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• W2105280204 abstract "Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3′-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease.Background: Meprin α is a metalloproteinase implicated in the pathogenesis of inflammatory bowel disease (IBD).Results: RNA-binding proteins post-transcriptionally regulate meprin α, and RNA stability decreases after induction of an inflammatory response.Conclusion: Inflammatory stimuli downregulate meprin α by inducing TTP expression and binding to the transcript.Significance: Determining how inflammation alters meprin α regulation is crucial to understanding its role in IBD. Meprins are multimeric proteases that are implicated in inflammatory bowel disease by both genetic association studies and functional studies in knock-out mice. Patients with inflammatory bowel disease show decreased colonic expression of meprin α, although regulation of expression, particularly under inflammatory stimuli, has not been studied. The studies herein demonstrate that the human meprin α transcript is bound and stabilized by Hu antigen R at baseline, and that treatment with the inflammatory stimulus phorbol 12-myristate 13-acetate downregulates meprin α expression by inducing tristetraprolin. The enhanced binding of tristetraprolin to the MEP1A 3′-UTR results in destabilization of the transcript and occurs at a discrete site from Hu antigen R. This is the first report to describe a mechanism for post-transcriptional regulation of meprin α and will help clarify the role of meprins in the inflammatory response and disease. Background: Meprin α is a metalloproteinase implicated in the pathogenesis of inflammatory bowel disease (IBD). Results: RNA-binding proteins post-transcriptionally regulate meprin α, and RNA stability decreases after induction of an inflammatory response. Conclusion: Inflammatory stimuli downregulate meprin α by inducing TTP expression and binding to the transcript. Significance: Determining how inflammation alters meprin α regulation is crucial to understanding its role in IBD. Meprins are zinc metalloproteases that are members of the astacin family of proteinases (1Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29: 309-328Crossref PubMed Scopus (160) Google Scholar, 2Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar). The two subunits, encoded by the human genes MEP1A (meprin α; chromosome 6p) and MEP1B (meprin β; chromosome 18q), are evolutionarily related and highly conserved at the amino acid level (2Bond J.S. Beynon R.J. The astacin family of metalloendopeptidases.Protein Sci. 1995; 4: 1247-1261Crossref PubMed Scopus (353) Google Scholar). Both subunits have similar domain structure and organization (1Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29: 309-328Crossref PubMed Scopus (160) Google Scholar), with the exception that meprin α has an inserted domain with a proteolytic cleavage site that results in secretion from the cell, whereas meprin β remains bound to the plasma membrane (3Hahn D. Lottaz D. Sterchi E.E. C-cytosolic and transmembrane domains of the N-benzoyl-l-tyrosyl-p-aminobenzoic acid hydrolase α subunit (human meprin α) are essential for its retention in the endoplasmic reticulum and C-terminal processing.Eur. J. Biochem. 1997; 247: 933-941Crossref PubMed Scopus (24) Google Scholar, 4Marchand P. Tang J. Johnson G.D. Bond J.S. COOH-terminal proteolytic processing of secreted and membrane forms of the α subunit of the metalloprotease meprin A. Requirement of the I domain for processing in the endoplasmic reticulum.J. Biol. Chem. 1995; 270: 5449-5456Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The β subunit can form disulfide-linked dimers at the cell surface that are referred to as meprin B. The α subunit also forms disulfide-linked homodimers (homomeric meprin A), and can further oligomerize to form large species. In addition, the α and β subunits can associate to form heterotetramers that are linked to the cell surface (heteromeric meprin A). Meprins are highly expressed in the brush border membrane of the kidney and intestinal epithelium (5Jiang W. Le B. Structure and expression of the human MEP1A gene encoding the α subunit of metalloendopeptidase meprin A.Arch. Biochem. Biophys. 2000; 379: 183-187Crossref PubMed Scopus (13) Google Scholar, 6Lottaz D. Hahn D. Müller S. Müller C. Sterchi E.E. Secretion of human meprin from intestinal epithelial cells depends on differential expression of the α and β subunits.Eur. J. Biochem. 1999; 259: 496-504Crossref PubMed Scopus (68) Google Scholar), and have also been identified in other tissue and cell types, such as liver, epidermis, and leukocytes (7Becker-Pauly C. Höwel M. Walker T. Vlad A. Aufenvenne K. Oji V. Lottaz D. Sterchi E.E. Debela M. Magdolen V. Traupe H. Stöcker W. The α and β subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation.J. Invest. Dermatol. 2007; 127: 1115-1125Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 8Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (75) Google Scholar, 9Crisman J.M. Zhang B. Norman L.P. Bond J.S. Deletion of the mouse meprin β metalloprotease gene diminishes the ability of leukocytes to disseminate through extracellular matrix.J. Immunol. 2004; 172: 4510-4519Crossref PubMed Scopus (68) Google Scholar). Their predominant location at sites where there is a barrier between the host and the outside and expression in leukocytes are suggestive of a function in host defense and the immune response (8Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (75) Google Scholar, 10Yura R.E. Bradley S.G. Ramesh G. Reeves W.B. Bond J.S. Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge.Am. J. Physiol. Renal Physiol. 2009; 296: F135-F144Crossref PubMed Scopus (41) Google Scholar). Meprins are capable of cleaving a wide variety of substrates, including gastrointestinal peptides, extracellular matrix proteins, cell junction molecules, cytokines, and chemokines (1Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29: 309-328Crossref PubMed Scopus (160) Google Scholar, 11Kronenberg D. Bruns B.C. Moali C. Vadon-Le Goff S. Sterchi E.E. Traupe H. Böhm M. Hulmes D.J. Stöcker W. Becker-Pauly C. Processing of procollagen III by meprins: new players in extracellular matrix assembly?.J. Invest. Dermatol. 2010; 130: 2727-2735Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 12Huguenin M. Müller E.J. Trachsel-Rösmann S. Oneda B. Ambort D. Sterchi E.E. Lottaz D. The metalloprotease meprinβ processes E-cadherin and weakens intercellular adhesion.PLoS One. 2008; 3: e2153Crossref PubMed Scopus (62) Google Scholar). Thus, meprins have the potential to modulate the immune response by generation of either active or inactive protein fragments (1Sterchi E.E. Stöcker W. Bond J.S. Meprins, membrane-bound and secreted astacin metalloproteinases.Mol. Aspects Med. 2008; 29: 309-328Crossref PubMed Scopus (160) Google Scholar, 13Banerjee S. Bond J.S. Prointerleukin-18 is activated by meprin β in vitro and in vivo in intestinal inflammation.J. Biol. Chem. 2008; 283: 31371-31377Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Dysregulation and abnormal meprin expression have been implicated in urinary tract infection, kidney disease, IBD, 2The abbreviations used are: IBDinflammatory bowel diseasePMAphorbol 12-myristate 13-acetatePTRpost-transcriptional regulationRBPRNA-binding proteinAREAU-rich elementRNPribonucleoproteinIPimmunoprecipitationHuRHu antigen RTTPtristetraprolinqRT-PCRquantitative real time PCRFlucfirefly luciferase. and cancer (8Bond J.S. Matters G.L. Banerjee S. Dusheck R.E. Meprin metalloprotease expression and regulation in kidney, intestine, urinary tract infections and cancer.FEBS Lett. 2005; 579: 3317-3322Crossref PubMed Scopus (75) Google Scholar, 10Yura R.E. Bradley S.G. Ramesh G. Reeves W.B. Bond J.S. Meprin A metalloproteases enhance renal damage and bladder inflammation after LPS challenge.Am. J. Physiol. Renal Physiol. 2009; 296: F135-F144Crossref PubMed Scopus (41) Google Scholar, 14Lottaz D. Maurer C.A. Hahn D. Büchler M.W. Sterchi E.E. Nonpolarized secretion of human meprin α in colorectal cancer generates an increased proteolytic potential in the stroma.Cancer Res. 1999; 59: 1127-1133PubMed Google Scholar, 15Banerjee S. Jin G. Bradley S.G. Matters G.L. Gailey R.D. Crisman J.M. Bond J.S. Balance of meprin A and B in mice affects the progression of experimental inflammatory bowel disease.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G273-G282Crossref PubMed Scopus (35) Google Scholar). inflammatory bowel disease phorbol 12-myristate 13-acetate post-transcriptional regulation RNA-binding protein AU-rich element ribonucleoprotein immunoprecipitation Hu antigen R tristetraprolin quantitative real time PCR firefly luciferase. IBD is a group of illnesses characterized by aberrant inflammation within the gastrointestinal tract, and several lines of evidence suggest a role for meprin α in the disease. Studies with meprin knock-out (KO) mice indicate that meprin α expression in the intestine may be protective, as both αKO and αβKO mice developed a more severe dextran sulfate sodium-induced colitis compared with wild-type (WT) or βKO mice (15Banerjee S. Jin G. Bradley S.G. Matters G.L. Gailey R.D. Crisman J.M. Bond J.S. Balance of meprin A and B in mice affects the progression of experimental inflammatory bowel disease.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G273-G282Crossref PubMed Scopus (35) Google Scholar, 16Banerjee S. Oneda B. Yap L.M. Jewell D.P. Matters G.L. Fitzpatrick L.R. Seibold F. Sterchi E.E. Ahmad T. Lottaz D. Bond J.S. MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.Mucosal Immunol. 2009; 2: 220-231Crossref PubMed Scopus (58) Google Scholar). In addition, there is a strong correlation between decreased MEP1A mRNA expression in inflamed mucosa of patients with IBD relative to unaffected tissue from all groups (16Banerjee S. Oneda B. Yap L.M. Jewell D.P. Matters G.L. Fitzpatrick L.R. Seibold F. Sterchi E.E. Ahmad T. Lottaz D. Bond J.S. MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.Mucosal Immunol. 2009; 2: 220-231Crossref PubMed Scopus (58) Google Scholar). There is strong evidence for a genetic component in IBD (17Xavier R.J. Podolsky D.K. Unravelling the pathogenesis of inflammatory bowel disease.Nature. 2007; 448: 427-434Crossref PubMed Scopus (3254) Google Scholar), and genome-wide associations have consistently identified chromosome 6p as an IBD susceptibility locus (18Hampe J. Schreiber S. Shaw S.H. Lau K.F. Bridger S. Macpherson A.J. Cardon L.R. Sakul H. Harris T.J. Buckler A. Hall J. Stokkers P. van Deventer S.J. Nürnberg P. Mirza M.M. Lee J.C. Lennard-Jones J.E. Mathew C.G. Curran M.E. A genomewide analysis provides evidence for novel linkages in inflammatory bowel disease in a large European cohort.Am. J. Hum. Genet. 1999; 64: 808-816Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 19Hampe J. Shaw S.H. Saiz R. Leysens N. Lantermann A. Mascheretti S. Lynch N.J. MacPherson A.J. Bridger S. van Deventer S. Stokkers P. Morin P. Mirza M.M. Forbes A. Lennard-Jones J.E. Mathew C.G. Curran M.E. Schreiber S. Linkage of inflammatory bowel disease to human chromosome 6p.Am. J. Hum. Genet. 1999; 65: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar, 20van Heel D.A. Fisher S.A. Kirby A. Daly M.J. Rioux J.D. Lewis C.M. the Genome Scan Meta-Analysis Group of the IBD International Genetics ConsortiumInflammatory bowel disease susceptibility loci defined by genome scan meta-analysis of 1952 affected relative pairs.Hum. Mol. Genet. 2004; 13: 763-770Crossref PubMed Scopus (227) Google Scholar). Two recent reports have established that the MEP1A gene is associated with IBD (15Banerjee S. Jin G. Bradley S.G. Matters G.L. Gailey R.D. Crisman J.M. Bond J.S. Balance of meprin A and B in mice affects the progression of experimental inflammatory bowel disease.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 300: G273-G282Crossref PubMed Scopus (35) Google Scholar, 16Banerjee S. Oneda B. Yap L.M. Jewell D.P. Matters G.L. Fitzpatrick L.R. Seibold F. Sterchi E.E. Ahmad T. Lottaz D. Bond J.S. MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.Mucosal Immunol. 2009; 2: 220-231Crossref PubMed Scopus (58) Google Scholar). One of these studies found a significant association between a single nucleotide polymorphism (SNP) in the 3′-UTR and IBD (16Banerjee S. Oneda B. Yap L.M. Jewell D.P. Matters G.L. Fitzpatrick L.R. Seibold F. Sterchi E.E. Ahmad T. Lottaz D. Bond J.S. MEP1A allele for meprin A metalloprotease is a susceptibility gene for inflammatory bowel disease.Mucosal Immunol. 2009; 2: 220-231Crossref PubMed Scopus (58) Google Scholar), raising the question of whether post-transcriptional regulation of meprin α might play a role in IBD. Post-transcriptional regulation (PTR) is recognized as an important means by which cells can rapidly adjust to changing conditions, as well as an additional level of control for genes whose expression must be transient (21Khabar K.S. The AU-rich transcriptome: more than interferons and cytokines, and its role in disease.J. Interferon Cytokine Res. 2005; 25: 1-10Crossref PubMed Scopus (142) Google Scholar, 22Abdelmohsen K. Kuwano Y. Kim H.H. Gorospe M. Posttranscriptional gene regulation by RNA-binding proteins during oxidative stress: implications for cellular senescence.Biol. Chem. 2008; 389: 243-255Crossref PubMed Scopus (207) Google Scholar). This form of regulation is known to be effected through RNA-binding proteins (RBPs) and regulatory sequences in the 3′-UTR of target mRNA broadly categorized as AU-rich elements (AREs) (23Kishore S. Luber S. Zavolan M. Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression.Brief. Funct. Genomics. 2010; 9: 391-404Crossref PubMed Scopus (119) Google Scholar, 24Barreau C. Paillard L. Osborne H.B. AU-rich elements and associated factors: are there unifying principles?.Nucleic Acids Res. 2005; 33: 7138-7150Crossref PubMed Scopus (783) Google Scholar). The consequences of PTR may be a change in RNA shuttling, stability, and/or translation efficiency depending on the protein(s) bound (23Kishore S. Luber S. Zavolan M. Deciphering the role of RNA-binding proteins in the post-transcriptional control of gene expression.Brief. Funct. Genomics. 2010; 9: 391-404Crossref PubMed Scopus (119) Google Scholar, 25Jing Q. Huang S. Guth S. Zarubin T. Motoyama A. Chen J. Di Padova F. Lin S.C. Gram H. Han J. Involvement of microRNA in AU-rich element-mediated mRNA instability.Cell. 2005; 120: 623-634Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar). Two ubiquitously expressed RBPs are TTP, which is known to destabilize transcripts directly by promoting their deadenylation (26Lai W.S. Kennington E.A. Blackshear P.J. Tristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly(A) ribonuclease.Mol. Cell. Biol. 2003; 23: 3798-3812Crossref PubMed Scopus (195) Google Scholar), and ELAVL1/HuR (27Peng S.S. Chen C.Y. Xu N. Shyu A.B. RNA stabilization by the AU-rich element binding protein, HuR, an ELAV protein.EMBO J. 1998; 17: 3461-3470Crossref PubMed Scopus (654) Google Scholar, 28Brennan C.M. Steitz J.A. HuR and mRNA stability.Cell. Mol. Life Sci. 2001; 58: 266-277Crossref PubMed Scopus (879) Google Scholar), which often functions to counteract the actions of destabilizing proteins by promoting adenylation, competing with other RBPs for binding sites, or enhancing translation (29Meisner N.C. Hintersteiner M. Seifert J.M. Bauer R. Benoit R.M. Widmer A. Schindler T. Uhl V. Lang M. Gstach H. Auer M. Terminal adenosyl transferase activity of posttranscriptional regulator HuR revealed by confocal on-bead screening.J. Mol. Biol. 2009; 386: 435-450Crossref PubMed Scopus (33) Google Scholar, 30Pan Y.X. Chen H. Kilberg M.S. Interaction of RNA-binding proteins HuR and AUF1 with the human ATF3 mRNA 3′-untranslated region regulates its amino acid limitation-induced stabilization.J. Biol. Chem. 2005; 280: 34609-34616Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Sureban S.M. Murmu N. Rodriguez P. May R. Maheshwari R. Dieckgraefe B.K. Houchen C.W. Anant S. Functional antagonism between RNA binding proteins HuR and CUGBP2 determines the fate of COX-2 mRNA translation.Gastroenterology. 2007; 132: 1055-1065Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Both HuR and TTP have been demonstrated to regulate a number of cytokines and chemokines, and post-transcriptional regulation is recognized to be important in the inflammatory response (32Khabar K. Post-transcriptional control during chronic inflammation and cancer: a focus on AU-rich elements.Cell. Mol. Life Sci. 2010; 67: 2937-2955Crossref PubMed Scopus (131) Google Scholar, 33Stoecklin G. Anderson P. Posttranscriptional mechanisms regulating the inflammatory response.Adv. Immunol. 2006; 89: 1-37Crossref PubMed Scopus (85) Google Scholar). Mice with a deletion of the tumor necrosis factor (TNF) ARE develop both chronic inflammatory arthritis and IBD (34Kontoyiannis D. Pasparakis M. Pizarro T.T. Cominelli F. Kollias G. Impaired on/off regulation of TNF biosynthesis in mice lacking TNF AU-rich elements: implications for joint and gut-associated immunopathologies.Immunity. 1999; 10: 387-398Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar), and TTP knockout mice develop severe inflammatory arthritis and autoimmunity (35Carrick D.M. Lai W.S. Blackshear P.J. The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis.Arthritis Res. Ther. 2004; 6: 248-264Crossref PubMed Scopus (141) Google Scholar). Thus, dysregulation of ARE-binding proteins has implications for multiple disease processes, including IBD and cancer (21Khabar K.S. The AU-rich transcriptome: more than interferons and cytokines, and its role in disease.J. Interferon Cytokine Res. 2005; 25: 1-10Crossref PubMed Scopus (142) Google Scholar, 36López de Silanes I. Lal A. Gorospe M. HuR: post-transcriptional paths to malignancy.RNA Biol. 2005; 2: 11-13Crossref PubMed Google Scholar, 37Taylor G.A. Carballo E. Lee D.M. Lai W.S. Thompson M.J. Patel D.D. Schenkman D.I. Gilkeson G.S. Broxmeyer H.E. Haynes B.F. Blackshear P.J. A pathogenetic role for TNFα in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (TTP) deficiency.Immunity. 1996; 4: 445-454Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). The studies herein were conducted to determine whether expression of meprin α is subject to PTR and, using a tissue culture model of IBD, whether this regulation is altered in response to inflammatory stimuli. Caco-2 human colorectal adenocarcinoma cells (ATCC) were maintained in a 37 °C incubator with 5% CO2 and cultured in Eagle's minimum essential medium (ATCC) supplemented with 20% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). A549 human lung carcinoma cells (ATCC) were grown under similar conditions in F12K medium (ATCC) supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. Where indicated, cells were treated with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Acros Organics), 5 μg/ml Actinomycin D (Acros Organics), or vehicle control (DMSO or methanol, respectively). The luciferase reporter vector pmirGLO was purchased from Promega for expression of both firefly and Renilla luciferase from the same plasmid under the control of separate promoters. This reporter was used as both a reference control in transfection experiments (described below) and as the backbone vector for the hMEP1A 3′-UTR construct. The vector pSGG-MEP1A-3UTR (SwitchGear Genomics) was used as template for amplification of the target sequence by Phusion DNA polymerase (Finnzymes Oy) and subcloning into the pmirGLO vector. The following primers were purchased from Integrated DNA Technologies and used to introduce restriction endonuclease sites for DraI and XbaI (bases changed/added in lowercase; recognition sites underlined): 5′-aaatttAAAGGCCAAGGAAGTGACCTG-3′ and 5′-aatctagAAGGTGCTAACTCAAT-3′. Plasmid DNA was isolated from multiple colonies using the Zyppy Plasmid Miniprep kit (Zymo Research) for screening via restriction analysis, and all positive clones were confirmed by sequencing at the Penn State University Nucleic Acid Facility. The final construct, pmG-hMEP1A-3UTR-WT, consisted of the pmirGLO vector with the complete human MEP1A 3′-UTR sequence (GenBank accession number NM_005588.2) cloned downstream of the firefly luciferase coding region. To generate the TTP expression construct, Caco-2 cDNA was used as template for amplification of the TTP coding sequence by Phusion DNA polymerase (Finnzymes Oy) and subcloning into the mammalian expression vector pcDNA3.1/V5-His (Invitrogen). The following primers were purchased from Integrated DNA Technologies to first amplify TTP cDNA: 5′-acttcagcgctcccactctc-3′ and 5′-cttgattactgtcccccaagtc-3′. A second set of primers was then used to introduce restriction endonuclease sites for KpnI and SacII (bases changed/added in lowercase; recognition sites underlined): 5′-aaaaATGGggtACCGTTACACC-3′ and 5′-aaaaccgCGGGCAGTCACTTTG-3′. Plasmid DNA was isolated from multiple colonies using the Zyppy Plasmid Miniprep kit (Zymo Research) for screening via restriction analysis, and all positive clones were confirmed by sequencing at the Penn State University Nucleic Acid Facility. The final construct, pcDNA-hTTP, includes the ORF from GenBank accession number NM_003407.2 with an intact stop codon such that the V5-His tag is not translated. Silencing experiments were conducted with TTP, HuR, or negative control shRNA constructs obtained from Genecopoeia. For each target, three shRNA constructs were tested. Cells were grown to 70–80% confluence in 6-well plates, and 2 μg of each plasmid was transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. Medium was changed after 16 h, and cells were harvested at 48 h post-transfection for isolation of both RNA and protein. Efficacy of silencing was assessed by Western blot for either HuR or TTP. The shRNA construct that gave the greatest silencing was used in subsequent experiments. For reporter mRNA stability assays, A549 cells were grown to 70% confluence in 6-well plates and cotransfected by Attractene reagent with 50 ng of either pmirGLO or pmG-hMEP1A-3UTR-WT and 200 ng of either negative control shRNA, HuR shRNA2, pUC19, or TTP expression plasmid where appropriate. Actinomycin D assays were performed 24 h post-transfection, and half-lives were calculated from the decay curves generated. Total RNA was isolated using TRIzol reagent (Invitrogen) as described by the manufacturer and quantitated by UV absorbance at 260 nm. Within an experiment, equivalent amounts of RNA were reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer's instructions. Quantitative real time PCR was performed using template cDNA, 2× SensiMix SYBR Master Mix with fluorescein (Bioline), and the gene-specific primers in Table 1. All reactions were run in 96-well plates on the MyiQ2 Two-Color Real-Time Detection System (Bio-Rad) using the following program: 2-min hot start at 95 °C, 40 cycles of 15 s at 95 °C and 60 s at 60 °C, and 2 min at 10 °C. Data were analyzed by the ΔΔCt method with normalization to the endogenous control, GAPDH.TABLE 1Primers used for quantitative real time PCRTargetPrimer sequenceAmplicon lengthbpGAPDH5′-GAG TCA ACG GAT TTG GTC GT-3′2385′-TTG ATT TTG GAG GGA TCT CG-3′MEP1A5′-CAG GTG GAC GTT CCC CAT TC-3′1675′-AGC ACC CAT CAA ACT GTT GAA AT-3′HuR5′-GGC TGG TGC ATT TTC ATC TAC-3′1755′-CCA TCG CGG CTT CTT CAT AG-3′CCL25′-GCT CAG CCA GAT GCA ATC A-3′1575′-AGA TCT CCT TGG CCA CAA TG-3′TTP5′-GAA GGG CCA CTC CTA TCA GC-3′2175′-TCA GAA ACA GAG ATG CGA TTG A-3′Fluc5′-CAG CAA GGA GGT AGG TGA GG-3′2815′-CGA TGA GAG CGT TTG TAG CC-3′ Open table in a new tab To generate the biotinylated RNA oligos for biotin pulldown experiments, cDNA templates were first amplified using the primers shown in Table 2 to add a T7 site for RNA polymerase. Templates were then in vitro transcribed to RNA using the MAXIscript T7 kit and a ratio of 4:1 CTP:biotin-CTP, followed by digestion with DNase I and reaction cleanup using NucAway spin columns as described by the manufacturer (both from Ambion). RNA integrity was confirmed via agarose gel electrophoresis, and concentrations were determined via spectrophotometry. Biotin affinity pulldown experiments were performed as described previously (38Ishmael F.T. Fang X. Galdiero M.R. Atasoy U. Rigby W.F. Gorospe M. Cheadle C. Stellato C. Role of the RNA-binding protein tristetraprolin in glucocorticoid-mediated gene regulation.J. Immunol. 2008; 180: 8342-8353Crossref PubMed Scopus (70) Google Scholar, 39Fan J. Ishmael F.T. Fang X. Myers A. Cheadle C. Huang S.-K. Atasoy U. Gorospe M. Stellato C. Chemokine transcripts as targets of the RNA-binding protein HuR in human airway epithelium.J. Immunol. 2011; 186: 2482-2494Crossref PubMed Scopus (66) Google Scholar).TABLE 2Primers used to generate cDNA templates for in vitro transcription of biotinylated RNATargetPrimer sequenceaUppercase letters indicate T7 start site (common to all); lowercase letters indicate target-specific sequences.MEP1A 3′-UTR (full-length)5′-CCA AGC TTC TAA TAC GAC TCA CTA TAG GGA GAt gcc tgc tgg cat tgg-3′5′-ggt gct aac tca ata ttt att aaa ttt att aaa gg-3′MEP1A 3′-UTR (segment A)5′-CCA AGC TTC TAA TAC GAC TCA CTA TAG GGA GAt gcc tgc tgg cat tgg-3′5′-atg aag tgt gat gag tgt tgt cac-3′MEP1A 3′-UTR (segment B)5′-CCA AGC TTC TAA TAC GAC TCA CTA TAG GGA GAg tgt cct gtg aca aca ctc atc-3′5′-gtt gag tgc caa caa tgt gc-3′MEP1A 3′-UTR (segment C)5′-CCA AGC TTC TAA TAC GAC TCA CTA TAG GGA GAc atg gct ggc aca ttg tt-3′5′-ggt gct aac tca ata ttt att aaa ttt att aaa gg-3′CCL5 3′-UTR5′-CCA AGC TTC TAA TAC GAC TCA CTA TAG GGA GAc tgc aga gga ttc ctg ca-3′5′-tgg ttg cat tga gaa ctt taa tgg-3′a Uppercase letters indicate T7 start site (common to all); lowercase letters indicate target-specific sequences. Open table in a new tab Cell pellets were lysed in 1× PLB (100 mm KCl, 5 mm MgCl2, 10 mm HEPES, pH 7.0, 0.5% Nonidet P-40) for 10 min on ice in the presence of EDTA-free protease inhibitors (Roche Applied Science), then centrifuged for 5 min at maximum speed (4 °C) to clear cellular debris. Samples were prepared for Western blotting by boiling for 5 min in SDS-PAGE sample buffer and subjected to denaturing polyacrylamide gel electrophoresis using precast 4–20% Ready Gels (Bio-Rad) for ∼45 min at 200 V or 10% SDS-polyacrylamide gels for 45 min at 45 mA. The following antibodies were used at a 1:1000 dilution: HuR (sc-5261; Santa Cruz Biotechnology), TTP (T5327; Sigma), β-actin (A5441; Sigma), donkey anti-rabbit IgG (NA934; GE Healthcare), and donkey anti-mouse IgG (sc-2314; Santa Cruz Biotechnology). For detection of secreted meprin A, 1 ml of medium was collected from Caco-2 cells stimulated with PMA (50 ng/ml) or control for 24 h. Samples were dialyzed into a 0.01% SDS solution using a 100-kDa-molecular mass-cutoff cellulose ester membrane (Spectrum Laboratories) and concentrated 100-fold by vacuum drying (SpeedVac concentrator, Savant). The SDS solution was necessary to prevent protein from precipitating during concentration. After addition of SDS-PAGE loading buffer containing 100 mm DTT, the sample was separated by SDS-PAGE using an 8% acrylamide gel and subjected to Western blot as described previously (40Bertenshaw G.P. Norcum M.T. Bond J.S. Structure of homo- and hetero-oligomeric meprin metalloproteases. Dimers, tetramers, and high molecular mass multimers.J. Biol. Chem. 2003; 278: 2522-2532Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Blots were scanned and analyzed by ImageJ for densitometric analysis of relative protein expression as indicate" @default.
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• W2105280204 date "2013-02-01" @default.
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• W2105280204 title "Post-transcriptional Regulation of Meprin α by the RNA-binding Proteins Hu Antigen R (HuR) and Tristetraprolin (TTP)" @default.
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