verify its authenticity.
Competent E. coli BL21 cells, transformed with pGEX-2TK harboring the PGK cDNA, were grown to 600-nm optical density =
0.5 in 1 l of Luria Bertani (LB) medium supplemented with 100 µg/ml ampicillin and induced with 0.1 mM IPTG. After an overnight induction at room temperature, the cells were collected by centrifugation and homogenized in 20 ml of ice-cold lysis buffer (20 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 1 mM EDTA) using an Aminco French Press. Twenty microliters of 10 mg/ml aprotinin (a protease inhibitor) were added to the crude homogenate. The crude homogenate was centrifuged at 10,000 g for 30 min at 4°C. The supernatant was fractionated with 0.5 ml of glutathione-Sepharose, and the bound GST fusion protein was treated with 2 ml of a thrombin digestion buffer (50 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2) containing 5 U/ml bovine thrombin. After a 30-min incubation at room temperature with constant agitation, the preparation was subjected to centrifugation. The supernatant containing recombinant PGK was collected and subjected to SDS-PAGE and enzymatic analysis as described below.
Generation, expression, and purification of point-mutated PGKs.
The QuikChange site-directed mutagenesis kit from Stratagene was used for the generation of point-mutated PGKs, P204H, and D219A. In brief, wild-type PGK cDNA packaged in pGEX-2TK prokaryotic expression vector was used as the template in conjunction with specific mutagenic primers. To prepare the P204H mutant, the mutagenic oligonucleotide primer set 5'-GCA AAG GCC TTG GAG AGC CAC GAG CGA CCC TTC CTG GCC-3' and 5'-GGC CAG GAA GGG TCG CTC GTG GCT CTC CAA GGC CTT TGC-3' was used. Similarly, the D219A mutant was prepared using the mutagenic primer set 5'-GGC GGA GCT AAA GTT GCA GCC AAG ATC CAG CTC ATC AAT-3' and 5'-ATT GAT GAG CTG GAT CTT GGC TGC AAC TTT AGC TCC GCC-3'. The amplification conditions were 12 cycles of 30 s at 95°C, 1 min at 55°C, and 15 min at 68°C. The P204H and D219A mutant sequences were verified by nucleotide sequencing (23). pGEX-2TK vector harboring P204H or D219A mutant sequence was transformed into competent BL21 E. colicells. The transformed cells, grown to an optical density of 600 nm =
0.5 in 1 l of LB medium supplemented with 100 µg/ml ampicillin and induced with 0.1 mM IPTG overnight at room temperature, were collected by centrifugation and processed for the purification of recombinant PGK mutant enzyme by the same procedure for the wild-type PGK as described above and subjected to PGK catalytic activity assay as described below.
3-PGK activity assay.
3-PGK activity was determined spectrophotometrically following the oxidation of
-NADH at an optical density of 340 nm. The activity of the 3-PGK was assayed in the reverse direction of glycolysis in conditions described by Adam (1). The final volume of the assay mixture containing 20 mM Tris · Cl, pH 7.4 and 1 mM ATP was 1.5 ml. The final volume of the assay mixture was 1.5 ml. Controls contained all additions except 3-phosphoglycerate. The baseline was monitored at an optical density of 340 nm until constant. One unit of 3-PGK is defined as the amount of enzyme that converts 1.0 µmol of 3-phosphoglycerate to 1,3-bisphosphoglycerate per minute at 45°C under the assay conditions described above, assuming that the ratio of
-NADH oxidized to 3-phosphoglycerate utilized is unity.
Transfection of H157 cells.
The H157 cells were transfected with or without wild-type or mutant PGK cDNAs in pcDNA3.1D/V5-His-TOPO or empty vector by lipofection, as we previously described (41). Stable cell lines were generated by antibiotic selection, after which the cells were cultured in large amounts and expression was confirmed by Western blotting using an anti-V5 antibody.
Gel mobility shift assay.
Eluates purified from a glutathione-Sepharose column of lysates of bacterial cells transfected with cDNAs coding for wild-type or mutant PGK or human plasminogen activator inhibitor (PAI)-1 mRNA binding protein or zebra fish sulfotransferase proteins were incubated with 2 x 104 cpm of 32P-labeled transcript corresponding to the 51-nt uPAR mRNA CDR determinant (41) in a mixture containing 15 mM KCl, 5 mM MgCl2, 0.25 mM DTT, 12 mM HEPES (pH 7.9), 10% glycerol, and E. coli tRNA (200 ng/µl) in a total volume of 20 µl at 30°C for 30 min. The reaction mixture were treated with 50 units of RNase T1 and incubated at 37°C for 30 min. To avoid nonspecific binding, we added heparin (5 mg/ml) per ml, and the mixture was incubated at room temperature for an additional 10 min. Samples were separated by electrophoresis on a 5% native polyacrylamide gel using 0.25x Tris-borate-EDTA running buffer. The gels were dried and developed by autoradiography at 70°C.
Northwestern assay.
We alternatively confirmed the uPAR mRNA-PGK interaction by Northwestern assay. Protein eluates from the Nickel column were separated on 8% SDS-PAGE and then blotted to a nitrocellulose membrane. The membrane was blocked with gel shift buffer containing 1% BSA and 20 µg of rRNA for 1 h. The membrane was replaced with fresh buffer containing the same 32P-labeled uPAR mRNA CDR determinant used in the gel shift analyses described above (2 x 105 cpm/ml) and was incubated for an additional 1 h at room temperature (41). The membrane was later washed three times with 50 ml of gel shift buffer for 10 min each, air-dried, and exposed to X-ray film. The membrane was later stripped and developed by Western blotting using an anti-PGK polyclonal antibody as a control for equal loading. In separate experiments, we also performed UV cross-linking assays using 32P-labeled uPAR 51 nt. uPAR mRNA CDR determinant (41).
Total cellular membrane extraction and Western blotting.
Polyclonal stable H157 cells of PGK or vector cDNA transfected cells grown to confluence were serum starved overnight with RPMI-glutamine media containing 0.5% BSA. The cells were washed with PBS, and receptor-bound uPA was removed by glycine-HCl treatment as previously described (36, 40). We used SDS gel electrophoresis and Western blotting to measure functional uPAR at the cell surface. Membrane proteins were isolated as described (37, 38), 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 and washed, and uPAR proteins were detected by enhanced chemiluminescence. The same membrane was stripped and developed with anti-
-actin antibody.
Random priming of uPAR cDNA.
The full-length template of uPAR was released with HindIII or XbaI, 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. H157 cells transfected with or without various PGK cDNA constructs grown to confluence were serum starved overnight in RPMI-BSA media. Total RNA was isolated with 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: 2x SSC, 1% SDS; 1x 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
-actin.
We also measured the stability of uPAR mRNA in these cell lines by transcription chase experiments as we previously described (39).
DNA synthesis assay.
H157 and stable cell lines transfected with or without wild-type or mutant PGK cDNAs in pcDNA3.1 or vector alone were grown to subconfluence in 24-well plates. The cells were serum starved overnight in RPMI containing 0.5% BSA. [3H]thymidine (1 µCi/ml, 20.3 mmol Ci) was later added to the same media and incubated for an additional 6 h. The cells were washed with ice-cold PBS three times and five times with ice-cold 5% TCA. The cells were lysed in 0.2 N NaOH, and the incorporated [3H]thymidine was then counted with a scintillation counter.
Cell migration-invasion assay.
Cell migration assays were performed in modified Boyden chambers with 6.5 mm diameter, 10 µm thickness, and a porous (8.0 µm) polycarbonate membrane separating two chambers (Transwell; Costar, Cambridge MA). Cells were added at a final concentration of 10,000 cells to upper chamber in RPMI (10% FCS). The cells were incubated for 72 h at 37°C. At the end of the assay the cells from the upper and lower chamber were detached by trypsin/EDTA. The cells that migrated to the lower chamber and those remaining in the upper chamber were counted with a Coulter counter. The percentage of cells in the upper and lower chambers was calculated based on the total number of cells present in these chambers. The invasion-migration ratio was calculated as a ratio of the percentage of cells in the lower chamber vs. the upper chamber, as we previously described (37, 41).
Statistical analysis.
In selected experiments, we evaluated differences between wild-type PGK, mutant PGK cDNA transfected H157 cells, and corresponding vector (pcDNA3.1) cDNA transfected controls by Student's t-test. Data were evaluated in a randomized block analysis of variance.
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RESULTS
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Cloning and expression of mutant PGK.
The PGK coding region and a 1272-nt clone were amplified by PCR, and the product was sequenced to confirm its orientation. This product was later subcloned in a prokaryotic expression pET vector. PGK mutants (P204H and D219A) were generated by PCR-based site directed mutagenesis. The wild-type and two mutant constructs were transfected into prokaryotic (BL21) cells, and the overexpressed mutant fusion proteins were affinity purified. The proteins were later subjected to SDS-PAGE and developed by Coomassie staining (Fig. 1A). We assayed the specific activities of the wild-type and mutant PGK proteins by measuring the absorbance of NADH at 340 nm spectrophotometrically. Mutations at PGK P204H and D219A reduced the PGK activity by 93 and 83%, respectively (Fig. 1B).

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Fig. 1. Expression and purification of wild-type and mutant phosphoglycerate kinase (PGK). A: recombinant wild-type and mutant PGK expressed in bacteria were affinity purified with a glutathione-Sepharose column. The purified proteins were separated on SDS-PAGE and stained with Coomassie blue. Lanes: PGK Wt, wild-type PGK; PGK Mt1 and PGK Mt2, mutant PGK. B: corresponding samples were subjected to PGK enzymatic activity analyses. Specific activity of the wild-type PGK is expressed as 100%.
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uPAR mRNA binding activity by gel mobility shift assay.
Wild-type and mutant PGK proteins were subjected to gel mobility shift and Northwestern assays to confirm uPAR mRNA binding activity. The wild-type as well as mutant PGK proteins bound to uPAR mRNA as determined by gel mobility (Fig. 2A) and Northwestern assays (Fig. 2C), whereas a 50-kDa recombinant human PAI-1 mRNA binding protein or similarly sized sulfotransferase protein from zebra fish failed to interact with uPAR mRNA (Fig. 2B), indicating that PGK-uPAR mRNA forms a specific complex with uPAR mRNA. The specificity of the interaction of PGK with its uPAR mRNA CDR determinant has also been described in our previous report (41).

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Fig. 2. Urokinase receptor mRNA binding activity of recombinant wild-type as well as mutant PGK by gel mobility shift assay and Northwestern analyses. A: urokinase-type plasminogen activator receptor (uPAR) mRNA binding activity demonstrated by gel mobility shift assay. Bacterial lysates from isopropyl -D-1-thiogalactoside (IPTG)-induced BL21 cells transfected with PGK cDNAs in pET vector. PGK protein was purified from prokaryotic expression vector with a glutathione-Sepharose column. Five micrograms of each protein were used. A: PGK protein purified from cell extracts of bacterial cells transfected with PGK Wt cDNA or PGK Mt1 or Mt2 and 32P-labeled uPAR mRNA alone (Fp). Arrow indicates uPAR mRNA-PGK complex. The data are illustrative of the findings in 5 independent experiments. B: specificity of PGK interaction with uPAR mRNA by gel mobility shift assay. PGK Wt, plasminogen activator inhibitor (PAI)-1 mRNA binding protein (PAI-1 mRNABp), and Zebra fish sulfotransferase (S-transferase) proteins purified on a glutathione-Sepharose column were subjected to uPAR mRNA binding. Arrow indicates uPAR mRNA-PGK complex, 32P-labeled uPAR mRNA alone (Fp). C: corresponding proteins (from A) were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were later developed by Northwestern assay using 32P-labeled uPAR mRNA coding region transcript and autoradiography. Protein eluate of extracts of cells transfected with PGK Wt cDNA or PGK Mt1 and 2 was purified on a glutathione-Sepharose column and incubated with [32P]uPAR mRNA. The PGK-bound uPAR mRNA was detected by autoradiography. The data are illustrative of the findings in 6 independent experiments.
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Effect of mutant PGK on cell surface uPAR expression.
Because mutant PGK proteins lacking enzymatic activity specifically interacted with uPAR mRNA, we next wanted to determine whether this interaction regulates cell surface uPAR expression. To confirm our hypothesis that it does, we cultured stable H157 cells transfected with empty vector alone or wild-type or mutant PGK cDNAs in eukaryotic expression vector pcDNA3.1 separately in culture dishes. We also used H157 cells without any transfection as controls. As shown in Fig. 3A, transfection of mutant PGK cDNAs reduced the cell surface uPAR expression compared with vector transfected or control naïve H157 cells. Similarly, mutant PGK also inhibited cell surface uPAR expression as much as wild-type PGK. These data strongly suggest the likelihood that the inhibitory effect of PGK on cell surface uPAR expression in H157 cells is independent of PGK enzymatic activity.

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Fig. 3. Inhibition of uPAR and mRNA expression by PGK in H157 cells. A: urokinase receptor expression in wild-type and mutant PGK-transfected H157 cells. Control H157 cells (H157) or stable H157 cells transfected with pcDNA 3.1 alone (pcDNA3.1) or PGK Wt cDNA or PGK Mt1 or Mt2 in pcDNA 3.1 were grown to confluence. The membrane proteins isolated from these cells were separated on 8% SDS-PAGE and electroblotted to nitrocellulose membranes. The membranes were subjected to Western blotting with a urokinase receptor monoclonal antibody. The data shown are a representative of 5 independent experiments. Error bars represent SE. B: urokinase receptor mRNA expression in PGK-transfected H157 cells. H157 cell lines as described in A were grown to confluence in RPMI-1640 with or without G418. Total RNA was isolated and uPAR mRNA was measured by Northern blot using 32P-labeled uPAR cDNA. The data illustrated are representative of 4 independent experiments. Error bars represent SE.
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We next sought to confirm our inference that downregulation of cell surface uPAR expression in H157 cells occurs via regulation of cellular uPAR mRNA. To confirm this inference, we next determined the effect of PGK on uPAR mRNA expression in these cells. As shown in Fig. 3B, H157 cells overexpressing wild-type and mutant PGK cDNAs significantly (P < 0.05) inhibited uPAR mRNA expression compared with control H157 cells or H157 cells transfected with empty vector. These data show that PGK-mediated inhibition of cell surface uPAR expression is due to decreased uPAR mRNA expression. However, our experiments do not exclude the possibility that associated changes in the half-life of the uPAR protein could contribute to the findings.
Effect of PGK on regulation of uPAR mRNA expression.
We next wanted to determine whether PGK-mediated inhibition of uPAR mRNA expression in H157 cells is due to decreased mRNA synthesis or enhanced mRNA degradation. We therefore cultured these cell lines in T170 flasks and isolated intact nuclei that were then subjected to nuclear run-on assays (38, 41). The results of nuclear run-on assays indicate that both mutant as well as wild-type PGK failed to alter the rate of uPAR mRNA transcription in PGK cDNA transfected cells (Fig. 4). Because both wild-type and mutant PGK proteins bind to uPAR mRNA and PGK overexpression did not alter the rate of transcription, we inferred that PGK probably regulates uPAR expression at the posttranscriptional level. To confirm this inference, we next treated confluent H157 cells with 5,6-dichloro-1
-D-ribofuranosylbenzamidazole (10 µg/ml) to inhibit ongoing transcription and analyzed uPAR mRNA expression by Northern blotting. As shown in Fig. 5, uPAR mRNA is quite stable in vector cDNA (pcDNA3.1)-transfected or naïve H157 cells with a half-life of 68 h. Conversely, uPAR mRNA was degraded significantly faster in wild-type (pcDNA3.1 vs. wild-type PGK, P < 0.01) and mutant PGK (pcDNA3.1 vs. mutant 1ormutant 2 PGK, P < 0.05)-overexpressing cells.

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Fig. 4. Effect of PGK on the rate of transcription of uPAR mRNA in H157 cells. Nuclei isolated from H157 cell (H157) or stable cell lines transfected with empty vector (pcDNA 3.1) or PGK Wt or PGK Mt1 or Mt2 cDNAs in pcDNA 3.1 as described above were subjected to the transcription reaction in the presence of [32P]UTP at 30°C for 30 min. 32P-labeled nuclear RNA was hybridized with uPAR cDNA immobilized on nitrocellulose membrane. -Actin and plasmid University of California (pUC) 18 cDNAs were used as positive and negative loading controls, respectively. The experiments were repeated 4 times, and a representative experiment is illustrated.
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Fig. 5. Effect of PGK on uPAR mRNA stability. Ongoing transcription of H157 cells (H157), or stable cell lines transfected with empty vector (pcDNA 3.1) or PGK Wt or PGK Mt1 or Mt2 cDNAs in pcDNA 3.1 as described above was inhibited by treating the confluent cells with 5,6-dichloro-1 -D-ribofuranosylbenzamidazole (10 µg/ml) for 0, 3, 6, 12, and 24 h in the same medium. Total RNA was isolated, and uPAR was analyzed by Northern blot. Densitometric scanning of an individual experiment is shown by line graph. Experiments were repeated at least 4 times.
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Effect of PGK activity on proliferation and migration-invasion.
Because cellular proliferation of H157 cells is influenced by differences in uPAR expression (37, 38), we next assessed the effect of mutant or wild-type PGK overexpression on the rate of proliferation of H157 cells by [3H]thymidine incorporation. [3H]thymidine uptake by subconfluent monolayers of the cells transfected with vector alone or wild-type or mutant PGK cDNAs was compared with control H157 cells. As shown in Fig. 6A, mutant PGK overexpression significantly (P < 0.05) inhibited [3H]thymidine uptake in H157 cells compared with vector-transfected or control H157 cells. We also found a significant difference (P < 0.05) in [3H]thymidine uptake between wild-type PGK and mutant 2 PGK (D219A) overexpressed in H157 cells. This observation indicates that PGK inactivation achieved by replacement of aspartic acid by alanine could decrease DNA synthesis. Having confirmed that inactive PGK regulates proliferation of H157 cells, we lastly studied these cells in invasion-migration assays, which are likewise influenced by cellular uPAR expression (37, 38, 41). As shown in Fig. 6B, mutant PGK-overexpressing cells exhibited decreased migration compared with control cells used in the assays.

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Fig. 6. A: inhibition of H157 cells [3H]thymidine uptake by PGK. H157 cell lines were serum starved overnight and treated with [3H]thymidine for 6 h. The cells were washed, and the rate of DNA synthesis was determined by measuring [3H]thymidine incorporation. The data from 4 independent experiments are illustrated, and bars represent SE. B: effect of PGK expression on invasion-migration ratio of H157 cells. The H157 cells grown to confluence were transferred to the upper chamber of Transwell plates for assessment in the invasion-migration assay. The cells were incubated for 72 h at 37°C, after which the number of cells present in the upper and the lower chambers were counted, and the invasion-migration ratios were calculated based on the total number of cells present in both the upper and lower chambers. The mean data from 4 independent experiments are illustrated, and bars represent SE.
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DISCUSSION
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The uPA and uPAR interaction is involved in the proteolytic cascade required for mediating tissue remodeling, tumor cell invasion, and metastasis (8, 34). These are highly expressed in many human tumors. We and other investigators previously reported that uPA is able to increase the expression of uPAR at the posttranscriptional level (23, 38). Many biological activities of uPA depend on association with its receptor. uPAR plays a principal role in localizing uPA-mediated plasminogen activation and cellular signaling (9, 19, 43). The uPAR is also involved in the regulation of cell adhesion and migration independently of the enzymatic activity of its ligand (4). In addition, outcome studies in cancer patients have shown that high levels of either uPA and uPAR in tumors correlate with poor prognosis (28, 29). Therefore, regulation of uPAR could thus influence a broad range of uPA-mediated effects on cellular pathophysiology that are relevant to the pathogenesis of solid tumors such as lung carcinomas.
We previously reported that overexpression of uPAR appears to increase the invasiveness of lung carcinomas, which could strongly influence their clinical behavior (28, 29). Expression of uPAR by nonmalignant lung epithelial and carcinoma cells is now known to be controlled both at transcriptional and posttranscriptional levels by a variety of hormones, cytokines, and hypoxia (17, 18, 23, 39, 46). We recently confirmed that PGK specifically binds to uPAR mRNA and regulates uPAR expression at the posttranscriptional level (41). PGK is a ubiquitous cellular enzyme that catalyzes the reversible transfer of the acyl phosphate group of 1,3-bisphosphate-D-glycerate to Mg-ADP, resulting in the formation of 3-phospho-D-glycerate and Mg-ATP (21). The reaction forms part of the glycolytic and gluconeogenic pathways. We now extend our observations to further elucidate whether PGK enzymatic activity is involved in the regulation of uPAR expression in these cells. The catalytic mechanism of PGK involves a hinge-bending domain motion that brings the substrates together to allow phosphoryl transfer (14, 20). Site-specific mutants (14, 20) were produced to investigate the role of PGK activity on uPAR mRNA binding and its ability to regulate cell surface uPAR expression. The residue was replaced by a histidine (Pro204His) and an alanine (D219A), and the resulting proteins lacked PGK enzymatic activity.
In a previous report, we showed that PGK interacts with uPAR mRNA and regulates its cell surface expression by regulating the stability of the mRNA (41). Proinflammatory agents that are known to enhance uPAR mRNA stability (11) induce tyrosine phosphorylation of PGK without altering its basal level of expression (35). Inhibition of tyrosine phosphorylation by a tyrosine kinase inhibitor decreases uPAR expression by destabilization of uPAR mRNA. Our data now show that the interaction of PGK with uPAR mRNA does not require PGK enzymatic activity. Overexpression of cell surface uPAR in H157 cells is correlated with the lower affinity of endogenous uPAR mRNA to PGK (41), indicating that PGK-mediated posttranscriptional regulation of uPAR could influence a wide range of pathophysiological responses germane to either lung inflammation or cancer.
The increased cell surface uPAR expression in lung-derived epithelial and mesothelioma cells correlates with increased uPAR mRNA stability. 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'-UTR (46). The present study is the first to elucidate the role of PGK that is independent of its enzymatic activity in the regulatory mechanism that controls uPAR expression at the posttranscriptional level. In addition, mutant PGK regulates the destabilization of uPAR mRNA. Its overexpression decreases uPAR expression at the cell surface and influences pathophysiological responses that are likewise contingent on the expression of this receptor by H157 lung carcinoma cells.
Our results provide a novel example of a functional and mRNA binding activity, linking newly recognized mRNA binding by PGK independently of its previously known glycolytic enzyme activity (14, 20, 21). The physiological relevance of the capacity of PGK to exert bifunctional roles in both uPAR mRNA decay and glycolysis remains an enigma at this time. Indeed, many other proteins involved in the regulation of the turnover and translation of mRNA may serve additional and quite disparate roles, as further exemplified by glutamate dehydrogenase, NAD+-dependent isocitrate dehydrogenase, thymidylate synthase, dihyrofolate reductase, catalase, thiolase, glyceraldehyde phosphate dehydrogenase, lactate dehydrogenase, and tumor suppressor protein p53 (57, 12, 15, 2427, 3133).
In summary, we confirmed that both catalytically active as well as inactive PGK regulates uPAR mRNA expression at the posttranscriptional level. This newly recognized pathway involves interaction of PGK with the uPAR mRNA CDR and regulates expression of uPAR at the cell surface. This mechanism represents a 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.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-62453 and R01-HL-71147.
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ACKNOWLEDGMENTS
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We are grateful to Brad Low and Dr. Ruth Zang for technical assistance.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. Shetty, Univ. of Texas Health Center at Tyler, 11937 US Hwy. 271, Lab C-6, Tyler, TX 75708 (e-mail: sreerama.shetty{at}uthct.edu)
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