Comment[ArrayExpressAccession] E-GEOD-25264 Public Release Date 2010-11-10 Investigation Title Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium Comment[Submitted Name] Chemokine Transcripts as Targets of the RNA-Binding Protein HuR in Human Airway Epithelium Experiment Description HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements (ARE) and related motifs present in the 3’untranslated region (UTR) of mRNAs. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNFa plus IFNg, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein (RNP) complexes from resting and cytokine-treated cells were immunoprecipitated (IP) using anti-HuR and isotype-control antibody, and eluted mRNAs were reverse-transcribed and hybridized to an inflammatory-focused gene array. The chemokines CCL2, CCL8, CXCL1 and CXCL2 ranked highest among 27 signaling and inflammatory genes significantly enriched in the HuR RNP-IP from stimulated cells over the control IP. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene RNP-IP, and shown to be 3’UTR-dependent by biotin pull-down assay. Cytokine treatment increased mRNA stability only for CCL2 and CCL8, and transient silencing and overexpression of HuR affected only CCL2 and CCL8 expression in primary and transformed epithelial cells. Cytokine-induced CCL2 mRNA was predominantly cytoplasmic; conversely, CXCL1 mRNA remained mostly nuclear and unaffected, as CXCL2, by changes in HuR levels. Increase in cytoplasmic HuR and HuR target expression partially relied on the inhibition of AMP-dependent kinase, a negative regulator of HuR nucleocytoplasmic shuttling. We postulate that HuR critically regulates the epithelial response, by associating with multiple adenylate-uridylate-rich elements (ARE)-bearing, functionally related inflammatory transcripts. On the basis of genome-wide studies probing the relationship between RNA-binding proteins and the functional profile of their associated transcripts, we combined the specific HuR immunoprecipitation of RNPs and the genome-scale microarray to profile the target mRNAs of HuR in the human airway epithelial cell line BEAS-2B challenged with very strong inflammatory stimulation, TNFa plus IFNG. The array platform we used is the Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD). To validate the association of HuR and its target mRNAs, we planned to apply biotin pull-down assay. HuR overexpression and knockdown assays were also planned to further verify the association of HuR and its target mRNAs. Overall, we did three biological replicates for the IP array experiments, which include both IgG1 control IP and HuR IP arrays. Date of Experiment Term Source Name EFO Term Source Version Term Source File http://efo.svn.sourceforge.net/viewvc/efo/trunk/src/efoinowl/efo.owl Person Last Name Fan Fan Ishmael Fang Cheadle Myers Huang Atasoy Gorospe Stellato Person First Name Jinshui Jinshui Faoud Xi Chris Allen Shau-Ku Ulus Myriam Cristiana Person Mid Initials T Person Email jfan8@jhmil.edu Person Affiliation Johns Hopkins University Person Phone Person Fax Person Address Division of Allergy & Clinical Immunology, Johns Hopkins University, 5200 Eastern Avenue, Baltimore, MD, USA Person Roles submitter Person Roles Term Source REF Person Roles Term Accession Number Normalization Type Normalization Term Accession Number Normalization Term Source REF Replicate Type Replicate Term Accession Number Replicate Term Source REF Experimental Design Experimental Design Term Accession Number Experimental Design Term Source REF Quality Control Type Quality Control Term Accession Number Quality Control Term Source REF Protocol Name P-GSE25264-1 P-GSE25264-53 P-GSE25264-52 P-GSE25264-31 P-GSE25264-29 P-GSE25264-30 P-GSE25264-28 P-GSE25264-57 P-GSE25264-55 P-GSE25264-58 P-GSE25264-56 P-GSE25264-54 P-GSE25264-14 P-GSE25264-18 P-GSE25264-9 P-GSE25264-13 P-GSE25264-26 P-GSE25264-20 P-GSE25264-7 P-GSE25264-19 P-GSE25264-24 P-GSE25264-3 P-GSE25264-10 P-GSE25264-2 P-GSE25264-5 P-GSE25264-8 P-GSE25264-16 P-GSE25264-6 P-GSE25264-4 P-GSE25264-21 P-GSE25264-11 P-GSE25264-15 P-GSE25264-27 P-GSE25264-25 P-GSE25264-23 P-GSE25264-17 P-GSE25264-12 P-GSE25264-22 P-GSE25264-34 P-GSE25264-41 P-GSE25264-49 P-GSE25264-36 P-GSE25264-43 P-GSE25264-42 P-GSE25264-40 P-GSE25264-47 P-GSE25264-45 P-GSE25264-48 P-GSE25264-39 P-GSE25264-37 P-GSE25264-32 P-GSE25264-33 P-GSE25264-38 P-GSE25264-35 P-GSE25264-46 P-GSE25264-44 P-GSE25264-50 P-GSE25264-51 Protocol Description ID_REF =
VALUE = normalized signal intensity which contains full-length cDNAs for 364 known human genes. Labeled probes were hybridized following the manufacturer’s instructions against a GEArrayTM S series Human Autoimmune and Inflammatory Response Gene Array (SuperArray Bioscience, Frederick, MD), used from passages 36 to 45. All cells were cultured at 37°C in humidified air containing 5% CO2. Primary bronchial epithelial cells (PBECs) were isolated by pronase digestion from bronchi of cadaveric lungs. The purity of these preparations was confirmed by immunohistochemical staining for cytokeratin. PBECs were cultured on collagen-coated flasks and maintained in serum-free LHC-9 medium (Biofluids, Rockville, MD). Cultures of PBEC were used during the first passage only, while BEAS-2B cells were The BEAS-2B cells were cultured in F12/DMEM (Gibco/Invitrogen, Frederick, MD) containing 5% heat-inactivated fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml). antibody and IgG matched replicates within each set and tested for significance using the paired t test. Genome Ontology analysis was performed using the DAVID gene functional classification tool (NIAID, NIH, 2008). Analysis was performed with the P-SCAN analysis software using a custom array option available. Data were analyzed using repeated measures ANOVA test with post-hoc analysis or paired T test when appropriate. Arrays were normalized within each treatment/control set using a subset of genes which were reproducibly precipitated by IgG control only. Fold changes were independently calculated between HuR The results were collected by exposure to a phosphoimager screen and then scanning by a Typhoon 8600 Variable Mode Image (GE Healthcare). a horseradish peroxidase-conjugated goat anti-mouse secondary Ab. After a final washing step (1x PBS containing 0.1% Tween, 10 min, RT), immunoreactive bands were visualized by enhanced Transcription Kit, Ambion, Austin, TX). Biotinylated transcripts were amplified by RT-PCR using specific primers. Cytoplasmic fractions of unstimulated BEAS-2B cells (40 µg) were incubated for 1 h at RT with (Life Technologies, Carlsbad, CA) and allowed to complex with 1 µg of plasmid DNA for 15 min at RT. The plasmid/FuGene mixture was then overlayed on the cells in a final volume of 2 ml of complete medium. Signaling, Danvers, MA) in 1x PBS containing 1% Tween for 1 h at RT with continuous shaking. After washes with 1x PBS containing 0.1% Tween (10 min, RT), membranes were incubated for 1 h at RT with CA). A Zeiss Axiovert 200 M microscope (63X lens) was used for visualization employing separate channels for the analysis of phase-contrast images, red fluorescence and blue fluorescence. Images standard Western blot analysis using the mouse monoclonal anti-HuR antibody 3A2 (Santa Cruz Biotechnology) and the enhanced chemiluminescence protocol (GE Healthcare). (5’-GCCAAUUCAUCAGCAAUGG) (Qiagen, Valencia, CA). Cells were seeded in 6-well plates at a density of ~250,000/well and transfected 24 h later, when cells reached ~50 % confluence, using the non-liposomal 1 µg of biotinylated transcripts, then ribonucleoprotein complexes were isolated with streptavidin-conjugated Dynabeads (Invitrogen/Life Technologies). The presence of HuR in the pull-down pellet was verified by blocking buffer, samples were incubated with a mixture of horse anti-mouse Texas Red (1:200; Jackson Immunoresearch Laboratories, West Grove, PA) and Hoechst 33342 (1:5,000; Molecular Probes/Life Cells were cultured in six-well plates in serum-free DMEM, and infections were carried out at 80% cell confluency. Titration experiments showed that significant upregulation of HuR levels was achieved with After incubation for 48 h at 37°C, cells were treated according to the experimental protocols. The efficiency of the transient transfection of cells, assessed with a CMV promoter-driven expression vector coding for (1) Vector transfection: HuR was overexpressed in PBEC and BEAS-2B cells by infection with an adenoviral vector (AdCMV-HuR) in parallel with the empty vector (Ad-EV) (ViraQuest, Inc, North Liberty, IA). form of the enzyme. cationic vehicle FuGene (Roche Applied Science, Indianapolis, IN) according to the manufacturer's specifications. Briefly, 3 µl of FuGene per sample were resuspended (5 min at RT) in serum-free Opti-MEM (4) In vitro biotin pull-down assay: To generate the labeled RNA probes, PCR products encompassing the coding regions or 3’UTR of the selected HuR targets were generated by RT-PCR of BEAS-2B RNA using (2) siRNA transfection: For HuR silencing, PBEC and BEAS-2B cells were transiently transfected with a previously described siRNA (5’-aaGAGUGAAGGAGUUGAAACU-3’) or a scrambled control 6.25 PFU/cell. The same protocol was used for transduction of Ad (CA)AMPK, expressing a cDNA encoding residues 1 to 312 of the 1 subunit of AMPK, which encodes for a constitutively active (5) Immunofluorescence: BEAS-2B cells and PBEC were seeded at 40-50% confluency on sterile coverslips and incubated in complete medium overnight. Following treatment, cells were fixed for 15 min in phosphate-buffered green fluorescence protein (Promega, Madison, WI), was ~ 22% of total cell count performed by nuclear staining with Hoechst dye. Changes in HuR levels were determined by Western blot 48 h after transfection. chemiluminescence (Amersham Biosciences, Piscataway, NJ). were then processed with the Zeiss AxioVision program, version 3.0. Technologies) or DAPI nuclear staining (1 ug/ml, Roche Applied Science, Indianapolis, IN) for 1 h. After washes with blocking buffer, coverslips were mounted in Vectashield (Vector Laboratories, Burlingame, 2% bovine serum albumin and 0.1% Tween 20) for 16 h, coverslips were incubated in a 1:500 dilution of mouse anti-HuR antibody (Santa Cruz Biotechnology) in blocking buffer for 1 h. Following washes in corresponding primers and purified from agarose gels. The resulting cDNAs were used as templates for the synthesis of biotinylated RNAs using T7 RNA polymerase and biotin-conjugated CTP (MaxiScript T7 In Vitro (3) Western blot: Western blot was performed using primary antibodies 2 µg/ml of mouse monoclonal anti-HuR 3A2 or 0.3 µg/ml of anti-alpha-tubulin (both from Santa Cruz), or a 1:1000 dilution of the AMPK or pAMPK abs (Cell saline (PBS) containing 4% paraformaldehyde for a period of 15 min, followed by permeabilization in PBS containing 0.4% TritonX-100 for an additional 15 min. After incubation in blocking buffer (PBS containing An equal number of cells per condition (30 x 106) was pelleted and resuspended in approximately two cell-pellet volumes of polysome lysis buffer (PLB) containing 100 mM KCl, 5 mM MgCl2, 10 mM Hepes, ul of NT2 buffer supplemented with 100 Units/ml RNaseOUT, 0.2% vanadyl-ribonucleoside complex, 1 mM DTT, and 20 mM EDTA. The beads were vvortexed briefly, and 100 ul of the mRNP cell lysate was (Applied Biosystems/LifeTechnologies) and quantified using the comparative cycle threshold (CT) method. mg/ml leupeptin added fresh immediately before use. Cell lysates were then frozen at -80°C for storage. At time of use, cell lysates were thawed and centrifuged at 16,000 x g for 10 min at 4°C. The cell lysate buffer. Washed beads were resuspended in 100 ul NT2 buffer supplemented with 0.1% SDS and 30 ug proteinase K, incubated for 30 min at 55°C, then cytoplasmic RNA was extracted using phenol- added and immediately centrifuged. A 100-ul aliquot was removed to represent total cellular RNA. The IP reactions were tumbled at room temperature for 2 h and then washed six times with ice-cold NT2 Biotechnology, Santa Cruz, CA) (55) or an IgG1 isotype control antibody (B&D Life Sciences, Franklin Lanes, NJ). The antibody-coated beads were washed with ice-cold NT2 buffer and resuspended in 900 and GAPDH, used for normalization, are listed in Table 1. The conditions for amplification by real-time PCR for all targets were as follows: 50 °C, 2 minutes for UNG activation, then 95 °C, 10 min (1 cycle each, hot-start (2) RNA Isolation and Analysis: Total RNA was extracted using TRIzol (Invitrogen/ Life Technologies). Cytoplasmic RNA was isolated and DNase-treated using the RNAeasy kit (Qiagen). RNA was reverse-transcribed to activate the Taq polymerase), then 95 °C, 15 sec followed by 63 °C, 1 min, for 40 cycles. All samples were run in triplicate (SD <0.1) for real-time fluorimetric determination in an ABI 7300 sequence detector protein-A bead slurry was used for IP reaction and incubated 4 h at RT with excess immunoprecipitating antibody (30 ug), using either a mouse monoclonal antibody specific for HuR, 3A2 (Santa Cruz contained approximately 30 mg/ml total protein and was immunoprecipitated with anti-HuR or the irrelevant antibody, according to adaptation of an established protocol. Briefly, Protein-A Sepharose (1) IP and RNA extraction: BEAS-2B cell monolayers cultured in T-175 flasks were stimulated at ~70% confluence in the absence or presence of TNFalpha (50 ng/ml) in combination with IFNgamma at 50 ng/ml (R&D Systems, Minneapolis, MN). Cytokine stimulation did not affect cell viability, measured by trypan blue exclusion. Cells were then harvested by trypsinization and their viability was assessed by trypan blue exclusion (consistently >95%). beads (Sigma-Aldrich, St. Louis, MO) were swollen 1:1 v/v in NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2/0.05% Nonidet P40) supplemented with 5% BSA. A 100 ul aliquot of the preswollen pH 7.0, 0.5% Nonidet P-40 with 1 mM DTT, 100 Units/ml RNaseOUT (Gibco BRL), 0.2% vanadyl-ribonucleoside complex (Gibco BRL), 0.2 mM PMSF, 1 mg/ml pepstatin A, 5 mg/ml bestatin, and 20 using the Gene Amp Kit (Perkin-Elmer, Waltham, MA) and PCR amplified using the SYBR Green reagent Kit (Perkin-Elmer). The PCR primers (forward/reverse) for detection of CCL2, CCL8, CCL13, CXCL1, CXCL2 chlorophorm-isoamylalcohol and precipitation in ethanol. The IP RNA was reverse-transcribed according to an established labeling protocol to generate 33P-labeled probes (for detail, please refer to Fan et al. Global analysis of stress-regulated mRNA turnover by using cDNA arrays. Proceedings Natl Acad Sci USA 2002; 99:10611-16). Protocol Software Protocol Hardware Protocol Contact Protocol Type bioassay_data_transformation hybridization hybridization grow grow grow grow feature_extraction feature_extraction feature_extraction feature_extraction image_aquisition specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction nucleic_acid_extraction labeling labeling Protocol Term Source REF Protocol Term Accession Number Experimental Factor Name TIME ANTIBODY VENDOR AGENT ANTIBODY Experimental Factor Type time antibody vendor agent antibody Experimental Factor Term Source REF Experimental Factor Term Accession Number Publication Title Publication Author List PubMed ID Publication DOI Publication Status Publication Status Term Source REF Publication Status Term Accession Number Comment[SecondaryAccession] GSE25264 Comment[GEOLastUpdateDate] 2010-11-10 Comment[AEExperimentType] transcription profiling by array Comment[GEOReleaseDate] 2010-11-10 Comment[ArrayExpressSubmissionDate] 2010-11-10 SDRF File E-GEOD-25264.sdrf.txt