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Distinct Roles of Heterogeneous Nuclear Ribonuclear Protein K and microRNA-16 in Cyclooxygenase-2 RNA Stability Induced by S100b, a Ligand of the Receptor for Advanced Glycation End Products*

2008; Elsevier BV; Volume: 283; Issue: 52 Linguagem: Inglês

10.1074/jbc.m806322200

ISSN

1083-351X

Autores

Narkunaraja Shanmugam, Marpadga A. Reddy, Rama Natarajan,

Tópico(s)

S100 Proteins and Annexins

Resumo

Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3′-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3′-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3′-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3′-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs. Advanced glycation end products play major roles in diabetic complications. They act via their receptor RAGE to induce inflammatory genes such as cyclooxygenase-2 (COX-2). We examined the molecular mechanisms by which the RAGE ligand, S100b, induces COX-2 in monocytes. S100b significantly increased COX-2 mRNA accumulation in THP-1 monocytes at 2 h via mRNA stability. This was further confirmed by showing that S100b increased stability of luciferase-COX-2 3′-UTR mRNA. Chromatin immunoprecipitation and RNA immunoprecipitation revealed that S100b decreased occupancy of the DNA/RNA-binding protein, heterogeneous nuclear ribonuclear protein K (hnRNPK), at the COX-2 promoter but simultaneously increased its binding to the COX-2 3′-UTR. S100b treatment promoted the translocation of nuclear hnRNPK to cytoplasm, whereas a cytoplasmic translocation-deficient hnRNPK mutant inhibited S100b-induced COX-2 mRNA stability. Small interfering RNA-mediated specific knockdown of hnRNPK blocked S100b-induced COX-2 mRNA stability, whereas on the other hand, overexpression of hnRNPK increased S100b-induced COX-2 mRNA stability. S100b promoted the release of entrapped COX-2 mRNA from cytoplasmic processing bodies, sites of mRNA degradation. Furthermore, S100b significantly down-regulated the expression of a key microRNA, miR-16, which can destabilize COX-2 mRNA by binding to its 3′-UTR. MiR-16 inhibitor oligonucleotides increased, whereas, conversely, miR-16 mimic oligonucleotides decreased COX-2 mRNA stability in monocytes, further supporting the inhibitory effects of miR-16. Interestingly, hnRNPK knockdown increased miR-16 binding to COX-2 3′-UTR, indicating a cross-talk between them. These new results demonstrate that diabetic stimuli can efficiently stabilize inflammatory genes via opposing actions of key RNA-binding proteins and miRs. The cyclooxygenase-2 (COX-2) 3The abbreviations used are: COX-2cyclooxygenase-2UTRuntranslated regionRNPribonuclear proteinhnRNPKheterogeneous nuclear RNP KsiRNAsmall interfering RNAmiRmicroRNAAGEadvanced glycation end productRAGEreceptor for advanced glycation end productsPBMCperipheral blood mononuclear cellAREAU-rich elementsRT-PCRreverse transcription-PCRQPCRquantitative PCRRT-QPCRreal-time QPCRLucluciferaseNGnormal glucoseRNA-EMSARNA-electrophoretic mobility shift assayChIPchromatin immunoprecipitationRNA-IPRNA-immunoprecipitationCEcytoplasmic extractsAct Dactinomycin DGFPgreen fluorescent proteinERKextracellular signal-regulated kinaseMEKmitogen-activated protein kinase/ERK kinaseP-bodiesprocessing bodiesDAPI4′,6-diamidino-2-phenylindolentnucleotideCtrlcontrolk/dknock-down enzyme catalyzes the conversion of arachidonic acid to prostaglandins and related eicosanoids. COX-2 is significantly induced by cytokines and growth factors and can lead to the formation of potent inflammatory prostaglandins such as prostaglandin E2 as well as the vasodilatory and protective prostacyclin (1Vane J.R. Bakhle Y.S. Botting R.M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 97-120Crossref PubMed Scopus (2620) Google Scholar, 2Crofford L.J. Wilder R.L. Ristimaki A.P. Sano H. Remmers E.F. Epps H.R. Hla T. J. Clin. Investig. 1994; 93: 1095-1101Crossref PubMed Scopus (663) Google Scholar). COX-2 and its proinflammatory products have been implicated in the pathogenesis of atherosclerosis and islet dysfunction related to diabetes (3Schonbeck U. Sukhova G.K. Graber P. Coulter S. Libby P. Am. J. Pathol. 1999; 155: 1281-1291Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar, 4Burleigh M.E. Babaev V.R. Oates J.A. Harris R.C. Gautam S. Riendeau D. Marnett L.J. Morrow J.D. Fazio S. Linton M.F. 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Metab. 2008; 4: 285-293Crossref PubMed Scopus (357) Google Scholar). cyclooxygenase-2 untranslated region ribonuclear protein heterogeneous nuclear RNP K small interfering RNA microRNA advanced glycation end product receptor for advanced glycation end products peripheral blood mononuclear cell AU-rich elements reverse transcription-PCR quantitative PCR real-time QPCR luciferase normal glucose RNA-electrophoretic mobility shift assay chromatin immunoprecipitation RNA-immunoprecipitation cytoplasmic extracts actinomycin D green fluorescent protein extracellular signal-regulated kinase mitogen-activated protein kinase/ERK kinase processing bodies 4′,6-diamidino-2-phenylindole nucleotide control knock-down Previously, we reported that RAGE ligation by exogenous AGEs or by S100b can increase COX-2 expression and activity in THP-1 monocytic cells as well as in primary human blood monocytes (PBMC). COX-2 induction by S100b after 4 h of treatment was transcriptionally regulated and involved key NF-κB elements in the distal COX-2 promoter (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). However, in those studies, we noted that S100b could also increase COX-2 mRNA expression at an early time point of 2 h via unknown post-transcriptional mechanisms and were therefore investigated in the current study. Transcription of the COX-2 gene is regulated via cis-acting elements that bind to transcription factors such as NF-κB (19Appleby S.B. Ristimaki A. Neilson K. Narko K. Hla T. Biochem. J. 1994; 302: 723-727Crossref PubMed Scopus (460) Google Scholar, 20Tanabe T. Tohnai N. Prostaglandins Other Lipid Mediat. 2002; 68–69: 95-114Crossref PubMed Scopus (375) Google Scholar). However, COX-2 can also be regulated by post-transcriptional mechanisms such as mRNA stabilization (21Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 22Tamura M. Sebastian S. Yang S. Gurates B. Fang Z. Bulun S.E. J. Clin. Endocrinol. Metab. 2002; 87: 3263-3273Crossref PubMed Scopus (116) Google Scholar). In mammalian cells, conserved adenosine- and uridine-rich (AU-rich) elements (AREs) found in the 3′-untranslated regions (UTRs) of many inflammatory genes, including COX-2, can promote rapid mRNA destabilization and degradation (23Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar). AREs present within the COX-2 3′-untranslated region (COX-2 3′-UTR) regulate COX-2 mRNA decay through association with specific RNA-binding proteins such as RNA stability factor HuR and the translational repressor protein TIA-1 that target specific AREs. These RNA-protein (RNP) complexes regulate COX-2 mRNA degradation, stabilization, and translation (24Cok S.J. Acton S.J. Morrison A.R. J. Biol. Chem. 2003; 278: 36157-36162Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 25Tebo J. Der S. Frevel M. Khabar K.S. Williams B.R. Hamilton T.A. J. Biol. Chem. 2003; 278: 12085-12093Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 26Dixon D.A. Tolley N.D. King P.H. Nabors L.B. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Clin. Investig. 2001; 108: 1657-1665Crossref PubMed Scopus (376) Google Scholar, 27Sureban S.M. Murmu N. Rodriguez P. May R. Maheshwari R. Dieckgraefe B.K. Houchen C.W. Anant S. Gastroenterology. 2007; 132: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Increasing evidence suggests that microRNAs (miRs) also play key roles in ARE-mediated destabilization of inflammatory gene mRNAs (28Vasudevan S. Steitz J.A. Cell. 2007; 128: 1105-1118Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar). Binding of miR-16 to AREs of mRNA transcripts like tumor necrosis factor-α, interleukin-6, and COX-2 could promote their degradation (29Jing Q. Huang S. Guth S. Zarubin T. Motoyama A. Chen J. Di Padova F. Lin S.C. Gram H. Han J. Cell. 2005; 120: 623-634Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). Although several studies have examined transcriptional mechanisms regulating pathological genes under diabetic conditions, much less is known about the mechanisms involved in mRNA stability or the role of miRs in regulating ARE-containing mRNAs. In the present study, we show for the first time that miR-16 and a key DNA/RNA-binding protein, namely heterogeneous nuclear ribonuclear protein K (hnRNPK), play opposing roles in S100b-induced COX-2 mRNA stabilization. Our results suggest that, at early time points after treatment of monocytes with S100b, increased stabilization of COX-2 mRNA occurs via movement of hnRNPK from the COX-2 promoter to the 3′-UTR, whereas miR-16 binding to the 3′-UTR is simultaneously reduced. These data illustrate an efficient mechanism by which diabetic conditions can rapidly augment the stability of inflammatory genes without the need for increased transcription. Materials—The following antibodies were used for the co-immunoprecipitation and Western blotting analyses: anti-GFP, anti-hnRNPK (Cell Signaling, Danvers, MA), anti-luciferase, and anti-α-actin (Sigma). miR-16 siRNAs, inhibitor and mimics were from Dharmacon (Lafayette, CO). Control and hnRNPK siRNAs were from Ambion (Austin, TX). Bovine brain S100b peptide was obtained from Calbiochem. RAGE antibody was from Dr. A. M. Schmidt, Columbia University, New York, NY). Cell Culture and Treatments—Human THP-1 monocytic cells were obtained from the American Type Culture Collection or from the National Cell Culture Center (Minneapolis, MN) and cultured as described (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) in RPMI 1640 medium supplemented with heat-inactivated 10% fetal calf serum and 5.5 mm d-glucose (normal glucose, NG). Where indicated, THP-1 cells were treated with S100b (40 μg/ml) protein. Isolation of PBMC—Human PBMC were isolated as described earlier using an Institutional Review Board-approved protocol (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). PBMC were plated in 6-well dishes (1 × 105 cells/well) and treated with S100b as indicated, and total RNA were isolated as described below. RNA Preparation and Relative RT-PCR—Total RNA was isolated and reverse-transcribed, and relative RT-PCR was performed using gene-specific primers paired with QuantumRNA 18 S internal standards as described earlier (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 30Shanmugam N. Gaw Gonzalo I.T. Natarajan R. Diabetes. 2004; 53: 795-802Crossref PubMed Scopus (87) Google Scholar). The 5′ and 3′ primers for firefly luciferase (Luc) mRNA were 5′-ACGGATTACCAGGGATTTCAGTC-3′ and 5′-AGGCTCCTCAGAAACAGCTCTTC-3′, respectively. Gene expression was expressed as -fold stimulation over NG after normalizing with paired 18 S RNA levels. Analysis of microRNA (miR) Expression—The microRNAs were isolated using the mirVana quantitative RT-PCR miRNA detection kit as per the manufacturer's protocol (Ambion, Austin, TX). MiR-16 levels were quantified with Has-miR-16 quantitative RT-PCR primer sets (Ambion). For Northern blot detection of miR-16, total RNA (40 μg) was fractionated on 12.5% 7 m urea gel at 125 volts for 100 min. RNAs were transferred overnight using capillary transfer on to a Zeta probe membrane (Bio-Rad) in 2× SSC solution. The membranes were covalently cross-linked by UV irradiation for 10 min with UV Stratalinker 1800 (Stratagene, San Diego, CA). An antisense miR-16 oligonucleotide probe was labeled using T4-Poly nucleotide kinase and [γ-32P]ATP to be used as probe. Membranes were hybridized overnight with the probe in 7% SDS, 0.2 m Na2PO4, pH 7.0, at 35 °C, washed three times with 2× SSC containing 0.1% SDS at 37 °C, once with 1× SSC containing 0.1% SDS, and once with 0.5× SSC containing 0.1% SDS at 37 °C. Washed membranes were exposed to x-ray film at –70 °C for overnight. Measurement of mRNA Stability—For Luc mRNA stability assays, Luc reporter constructs containing the indicated COX-2 3′-UTR regions (21Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) were transiently transfected into THP-1 cells using Nucleofection equipment (Amaxa). Cells were allowed to recover for 1 day and treated without or with S100b (40 μg/ml) for 2 h, and then transcription was stopped by adding actinomycin D (Act D, 10 μg/ml). Samples (5 × 106 cells/ml) were collected at the indicated time intervals in RNA-STAT60 reagent. Total RNA was isolated, and Luc mRNA levels were quantified by real-time PCR. Real-time Quantitative PCR—Real-time quantitative PCRs (QPCRs) were performed using SYBR green reagent kits with gene-specific primers on the Applied Biosystems 7300 real-time PCR system (Foster City, CA). The 5′ and 3′ primers for Luc mRNA amplification were 5′-TCTCTGGCATGCGAGAATCT-3′ and 5′-ACGGATTACCAGGGATTTCAGTC-3′, respectively. Primers for control GAPDH were forward, 5′-GGTGAAGGTCGGAGTCAACG-3′, and reverse, 5′-CACCATTCTCGCTCCTGGAAGATGGTG-3′. Each sample was run in triplicate. The relative RNA amount was calculated after normalization with the internal control glyceraldehyde-3-phosphate dehydrogenase according to the method described earlier (29Jing Q. Huang S. Guth S. Zarubin T. Motoyama A. Chen J. Di Padova F. Lin S.C. Gram H. Han J. Cell. 2005; 120: 623-634Abstract Full Text Full Text PDF PubMed Scopus (712) Google Scholar). Preparation of Cytoplasmic and Nuclear Extracts—Cytoplasmic and nuclear extracts from control (NG) and S100b (40μg/ml)-treated THP-1 cells were prepared as described before (30Shanmugam N. Gaw Gonzalo I.T. Natarajan R. Diabetes. 2004; 53: 795-802Crossref PubMed Scopus (87) Google Scholar). Plasmid DNA/siRNA Transfections and Luc Assays—THP-1 cells (1.2 × 106/transfection) were transfected with indicated siRNA or miR oligonucleotides (1 nm each) and plasmids (1 μg each) using an Amaxa Nucleofector (Amaxa Biosystem) according to the manufacturer's protocols and plated in six-well plates. Following an overnight recovery period, the transfected cells were cultured in either medium alone (NG) or medium containing S100b (40 μg/ml) as indicated. Luc activity in cell lysates (20 ul) was determined using a Luc assay kit (Promega, Madison, WI) according to the manufacturer's instructions. RNA-Electrophoretic Mobility Shift Assay (RNA-EMSA) and UV Cross-linking—RNA-EMSA and UV-cross-linking were performed as described earlier (31Shanmugam N. Ransohoff R.M. Natarajan R. J. Biol. Chem. 2006; 281: 31212-31221Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). In vitro transcribed 32P-labeled COX-2 3′-UTR RNA and its deletion fragments (115, 420, and Δ1000 bp) were used as probes. After the UV cross-linking, the RNA body was digested with RNases (RNase A and RNase U2), and the proteins were fractionated on 12% SDS-PAGE. 32P-labeled RNP complexes visualized by autoradiography. Chromatin Immunoprecipitation (ChIP) Assays—ChIP assays were performed as described earlier (32Miao F. Natarajan R. Mol. Cell. Biol. 2005; 25: 4650-4661Crossref PubMed Scopus (96) Google Scholar). ChIP-enriched DNA was analyzed by PCR and QPCR using COX-2 promoter-specific primers. Distal promoter region primers were: forward primer, 5′-CCTATTAAGCGTCGTCACTA-3′, and reverse primer, 5′-CGAGAGCCAGTTCGGACTG-3′. Proximal promoter region primers were: forward primer, 5′-TGTGCGCCTGGGGCGGTGGA-3′, and reverse primer, 5′-GTCGCTAACCGAGAGAACCT-3′. Data were normalized using input samples. RNA-Immunoprecipitation (RNA-IP)—THP-1 cells were lysed using Buffer A (10 mm Tris-Cl, pH 7.9, 60 mm KCl, 1 mm EDTA, and 1 mm dithiothreitol) containing 0.1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, and Complete protease inhibitors (Roche Applied Science). Lysates were clarified by centrifugation for 10 min at 12,000 rpm, and RNasin (Promega, 40 units/ml) was added. Cell lysates were immunoprecipitated using GFP or hnRNPK antibodies (2 μg each) and 30 μl of a 50% suspension of protein A-agarose (Upstate Biotechnology) for 2 h at RT. Then protein A agarose beads were collected by centrifugation at 1000 × g and washed four times with phosphate-buffered saline containing 150 mm NaCl. RNA was extracted from the beads by phenol-chloroform extraction and reverse-transcribed using a reverse transcriptase kit (Applied Biosystems, Foster City, CA). The resulting cDNA was amplified using the following primers for human COX-2: forward, 5′-ATCTACCCTCCTCAAGTCCC-3′, and reverse, 5-′TACCAGAAGGGCAGGATACAG-3′. Immunofluorescent Staining—THP-1 cells were treated with or without S100b as indicated, and at the end of the incubation period, cells were washed and immobilized on a polylysine-coated glass slide for 30 min. Cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with phosphate-buffered saline containing 0.05% Triton X-100. The slides were then blocked (10 min) in phosphate-buffered saline containing 4% human albumin and immunostained with hnRNPK antibody (1:250 dilution) followed by appropriate Texas Red-conjugated secondary antibody (1:200 dilution). Slides were viewed using a fluorescent microscope (Olympus IX50 with DP70 camera). In Situ Hybridization—THP-1 cells were transfected with GFP-Dcp1a expression plasmid and immobilized on a polylysine glass slide for 30 min. After fixing with paraformaldehyde (4%), cells were treated with 70% ethanol, rehydrated in 2× SSC, and used for hybridization. Cells were hybridized with 15 ng of the fluorescent oligonucleotides complementary to the COX-2 coding region in 50 μl of hybridization solution containing 2× SSC, 30% formamide, 10% dextran sulfate, 0.2 mg/ml bovine serum albumin, and 250 μg/ml yeast tRNA. The sequence of the probe used was 5′-gT*actggaattgtttgttgaaaagT*agttctgggtcaaattT*c-3′. Residues marked as T* are the amino-modified thymidines that were conjugated to Alexa Fluor 555. Slides were washed three times with 2× SSC and 30% formamide for 30 min at 37 °C. Slides were counterstained for DNA with DAPI (Sigma), mounted using antifade Vectashield (Vector Laboratories, Burlingame, California), and viewed under a fluorescence microscope. In Vitro Pull-down Assays to Evaluate miR-16 Binding—Biotin UTP-labeled RNA of COX2–3′-UTRs were prepared by in vitro transcription using T7 RNA polymerase for 2 h at 37°C with 1:10 ratio of biotin-labeled UTP:UTP (Applied Biosystems, Foster City, CA). The RNA samples were then run on a 5% 7 m urea PAGE. RNA was extracted from the excised gel samples followed by phenol:chloroform extraction and precipitated with ethanol. The biotin-labeled RNA was further purified by another round of phenol:chloroform extraction and ethanol precipitation. MiR-16 and antisense miR-16 were 32P-labeled at the 5′ end with T4 polynucleotide kinase. These labeled miRs (5 × 106 cpm) were each added to a 30-μl reaction mixture containing 15 μl of THP-1 cell cytoplasmic extracts (CE), 1 mm ATP, 0.2 mm GTP, 40 units/ml RNasin, 5 mm EGTA, 30 μg/ml creatine kinase, 25 mm creatine phosphate, and 200 pmol of biotin-labeled RNA. The binding reaction was carried out for 10 min at 30 °C. The biotin-labeled RNA was pulled down with streptavidin-coated Dynabeads that had been preblocked with CEs. The amount of miR bound to the RNA was determined using a scintillation counter. Data Analyses—Data are expressed as mean ± S.E. of multiple experiments. Paired Student's t tests were used to compare two groups and analysis of variance with Dunnett's post tests for multiple comparisons. COX-2–3′-UTR Plays an Important Role in S100b-mediated Stabilization of COX-2 mRNA—We previously reported that S100b could induce COX-2 expression at 4 h in human monocytes through NF-κB-mediated transcriptional activation of the COX-2 promoter (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) and at 2 h due to mRNA stability regulated via unknown mechanisms. We therefore evaluated the mechanisms involved in S100b-induced COX-2 mRNA stability. Human THP-1 monocytic cells were first transiently transfected with plasmids containing Luc reporter under the control of human COX-2 promoter regions (–7140/+127, –1430/+127, and –860/+127), stimulation with S100b for 2 h, and Luc activities were determined. S100b did not induce COX-2 promoter transcriptional activation at 2 h with any of these constructs (Fig. 1A), unlike at 4 h (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Evidence shows that COX-2 mRNA stability can be regulated by the presence of a 3′-UTR with multiple copies of AREs targeted by RNA-interacting factors (21Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 24Cok S.J. Acton S.J. Morrison A.R. J. Biol. Chem. 2003; 278: 36157-36162Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 25Tebo J. Der S. Frevel M. Khabar K.S. Williams B.R. Hamilton T.A. J. Biol. Chem. 2003; 278: 12085-12093Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 26Dixon D.A. Tolley N.D. King P.H. Nabors L.B. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Clin. Investig. 2001; 108: 1657-1665Crossref PubMed Scopus (376) Google Scholar, 27Sureban S.M. Murmu N. Rodriguez P. May R. Maheshwari R. Dieckgraefe B.K. Houchen C.W. Anant S. Gastroenterology. 2007; 132: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We therefore hypothesized that the S100b-induced COX-2 stabilization may involve similar mechanisms. Because COX-2 mRNA can be increased by both stability (at 2 h) as well as promoter activation at > 4 h (18Shanmugam N. Kim Y.S. Lanting L. Natarajan R. J. Biol. Chem. 2003; 278: 34834-34844Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), measuring COX-2 mRNA will not reflect the true RNA stability component. Therefore, we used reporter plasmids containing the COX-2 3′-UTR at the 3′ end of the Luc gene expressed from a cytomegalovirus promoter (21Dixon D.A. Kaplan C.D. McIntyre T.M. Zimmerman G.A. Prescott S.M. J. Biol. Chem. 2000; 275: 11750-11757Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) and used Luc as a surrogate for the COX-2. Under these conditions, measuring Luc mRNA half-life (steady-state Luc mRNA levels) after treatment with Act D to stop transcription would reflect COX-2 mRNA stabilization by S100b due to the COX-2 3′-UTR. THP-1 cells were transfected with indicated Luc-COX-2 3′-UTR plasmids (Fig. 1B), treated with or without S100b, and Luc activities in lysates were measured. Results showed that basal Luc activity was dramatically reduced in cells expressing Luc containing full-length COX-2 3′-UTR (pZeo/Luc-COX-2–1477) and 420 bp (pZeo/Luc-COX-2–420) when compared with those transfected without COX-2 3′-UTR (pZeo/Luc) (Fig. 1C), demonstrating the destabilizing effect of the COX-2 3′-UTR on Luc mRNA (or COX-2 mRNA). S100b treatment reversed this effect and significantly increased Luc activity of both pZeo/Luc-COX-2–1477 and pZeo/Luc-COX-2–420 by 3-fold with no effect on pZeo/Luc transfected cells (Fig. 1C). In contrast, Luc with COX-2 3′-UTR region in the antisense orientation (pZeo/Luc-COX-2–420AS) or with 3′-UTR harboring a deletion at the 5′end (pZeo/Luc-COX-2-Δ1000) displayed higher basal Luc activity when compared with the 1477- and 420-bp constructs. S100b treatment also failed to increase their Luc activities (Fig. 1C). These results demonstrate that the cis-elements located within 5′ end 420 bp of the 3′-UTR are involved in S100b-induced COX-2 mRNA stability in monocytes. Furthermore, S100b-induced Luc activity at 2 h in THP-1 cells transfected with pZeo/Luc-COX-2–420 was significantly blocked by pretreatment with a RAGE antibody relative to IgG alone (data not shown), demonstrating the role of RAGE-mediated signaling in COX-2 stability. Immunoblotting of transfected cell lysates with Luc antibody showed increased Luc protein levels in S100b-treated THP-1 cells transfected with pZeo/Luc-COX-2–1477 and pZeo/Luc-COX-2–420 when compared with the respective untreated cells (Fig. 1D) but not in cells transfected with pZeo/Luc or pZeo/Luc-COX-2–420AS (Fig. 1D). S100b also significantly increased levels of Luc-COX-2–1477 mRNA in THP-1 cells (Fig. 1E). These results confirm that the observed changes in Luc activity reflect Luc protein and mRNA levels in transfected cells. Next, we further confirmed the involvement of the COX-2 3′-UTR in Luc mRNA (or COX-2 mRNA) stability in the THP-1 cells treated with S100b. THP-1 cells transfected with pZeo/Luc-COX-2–1477 were treated with S100b for 2 h, and transcription was inhibited by treatment with Act D. Luc mRNA levels were then determined by RT-QPCR at various time intervals up to 2 h

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