Abstract
Human inducible nitric oxide synthase (iNOS) expression is regulated by transcriptional and post-transcriptional mechanisms. We have recently shown that the multifunctional RNA-binding proteins KH-type splicing regulatory protein and tristetraprolin are critically involved in the post-transcriptional regulation of human iNOS expression. Several reports have shown that KH-type splicing regulatory protein colocalizes with the polypyrimidine tract-binding protein (PTB), and both RNA-binding proteins seem to interact with the same mRNAs. Therefore we analyzed the involvement of PTB in human iNOS expression. In human DLD-1 cells, cytokine incubation necessary to induce iNOS expression did not change PTB localization or expression. However, intracellular binding of PTB to the human iNOS mRNA increased after cytokine stimulation. Overexpression of PTB resulted in enhanced cytokine-induced iNOS expression. Accordingly, small interfering RNA-mediated knock down of PTB reduced cytokine-dependent iNOS expression. Recombinant PTB displayed binding to an UC-rich sequence in the 3'-untranslated region of the human iNOS mRNA. Transfection experiments showed that PTB mediates its effect on iNOS expression via binding to this region. The underlying mechanism is based on a modulation of iNOS mRNA stability. In summary, human iNOS is the first example of a human pro-inflammatory gene regulated by PTB on the level of mRNA stability.
Highlights
The polypyrimidine tract-binding protein (PTB), known as hnRNP I, is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing [6], internal ribosome entry site-driven translation [7], hepatitis C virus replication [8], mRNA localization [9], and polyadenylation [10]
We showed that KSRP, HuR, and tristetraprolin are essentially involved in furanosylbenzimidazol; hnRNP, heteronuclear ribonucleoprotein; KSRP, KH-type splicing regulatory protein; NO, nitric oxide; isoform of nitric oxide synthase (iNOS), inducible NO synthase; PTB, polypyrimidine tract-binding protein; Quantitative Reverse Transcription-PCR (qRT-PCR), quantitative real-time reverse transcription-PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; GST, glutathione S-transferase; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Luc, luciferase
Cytokine Incubation Does Not Change PTB Expression or Localization in DLD-1 Cells—In previous studies, we demonstrated that KSRP is critically involved in the post-transcriptional regulation of human iNOS expression in interplay with other RNA-binding proteins [24, 25]
Summary
Eighteen hours before cytokine induction, the cells were washed with phosphate-buffered saline and incubated with Dulbecco’s modified Eagle’s medium containing 2 mM L-glutamine in the absence of serum and phenol red. Establishment of Cell Lines Expressing an EGFP1⁄7PTB Fusion Protein—To generate DLD-1 cells overexpressing an EGFP1⁄7 PTB fusion protein, cells were transfected with 5 g of pEGFPPTB [28] with FuGENE (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s recommendations. Real-time qRT-PCR was performed according to the manufacturer’s recommendations using the oligonucleotides listed (all from MWG-Biotech, Ebersberg, Germany)
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