Received for publication October 28, 2004.
Revised March 2, 2005.
Accepted for publication March 18, 2005.

Tristetraprolin regulates the expression of the human inducible nitric oxide synthase gene

Abstract

The expression of human inducible NO synthase (iNOS) is regulated both by transcriptional and post-transcriptional mechanisms. Stabilization of mRNAs often depends on activation of p38 mitogene activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by compound SB203580 or by overexpression of a dominant negative p38 MAPK protein resulted in a reduction of human iNOS mRNA and protein expression, whereas human iNOS promoter activity was not affected. An important RNA binding protein regulated by the p38 MAPK pathway and involved in the regulation of the stability of several mRNAs is tristetraprolin. RNase protection, qRT-PCR and Western blot experiments showed that cytokines used to induce iNOS expression in DLD-1 cells also enhanced tristetraprolin expression. SB203580-incubation reduced cytokine-mediated enhancement of tristetraprolin expression. Consistently, overexpression or down-regulation of tristetraprolin in stably transfected DLD-1- or A549/8 cells resulted in enhanced or reduced iNOS expression by modulating iNOS-mRNA stability, respectively. In UV-crosslinking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, co-immunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP) which is known to recruit AU-rich element containing mRNAs to the exosome for degradation. This tristetraprolin-KSRP interaction was enhanced by cytokines and reduced by SB203580-treatment. We conclude that tristetraprolin positively regulates human iNOS expression by enhancing the stability of human iNOS mRNA. Since tristetraprolin does not directly bind to the human iNOS mRNA but interacts with KSRP, tristetraprolin is likely to stabilize iNOS mRNA by capturing the KSRP-exosome complex.

Key words: Nitric oxide, Nitric oxide synthases, P38 MAP Kinase, Promoter analysis, Regulation - post-transcriptional, Regulation - transcriptional, RNA/siRNA

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