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• W2092440892 abstract "We sought to determine if urokinase expression is regulated at the post-transcriptional level in cultured lung epithelial cells. We also sought to determine if differences in urokinase expression by cultured human lung carcinoma and non-malignant lung epithelial subtypes were attributable to post-transcriptional regulatory mechanisms. Urokinase was expressed by phenotypically diverse lung carcinoma cell lines as well as non-malignant small airway epithelial cells and bronchial epithelial cells. Using gel mobility shift and UV cross-linking assays, we identified a 30-kDa urokinase mRNA-binding protein that selectively bound to a 66-nucleotide protein-binding fragment of urokinase mRNA. The urokinase mRNA-binding protein is found in the cytosolic but not nuclear extracts of non-malignant lung epithelial cells; whereas, it is found in the nuclear but not cytosolic extracts of selected malignant carcinoma-derived cells that express relatively large amounts of urokinase. Chimeric β-globin/urokinase cDNA containing the urokinase mRNA-binding protein binding sequence destabilized otherwise stable β-globin mRNA. Our results demonstrate that urokinase gene expression in lung epithelial and lung carcinoma-derived cells is regulated at the post-transcriptional level. The mechanism involves an interaction between a 66-nucleotide sequence of the urokinase mRNA 3′-untranslated region with a newly recognized urokinase mRNA-binding protein to regulate urokinase mRNA stability. We sought to determine if urokinase expression is regulated at the post-transcriptional level in cultured lung epithelial cells. We also sought to determine if differences in urokinase expression by cultured human lung carcinoma and non-malignant lung epithelial subtypes were attributable to post-transcriptional regulatory mechanisms. Urokinase was expressed by phenotypically diverse lung carcinoma cell lines as well as non-malignant small airway epithelial cells and bronchial epithelial cells. Using gel mobility shift and UV cross-linking assays, we identified a 30-kDa urokinase mRNA-binding protein that selectively bound to a 66-nucleotide protein-binding fragment of urokinase mRNA. The urokinase mRNA-binding protein is found in the cytosolic but not nuclear extracts of non-malignant lung epithelial cells; whereas, it is found in the nuclear but not cytosolic extracts of selected malignant carcinoma-derived cells that express relatively large amounts of urokinase. Chimeric β-globin/urokinase cDNA containing the urokinase mRNA-binding protein binding sequence destabilized otherwise stable β-globin mRNA. Our results demonstrate that urokinase gene expression in lung epithelial and lung carcinoma-derived cells is regulated at the post-transcriptional level. The mechanism involves an interaction between a 66-nucleotide sequence of the urokinase mRNA 3′-untranslated region with a newly recognized urokinase mRNA-binding protein to regulate urokinase mRNA stability. untranslated region urokinase-type plasminogen activator small airway epithelial cells uPA mRNA-binding protein ammonium persulfate dichloro-1-β-d-ribofuranosyl benzamidazole polyacrylamide gel nucleotide(s) kilobase(s) base pair(s) tracheal epithelial The regulation of mRNA stability is thought to play an important role in eukaryotic gene expression by modulating the cytoplasmic abundance of several mRNAs. Different mRNAs have different half-lives in a given cell, and the stability of the same mRNA may be modulated by extracellular stimuli or environmental factors. Relatively little is known about the mechanisms of mRNA degradation: post-transcriptional regulation. A few instability determinants have been identified in the coding and 3′-untranslated region (UTR)1 of diverse mRNAs. These determinants include 3′-UTR AU-rich sequences in various oncogenes, lymphokine, and cytokine mRNAs (1.Akashi M. Shaw G. Gross M. Saito M. Koeffler H.P. Blood. 1991; 78: 2005-2012Crossref PubMed Google Scholar, 2.Caput D. Beutler B. Hortog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1208) Google Scholar, 3.Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3107) Google Scholar, 4.Jones T.R. Cole M.D. Mol. Cell. Biol. 1987; 7: 4513-4521Crossref PubMed Scopus (213) Google Scholar, 5.Wilson T. Triesman R. Nature. 1988; 336: 396-399Crossref PubMed Scopus (504) Google Scholar) and an iron-responsive element in transferrin receptor mRNA (6.Casey J.L. Koeller D.M. Ramin V.C. Klausner R.D. Harford J.B. EMBO J. 1989; 12: 3693-3699Crossref Scopus (257) Google Scholar, 7.Mullner E.W. Kuhn L.C. Cell. 1988; 53: 815-825Abstract Full Text PDF PubMed Scopus (374) Google Scholar). Other such determinants include a cell cycle-dependent regulatory element in histone mRNA (8.Levine B.J. Chodchoy N. Marzluff W.F. Skoultchi A.I. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6189-6193Crossref PubMed Scopus (80) Google Scholar), a calcium-sensitive regulatory element in granulocyte macrophage- colony stimulating factor mRNA (9.Iwai Y. Akahane K. Pluznik D.H. Cohen R.B. J. Immunol. 1993; 150: 4386-4394PubMed Google Scholar), and a coding region instability element of MATα1 (10.Parkar R. Jacobson A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2780-2784Crossref PubMed Scopus (88) Google Scholar).While some of these mRNA stability determinants have been purified or characterized (11.Hamilton B.J. Nagy E. Malter J.S. Arrick B.A. Rigby W.F.C. J. Biol. Chem. 1993; 268: 8881-8887Abstract Full Text PDF PubMed Google Scholar, 12.Kaptin S. Downey W.E. Tang C. Phipott C. Haile D. Orloff D.G. Harford J.B. Rouault T.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10109-10113Crossref PubMed Scopus (157) Google Scholar, 13.Levine T.D. Gao F. King P.H. Andrews L.G. Keene J.D. Mol. Cell. Biol. 1993; 13: 3494-3504Crossref PubMed Scopus (334) Google Scholar, 14.Nanbu R. Kubo T. Hashimato T. Natori S. J. Biochem. (Tokyo). 1993; 114: 432-437Crossref PubMed Scopus (33) Google Scholar, 15.Shetty S. Idell S. Am. J. Physiol. 1998; 274: 871-882PubMed Google Scholar), their precise role in the mRNA degradation process has not been clearly delineated. A regulatory role of the poly(A) tail has been suggested, because removal of this tail precedes mRNA degradation (5.Wilson T. Triesman R. Nature. 1988; 336: 396-399Crossref PubMed Scopus (504) Google Scholar, 16.Brewer G. Ross J. Mol. Cell. Biol. 1988; 8: 1697-1708Crossref PubMed Scopus (215) Google Scholar, 17.Shyu A.B. Belasco J.G. Greenberg M.E. Genes Dev. 1991; 3: 60-70Crossref Scopus (448) Google Scholar). A poly(A) degrading enzyme was identified in mammalian cells and the cDNA was cloned from yeast (18.Astrom J. Astrom A. Virtanen A. EMBO J. 1991; 10: 3067-3071Crossref PubMed Scopus (75) Google Scholar, 19.Sachs A.B. Deardorff J.A. Cell. 1992; 70: 961-973Abstract Full Text PDF PubMed Scopus (159) Google Scholar). To our knowledge, no RNase that specifically degrades mRNAs in eukaryotic cells has yet been identified.We recently characterized a post-transcriptional regulatory mechanism that governs the expression of urokinase receptor in human mesothelioma cells (20.Shetty S. Kumar A. Idell S. Mol. Cell. Biol. 1997; 17: 1075-1083Crossref PubMed Scopus (106) Google Scholar). In these cells, urokinase-type plasminogen activator receptor mRNA is post-transcriptionally stabilized by phorbol myristate acetate or a variety of proinflammatory cytokines (20.Shetty S. Kumar A. Idell S. Mol. Cell. Biol. 1997; 17: 1075-1083Crossref PubMed Scopus (106) Google Scholar). A specific mRNA-binding protein was identified and purified (15.Shetty S. Idell S. Am. J. Physiol. 1998; 274: 871-882PubMed Google Scholar). We also found that the same mechanism is operative in human lung epithelial cells, rabbit lung fibroblasts, and pleural mesothelial cells (15.Shetty S. Idell S. Am. J. Physiol. 1998; 274: 871-882PubMed Google Scholar, 21.Shetty S. Idell S. Arch. Biochem. Biophys. 1998; 356: 265-279Crossref PubMed Scopus (32) Google Scholar). Previous studies have shown that urokinase (uPA) mRNA stability was modulated by phorbol myristate acetate and cycloheximide in porcine renal epithelial cells (22.Altus M.S. Nagamine Y. J. Biol. Chem. 1991; 266: 21190-21196Abstract Full Text PDF PubMed Google Scholar, 23.Ziegler A. Knesel J. Fabbro D. Nagamine Y. J. Biol. Chem. 1991; 266: 21067-21074Abstract Full Text PDF PubMed Google Scholar) or rat mammary tumor cells (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar, 25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar). In this report, we provide new evidence that uPA gene expression occurs at the post-transcriptional level in human lung epithelial cells and in lung carcinoma-derived cells in vitro.In these studies, we used the Beas2B lung epithelial cell line and primary cultures of human small airway epithelial cells (SAEC) as well as lung carcinoma-derived epithelial cell lines to define the post-transcriptional mechanism by which uPA gene expression is regulated. We now report that uPA mRNA has a half-life of 1–2 h in nonmalignant lung epithelial cells and that its stability is altered by inhibitors of protein synthesis. We further demonstrate that the post-transcriptional regulatory mechanism involves a cis-trans interaction between a sequence within the 3′-UTR of uPA mRNA and a newly recognized uPA mRNA-binding protein (uPA mRNABp). Overexpression of a chimeric β-globin/uPA gene containing a 66-nt uPA mRNABp binding sequence inserted into the β-globin 3′-UTR produced a destabilized chimeric mRNA transcript. We therefore conclude that uPA gene expression is regulated at the post-transcriptional level and that the mechanism involves a newly recognized interaction between a uPA mRNABp and a uPA mRNA 3′-UTR binding region that contains regulatory information capable of destabilizing uPA mRNA.EXPERIMENTAL PROCEDURESMaterialsCulture media, penicillin, streptomycin, fetal bovine serum, and RNase T1 were purchased from Life Technologies, Inc. (Grand Island, NY); tissue culture plastics were from Becton Dickinson Labware (Linclon Park, NJ). Tris base, aprotinin, dithiothreitol, phenylmethylsulfonyl fluoride, heparin, and ammonium persulfate were from Sigma. Acrylamide, bisacrylamide, and nitrocellulose were from Bio-Rad. X-AR x-ray film was from Eastman Kodak (Rochester, NY).In vitro transcription kits and 5,6-dichloro-1-β-d-ribofuranosyl benzamidazole (DRB) were purchased from Ambion (Austin TX) and Calbiochem (La Jolla, CA), respectively. HEPES and other reagents were from Fisher Scientific (Pittsburg, PA). Restriction enzymes were from New England Biolabs (Beverly, MA) and [32P]UTP was from Dupont (Wilmington, DE).Cell CulturesHuman SAEC were obtained from Clonetics (San Diego, CA) and Beas2B lung epithelial cells were obtained from the ATCC. These cells were maintained in Small Airway Growth Medium and LHC-9 media from Clonetics and Biofluids, Rockville, MD. H460 large cell-derived, A549 adenocarcinoma-derived, H157 squamous cell-derived, H1395 non-small cell-derived, and H146 small cell-derived lung carcinoma cell lines were obtained from the ATCC. These cells were maintained in RPMI media containing 10% fetal bovine serum.Western BlottingWe used SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to measure the expression of antigenic uPA by cells and conditioned media. Cell lysates and conditioned media prepared from cells were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked with 1% bovine serum albumin for 1 h at room temperature followed by overnight hybridization with uPA monoclonal antibodies, after which the membrane was washed and the blot developed using a horseradish peroxidase-conjugated secondary antibody.Treatment with Translation InhibitorsConfluent monolayers of cells in T75 flasks were switched to serum-free media containing 1% glutamine and 0.5% bovine serum albumin for 12 h. The cells were treated with or without cycloheximide, emitine, or anisomycin for 12 h at 37 °C in the same medium, using conditions and concentrations that we previously found to be effective in inducing uPA mRNA.Determination of uPA mRNA StabilityTotal RNA and RNA stability was measured by transcription-chase experiments. In this method, cells stimulated with selected agonists are then treated with DRB to inhibit ongoing transcription, after which total RNA is isolated at selected time points as described previously.Plasmid ConstructionPlasmid uPA cDNA (2.4 kb) was obtained by polymerase chain reaction amplification of a human lung cDNA library. The cDNA corresponding to the coding region (1.3 kb) and 3′-UTR (1.1 kb) were separately amplified. The human uPA mRNA template containing a complete sequence of uPA cDNA was subcloned separately to pBluescript at HindIII and XbaI sites. We made a series of enzyme-based overlapping deletions, subcloned to pBluescript SK(+) and transcribed in vitro. A deletion product of the uPA 3′-UTR containing the uPA mRNABp binding sequence (1633–1699) was created by polymerase chain reaction using prospective forward and reverse primers. The polymerase chain reaction product was cloned directly to the TA cloning vector PCRII (Invitrogen). The orientation and sequence of the clones were confirmed by sequencing. The uPA templates were linearized by HindIII or XbaI, purified on 1% agarose gels, extracted with phenol/chloroform, and used as templates for in vitro transcription.In Vitro TranscriptionThe full-length template or deletion product of uPA, containing the uPA mRNABp binding sequence, was linearized withHindIII or XbaI, purified on 1% agarose gels, and transcribed in vitro with T3 or T7 polymerase for sense or antisense mRNA according to the supplier's protocol. We modified this protocol slightly, so that 50 μCi of [32P]UTP (800 Ci/mmol) was substituted for unlabeled UTP in the reaction mixture using an Ambion in vitro transcription kit. Passage through a Sephadex G-25 column removed unincorporated radioactivity. The specific activity of the product was 4.0–5.0 × 108 cpm/μg. Sizes of labeled mRNA transcripts were confirmed by electrophoresis on 5% urea gels.Steady-state mRNA Assessment by Northern BlottingA Northern blotting assay was used to assess the steady-state level of uPA mRNA. Total RNA was isolated from non-malignant lung epithelial cells and lung carcinoma-derived cells grown to confluence in T75 flasks using TRI reagent. RNA (20 μg) was separated on agarose/formaldehyde gels, after which it was transferred to Hybond N+ according to the instructions of the manufacturer. Prehybridization and hybridization were done at 65 °C in NaCl (1 m)/SDS (1%) and 100 μg/ml salmon sperm DNA. Hybridization was performed with uPA cDNA probes (1 ng/μl) labeled to approximately 6 × 108 cpm/μg of DNA overnight by random primer method. After hybridization, the filters were washed twice for 15 min at 65 °C, with: 2 × SSC, 1% SDS; 1 × SSC, 1% SDS, and 0.1% SSC, 1% SDS, respectively. The membranes were exposed to x-ray film at −70 °C overnight. The intensity of the bands was measured densitometrically and normalized against that of β-actin.Preparation of Cytosolic ExtractsT175 flasks containing non-malignant lung epithelial cells and lung carcinoma-derived cells were serum starved overnight. The cells were detached with trypsin/EDTA, homogenized with 10 volumes of extraction buffer (25 mm Tris-HCl, pH 7.9, 0.5 mm EDTA, and 0.1 mm phenylmethylsulfonyl fluoride), after which the homogenates were centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatants were collected and centrifuged at 36,000 rpm for 4 h at 4 °C in a Beckman ultracentrifuge. The protein contents of these extracts were measured with a Pierce BCA protein assay kit using serum albumin as standards and these supernatants were used as cytosolic extracts. Intact nuclei from non-malignant lung epithelial cells and lung carcinoma-derived cells were isolated by the protocol of Antalis and Godbolt (26.Antalis T.M. Godbolt D. Nucleic Acids Res. 1991; 19: 4301Crossref PubMed Scopus (57) Google Scholar) and nuclear extracts were prepared.RNA-Protein Binding AssaysGel Mobility Shift AnalysesBinding assays were performed using uniformly 32P-labeled transcripts corresponding to the uPA coding and 3′-untranslated regions of uPA mRNA (20.Shetty S. Kumar A. Idell S. Mol. Cell. Biol. 1997; 17: 1075-1083Crossref PubMed Scopus (106) Google Scholar). Reactions were performed at 30 °C by incubating these transcripts (20,000 cpm) with cytosolic extracts (20 μg) in 15 mmKCl, 5 mm MgCl2, 0.25 mm EDTA, 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 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 was added and the mixture was incubated at room temperature for an additional 10 min. Samples were then separated by electrophoresis on 5% native polyacrylamide gels with 0.25 × TBE running buffer. The gels were dried and autoradiographed at −70 °C using Kodak X-AR film.UV Cross-linking AssayAlternatively, RNA-protein binding reactions were done as described above and then characterized as follows. After the addition of heparin, reaction mixtures were transferred to a 96-well microtiter plate and irradiated at 4 °C at 2500 μJ for 10 min with a UV-stratalinker chamber apparatus (Stratagene). The samples were then boiled for 5 min and separated on an 8% SDS-polyacrylamide gel under nonreducing conditions. The gels were dried and 32P-labeled proteins were visualized by autoradiography.Competitive Inhibition by Sense and Antisense mRNA or PolyribonucleotidesCytosolic extracts were incubated with various amounts (0–400-fold excess) of unlabeled uPA sense or antisense mRNA at 30 °C for 30 min and then treated with RNase T1 and heparin as described above, and the reaction mixtures were run on 5% native gels, dried, and autoradiographed. In separate experiments to determine the specificity of the RNA-protein complex, cytosolic extracts were pretreated with a molar excess of ribonucleotide poly(A), poly(C), poly(G), or poly(U) for 30 min at 30 °C prior to the32P-labeled uPA mRNA and RNase T1steps.Effects of SDS and Proteinase KCytosolic extracts were treated with SDS (0.1%) or proteinase K (2.5 mg/ml) for 30 min at 30 °C prior to addition of32P-labeled uPA mRNA. The reaction mixtures were subjected to RNase T1 and heparin digestion as described above, after which the complexes were resolved on 5% native gels that were then dried and exposed to x-ray film at −70 °C. In separate experiments, pre-formed complexes were treated with SDS and proteinase K before separation on polyacrylamide/TBE gels.Determination of uPA mRNABp-binding Site on uPA mRNADifferent size uPA fragments were made by deletion. These fragments were cloned into pBluescript and transcribed in vitro, then radiolabeled using [32P]UTP. Deletion transcripts were subsequently used as probes for gel mobility shift and UV-cross-linking studies to localize the protein binding sequence.Construction of β-Globin/uPA Chimeric MessageThe plasmid (pSP6βc) containing the complete human β-globin cDNA was generously provided by Dr. Richard A. Spritz (University of Wisconsin). The complete human β-globin cDNA was excised from pSP6βc and inserted into the HindIII-XbaI site of the plasmid pBluescript. Two 66-bp DNA fragments, one corresponding to the uPA mRNABp binding and the other corresponding to the control non-binding sequence, were prepared from uPA cDNA. Each of these cDNA fragments were inserted into β-globin cDNA at the 3′ XhoI site. The orientations and sequences of β-globin/uPA chimeric clones were verified by sequencing. The clones were then inserted into a eukaryotic expression vector containing the cytomegalovirus promoter, pcDNA3 (Invitrogen). Beas2B cells were transfected with the prepared chimeric plasmid constructs by lipofection using LipofectAMINE (Life Technologies, Inc.), and transient transfectants were grown in culture flasks. Total RNA was isolated at various time points after the inhibition of transcription by DRB. Chimeric β-globin/uPA mRNA was then measured by Northern blotting using 32P-labeled cDNA. The half-life of the mRNA at each interval was determined by densitometry, normalized to the β-actin control mRNA of the sample, and subsequently compared with the densitometric values of samples determined at the 0 h baseline of each experiment.DISCUSSIONAlterations that bypass normal control of cell growth and motility are central elements of the pathogenesis of cancer. The ability of tumor cells to invade normal tissue and metastasize to distant sites continues to present a major barrier to the treatment of human cancer. The plasmin/plasminogen activator (PA) cascade has been implicated in neoplastic spread. uPA, which is mainly responsible for initiation of extravascular fibrinolysis, has, in particular, been causally linked to tumor cell invasion and metastasis. uPA gene expression is generally increased in metastatic versus nonmetastatic cells, due to a combined increase in gene transcription and mRNA stability (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar,25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar).In previous studies, phorbol myristate acetate and tumor necrosis factor-α were found to increase uPA expression severalfold (27.Marshall B.C. Xu Q.-P. Rao N.V. Brown B.R. Hoidal J.R. J. Biol. Chem. 1990; 267: 11462-11469Abstract Full Text PDF Google Scholar). Elevated levels of uPA mRNA in phorbol myristate acetate and tumor necrosis factor-α stimulated cells could be due to induction of growth factors (28.Makela T.P. Alitalo R. Paulsson Y. Westermark B. Heldin C.-H. Alitalo K. Mol. Cell. Biol. 1987; 7: 3656-3662Crossref PubMed Scopus (58) Google Scholar), the activation of protein kinase C (29.Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4018) Google Scholar), increased transcription factor AP1 (30.Angel P. Imagawa M. Chui R. Stein B. Imbra R.J. Rahmsdorf H.J. Jonat C. Herrlich P. Karin M. Cell. 1987; 49: 729-739Abstract Full Text PDF PubMed Scopus (2149) Google Scholar), by increased uPA mRNA half-life, or by post-transcriptional regulation combined with other mechanisms. Recently, Nanbu et al. (31.Nanbu R. Menoud P.-A. Nagamine Y. Mol. Cell. Biol. 1994; 14: 4920-4928Crossref PubMed Google Scholar) reported post-transcriptional regulation of uPA gene expression in porcine kidney epithelial (LLC-PK1) cells. Henderson et al. (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar, 25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar) similarly reported a similar post-transcriptional regulation of uPA gene expression in rat mammary DMBA-8 cell lines. In addition, uPA gene expression has been found to be regulated at least in part by post-transcriptional mechanisms in different cell lines (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar, 25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar, 32.Altus M.S. Pearson D. Horiuchi A. Nagamine Y. Biochem. J. 1987; 242: 387-392Crossref PubMed Scopus (43) Google Scholar), since translational inhibitors increase the levels of uPA mRNA. uPA mRNA stability is also reported to be determined by multiple instability determinants present in the 3′-untranslated region of pig uPA mRNA (31.Nanbu R. Menoud P.-A. Nagamine Y. Mol. Cell. Biol. 1994; 14: 4920-4928Crossref PubMed Google Scholar).We tested the hypothesis that the increase in uPA levels in selected lung tumor cells in vitro is due to increased uPA mRNA levels. Large cell carcinoma, squamous cell carcinoma, and adenocarcinoma cells all expressed increased amounts of uPA mRNA compared with cultured normal human small airway epithelial cells or Beas2B cells. In this study, we found that uPA mRNA is differentially up-regulated in certain lung carcinoma-derived cellsversus non-malignant lung epithelial cells. We found that uPA was post-transcriptionally regulated in these cells by a newly recognized mechanism that was shown to be able to influence uPA mRNA stability.A potential link between mRNA degradation and translation emerged from prior in vitro studies showing that protein synthesis inhibitors superinduced uPA mRNA. These observations suggested the participation of a labile trans-acting protein in uPA mRNA degradation. We found that non-malignant lung epithelial cells and lung carcinoma-derived cells respond differently to translational inhibitors. Similar results have been observed in mammary adenocarcinoma (DMBA-8) cells where cycloheximide induced uPA mRNA by 8-fold, while translational inhibition has a lesser effect on uPA mRNA in metastatic cells (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar, 25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar). In porcine renal epithelial (LLC-PK1) cells, uPA mRNA stability is modulated by inhibition of protein synthesis (22.Altus M.S. Nagamine Y. J. Biol. Chem. 1991; 266: 21190-21196Abstract Full Text PDF PubMed Google Scholar, 32.Altus M.S. Pearson D. Horiuchi A. Nagamine Y. Biochem. J. 1987; 242: 387-392Crossref PubMed Scopus (43) Google Scholar), PKC down-regulation (23.Ziegler A. Knesel J. Fabbro D. Nagamine Y. J. Biol. Chem. 1991; 266: 21067-21074Abstract Full Text PDF PubMed Google Scholar), and calcium ions (33.Ziegler A. Hagmann J. Kiefer B. Nagamine Y. J. Biol. Chem. 1990; 265: 21194-21201Abstract Full Text PDF PubMed Google Scholar). These results suggest the possibility that short-lived protein(s) may account for uPA mRNA stability but do not exclude the alternative possibility that stimulation of signal transduction intermediates by the translational inhibitors could account for up-regulation of uPA.We assumed that some protein factor(s) interacts with uPA mRNA to alter its stability and confirmed this assumption by identifying a cytosolic protein in non-malignant lung epithelial cells that specifically binds to uPA mRNA 3′-untranslated region transcripts. Cytosolic extracts of the lung carcinoma-derived cells failed to form specific complexes. The cytosolic protein-RNA complex is resistant to RNase T1 digestion. Nuclear extracts of the non-malignant lung epithelial cells also failed to form a specific complex. However, a similar uPA mRNA-uPA mRNABp complex was found in the nuclear extracts of selected lung tumor cells. The specificity of the uPA mRNABp was assessed by competition experiments in which an unlabeled uPA sense probe was effectively competed by its labeled analog. An antisense transcript had no effect. Furthermore, molar excess of homoribonucleotide polymers (poly(A), poly(U), poly(C), or poly(G)), did not compete for specific probe binding, indicating that the binding of the uPA mRNABp requires a specific sequence. The involvement of a specific protein factor is further indicated by the finding that pretreatment with either SDS or proteinase K completely abolished the complex.Our experiments extend prior reports that uPA mRNA is regulated by a post-transcriptional mechanism (24.Henderson B.R. McDonald D.A. Kefford R.F. Int. J. Cancer. 1992; 50: 918-923Crossref PubMed Scopus (11) Google Scholar, 25.Henderson B.R. Tansey W.P. Phillips S.M. Ramshaw I.A. Kefford R.F. Cancer Res. 1992; 52: 2489-2496PubMed Google Scholar, 27.Marshall B.C. Xu Q.-P. Rao N.V. Brown B.R. Hoidal J.R. J. Biol. Chem. 1990; 267: 11462-11469Abstract Full Text PDF Google Scholar, 31.Nanbu R. Menoud P.-A. Nagamine Y. Mol. Cell. Biol. 1994; 14: 4920-4928Crossref PubMed Google Scholar). Cross-linking experiments with UV light indicate that the binding protein has an approximate molecular mass of 30 kDa and is distinct from previously reported AU-rich element binding proteins. Other authors described several 3′-untranslated region AUUUA multimer sequence binding proteins (34.Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (399) Google Scholar). We identified a cytoplasmic RNA binding factor that binds to newly recognized uPA 3′-UTR sequences. This protein is present in the cytoplasm of non-malignant epithelial cells and the same protein is located in the nucleus of selected lung carcinoma-derived cells. Some malignant cell types, H1395 and H146, likely degrade uPA mRNA via an alternate pathway, since we could not detect the uPA mRNABp-uPA mRNA interaction using these cells. In cells in which the interaction was detectable, binding of uPA mRNABp to cytoplasmic uPA mRNA somehow destabilizes uPA mRNA via a mechanism that remains to be elucidated. The observed increase of uPA mRNA by lung carcinoma-derived cells probably results, at least in part, from down-regulation of cytoplasmic uPA mRNABp and a consequent decrement in the uPA mRNABp-uPA mRNA interaction.The half-lives of most mRNAs are influenced by the 3′-UTR. Most studies dealing with mRNA stability determinants have identified an mRNA decay signal in the 3′-UTR. The full-length uPA mRNA transcript used in the study contains a 1300-nt coding sequence and a 1100-nt 3′-UTR sequence. The results of deletion experiments showed no 30-kDa uPA mRNABp binding sequence in the coding region or elsewhere in the 1100" @default.
• W2092440892 created "2016-06-24" @default.
• W2092440892 creator A5062403613 @default.
• W2092440892 creator A5064777131 @default.
• W2092440892 date "2000-05-01" @default.
• W2092440892 modified "2023-10-01" @default.
• W2092440892 title "Post-transcriptional Regulation of Urokinase mRNA" @default.
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