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• W2082030103 abstract "Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability. Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5′-promoter region showed weak responses to ATPγS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3′-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPγS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3′-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPγS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability. The matrix metalloproteinases (MMPs) 1The abbreviations used are: MMPmatrix metalloproteinaseAREAU-rich elementELAVembryonic lethal abnormal visionECMextracellular matrixUTRuntranslated regionILinterleukinGAPDHglyceraldehyde-3-phosphate dehydrogenaseMCmesangial cellsEMSAelectromobility shift assayATPγSadenosine 5′-O-(thiotriphosphate)PBSphosphate-buffered salineRLUrelative light unitPKCprotein kinase CMAPKmitogen-activated protein kinase.1The abbreviations used are: MMPmatrix metalloproteinaseAREAU-rich elementELAVembryonic lethal abnormal visionECMextracellular matrixUTRuntranslated regionILinterleukinGAPDHglyceraldehyde-3-phosphate dehydrogenaseMCmesangial cellsEMSAelectromobility shift assayATPγSadenosine 5′-O-(thiotriphosphate)PBSphosphate-buffered salineRLUrelative light unitPKCprotein kinase CMAPKmitogen-activated protein kinase. are members of a family of zinc-dependent endopeptidases which specifically degrade components of the extracellular matrix (ECM). Therefore, MMPs have mainly been implicated in various diseases accompanied with an altered turnover of ECM. Besides the altered synthesis of single ECM components the increased expression and/or activity of MMPs seems of paramount importance for pathological remodeling processes within the kidney such as acute proliferative glomerulonephritis (1Edelstein C.L. Ling H. Schrier R.W. Kidney Int. 1997; 51: 1341-1351Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 2Lenz O. Elliot S.J. Stetler-Stevenson W.G. J. Am. Soc. Nephrol. 2000; 11: 574-581PubMed Google Scholar). Mainly the altered expression of MMP-2 and MMP-9, which are also denoted as gelatinases, is crucially involved in the progression of glomerular inflammatory processes (2Lenz O. Elliot S.J. Stetler-Stevenson W.G. J. Am. Soc. Nephrol. 2000; 11: 574-581PubMed Google Scholar, 3Davies M. Martin J. Thomas G.J. Lovett D.H. Kidney Int. 1992; 41: 671-678Abstract Full Text PDF PubMed Scopus (174) Google Scholar). In addition to various inflammatory cytokines, the expression of MMP-9 can be activated by many other stimuli such as mitogens, growth factors, and activators of the Ras oncogene (for review, see Refs. 4Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3081) Google Scholar and 5Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar). Although most of these stimuli can modulate gelatinolytic activity by influencing MMP-9 gene expression, the regulation of MMP-9 activity is also achieved by the processing of the inactive proenzyme by the action of other proteases and by an inhibition of the active enzyme by its endogenous inhibitors, the tissue inhibitors of MMPs (4Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3081) Google Scholar, 5Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3860) Google Scholar). Previously, we have demonstrated an additional mode of posttranscriptional regulation of MMP-9, which involves the reduction of cytokine-induced MMP-9 via reduction of mRNA stability exerted by exogenous and endogenouly produced NO (6Eberhardt W. Akool E.S. Rebhan J. Frank S. Beck K.F. Franzen R. Hamada F.M. Pfeilschifter J. J. Biol. Chem. 2002; 277: 33518-33528Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). A variety of inducible genes including proto-oncogenes, transcription factors, cell cycle-regulating proteins, and cytokines have been demonstrated to be regulated by a modulation of mRNA turnover. Recent evidence has revealed that AU-rich sequences also denoted as AU-rich elements (AREs) located in the 3′-untranslated regions (UTRs) of these genes comprise specific cis-regulating elements which target mRNAs for rapid degradation (for review, see Refs. 7Chen C.Y. Shyu A.B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1673) Google Scholar, 8Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar, 9Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar, 10Peng S.S. Chen C.Y. Xu N. Shyu A.B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (653) Google Scholar). Several studies have identified proteins specifically binding to AREs, among them RNA stabilizing factors of the embryonic lethal abnormal vision (ELAV) protein family especially the ELAV-like protein HuR (9Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar, 10Peng S.S. Chen C.Y. Xu N. Shyu A.B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (653) Google Scholar, 11Brennan C.M. Steitz J.A. Cell Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (875) Google Scholar). Overexpression and subsequent binding of HuR was shown to result in an efficient stabilization of ARE-containing mRNAs in vivo (9Fan X.C. Steitz J.A. EMBO J. 1998; 17: 3448-3460Crossref PubMed Scopus (743) Google Scholar). matrix metalloproteinase AU-rich element embryonic lethal abnormal vision extracellular matrix untranslated region interleukin glyceraldehyde-3-phosphate dehydrogenase mesangial cells electromobility shift assay adenosine 5′-O-(thiotriphosphate) phosphate-buffered saline relative light unit protein kinase C mitogen-activated protein kinase. matrix metalloproteinase AU-rich element embryonic lethal abnormal vision extracellular matrix untranslated region interleukin glyceraldehyde-3-phosphate dehydrogenase mesangial cells electromobility shift assay adenosine 5′-O-(thiotriphosphate) phosphate-buffered saline relative light unit protein kinase C mitogen-activated protein kinase. Besides the proinflammatory cytokines, MC are able to respond to a variety of other biological mediators including eicosanoids, growth factors, reactive oxygen species, NO, and to extracellular nucleotides such as ATP and UTP (12Pfeilschifter J. Merriweather C. Br. J. Pharmacol. 1993; 110: 847-853Crossref PubMed Scopus (55) Google Scholar, 13Pfeilschifter J. Eberhardt W. Beck K.F. Huwiler A. Nephron. 2003; 93: 23-26Google Scholar). Previously, we have reported that in renal mesangial cells, extracellular ATP via the P2Y2 receptor can cause a mobilization of intracellular calcium and subsequent activation of protein kinases C (14Pavenstädt H. Gloy J. Leipziger J. Klar B. Pfeilschifter J. Schollmeyer P. Greger R. Br. J. Pharmacol. 1993; 109: 953-959Crossref PubMed Scopus (67) Google Scholar, 15Pfeilschifter J. Huwiler A. J. Auton. Pharmacol. 1996; 16: 315-318Crossref PubMed Scopus (25) Google Scholar). The cellular long term responses toward ATP and UTP include a variety of pathophysiological key functions most importantly the inhibition of programmed cell death and an increase in cell proliferation (16Huwiler A. Rölz W. Dorsch S. Ren S. Pfeilschifter J. Br. J. Pharmacol. 2002; 136: 520-529Crossref PubMed Scopus (52) Google Scholar, 17Schulze-Lohoff E. Hugo C. Rost S. Arnold S. Gruber A. Brüne B. Sterzel R.B. Am. J. Physiol. 1998; 275: 962-971PubMed Google Scholar, 18Huwiler A. Pfeilschifter J. Br. J. Pharmacol. 1994; 113: 1455-1463Crossref PubMed Scopus (100) Google Scholar). Both processes, cell proliferation and the excessive ECM degradation, are hallmarks of many chronic progressive glomerular diseases (1Edelstein C.L. Ling H. Schrier R.W. Kidney Int. 1997; 51: 1341-1351Abstract Full Text PDF PubMed Scopus (223) Google Scholar, 2Lenz O. Elliot S.J. Stetler-Stevenson W.G. J. Am. Soc. Nephrol. 2000; 11: 574-581PubMed Google Scholar, 3Davies M. Martin J. Thomas G.J. Lovett D.H. Kidney Int. 1992; 41: 671-678Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 19Fogo A.B. Kidney Int. 2001; 59: 804-819Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). For this reason we tested whether extracellular nucleotides can influence the expression of MMP-9 in MC. Our data provide the first report that ATP and UTP can potentiate the cytokine-induced expression and activity of MMP-9. Furthermore, we implicate modulation of the nuclear-cytosolic shuttling of the RNA stabilizing factor HuR in the posttranscriptional regulation of MMP-9 by extracellular nucleotides. Reagents—Human recombinant IL-1β was from Cell Concept (Umkirch, Germany). All nucleotides were obtained from Sigma (Deisenhofen, Germany). Actinomycin D (from Streptomyces species) was purchased from Alexis Biochemicals (Laeufelfingen, Switzerland). Ribonucleotides, restriction enzymes, and modifying enzymes were purchased from Roche Diagnostics GmbH (Mannheim, Germany). RNA oligonucleotides were synthesized from Whatman-Biometra (Göttingen, Germany). Cell Culture—Rat glomerular MC were characterized as described previously (20Pfeilschifter J. Vosbeck K. Biochem. Biophys. Res. Commun. 1991; 175: 372-379Crossref PubMed Scopus (162) Google Scholar) and grown in RPMI 1640 supplemented with 10% fetal calf serum, 2 mm glutamine, 5 ng/ml insulin, 100 units/ml penicillin, and 100 μg/ml streptomycin. Serum-free preincubations were performed in Dulbecco's modified Eagle's medium supplemented with 0.1 mg/ml of fatty acid-free bovine serum albumin for 24 h before cytokine treatment. For experiments MC between passages 8 and 19 were used. All cell culture media and supplements were purchased from Invitrogen (Karlsruhe, Germany). A monoclonal anti-HuR and the anti-mouse horseradish peroxidase-linked antibodies were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). cDNA Clones and Plasmids—cDNA inserts for rat MMP-9 was generated as described recently (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). A cDNA insert from mouse 18 S rRNA was from Ambion (Austin, TX). A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA clone from rats was generated as described previously (25Akool E.S. Kleinert H. Hamada F.M.A. Abdelwahab M.H. Forstermann U. Pfeilschifter J. Eberhardt W. Mol. Cell. Biol. 2003; 23: 4901-4916Crossref PubMed Scopus (154) Google Scholar). Generation of Reporter Plasmids and Transient Transfection of MC— The MMP-9 reporter gene pGL3-MMP-9(1.3kb) encompassing a 1.3-kb fragment of the 5′-flanking region of the rat MMP-9 gene was cloned as described previously (22Eberhardt W. Schulze M. Engels C. Klasmeier E. Pfeilschifter J. Mol. Endocrinol. 2002; 16: 1752-1766Crossref PubMed Scopus (113) Google Scholar). The plasmid 3′-UTR-MMP-9 pGL3-MMP-9(1.3kb) was generated by cloning of a 662-bp fragment from the 3′-UTR of rat MMP-9 mRNA into the pGL3-MMP-9(1.3kb) promoter plasmid. The 3′-UTR sequence of the rat MMP-9 gene was generated by PCR from reverse transcriptase products using the XbaI-flanked (underlined) forward primer 5′-TATTCTAGACCAACCTTTACCAGCTACTCGAA-3′ and BamHI-flanked (underlined) reverse primer 5′-TATGGATCCATTCATT-TATTTAAAAAAGAGTGT-3, corresponding to a region from nucleotides 2315-2338 and 2954-2977 of the rat MMP-9 cDNA (GenBank™ accession number U24441). The PCR products subsequently were digested with XbaI and BamHI restriction enzymes and cloned into the BamHI/XbaI cut pGL3-MMP-9(1.3kb) plasmid, thereby allowing a forced insertion of the 3′-UTR of rat MMP-9 mRNA at the 3′-end of the luc+ coding region of pGL3-MMP-9(1.3kb). Introduction of triple-point mutation into ARE sites (ATTTA to ACCCA) to generate ΔARE mutations were performed using the following (sense) primer: 5′-TACCGGCCCTTTTAC-CCATTATGTATGTGG-3′ (corresponding to a region from 2494 to 2523) to generate “3′-UTR-ΔARE1-pGL3-MMP-9(1.3 kb)” 5′-TTCACACACAT-GTACCCAACCTATAGAATG-3′ (corresponding to a region from 2529 to 2558) to generate “3′-UTR-ΔARE2-pGL3-MMP-9(1.3kb)” 5′-TTAGGGA-CAGAGGAACCCATTGGATGTTGG-3′ (corresponding to a region from 2735 to 2764) to generate “3′-UTR-ΔARE4-pGL3-MMP-9(1.3kb).” All mutants were generated by use of the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla CA). The plasmid “3′-UTR-ΔARE1-2-4-pGL3-MMP-9(1.3kb)” in which all three AREs have been mutated was generated by generation of a construct bearing double-mutated AREs (“3′-UTR-ΔARE1-2-pGL3-MMP-9(1.3kb)”), which then served as a template. Transient transfections of MC were performed using the Effectene reagent (Quiagen, Hilden, Germany). Transfections were performed following the manufactorer's instructions. The transfections were done as triplicates and repeated at least three times to ensure reproducibility of the results. Transfection with pRL-CMV coding for Renilla luciferase was used to control for transfection efficiencies. Luciferase activities were measured with the dual reporter gene system (Promega) using an automated chemoluminescence detector (Berthold, Bad Wildbad, Germany). Northern Blot Analysis—Total cellular RNA was extracted from MC using the Tri reagent (Sigma), and RNA was hybridized following standard procedures as described previously (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Preparation of Cytoplasmic Extracts—Cytoplasmic lysates were separated as described previously (22Eberhardt W. Schulze M. Engels C. Klasmeier E. Pfeilschifter J. Mol. Endocrinol. 2002; 16: 1752-1766Crossref PubMed Scopus (113) Google Scholar). Briefly, cytoplasmic lysates were prepared from rat MC by lysis in a hypotone extraction buffer containing 10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 100 μg/ml phenylmethanesulfonyl fluoride. Nuclei were removed by centrifugation (1200 × g for 15 min) and the supernatants used as cytoplasmic fractions. RNA Electromobility Shift Assay (EMSA) and Supershift Analysis— Radiolabeled single-stranded RNA oligonucleotides were prepared by kinase reaction using T4 polynucleotide kinase. The labeled oligonucleotides were subsequently separated by Nick-Sephadex columns (Amersham Biosciences, Freiburg, Germany). The sequences for gene specific oligonucleotides are summerized in Table I. Approximately 30 fmol of the radiolabeled RNA oligonucleotide (∼30,000 cpm/reaction) were incubated with 6 μg of cytoplasmic extract and incubated at room temperature for 15 min in a buffer containing 10 mm Hepes, pH 7.6, 3 mm MgCl2, 40 mm KCl, 2 mm dithiothreitol, 5% glycerol, and 0.5% Nonidet P-40. To reduce nonspecific binding total yeast RNA (200 ng/ml final concentration) was added. The total volume of each reaction was 10 μl. RNA-protein complexes were separated in 6% nondenaturating polyacrylamide gels and run in Tris borate EDTA.Table IOligonucleotides used in EMSA The sequence of oligonucleotides was derived from the region encompassing AUUUA-rich motifs within the 3′-UTR region of the rat MMP-9 gene. The position corresponding to the rat MMP-9 gene (GeneBank™ accession number U24441) is indicated by numbers. The consensus sequence is indicated by bold letters, and the nucleotides changed for mutation are underlined. nt, nucleotide.OligonucleotideStarting ntEnding ntUTR-ARE-125005′-CCCUUUUAUUUAUUAUGUAUG-3′2520UTR-ΔARE-15′-CCCUUUUACCCAUUAUGUAUG-3′UTR-ARE-225365′-ACAUGUAUUUAACCUAUAGAA-3′2556UTR-ΔARE-25′-ACAUGUACCCAACCUAUAGAA-3′UTR-ARE-427425′-CAGAGGAAUUUAUUGGAUGUU-3′2762UTR-ΔARE-45′-CAGAGGAACCCAUUGGAUGUU-3′ Open table in a new tab Supershift analysis was done by addition of 200 ng of a monoclonal anti-HuR-specific antibody (Santa Cruz Biotechnology) 15 min after the addition of the radioactive labeled RNA oligonucleotide and incubated for a further 15 min at room temperature. In Vitro Degradation Assay—Degradation assays were performed as described previously with the following modifications (23Levy A.P. Levy N.S. Goldberg M.A. J. Biol. Chem. 1996; 271: 2746-2753Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar). Instead of using in vitro transcribed radioactively labeled MMP-9 mRNA, we used preparations from cytokine-induced total RNAs, thereby yielding a high amount of endogenous MMP-9 mRNA. 20 μg of total RNA from one pool were aliquoted and subsequently incubated at room temperature with 130 μg of cytoplasmic extract derived from untreated or alternatively from ATPγS-treated MC for different time points. RNA from these incubations was isolated by standard procedures and assessed by Northern blot analysis using a 32P-labeled cDNA insert specific for rat MMP-9 (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The remaining (undegraded) MMP-9 hybridized signals were analyzed and quantified on an imaging system from Fujifilm (Raytest, Struabenhardt, Germany). HuR Neutralization Experiments—The impact of HuR on the degradation of MMP-9 mRNA was tested by the addition of a neutralizing monoclonal anti-HuR antibody (Santa Cruz Biotechnology). 0.4 μg of the antibody was added to the cytoplasmic extracts from ATPγS-treated MC and preincubated at room temperature for 1 h before addition of the RNA samples. To exclude any unspecific inhibitory effects by the mouse serum, we tested in parallel normal mouse serum. Western Blot Analysis—The total cellular level of HuR protein was analyzed by Western blot analysis using total cellular extracts (50 μg) using a monoclonal antibody specifically raised against full-length human HuR (Santa Cruz Biotechnology). A polyclonal antibody specific for human α-actin was purchased from (Chemicon, Hofheim, Germany). The secondary antibodies conjugated to horseradish peroxidase were incubated for 45 min at room temperature and blots were developed using the ECL system (Amersham Biosciences). Immunocytochemistry of HuR—At ∼60-80% confluence, mesangial cells were rendered serum-free for 24 h and thereafter stimulated with nucleotides for the indicated time periods. Subsequently, cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated for 30min at -20 °C with methanol containing 0.02% (w/v) EDTA. Cells were then washed twice with PBS, blocked for 1h in PBS containing 3% (w/v) bovine serum albumin and incubated for 2 h with monoclonal anti-HuR antibody. Cells were then washed several times with PBS and incubated for 1 h with an anti-mouse-Alexa 488-coupled secondary antibody and thereafter washed again with PBS. Cellular fluorescence was monitored using confocal microscopy (MicroRadiation, Bio-Rad, Hertfordshire, UK). Statistical Analysis—Results are expressed as means ± S.E. The data are presented as x-fold induction compared with control conditions or compared with IL-1β-stimulated values. Statistical analysis was performed using Student's t test for significance. p values <0.05, <0.01, and <0.005 were considered significant. Extracellular Nucleotides Potentiate IL-1β-induced Lytic Activity and mRNA Steady-state Levels of MMP-9 —To evaluate possible effects of extracellular nucleotides on the activity of MMP-9, MC were treated with IL-1β for 24 h in the presence or absence of either ATP (30 μm) or UTP (30 μm). Furthermore, we tested for possible effects of adenosine a degradation product of ATP, as well as ATPγS, a stable ATP analog. The gelatinolytic contents in the conditioned medium of MC were tested by zymography using gelatin as a substrate (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). We chose the 24-h treatment to allow accumulation of extracellular MMP-9, which is high enough to be detected by gelatin zymography (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The cytokine-mediated gelatinolytic content of latent MMP-9, which is represented by one lytic band at 92 kDa, is strongly increased when MC were cotreated with ATPγS or UTP (Fig. 1A). No effect on the IL-1β-caused lytic content is seen with ATP and its degradation product adenosine (Fig. 1A). We suggest that under cell culture conditions the rapid degradation by ectonucleotidases removes ATP and therefore prevents sustained signaling by ATP. We observed that the levels of lytic bands migrating at 68 and 72 kDa, which corresponds to latent and active MMP-2, were not significantly changed, which is consistent with the constitutive expression of MMP-2 in rat MC (21Eberhardt W. Beeg T. Beck K.F. Walpen S. Gauer S. Böhles H. Pfeilschifter J. Kidney Int. 2000; 57: 59-69Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). In the absence of IL-1β, none of the tested nucleotides or nucleoside was able to induce MMP-9 activity (Fig. 1A). Furthermore, the stimulatory effect of ATPγS occurred in a dose-dependent manner with a maximal effect seen with 30 μm of ATPγS (Fig. 1B). In contrast, the higher concentrations did not further augment the cytokine-induced gelatinolytic content in the conditioned media (Fig. 1B, upper panel). To evaluate whether the increased level of gelatinolytic activity of MMP-9 is preceeded by an enhanced expression of MMP-9 mRNA, we performed Northern blot analyses using a cDNA from the rat MMP-9 gene. As shown in Fig. 1B (lower panel) similar to the changes in the gelatinolytic contents, ATPγS dose-dependently augmented the cytokine-induced MMP-9 mRNA level with a maximal effect seen at 30 μm ATPγS, whereas higher concentrations than 30 μm blunted the amplification of cytokine-induced MMP-9. No MMP-9 mRNA was detected in the absence of IL-1β when stimulating with the nucleotide alone (Fig. 1B, lower panel). To test the time-dependence of cytokine-induced MMP-9 by ATPγS we monitored the time course of MMP-9 induction by Northern blot analysis. As shown in Fig. 1C the indcution of MMP-9 mRNA as caused by the treatment of MC with either IL-1β or with IL-1β plus ATPγS did not occur at the early time points tested (4 and 12 h), thus indicating that ATPγS cannot induce MMP-9 mRNA expression by its own. Furthermore, these data indicate that the alterations of cytokine-induced gelatinolytic activity by ATPγS predominantly result from changes in the MMP-9 expression levels. Involvement of the P2Y2 Receptor in the Amplification Cascade of MMP-9 by ATP—Many of the physiologcial actions exerted by ATP involve the G-protein coupled P2Y2 subtype of purinoceptors. We therefore tested by a pharmacological approach for the involvement of the P2Y2 purinoreceptor in the amplification of cytokine-induced MMP-9 expression by use of suramin, a putative antagonist of P2Y2 but not of P2Y4 purinoceptors (24Boarder M.R. Hourani S.M. Trends Pharmacol. Sci. 1998; 19: 99-107Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). As shown in Fig. 2, suramin dose-dependently inhibited the ATPγS-mediated potentiation of IL-1β gelatinolytic activity of MMP-9 without affecting the cytokine-caused gelatinolytic content of MMP-9. This suggests an involvement of the P2Y2 subtype of purinoceptor in the ATP signaling of MMP-9 expression. Nucleotides Have No Effects on Cytokine-induced MMP-9 Promoter Activity—To further evaluate whether the ATP-mediated amplification of IL-1β-induced MMP-9 expression resulted from an increase in MMP-9 gene transcription we assessed promoter activites derived from a 1.3-kb fragment of the rat MMP-9 promoter region (pGL-MMP-9(1.3 kb)) by luciferase reporter gene assays. This promoter region contains several functional elements necessary for cytokine-mediated regulation of MMP-9 expression most important one NF-κB binding site and one AP-1 response element which is close to a functional Ets-1 binding site (22Eberhardt W. Schulze M. Engels C. Klasmeier E. Pfeilschifter J. Mol. Endocrinol. 2002; 16: 1752-1766Crossref PubMed Scopus (113) Google Scholar). Transient transfection of MC with pGL-MMP-9(1.3 kb), comprising the 1.3-kb promoter fragment fused to the luciferase reporter gene, was followed by a 24-h treatment with either vehicle, IL-1β (2 nm), with or without either ATPγS (30 μm), or UTP (30 μm) or with each nucleotide alone and subsequently assayed for luciferase activity (Fig. 3). IL-1β significantly stimulated luciferase activity (1.9-fold, p < 0.01), but the promoter activity was only weakly enhanced by either nucleotide alone. These data demonstrate that the amplification of cytokine-induced mRNA steady-state levels by ATPγS cannot be located to the upstream 1.3-kb MMP-9 promoter context. ATPγS Inhibits the Decay of Cytokine-induced MMP-9 mRNA—To test whether the ATP effects relay to some post-transcriptional events, we performed actinomycin D experiments. MC were stimulated for 20 h with IL-1β (2 nm) before transcription was blocked by actinomycin D (5 μg/ml). Subsequently, cells were either directly homogenized (vehicle, 0 h) or left untreated (vehicle, 12 h) or alternatively treated with the stable ATP analog ATPγS (30 μm). After 12 h cells were homogenized for the isolation of total RNA. The MMP-9 mRNA from untreated MC displayed a strong reduction by almost 80% (p < 0.005) in the mRNA steady-state level (Fig. 4A). Most interestingly, the decay of MMP-9 mRNA was completely blocked in the presence of ATPγS, thus indicating that ATPγS can stabilize the IL-1β-induced MMP-9 mRNA (Fig. 4A). Furthermore, time course experiments revealed that the mRNA stabilizing effects on MMP-9 transcripts by ATPγS already occurred at 4 h and were maximal 12 h after the blockade of transcription by actinomycin D (Fig. 4B). Recently it has been reported that transcription blockage by actinomycin D can induce a redistribution of the mRNA stabilization factor HuR from the nucleus to the cytoplasm and therefore may cause an increase in the mRNA half-life (10Peng S.S. Chen C.Y. Xu N. Shyu A.B. EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (653) Google Scholar). However, in our experiments all cells were equally treated with actinomycin D, and therefore, the net effect on MMP-9 decay exclusively depend on the presence or absence of extracellular ATPγS. In contrast, IL-1β by itself had no effect on the stability of cytokine-induced MMP-9 mRNA, since the addition of IL-1β (2 nm) after transcriptional blockade did not cause any changes in the mRNA decay of MMP-9 (Fig. 4C). In summary, these data indicate that ATPγS can augment the cytokine-induced MMP-9 expression by" @default.
• W2082030103 created "2016-06-24" @default.
• W2082030103 creator A5000091817 @default.
• W2082030103 creator A5000332488 @default.
• W2082030103 creator A5015539308 @default.
• W2082030103 creator A5046524304 @default.
• W2082030103 creator A5074909982 @default.
• W2082030103 creator A5088322762 @default.
• W2082030103 date "2003-12-01" @default.
• W2082030103 modified "2023-10-10" @default.
• W2082030103 title "ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR" @default.
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