Posttranscriptional regulation of IL-13 in T cells: Role of the RNA-binding protein HuR

Casolaro, Vincenzo; Fang, Xi; Tancowny, Brian; Fan, Jinshui; Wu, Fan; Srikantan, Subramanya; Asaki, S. Yukiko; De Fanis, Umberto; Huang, Shau-Ku; Gorospe, Myriam; Atasoy, Ulus X.; Stellato, Cristiana

doi:10.1016/j.jaci.2007.12.1166

« Previous Next »The Journal of Allergy and Clinical Immunology
Volume 121, Issue 4 , Pages 853-859.e4, April 2008

Posttranscriptional regulation of IL-13 in T cells: Role of the RNA-binding protein HuR

Vincenzo Casolaro, MD, PhD
- Johns Hopkins University School of Medicine, Baltimore, Md
Xi Fang, MD
- Johns Hopkins University School of Medicine, Baltimore, Md
Brian Tancowny, MS
- Johns Hopkins University School of Medicine, Baltimore, Md
Jinshui Fan, PhD
- Johns Hopkins University School of Medicine, Baltimore, Md
Fan Wu, MD
- Johns Hopkins University School of Medicine, Baltimore, Md
Subramanya Srikantan, PhD
- National Institute of Aging, National Institutes of Health, Baltimore, Md
S. Yukiko Asaki, MS
- Johns Hopkins University School of Medicine, Baltimore, Md
Umberto De Fanis, MD
- Johns Hopkins University School of Medicine, Baltimore, Md
Shau-Ku Huang, PhD
- Johns Hopkins University School of Medicine, Baltimore, Md
Myriam Gorospe, PhD
- National Institute of Aging, National Institutes of Health, Baltimore, Md
Ulus X. Atasoy, MD
- University of Missouri-Columbia, Columbia, Mo
Cristiana Stellato, MD, PhD
- Johns Hopkins University School of Medicine, Baltimore, Md
- Reprint requests: Cristiana Stellato, MD, PhD, Division of Allergy and Clinical Immunology, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

Received 25 April 2007; received in revised form 6 December 2007; accepted 19 December 2007. published online 15 February 2008.

Background

IL-13, a critical cytokine in allergy, is regulated by as-yet-elusive mechanisms.

Objective

We investigated IL-13 posttranscriptional regulation by HuR, a protein associating with adenylate-uridylate-rich elements in the 3′ untranslated regions (UTRs) of mRNA, promoting mRNA stability and translation.

Methods

IL-13 mRNA decay was monitored in human T_H2-skewed cells by using the transcriptional inhibitor actinomycin D. The IL-13 3′UTR was subcloned into an inducible β-globin reporter transiently expressed in H2 cells in the absence or presence of overexpressed HuR. Association of HuR with IL-13 mRNA was detected by means of immunoprecipitation of ribonucleoprotein complexes and a biotin pull-down assay. The effects of HuR transient overexpression and silencing on IL-13 expression were investigated.

Results

IL-13 mRNA half-life increased significantly in restimulated T_H2-skewed cells compared with baseline values. Decay of β-globin mRNA was significantly faster in H2 cells transfected with the IL-13 3′UTR-containing plasmid than in those carrying a control vector. HuR overexpression increased the β-globin IL-13 3′UTR reporter half-life. Significant enrichment of IL-13 mRNA was produced by means of immunoprecipitation of Jurkat cell ribonucleoprotein complexes with anti-HuR. HuR binding to the IL-13 3′UTR was confirmed by means of pull-down assay of biotin-labeled RNA probes spanning the IL-13 3′UTR. Two-dimensional Western blot analysis showed stimulus-induced posttranslational modification of HuR. In Jurkat cells mitogen-induced IL-13 mRNA was significantly affected by HuR overexpression and silencing.

Conclusions

Mitogen-induced IL-13 expression involves changes in transcript turnover and a change in phosphorylation of HuR and its association with the mRNA 3′UTR.

Key words: Asthma, inflammation, mRNA turnover, posttranscriptional gene regulation, RNA-binding proteins, IL-13, T_H2 cytokines, T cells