Chemokine transcripts as targets of the RNA-binding protein HuR in human airway epithelium

Jinshui Fan, Faoud T. Ishmael, Xi Fang, Allen Myers, Chris Cheadle, Shau Ku Huang, Ulus Atasoy, Myriam Gorospe, Cristiana Stellato

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49 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2482-2494
Number of pages13
JournalJournal of Immunology
Volume186
Issue number4
DOIs
Publication statusPublished - Feb 15 2011

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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    Fan, J., Ishmael, F. T., Fang, X., Myers, A., Cheadle, C., Huang, S. K., Atasoy, U., Gorospe, M., & Stellato, C. (2011). Chemokine transcripts as targets of the RNA-binding protein HuR in human airway epithelium. Journal of Immunology, 186(4), 2482-2494. https://doi.org/10.4049/jimmunol.0903634