cGMP-independent nitric oxide signaling and regulation of the cell cycle.
BACKGROUND
Regulatory functions of nitric oxide (NO*) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO* on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO* donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO*-induced decreases in cAMP.


RESULTS
NO* regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO*, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO* and Bt2cAMP effects showed that NO* regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO* annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO*, as was E2F1 binding to E2F promoter elements. NO* activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M.


CONCLUSIONS
NO* coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO* may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO* regulates the transcriptome.
Cui
Xiaolin
X
Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland, USA. xcuia2003@yahoo.com
Zhang
Jianhua
J
Ma
Penglin
P
Myers
Daniela E
DE
Goldberg
Ilana G
IG
Sittler
Kelly J
KJ
Barb
Jennifer J
JJ
Munson
Peter J
PJ
Cintron
Ana del Pilar
Adel P
McCoy
J Philip
JP
Wang
Shuibang
S
Danner
Robert L
RL
D015153:Blotting, Western; D002453:Cell Cycle; D049109:Cell Proliferation; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050687:E2F1 Transcription Factor / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D015233:Models, Statistical; D009569:Nitric Oxide / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026422:S-Nitrosoglutathione / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014158:Transcription, Genetic; D020298:U937 Cells; D015854:Up-Regulation
D050759:Cyclin-Dependent Kinase Inhibitor p21; D050687:E2F1 Transcription Factor; D012333:RNA, Messenger; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D012313:RNA; D000242:Cyclic AMP; D005978:Glutathione; D006152:Cyclic GMP