Cytokines stimulate expression of inducible nitric oxide synthase in DLD-1 human adenocarcinoma cells by activating poly(A) polymerase.
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.


CONCLUSIONS
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.
Murthy
K G K
KG
Inotek Pharmaceuticals Corporation, 100 Cummings Center, Suite 419E, Beverly, MA 01915, USA. kmurthy@inotekcorp.com
Szabó
C
C
Salzman
A L
AL
D015153:Blotting, Western; D045744:Cell Line, Tumor; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007375:Interleukin-1 / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D011062:Polynucleotide Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007375:Interleukin-1; D012333:RNA, Messenger; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D011062:Polynucleotide Adenylyltransferase