title: Chemokine transcripts as targets of the RNA-binding protein HuR in human airway epithelium.
abstract: HuR is a regulator of mRNA turnover or translation of inflammatory genes through binding to adenylate-uridylate-rich elements and related motifs present in the 3'untranslated region (UTR) of mRNAs. We postulate that HuR critically regulates the epithelial response by associating with multiple ARE-bearing, functionally related inflammatory transcripts. We aimed to identify HuR targets in the human airway epithelial cell line BEAS-2B challenged with TNF-α plus IFN-γ, a strong stimulus for inflammatory epithelial responses. Ribonucleoprotein complexes from resting and cytokine-treated cells were immunoprecipitated using anti-HuR and isotype-control Ab, 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 immunoprecipitation. Among these, 20 displayed published HuR binding motifs. Association of HuR with the four endogenous chemokine mRNAs was validated by single-gene ribonucleoprotein-immunoprecipitation 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. HuR-mediated regulation in airway epithelium appears broader than previously appreciated, coordinating numerous inflammatory genes through multiple posttranscriptional mechanisms.
authors.affiliation: Division of Allergy and Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA.
authors.forename: Jinshui
authors.initials: J
authors.lastname: Fan
authors.forename: Faoud T
authors.initials: FT
authors.lastname: Ishmael
authors.forename: Xi
authors.initials: X
authors.lastname: Fang
authors.forename: Allen
authors.initials: A
authors.lastname: Myers
authors.forename: Chris
authors.initials: C
authors.lastname: Cheadle
authors.forename: Shau-Ku
authors.initials: SK
authors.lastname: Huang
authors.forename: Ulus
authors.initials: U
authors.lastname: Atasoy
authors.forename: Myriam
authors.initials: M
authors.lastname: Gorospe
authors.forename: Cristiana
authors.initials: C
authors.lastname: Stellato
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000502:physiology; D000954:Antigens, Surface / Q000235:genetics* / Q000378:metabolism*; D019921:Biotinylation; D001980:Bronchi / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002461:Cell Line, Transformed; D018932:Chemokine CCL2 / Q000235:genetics / Q000378:metabolism; D054412:Chemokine CCL8 / Q000235:genetics / Q000378:metabolism; D054360:Chemokine CXCL1 / Q000235:genetics / Q000378:metabolism; D054426:Chemokine CXCL2 / Q000235:genetics / Q000378:metabolism; D018925:Chemokines / Q000235:genetics / Q000378:metabolism*; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D006801:Humans; D018836:Inflammation Mediators / Q000502:physiology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D020871:RNA Stability / Q000235:genetics / Q000276:immunology; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015203:Reproducibility of Results; D020545:Respiratory Mucosa / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D014132:Trachea / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D014158:Transcription, Genetic / Q000276:immunology
chemical_list: D000954:Antigens, Surface; C111835:CCL2 protein, human; C515453:CCL8 protein, human; C475829:CXCL1 protein, human; C499706:CXCL2 protein, human; D018932:Chemokine CCL2; D054412:Chemokine CCL8; D054360:Chemokine CXCL1; D054426:Chemokine CXCL2; D018925:Chemokines; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D018836:Inflammation Mediators; D016601:RNA-Binding Proteins; D055372:AMP-Activated Protein Kinases