Regulation of eotaxin gene expression by TNF-alpha and IL-4 through mRNA stabilization: involvement of the RNA-binding protein HuR.
During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-alpha and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-alpha or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3' untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible beta-globin reporter linked to the eotaxin 3' untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-alpha and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-alpha and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-alpha and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.
Atasoy
Ulus
U
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Curry
Stephanie L
SL
López de Silanes
Isabel
I
Shyu
Ann-Bin
AB
Casolaro
Vincenzo
V
Gorospe
Myriam
M
Stellato
Cristiana
C
D020413:3' Untranslated Regions / Q000502:physiology; D000818:Animals; D000954:Antigens, Surface; D001483:Base Sequence; D001980:Bronchi; D002461:Cell Line, Transformed; D054413:Chemokine CCL11; D019742:Chemokines, CC / Q000096:biosynthesis* / Q000235:genetics; D004338:Drug Combinations; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D015847:Interleukin-4 / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D020871:RNA Stability / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000378:metabolism* / Q000502:physiology; D016601:RNA-Binding Proteins / Q000502:physiology*; D020545:Respiratory Mucosa / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D015854:Up-Regulation / Q000235:genetics / Q000276:immunology
D020413:3' Untranslated Regions; D000954:Antigens, Surface; C515456:CCL11 protein, human; C515457:Ccl11 protein, mouse; D054413:Chemokine CCL11; D019742:Chemokines, CC; D004338:Drug Combinations; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014409:Tumor Necrosis Factor-alpha; D015847:Interleukin-4