Cytokines stimulate expression of inducible nitric oxide synthase in DLD-1 human adenocarcinoma cells by activating poly(A) polymerase
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Objective: The stimulation of epithelial cells by cytokines or lipopolysaccharide results in a marked increase in cellular mRNA and protein levels of inducible nitric oxide synthase (iNOS) disproportionate to the small upregulation in transcriptional activity. The molecular mechanisms by which cytokines increase iNOS expression are not well characterized.
Methods: DLD-1 cells were treated with cytokines and we studied the expression patterns of various genes by using western blot analysis and RT-PCR assay method.
Results: Expression levels of iNOS protein were detected after 4 h of incubation with cytokines and reached a peak at 10 h. After cytokine treatment, iNOS mRNA molecules received longer poly(A) tails (200–500 adenosine residues) and total iNOS mRNA levels also increased significantly. Western blot analysis revealed that poly(A) polymerase (PAP) undergoes a significant dephosphorylation process. At the same time, cytokines have no significant effect on the expression pattern of other factors involved in polyadenylation.
Conclusion: Cytokines appear to induce elongation of iNOS mRNA poly(A) tail length by activating PAP. These results indicate a novel link between mRNA 3′ end formation and iNOS gene expression.
Key words.Poly(A) polymerase iNOS Polyadenylation Cytokines Posttranscription
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