A urokinase receptor mRNA binding protein from rabbit lung fibroblasts and mesothelial cells

Sreerama Shetty and Steven Idell

Department of Medical Specialties, The University of Texas Health Center at Tyler, Tyler, Texas 75710

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The urokinase receptor (uPAR) influences several biological functions relevant to lung injury and repair, including proteolysis, cell migration, and adhesion. In malignant mesothelioma cells, we recently found that a posttranscriptional mechanism involving a cis-trans interaction between a uPAR mRNA sequence and a cytoplasmic uPAR mRNA binding protein (mRNABP) regulates uPAR gene expression (S. Shetty, A. Kumar, and S. Idell. Mol. Cell Biol. 17: 1075-1083, 1997). In this study, we sought to determine if uPAR expression in lung and pleural cells involves a similar posttranscriptional pathway. We first identified and characterized the uPAR mRNABP in rabbit tissues using gel mobility shift, ultraviolet (UV) cross-linking, and RNase protection assays and detected it in liver, heart, brain, spleen, colon, and lung. Phorbol 12-myristate 13-acetate, lipopolysaccharide, transforming growth factor-beta , tumor necrosis factor-alpha , or cycloheximide induced uPAR and uPAR mRNA expression in cultured rabbit pleural mesothelial cells and lung fibroblasts and concurrently reduced the uPAR mRNA-uPAR mRNABP interaction. Using conventional and affinity chromatography, we purified a 50-kDa uPAR mRNABP that selectively binds to a 51-nucleotide fragment of the uPAR coding region. This protein migrates as a monomer when analyzed by SDS-PAGE and UV cross-linking and does not possess intrinsic RNase activity in vitro. A uPAR mRNABP physicochemically and functionally similar to that of human malignant mesothelioma is constitutively expressed in the rabbit lung and other nonneoplastic tissues. In rabbit lung fibroblasts and mesothelial cells, expression of uPAR involves posttranscriptional regulation whereby the uPAR mRNABP appears to interact with a specific coding region cis-element to decrease the stability of uPAR mRNA.

messenger ribonucleic acid binding protein

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NORMAL AND NEOPLASTIC CELLS express cell surface receptors for the urokinase-type plasminogen activator (uPA). The uPA receptor (uPAR) binds both uPA and its proenzyme (9). Many of the biological functions of uPA are importantly influenced by association with uPAR. For example, uPAR plays a central role in mitogenesis, cell motility, and localization of uPA-mediated plasminogen activation at the cell surface (8-10, 13, 28, 33, 36). Regulation of uPAR could thus influence a broad range of pathophysiological events relevant to tissue injury and repair. In the lung, uPAR expression could be particularly important in modulating local proteolysis and fibroblast proliferation (32), events associated with tissue remodeling and fibrosis in the adult respiratory distress syndrome or interstitial lung diseases.

The control of gene expression can occur by transcriptional and posttranscriptional regulation, which can independently influence the turnover rate of mRNA. The half-lives of many mRNAs further seem to be the major determinants of their abundance; that is, mRNA levels correlate directly with persistence of cytoplasmic mRNA rather than the rapidity of synthesis (12). Regulation of mRNA decay is a potentially important process for determining the level of gene expression. For instance, high-grade lability of an mRNA promotes rapid downregulation of protein synthesis after transcription blockade and thus provides an efficient mechanism for transient expression. Mechanisms that regulate mRNA decay involve cis-acting elements found in either the 3'-untranslated regions (UTR) or coding regions of selected mRNAs (12). A common cis element found in the 3'-UTR of rapidly decaying mRNA is an adenosine-uridine (AU)-rich element (ARE; see Refs. 7, 30). Different transcripts with similar yet distinct AREs, such as granulocyte macrophage colony-stimulating factor (GM-CSF) and c-myc mRNAs, are regulated differentially, suggesting that different trans-acting proteins are involved (29). This type of regulation is a key feature of normal physiological processes (1, 27), and downregulation of such control mechanisms could contribute to neoplastic transformation (16, 26). These studies suggested the possibility that cell surface expression of uPAR could also involve posttranscriptional regulation.

In human malignant mesothelioma cells, we recently confirmed that increased specific binding capacity for uPA and expression of uPAR mRNA induced by proinflammatory agents (32, 33) involved a posttranscriptional mechanism (31). In this pathway, a 50-kDa uPAR mRNA binding protein (mRNABP) interacts with cis-elements within a 51-nucleotide (nt) sequence of the uPAR mRNA coding region to regulate uPAR message stability (31). Binding of uPAR mRNABP to uPAR mRNA was abolished after treatment with cycloheximide and was rapidly downregulated by other agents, including phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), transforming growth factor-beta (TGF-beta ), and tumor necrosis factor-alpha (TNF-alpha ), that increased the half-life of uPAR mRNA. Picone et al. (25) and others (20, 32, 33, 35) showed that PMA and cycloheximide increased uPAR mRNA in various other cells. We therefore speculated that uPAR expression in the lung could involve posttranscriptional regulation and that this regulatory mechanism could similarly involve interaction between a uPAR mRNA fragment containing regulatory information for message stability and a cytoplasmic protein similar to that which we identified in malignant human pleural mesothelial cells (31). Rabbit tissues were chosen to expedite the analyses and to facilitate potential future efforts to determine how uPAR regulation influences the course of pleural injury in the rabbit.

We now report the identification and purification of a 50-kDa uPAR mRNABP from rabbit lung. This protein specifically binds to the same fragment of the uPAR coding sequence (195-246 nt) as does the human malignant mesothelioma uPAR mRNABP. Our results show that the rabbit lung uPAR mRNABP shares physical and functional properties with its counterpart derived from human malignant mesothelioma cells. This protein, the major uPAR mRNABP in lung cytoplasmic extracts, is also expressed by primary cultures of lung mesothelial cells and fibroblasts. To our knowledge, this study also includes the first isolation of a uPAR mRNA-specific binding protein from a eukaryotic system.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Tris base, dithiothreitol (DTT), heparin, phenylmethylsulfonyl fluoride (PMSF), ammonium persulfate, glycerol, and tRNA were from Sigma Chemical (St. Louis, MO). Acrylamide, bis-acrylamide, heparin-Sepharose, DEAE-Sepharose, blue Sepharose, Bio-Rex 70 resins, and other related chemicals were from Bio-Rad Laboratories (Richmond, CA). In vitro transcription kits and TA cloning kits were purchased from Ambion (Austin, TX) and Invitrogen (San Diego, CA), respectively. HEPES and other reagents were from Fisher Scientific (Pittsburgh, PA). Restriction enzymes were from New England Biolabs (Beverly, MA). [32P]UTP was from Dupont (Wilmington, DE). X-AR X-ray films were from Kodak (Rochester, NY). Biotin-labeled UTP was purchased from Boehringer Mannheim (Indianapolis, IN), and streptavidin-agarose was from GIBCO-BRL (Grand Island, NY).

Cell culture. Rabbit pleural mesothelial cells and lung fibroblasts were isolated from rabbit lung that was surgically removed and grown to confluence in RPMI media containing 10% FCS, 1% glutamine, and 1% antibiotics, as we described previously (32, 33). The cells were incubated at 37°C in an atmosphere of 95% air-5% CO2. Medium was replaced three times weekly, and the cells were subcultured when they reached confluence after being maintained for ~2 wk. Stock cultures were transferred to 24-well plates or T-75 or T-175 flasks as required for specific experiments and were used ~1 wk later when the cells became confluent.

125I-labeled uPA binding. Binding of 125I-labeled uPA was measured as we described previously (32, 33). Rabbit mesothelial cells and fibroblasts were treated with PMA (10 ng/ml), LPS (10 µg/ml), TGF-beta (2 ng/ml), and TNF-alpha (10 ng/ml). The cells were incubated for 24 h in serum-free media containing 0.5% BSA, and 125I-uPA binding was then performed. Nonspecific binding was determined in the presence of a 500-fold excess of cold uPA.

Ligand blotting. We used SDS-PAGE under nonreducing conditions and ligand blotting to identify cell surface uPAR, as we previously described (33).

Plasmid construction. Plasmid uPAR/pBluescript was obtained from American Type Culture Collection. The human uPAR mRNA template containing a complete sequence of uPAR cDNA (nt -16 to 1144) from uPAR/pBluescript was subcloned to Hind III and Xba I sites of pRC/CMV (Invitrogen). A deletion product of uPAR containing the uPAR mRNABP binding sequence (195-246) was created by PCR using perspective forward and reverse primers. The PCR product was cloned directly to the TA cloning vector PCRII (InVitrogen). The orientation and sequence of the clones were confirmed by sequencing. The uPAR template was linearized by Hind III or Xba I, purified on 1% agarose gels, extracted with phenol-chloroform, and used for in vitro transcription.

In vitro transcription. The full-length uPAR template or the deletion product of uPAR containing the uPAR mRNABP binding sequence was linearized with Hind III or Xba I, purified on 1% agarose gels, and transcribed in vitro with SP6 or T7 polymerase for sense or antisense mRNA according to the suppliers protocol, except that 50 µCi of [32P]UTP (800 Ci/mmol) were substituted for unlabeled UTP in the reaction mixture. Passage through a Sephadex G-25 column removed unincorporated radioactivity. The specific activity of the product was 4.9-5.2 × 108 counts · min-1 (cpm) · µg-1. A biotin-labeled uPAR sense transcript was synthesized in essentially the same manner as the 32P-labeled transcript using an Ambion in vitro transcription kit, except that ATP, GTP, CTP, and [32P]UTP were substituted by biotin RNA labeling mix containing biotin-16-UTP. For large-scale synthesis of biotin-labeled RNA, transcription reactions were performed with 10 µg linearized plasmid DNA in the presence of 1.5 mM ATP, CTP, GTP, and biotin-labeled UTP and 100 units T7 polymerase in a final volume of 100 µl for 2 h at 37°C. To increase the yield, the reaction was continued for another 2 h after addition of a fresh mixture of 50 µl of 1.5 mM ribonucleotide triphosphates together with 50 units of T7 polymerase. RNA transcripts with low specific activity were generated by the same procedure but included 10 µCi [32P]UTP in the reaction. The specific activity of these transcripts was 1.5 × 106 cpm/µg.

Preparation of a cytosolic extract from rabbit tissues. Rabbit lung, liver, stomach, heart, kidney, brain, spleen, colon, and pancreatic tissues were surgically obtained immediately after death. The animal protocols were approved by the Animal Committee of The University of Texas Health Center at Tyler. All tissues were chopped into small pieces with fine scissors and rinsed three times with PBS. They were then separately homogenized with 10 volumes of extraction buffer (25 mM Tris · HCl, pH 7.9, 0.5 mM EDTA, and 0.1 mM PMSF), and the homogenates were centrifuged at 15,000 rpm for 15 min at 4°C. The supernatants were next collected and centrifuged at 36,000 rpm for 4 h at 4°C in a Beckman ultracentrifuge. The postnuclear supernatants thus obtained were used as cytosolic extracts, and the protein content of these extracts was measured with a Bio-Rad protein assay kit using serum albumin standards.

RNA-protein binding assays. Gel shift binding assays were performed using a uniformly 32P-labeled transcript of the 51-nt 195-246 uPAR mRNA sequence (31). Reactions were performed at 30°C by incubating these transcripts (20,000 cpm) with cytosolic extracts as prepared above (10 µg) from rabbit tissues in 15 mM KCl, 5 mM MgCl2, 0.25 mM EDTA, 0.25 mM DTT, 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 mixtures were treated with 50 units of RNase T1 and incubated for an additional 30 min at 37°C. To avoid nonspecific protein binding, 5 mg/ml heparin were added, and the mixture was incubated at room temperature for an additional 10 min. Samples were then separated by electrophoresis on a 5% native polyacrylamide gel with 0.25× Tris-borate-EDTA running buffer. The gels were dried and autoradiographed at -70°C using Kodak X-AR film.

Alternatively, RNA-protein binding mixtures were created as described above, and the samples were then analyzed by ultraviolet (UV) cross-linking. After the addition of heparin, reaction mixtures were transferred to a 96-well microtiter plate and irradiated at 4°C at 2,500 µJ for 10 min with a UV-stratalinker chamber apparatus (Stratagene). The samples were then boiled for 5 min and separated on 10% SDS-PAGE under reducing or nonreducing conditions. The gels were dried, and 32P-labeled proteins were visualized by autoradiography. Rabbit pleural mesothelial cells and fibroblasts were grown in T-150 flasks and treated with PMA, LPS, TGF-beta , TNF-alpha , or 10 µg/ml cycloheximide for varying lengths of time in RPMI medium containing 0.5% BSA. The cells were detached from the flasks with trypsin-EDTA and washed three times with PBS. Cytosolic extracts of these cells were then prepared as described above. The protein contents were measured, and 10 µg protein from each time point were subjected to gel mobility shift assay as described above.

RNase protection assay. Rabbit mesothelial cells and lung fibroblasts grown to confluence were treated with or without PMA, LPS, TGF-beta , TNF-alpha (concentrations as mentioned above), or cycloheximide (10 µg/ml) for 12 h. Total RNA was isolated, and steady-state uPAR mRNA was quantitated by RNase protection assay using 32P-labeled uPAR antisense transcripts (31). Total RNA from rabbit lung, liver, stomach, heart, kidney, brain, spleen, colon, and pancreas were isolated by homogenizing these tissues separately in TRI-reagent, and uPAR mRNA was then analyzed by RNase protection assay as described above. The intensity of the bands was measured densitometrically and normalized against that of beta -actin. In separate experiments, mRNA decay was measured by blocking ongoing transcription by actinomycin D (10 µg/ml) for different time intervals after treatment of the cells with or without PMA, LPS, TGF-beta , TNF-alpha , or cycloheximide.

Purification of a uPAR mRNABP from rabbit lung. Rabbit lung cytosolic extract, prepared as described above, was added to solid (NH4)2SO4 crystals at 40% saturation, and the precipitated proteins were discarded after checking the uPAR mRNA binding activity. Solid (NH4)2SO4 crystals were added to the 40% (NH4)2SO4 supernatant to yield a final saturation of 60%. The precipitated proteins were collected, redissolved, and exhaustively dialyzed against extraction buffer containing 10% glycerol. RNA binding activity was assessed in an aliquot (10 µg protein) of this material. The 40-60% (NH4)2SO4 fraction was passed through a blue Sepharose column (90 ml) in the same buffer. After the unadsorbed protein was washed out in an extraction buffer containing 100 mM NaCl, uPAR mRNABP was eluted with a linear gradient (200 ml) of 100-1,000 mM NaCl. Positive fractions were pooled and concentrated using an Amicon concentrator.

This material (50 ml, 20 mg/ml) was loaded onto a heparin affigel column (90 ml) in the same buffer. After thorough washing of unbound material, uPAR mRNABP was eluted with a linear gradient (200 ml) of 100-1,000 mM NaCl in extraction buffer. The uPAR mRNA binding activity was eluted between 200 and 300 mM NaCl, which was collected and concentrated by dialysis against polyethylene glycol 8000.

The uPAR mRNA binding fractions from the heparin affigel column (99 mg/150 ml) were next loaded onto a Bio-Rex 70 column. Unbound proteins containing uPAR mRNABP were eluted with starting buffer and loaded onto a DEAE-Sepharose column (220 ml). The unadsorbed proteins were removed by washing with starting buffer containing 100 mM NaCl. uPAR mRNABP, which binds to the DEAE-Sepharose resin in this buffer, was eluted with a linear gradient (400 ml) of 100-1,000 mM NaCl in extraction buffer. Fractions containing uPAR mRNABP, which eluted between 400 and 500 mM NaCl, were pooled and concentrated using an Amicon concentrator.

For affinity purification, fractions containing uPAR mRNA binding activity from the DEAE-Sepharose column were diluted with gel shift buffer, supplemented with 30 µg/ml tRNA and 160 µg of biotin-labeled uPAR transcript in 800 µl of reaction mixture, and incubated at 30°C for 1 h. The reaction mixtures were applied to a streptavidin-agarose column that was equilibrated with gel shift buffer at 4°C and recycled at least five times. By including 106 cpm of low specific activity transcript, we found that >96% of the mRNA was bound under these conditions. The column was washed first with 20 volumes of gel shift buffer containing 10 mg/ml heparin and 30 mg/ml tRNA, followed by 20 volumes of gel shift buffer containing 30 mg/ml tRNA and 40 volumes of gel shift buffer alone. uPAR mRNA binding activity was eluted with 2 M KCl in gel shift buffer. Purified uPAR mRNABP was concentrated and equilibrated with gel shift buffer on a Centricon-10 microconcentrator. Aliquots were tested for mRNA binding activity by gel mobility shift assay and UV cross-linking. Homogeneity of the isolated protein was determined by SDS-PAGE and silver nitrate staining.

Fractions containing uPAR mRNABP from the streptavidin-agarose column were passed through a Mono-Q column connected to a fast-performance liquid chromatography (FPLC) system at a flow rate of 0.5 ml/min. The bound proteins were eluted by a linear gradient (40 ml) of 0-500 mM NaCl. Fractions containing uPAR mRNA binding activity were pooled and dialyzed against extraction buffer containing 500 mM NaCl and loaded onto a Sephadex G-150 column (550 ml). Chromatography was performed at a flow rate of 3 ml/h. Fractions having uPAR mRNABP were pooled and concentrated by dialysis. Aliquots were tested for homogeneity by SDS-PAGE and silver staining.

In vitro uPAR mRNA decay by uPAR mRNABP. A standard reaction mixture (50 µl) containing 100 mM potassium acetate, 2 mM magnesium acetate, 2 mM DTT, 30 µM all 20 common amino acids, 10 µM creatine phosphate, 1 µg of creatine kinase, 1 mM ATP, 0.4 µM GTP, 6 µg of tRNA, 0.1 mM spermine, 10 mM Tris · HCl, pH 7.6, and 0.5-2.5 µg of uPAR mRNABP (Sephadex G-150 fraction) was added to 0.5 µl (85,000 cpm) 32P-labeled uPAR sense transcript. In a parallel experiment, uPAR mRNABP was replaced with or without 0.5-2.5 µg RNase-free BSA. The reaction mixtures were incubated at 37°C for 16 h, and uPAR mRNA levels were measured by RNase protection assay and densitometric scanning.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue distribution of the uPAR mRNABP. A notable feature of the uPAR mRNABP that we identified in human pleural mesothelioma (MS-1) cells (31) is that it recognizes sequence(s) within a 51-nt sequence of the uPAR mRNA coding region. We initially sought to determine if a similar protein was present in rabbit tissues. To do this, we prepared cytosolic extracts of rabbit lung, liver, stomach, heart, kidney, brain, spleen, colon, and pancreas and screened these tissues for expression of uPAR mRNA-specific binding proteins. Figure 1 illustrates gel shift assay data indicating that rabbit lung and other tissue extracts contain the uPAR mRNABP, which hybridizes with the radiolabeled uPAR sense transcript. Addition of a molar excess of homologous unlabeled RNA to the binding reaction mixture markedly diminished the complex. Predigestion of cell lysates with proteinase K abolished the formation of the complex, and addition of SDS before the binding reaction likewise inhibited the RNA-protein interaction (data not shown). These data indicate that the properties of the uPAR mRNA-protein complex resembled that of an MS-1 cell cytosolic extract, as reported earlier (31).


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Fig. 1.   Identification of urokinase-type plasminogen activator receptor (uPAR) mRNA binding protein in rabbit tissues by gel mobility shift assay. Crude cytosolic extracts (10 µg protein) were hybridized with the 32P-labeled uPAR sense transcript at 30°C for 30 min and digested with RNase T1 at 37°C for 30 min and heparin at room temperature for 10 min. RNase-resistant complexes were resolved on 5% polyacrylamide gels, dried, and autoradiographed. Lanes 1-9, cytosolic extract of rabbit liver, stomach, heart, kidney, brain, spleen, pancreas, colon, and lung, respectively; lane 10, probe alone; lanes 11-19, same as in lanes 1-9 except corresponding rabbit tissue extracts were incubated with 100-fold molar excess of unlabeled sense transcript; lane 20, probe alone. Arrowhead indicates RNA-protein complex.

Molecular mass of the uPAR mRNABP. We next used UV-induced cross-linking of the RNA and protein complex to establish the apparent molecular mass of the uPAR mRNABP. The binding mixtures were analyzed on 10% SDS-PAGE after UV irradiation. A radioactive protein band (50 kDa) was detected by this method in rabbit lung extract, and treatment of UV cross-linked samples with beta -mercaptoethanol also yielded a single protein band of the same molecular mass (Fig. 2). The molecular mass of the protein is comparable to that present in a human mesothelioma cell (MS-1) extract, as reported earlier (31).


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Fig. 2.   Characterization of the uPAR mRNA binding protein (mRNABP) by ultraviolet (UV) cross-linking and SDS-PAGE. Crude cytosolic extracts from rabbit tissues were hybridized with 32P-labeled uPAR sense transcript, digested with RNase T1, and UV irradiated on ice at 2,500 µJ for 10 min. Samples were resolved by SDS-PAGE under nonreduced conditions, dried, and autoradiographed. Mr, protein standards; lanes 1-9, cytosolic extracts of rabbit kidney, pancreas, stomach, heart, brain, colon, liver, lung, and spleen, respectively; lanes 10-18, same as lanes 1-9 except that corresponding rabbit tissue extracts were incubated with 100-fold excess unlabeled sense transcript.

Expression of uPAR mRNA and uPAR in rabbit tissues. In complementary experiments, we found that the uPAR message and the receptor were coexpressed with uPAR mRNABP in these tissues. Total RNA isolated from the rabbit tissues was screened for uPAR mRNA by RNase protection assay using a uPAR antisense cRNA probe (Fig. 3). The autoradiographs indicated a hybridization signal for uPAR mRNA in each of the rabbit tissues and demonstrated that expression of uPAR mRNA was prominent in lung, stomach, colon, and kidney. Ligand blotting experiments were then done to confirm expression of uPAR in these tissues. These studies demonstrated the presence of a 50-kDa uPA binding protein [uPAR (data not shown), which exhibited the same molecular mass as human uPAR (32, 33)].


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Fig. 3.   RNase protection assay for uPAR mRNA in rabbit tissues. Total RNA (50 µg) was hybridized with 32P-labeled uPAR antisense probe and analyzed by RNase protection assay. RNase-resistant fragments were separated on 5% urea-polyacrylamide gels, dried, and autoradiographed. Lanes 1-9, RNA from rabbit liver, heart, kidney, brain, spleen, stomach, pancreas, colon, and lung, respectively, hybridized with 32P-labeled uPAR antisense mRNA (top). Corresponding RNA hybridized with 32P-labeled beta -actin antisense mRNA is shown (bottom).

Effects of inflammatory mediators on uPAR, uPAR mRNA, and the uPAR mRNABP expression. We next confirmed that rabbit pleural mesothelial and lung fibroblasts expressed uPAR at the cell surface by binding 125I-labeled uPA and used this assay system to elucidate the effects of selected mediators on cellular uPAR expression. Treatment with proinflammatory agents such as PMA, LPS, TGF-beta , and TNF-alpha increased 125I-labeled uPA binding two- to fivefold compared with untreated controls (Fig. 4). RNase protection assay data indicated that PMA, LPS, TGF-beta , TNF-alpha , or cycloheximide also increased uPAR mRNA 8- to 20-fold compared with unstimulated control mesothelial cells or lung fibroblasts (Fig. 4B). Transcriptional blockade with actinomycin D indicated that uPAR mRNA decreased with a half-life of 2-3 h in unstimulated mesothelial cells and lung fibroblasts. In follow-up experiments, cells treated with proinflammatory agents or cycloheximide were then subjected to actinomycin chase, after which the decay in uPAR mRNA was measured over time. The uPAR mRNA half-lives were extended at least three- to fivefold in both cell types treated with PMA, LPS, TGF-beta , TNF-alpha , or cycloheximide (Fig. 4, C and D). These data confirm that uPAR expression by rabbit mesothelial cells and lung fibroblasts is subject to posttranscriptional regulation.


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Fig. 4.   A: 125I-labeled urokinase-type plasminogen activator (uPA) binding to rabbit pleural mesothelial cells and fibroblasts. Rabbit pleural mesothelial cells and fibroblasts were treated with or without phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), transforming growth factor-beta (TGF-beta ), and tumor necrosis factor-alpha (TNF-alpha ) for 24 h. Receptor-bound uPA was removed by glycine hydrochloride treatment, as described earlier (35). Cells were treated with 125I-uPA at 4°C for 2 h, cell bound radioactivity was measured, and specific binding was calculated from difference between total and nonspecific binding (with a 500-fold excess of cold uPA). Values are means of 2 separate experiments. B: induction of uPAR mRNA by PMA, LPS, TGF-beta , TNF-alpha , or cycloheximide (Cyc D) in rabbit pleural mesothelial cells and fibroblasts by RNase protection assay. Rabbit pleural mesothelial cells and fibroblasts were treated with or without above agents for 12 h. Total RNA (20 µg) was isolated and hybridized with 32P-labeled uPAR antisense probe and analyzed by RNase protection assay. RNase-resistant fragments were separated on 5% urea-polyacrylamide gels, dried, and autoradiographed. uPAR mRNA was measured by spectrophotometric scanning of the autoradiograms and normalized against the corresponding amount of beta -actin mRNA in the sample. C and D: effects of PMA, LPS, TGF-beta , TNF-alpha , and cycloheximide on uPAR mRNA stability in rabbit pleural mesothelial cells (C) and rabbit fibroblasts (D). Cells were treated with or without PMA, LPS, TGF-beta , TNF-alpha , or cycloheximde for 12 h, after which actinomycin D (10 µg/ml) was added for various periods of time. uPAR mRNA was analyzed by RNase protection assay.

We next wanted to see whether posttranscriptional regulation of uPAR by these cell types involved a regulatory uPAR mRNABP-uPAR mRNA mechanism similar to that which we recently observed in malignant mesothelioma cells (31). We therefore tested whether treatment of pleural mesothelial cells and lung fibroblasts with PMA, LPS, TGF-beta , TNF-alpha , and cycloheximide altered the uPAR mRNA-uPAR mRNABP interaction. PMA and TGF-beta decreased the uPAR mRNA-uPAR mRNABP complex in a time-dependent fashion, with a maximal effect observed between 3 and 6 h after treatment. Prolonged exposure however did not reduce the complex further. Treatment of rabbit pleural mesothelial cells and fibroblasts with LPS, TNF-alpha , or cycloheximide totally abolished the RNA-protein complex by 12 h after the treatment (Fig. 5). The data confirm that parenchymal lung cells reciprocally downregulate the uPAR mRNABP-uPAR mRNA interaction in response to stimuli that increase both uPAR mRNA and receptor expression at the cell surface. These observations suggest that the rabbit uPAR mRNABP-uPAR mRNA interaction destabilizes the message via a mechanism similar to that which we recently described in malignant mesothelioma cells (31).


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Fig. 5.   Effects of PMA, LPS, TGF-beta , TNF-alpha , and cycloheximide on the uPAR mRNA-uPAR mRNABP interaction. Rabbit pleural mesothelial cells and rabbit fibroblasts were treated with PMA, LPS, TGF-beta , TNF-alpha , or cycloheximide for 0-24 h. Cytosolic extracts were hybridized with the labeled mRNA probe, digested with RNase T1, and analyzed by gel mobility shift assay as described in Fig. 1.

Purification of the uPAR mRNABP. Having confirmed that the uPAR mRNABP was present in rabbit lung extract and in resident lung parenchymal and pleural cells, we sought to purify this protein and characterize the purified material. The choice of rabbit lung as a source of uPAR mRNABP was influenced by our finding that this tissue has substantial quantities of uPAR mRNABP (Fig. 1), the molecular mass and RNA-binding specificity of which are identical to the protein present in human MS-1 mesothelioma cells (31). The cytoplasmic extract that we called crude extract was fractionated into 0-40%, 40-60%, and 60-100% ammonium sulfate derivatives by adding solid ammonium sulfate crystals. More than 90% of uPAR mRNA binding activity was found in the 40-60% ammonium sulfate precipitate. These data suggest that the uPAR mRNABP is hydrophilic in nature.

This fraction was later passed through a blue Sepharose column, which was chosen to remove albumin from the lung extract. Only ~40% of the total protein but >90% of uPAR mRNABP bound to this matrix, and almost none of the uPAR mRNABP dissociated upon repeated washing of the blue Sepharose beads. In an attempt to recover uPAR mRNABP at a specific salt concentration, we found a broad peak of elution between 300 and 500 mM NaCl that contained uPAR mRNA binding activity. These active fractions were later passed through a heparin affigel general affinity column, and uPAR mRNABP was eluted with a 100-1,000 mM NaCl gradient. Here, fractions showed that uPAR mRNA binding activity corresponded to 250 mM NaCl. There was also a fast migrating complex in the fractions probably formed due to proteolytic degradation during extraction/purification processes. However, this lower complex was not consistently observed in all of the preparations. These fractions were subsequently passed through a Bio-Rex 70 column, and 99% of uPAR mRNABP was present in the flow through. About 30% of contaminating proteins were removed by a Bio-Rex column, when the pH of the buffer was lowered to 7.0 from 7.9 and 90% of the uPAR mRNABP bound to the Bio-Rex 70 column resin (data not shown). The uPAR mRNABP fraction from a Bio-Rex 70 column was passed through a DEAE-Sepharose column. uPAR mRNABP was eluted in fractions 9-11 at ~400 mM NaCl (Fig. 6), indicating that the protein was anionic.


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Fig. 6.   A: chromatography of uPAR mRNABP DEAE-Sepharose column. uPAR mRNABP fraction from Bio-Rex 70 column was passed through DEAE-Sepharose column. Proteins bound to DEAE-Sepharose column were eluted with 100-1,000 mM NaCl. Fractions of 5 ml were collected, and the bar exhibits uPAR mRNA binding activity. B: uPAR mRNA binding activity of DEAE-Sepharose column fractions was determined by gel shift assay. Five microliters per fraction from DEAE-Sepharose column were assayed for uPAR mRNABP (lanes 2-20). Lanes 8-12, DEAE-Sepharose-bound fractions showing uPAR binding activity. Arrowhead indicates RNA-protein complex.

The DEAE fractions containing uPAR mRNABP were pooled, incubated with a biotin-labeled uPAR transcript, and passed through a streptavidin-agarose column containing biotin-labeled uPAR mRNA. The affinity column purified the uPAR mRNABP. The activity profile of this column (Fig. 7) shows that the uPAR mRNA binding activity coeluted with a protein of 50 kDa. This was one of the five proteins present in the streptavidin-agarose column fraction when visualized on a SDS-PAGE stained with silver. For affinity purification, we used biotin-labeled uPAR mRNA that demonstrates high-affinity binding to the streptavidin-agarose column. The biotin-labeled transcripts also bound with high affinity to uPAR mRNABP, as evident from gel shift assay (data not shown). We reasoned that the high affinity of the biotin-labeled uPAR mRNA-uPAR mRNABP interaction as measured by the gel shift assay would be sufficient for affinity purification.


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Fig. 7.   Purification of uPAR mRNABP by affinity chromatography. Protein fractions containing uPAR mRNABP from the DEAE-Sepharose column were incubated with biotin-labeled uPAR mRNA at 30°C for 30 min in the presence of E. coli tRNA. The reaction mixture was applied onto a streptavidin-agarose (1 ml) column 8 times at 4°C. Unbound proteins were washed with binding buffer containing 1,000 mM KCl. Bound proteins were eluted with 2 M KCl in binding buffer. uPAR mRNA binding activity of uPAR mRNABP by gel shift analysis from streptavidin-agarose flow-through (lane 1), 1 M KCl eluate of streptavidin-agarose column (lane 2), 2 M KCl eluate of streptavidin-agarose (lane 3), and free probe (lane 4). Lanes 5-7 are the same as lanes 1-3 except that eluates from the streptavidin-agarose column were incubated with 100-fold molar excess of unlabeled sense transcript. Lanes 8-10 are the same as in lanes 1-3 except that eluates are predigested with proteinase K before RNA-protein interaction. Arrowhead indicates RNA-protein complex.

One of the problems associated with attempting to purify an mRNA-binding protein by an RNA affinity column is the degradation of the RNA probe by endogenous RNases contained with the lysate. We found that, in chromatography with semipurified lysate at 4°C, the RNA transcripts that we used remained essentially intact. After interacting with the lysate, 70-80% of the probe was bound to the streptavidin-agarose beads. After binding of the RNA-protein complex to the beads, the beads were extensively washed. We screened different elution techniques by examining which treatments could dissociate the uPAR mRNA-uPAR mRNABP complex in solution and still allow recovery of binding activity after removal of the elution agent. This led us to choose 2 M NaCl as an efficient and reversible (after dialysis) eluent.

Fractions containing uPAR mRNABP were dialyzed and passed through a Mono-Q column connected to the FPLC system at a flow rate of 0.5 ml/min. The elution profile of uPAR mRNABP on the Mono-Q column was highly reproducible. Most of the uPAR mRNABP was eluted at 300 mM NaCl (Fig. 8). When the proteins were separated on SDS-PAGE and visualized with silver, there were three bands. However, the 50-kDa protein was the major band. The elution pattern of uPAR mRNABP from Sephadex G-150 suggests that the 50-kDa protein alone is the uPAR mRNABP.


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Fig. 8.   A: chromatography of uPAR mRNABP on a Mono-Q column. Proteins eluted from the uPAR mRNA affinity column in 2 M KCl were loaded onto a Mono-Q column, and the bound proteins were eluted with sample buffer containing a 0-500 mM linear NaCl gradient. Fractions (1 ml) containing protein were subjected to gel mobility shift assay. Bar indicates fractions showing uPAR mRNA binding activity. B: gel shift analysis of eluted fractions (lanes 10-28) as in A. Lanes 24-27, Mono-Q bound fractions having uPAR mRNA binding activity. Arrowhead shows uPAR mRNABP-uPAR mRNA complex.

To assess the purification, the protein fractions from each stage of the isolation procedure were analyzed by SDS-PAGE (Fig. 9A). The final fraction from the Sephadex G-150 column revealed one major protein band corresponding to a molecular mass of 50 kDa and two minor bands. Several lines of evidence indicate that the 50-kDa band corresponds to uPAR mRNABP. Gel shift (Fig. 9B) and UV cross-linking (Fig. 9C) experiments of each of the purification fractions identified only one uPAR mRNA-uPAR mRNABP complex. The enhancement of the uPAR mRNA-uPAR mRNABP signal indicated the enrichment of uPAR mRNABP. As shown in Fig. 9, the sole protein that is labeled is present in the eluate and comigrates with the complexes that are obtained from different steps of purification. This observation indicates that the protocol is isolating the major, if not the only, uPAR mRNABP in the lung lysate. The photoaffinity labeling of the uPAR mRNABP is specifically blocked by addition of excess unlabeled uPAR mRNA and is not affected by the presence of nonspecific (tissue factor) RNA competitor (Fig. 9).


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Fig. 9.   Sequential graphic of uPAR mRNABP purification. A: SDS-PAGE pattern of different purification steps of uPAR mRNABP. Crude rabbit lung extract (lane 1), 40-70% ammonium sulfate fraction (lane 2), blue Sepharose bound fraction (lane 3), heparin Sepharose bound (lane 4), DEAE-Sepharose bound (lane 5), streptavidin-agarose uPAR mRNA column bound (lane 6), Mono-Q bound (lane 7), and Sephadex G-150 column (lane 8). B: uPAR mRNA binding activity during purification as assessed by gel mobility shift assay of above fractions 1-8. C: UV cross-linking studies of uPAR mRNABPs during purification steps. D: uPAR mRNABP binding specificity by UV cross-linking. Lane 1, free 32P-labeled uPAR mRNA probe alone; lane 2, Sephadex G-150 purified protein; lane 3, Sephadex G-150 purified protein plus 100-fold excess unlabeled uPAR mRNA; lane 4: Sephadex G-150 purified protein plus 100-fold excess nonspecific (tissue factor) mRNA competitor.

Expression of RNase activity by the uPAR mRNABP. We next sought to determine if the isolated uPAR mRNABP expressed RNase activity, a putative mechanism by which the uPAR mRNABP-uPAR mRNA interaction could be disrupted. In vitro uPAR mRNA-uPAR mRNABP reaction mixtures were incubated at 37°C for 16 h, and uPAR mRNAs were analyzed by RNase protection assay. Intensities of uPAR mRNA-specific bands of uPAR mRNABP-treated samples were compared with or without BSA-treated controls. The density of uPAR mRNABP-treated samples was comparable to that of BSA-treated or untreated controls.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, we find that rabbit pleural mesothelial cells and lung fibroblasts increase uPAR expression in response to selected agents likely to mediate inflammatory reactions in the lung or pleural space. In rabbit lung fibroblasts and mesothelial cells, uPAR is induced by PMA, LPS, TGF-beta , and TNF-alpha . Rabbit pleural mesothelial cells express at least twofold more uPAR than rabbit fibroblasts. This finding is consistent with our earlier observation that human pleural mesothelial cells express more uPAR compared with normal human lung fibroblasts (32). The uPAR mRNA in rabbit mesothelial cells and fibroblasts increased by ~8- to 20-fold in response to PMA, LPS, TGF-beta , and TNF-alpha . By contrast, PMA increased uPAR mRNA by 40- to 50-fold in U-937 cells (20), a disparity that may relate to differences in the basal expression of uPAR by different cell types. We reasoned that the increase in uPAR mRNA under these circumstances could have been due to an increased rate of transcription and/or decreased posttranscriptional mRNA turnover.

We confirmed that the increase in uPAR levels in PMA-, LPS-, TGF-beta -, TNF-alpha -, and cycloheximide-treated rabbit mesothelial cells and fibroblasts involved posttranscriptional regulation. In these cells, uPAR mRNA half-life was prolonged by three- to fivefold compared with untreated control cells, as confirmed by the rate of decay of uPAR mRNA in actinomycin D-treated cells. These findings are consistent with those of previous studies in which uPAR expression by A549 cells derived from lung carcinoma (19) or MS-1 cells derived from human malignant mesothelioma was found to involve posttranscriptional regulation (31).

Because cycloheximide and inflammatory mediators prolonged the uPAR mRNA half-life, we assumed that some protein factor interacts with uPAR mRNA to alter its stability. We confirmed this assumption by identifying a cytosolic protein that specifically binds to the uPAR mRNA transcript. Treatment of rabbit mesothelial cells and fibroblasts with PMA or TGF-beta downregulated the uPAR mRNA-uPAR mRNABP complex by at least 60-70%, whereas LPS, TNF-alpha , or cycloheximide totally abolished the uPAR mRNA-protein complex. Increased longevity of uPAR mRNA in rabbit mesothelial cells and lung fibroblasts stimulated by proinflammatory agents could thus involve decreased complex formation between uPAR mRNA and the uPAR mRNABP. We previously correlated this finding with posttranscriptional regulation of uPAR in malignant mesothelioma cells (31).

In this study, we additionally purified and characterized a uPAR mRNABP that differs from previously reported mRNABPs (17, 22, 23, 38). Although uPAR mRNA was readily detectable in all of the rabbit tissues that we examined (Fig. 2), the uPAR mRNABP was increased in selected tissues, including lung (Fig. 1). We suspect that differences in the sensitivity of the independent assay systems may obviate any clear reciprocal relationship between the levels of uPAR mRNA and the binding protein. It is alternatively possible that message destabilization is achieved by more than one mechanism and that not all involve uPAR mRNABP-uPAR mRNA binding. We used rabbit lung as a tissue source to expedite purification of this protein and found that it is physically and functionally similar to the protein that we recently described in human malignant mesothelioma (MS-1) and transformed human mesothelial (MeT 5A) cells (31). The uPAR mRNABP was purified from rabbit lung by sequential steps of ultracentrifugation, ammonium sulfate fractionation, ion exchange chromatography on DEAE and Bio-Rex columns, and affinity chromatography on blue and heparin Sepharose. These steps were followed by passage through an mRNA affinity and Mono-Q column and then gel filtration on Sephadex G-150. Fractions were followed by assaying for uPAR mRNA binding. The 50-kDa uPAR mRNABP was enriched to >90% purity and consistently fractionated with uPAR mRNA binding activity. It is likely that the specificity demonstrated by this protein is due to its recognition of sequences within the 51-nt coding region of uPAR mRNA. Binding of uPAR mRNABP to a radiolabeled 51-nt transcript is competitively inhibited by adding an excess amount of an unlabeled sense but not antisense transcript (data not shown). The complex that is formed by the rabbit uPAR mRNABP is indistinguishable from that formed when MS-1 cell protein is incubated with the 51-nt uPAR mRNA sequence (31), strongly suggesting that this interaction is similarly involved in the control of uPAR message stability of these cells. We also detect a second fast migrating complex in some preparations incubated with the 51-nt uPAR sequence. However, this fast migrating complex is not highly reproducible, and its migration is not consistent, suggesting that it results from proteolytic degradation. Longer storage results in the quantitative increase of a fast migrating band and further suggests that it is a proteolytic fragment of uPAR mRNABP (data not shown).

Other mRNABPs have been implicated in the regulation of message expression. For instance, a polyadenylation and poly(A) binding protein (PABP; see Ref. 2) interaction protects the mRNA from rapid destruction, suggesting that PABP protects polyadenylated mRNA. Another protein that binds to AU-rich regions (AUBP) has the capacity to bind with high affinity to mRNA containing AU-rich and in some cases U-rich regions. Some observations suggest that AUBPs increase mRNA stability. For example, in peripheral blood mononuclear cells activated by phorbol esters, many lymphokine mRNAs are stabilized, and AUBP levels increase by as much as 15-fold (18, 21, 34, 37). The activity or abundance of AUBP correlates inversely with GM-CSF mRNA half-life in T cells activated by a phorbol ester plus an antibody to CD3 (3, 4). We find that uPAR expression by pleural mesothelial cells and lung fibroblasts is regulated by a different type of mRNABP-mRNA interaction.

In conclusion, we have identified and characterized a uPAR mRNABP that is distributed in several tissues, including rabbit lung. This protein appears to be involved in the regulation of cell surface uPAR expression and uPAR mRNA stability in primary cultures of cytokine-stimulated pleural mesothelial cells and lung fibroblasts by a posttranscriptional mechanism similar to that which we recently described in human malignant mesothelioma cells. We further describe the principal purification steps that can be applied to isolate this trans-acting RNA regulatory protein from cytosolic extracts of rabbit lung tissue. The purified uPAR mRNABP binds the same 51-nt sequence within the coding region of uPAR mRNA as does the mesothelioma uPAR mRNABP but does not demonstrate any intrinsic RNase activity. It may be that the uPAR message destabilization alternatively involves other intermediaries or structural requirements. Further studies will be required to determine the manner in which the uPAR mRNABP interacts with other components of the mRNA degradation machinery to effect this RNA processing pathway and to determine how expression of this protein in vivo contributes to uPAR-mediated remodeling in the lung or pleural compartments.

    ACKNOWLEDGEMENTS

We are grateful to Kathy Koenig for expert technical assistance.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-45018-06, the RGK Foundation, The Gina Sabatasse and Cindy Armstrong Brown Research grant awards, The Temple Chair in Pulmonary Fibrosis (S. Idell), and a grant from the American Heart Association, Texas Affiliate (S. Shetty).

Address for reprint requests: S. Shetty, The Univ. of Texas Health Center at Tyler, Routes 271 and 155, Tyler, TX 75710.

Received 2 September 1997; accepted in final form 26 February 1998.

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Abstract
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Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 274(6):L871-L882
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