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Molecular and Cellular Biology, May 2009, p. 2622-2635, Vol. 29, No. 10
0270-7306/09/$08.00+0     doi:10.1128/MCB.01495-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Analysis of Nitric Oxide-Stabilized mRNAs in Human Fibroblasts Reveals HuR-Dependent Heme Oxygenase 1 Upregulation{triangledown} ,{dagger}

Yuki Kuwano,1,{ddagger} Ariel Rabinovic,2,{ddagger} Subramanya Srikantan,1 Myriam Gorospe,1,§ and Bruce Demple2,§*

RNA Regulation Section, Laboratory of Cellular and Molecular Biology, National Institute on Aging-Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224,1 Department of Genetics and Complex Diseases, Room 509, Building 1, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 021302

Received 24 September 2008/ Returned for modification 6 November 2008/ Accepted 4 March 2009

We previously observed that nitric oxide (NO) exposure increases the stability of mRNAs encoding heme oxygenase 1 (HO-1) and TIEG-1 in human and mouse fibroblasts. Here, we have used microarrays to look broadly for changes in mRNA stability in response to NO treatment. Using human IMR-90 and mouse NIH 3T3 fibroblasts treated with actinomycin D to block de novo transcription, microarray analysis suggested that the stability of the majority of mRNAs was unaffected. Among the mRNAs that were stabilized by NO treatment, seven transcripts were found in both IMR-90 and NIH 3T3 cells (CHIC2, GADD45B, HO-1, PTGS2, RGS2, TIEG, and ID3) and were chosen for further analysis. All seven mRNAs showed at least one hit of a signature motif for the stabilizing RNA-binding protein (RBP) HuR; accordingly, ribonucleoprotein immunoprecipitation analysis revealed that all seven mRNAs associated with HuR. In keeping with a functional role of HuR in the response to NO, a measurable fraction of HuR increased in the cytoplasm following NO treatment. However, among the seven transcripts, only HO-1 mRNA showed a robust increase in the level of its association with HuR following NO treatment. In turn, HO-1 mRNA and protein levels were significantly reduced when HuR levels were silenced in IMR-90 cells, and they were elevated when HuR was overexpressed. In sum, our results indicate that NO stabilizes mRNA subsets in fibroblasts, identify HuR as an RBP implicated in the NO response, reveal that HuR alone is insufficient for stabilizing several mRNAs by NO, and show that HO-1 induction by NO is regulated by HuR.

* Corresponding author. Mailing address: Department of Genetics and Complex Diseases, Room 509, Building 1, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02130. Phone: (617) 432-3462. Fax: (617) 432-2590. E-mail: bdemple{at}hsph.harvard.edu

{triangledown} Published ahead of print on 16 March 2009.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Y.K. and A.R. are co-first authors.

§ M.G. and B.D. are co-senior authors.

Molecular and Cellular Biology, May 2009, p. 2622-2635, Vol. 29, No. 10
0270-7306/09/$08.00+0     doi:10.1128/MCB.01495-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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