We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3′-untranslated region (3′-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3′-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3′-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3′-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.
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 mitogen-activated protein kinase (p38 MAPK). In human DLD-1 cells, inhibition of p38 MAPK by the compound 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) or by overexpression of a dominantnegative 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, quantitative real-time polymerase chain reaction, 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.Overexpression or down-regulation of tristetraprolin in stably transfected DLD-1-or A549/8 cells consistently resulted in enhanced or reduced iNOS expression by modulating iNOSmRNA stability. In UV cross-linking experiments, recombinant tristetraprolin did not interact with the human iNOS mRNA. However, coimmunoprecipitation experiments showed interaction of tristetraprolin with the KH-type splicing regulatory protein (KSRP), which is known to recruit mRNAs containing AUrich elements 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. Because 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.
Human inducible nitric oxide synthase (iNOS) expression is regulated by transcriptional and post-transcriptional mechanisms. We have recently shown that the multifunctional RNAbinding 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 RNAbinding 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.Post-transcriptional mechanisms represent an important part of the regulation of gene expression. In particular, genes whose expression has to be controlled precisely (e.g. oncogenes, pro-inflammatory genes) are regulated at the post-transcriptional level, mostly by modulation of mRNA stability (1, 2). Inherently unstable mRNAs that code for cytokines, transcription factors, proto-oncogenes, and pro-inflammatory mediators often contain AU-rich elements (AREs) 3 in their 3Ј-untranslated regions (3Ј-UTRs). These AREs are targets for transacting proteins regulating mRNA stability and translation (1, 3). The family of ARE-binding proteins includes the embryonic lethal abnormal vision protein family members (most importantly HuR), the ARE/poly(U)-binding/degradation factor 1 (AUF-1, also named hnRNP D), the KH-type splicing regulatory protein (KSRP), tristetraprolin, the T cell-restricted intracellular antigen (TIA-1), and the T cell-restricted intracellular antigen-related protein (1, 3). In mammalian cells, the AREsequences mediate mRNA decay mainly by recruitment of the exosome (a multisubunit particle with 3Ј to 5Ј nuclease activity) to the mRNAs, thereby promoting their rapid degradation. However, the mammalian exosome does not seem to recognize the ARE-containing RNAs on its own but requires certain AREbinding proteins (like KSRP or tristetraprolin) for this interaction (4, 5). The polypyrimidine tract-binding protein (PTB), also 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 translatio...
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