BACKGROUND
nitric oxide  plays a pivotal role in vascular biology through both cgmp-dependent and -independent mechanisms.
in health, no• regulates vascular tone by activating soluble guanylate cyclase  <cit> .
however, other important effects of no• in the vasculature such as cytoprotection and anti-adhesion appear to occur independent of cgmp signaling  <cit> .
likewise, no• regulation of inflammation has frequently been associated with signal transduction events that do not involve cgmp  <cit> .
no• induces tnfα in human cells by decreasing intracellular levels of camp, thereby removing camp-mediated repression of the tnfα promoter through a proximal sp element  <cit> .
analogs of camp and sp site mutation both block, while antagonists of camp-dependent protein kinase simulate the effect of no• on tnfα.
<cit> .
in contrast to tnfα, no• induces interleukin- <dig>   <cit>  through a distinct post-transcriptional mechanism that is both cgmp- and camp-independent.
il- <dig> mrna is stabilized by no• activation of p <dig> mapk, increasing its half-life and translation  <cit> .
these and other reports  <cit> .
suggest that cgmp-independent gene regulation by no• occurs through multiple pathways.
similar to the regulation of blood pressure and inflammatory responses, no• regulation of cell proliferation is of central importance to circulatory health.
failure of this regulatory pathway has been linked to atherosclerosis and other forms of vascular dysfunction  <cit> .
despite extensive investigation, the relative contribution of cgmp-independent no• signaling in the regulation of cell cycle genes remains controversial.
in rats, no• has been shown to activate transcription through cgmp-dependent effects on ap- <dig> promoter sites  <cit> .
also in rodents, a no•-cgmp-pka-erk1/ <dig> signal transduction pathway has been described that inhibits cell proliferation  <cit>  and increases expression of p21/waf1/cip <dig>  <cit> .
a master regulatory gene, p <dig> directly inhibits cdk complexes  <cit>  and represses the transcription of many cell cycle genes through cde/chr  promoter elements  <cit> .
in contrast to rodents, no• regulation of cell cycle genes in humans, including regulation of p <dig>  appears to occur, at least in part, independent of cgmp  <cit> .
however, a global examination of cgmp-independent no• effects on the transcriptome in general or on cell cycle genes specifically has not been undertaken in either rodents or humans.
here, oligonucleotide microarrays and human u <dig> cells that lack soluble guanylate cyclase  <cit>  were used to globally characterize the cgmp-independent effects of no• on gene expression.
differentiation with pma was employed to render cells capable of cytokine production  <cit> .
this treatment also forced >80% of cells into the g0/g <dig> phase of the cell cycle, which facilitated the analysis of cell cycle gene regulation.
since no• lowers camp levels in u <dig> cells  <cit>  and camp is known to affect cell proliferation, no• effects were also tested in the absence and presence of a cell permeable camp analog.
for genes affected by no•-induced decreases in camp, camp analog would be expected to produce an apposite effect.
hypotheses generated from microarray results were further investigated by examining downstream changes in protein expression and signal transduction pathways.
RESULTS
functional distribution of no•-regulated genes and hypothesis generation
of  <dig> no•-responsive genes,  <dig> were induced, and  <dig> were repressed; the majority were not previously known to be no•-responsive.
both naïve and differentiated u <dig> cells lack no•-sensitive soluble guanylate cyclase  <cit> , and therefore gene regulation by no• in these cells can be attributed to cgmp-independent mechanisms.
genes were annotated into functional categories  .
no• had broad biological effects independent of cgmp.
heme oxygenase  <dig> , a known no•-responsive gene, had the second largest fold change among up-regulated genes.
tnfα and il- <dig>  cytokines previously associated with specific cgmp-independent mechanisms of no• regulation  <cit> , were also detected as differentially regulated by the microarray analysis.
no•-regulated genes annotated disproportionately to the cell cycle  biological process database; p =  <dig> ] .
in particular, a large majority of no• down-regulated genes annotated specifically to the cell cycle .
additional annotation using pubmed identified  <dig> of  <dig> genes  as cell cycle or cell proliferation related.
of  <dig> down-regulated transcripts,  <dig>  were ultimately annotated specifically to the cell cycle.
previously, we demonstrated that no• activates p <dig> mapk and thereby stabilizes il- <dig> mrna through effects on au-rich elements  in 3' untranslated regions   <cit> .
therefore, the are database  was used to identify are-containing genes among those regulated by no•.
twenty-two of  <dig> genes contained are  compared to  <dig> are genes of  <dig> on the microarray .
an additional  <dig> are-containing genes were identified in pubmed for a total of  <dig> .
nearly half of these genes  have been reported to be p <dig> mapk regulated .
importantly, for these  <dig> genes, p <dig> mapk activation produces responses that are in the same direction as those observed here for no•.
the broad influence of no• on cell cycle-related genes and are-containing transcripts independent of cgmp was unexpected, as was the strong association of these effects with p <dig> mapk.
therefore, further experiments were performed to confirm these results and to define underlying regulatory mechanisms that might link no• effects on the cell cycle with post-transcriptional gene regulation through are sites.
a as reported in the literature; citations shown in parentheses
validation of no•-regulated genes
to determine whether no• and camp effects on mrna, as measured by microarray, produced downstream effects on secreted protein, tnfα, il- <dig> and il-1β were measured in supernatants collected from parallel cell cultures incubated for  <dig> h. s-nitrosoglutathione  significantly increased tnfα, il- <dig>  and il-1β protein .
in contrast, camp decreased tnfα , increased il-1β , and had no significant effect on il- <dig>  no•-induced changes in transcript abundance as determined by microarray were consistent with these results  was used to validate no•-mediated changes in mrna levels .
of  <dig> selected genes,  <dig> were no• up-regulated, and  <dig> were down-regulated.
fold changes from microarray experiments strongly correlated with results from rt-pcr .
western blotting of key cell cycle genes regulated by no• was performed to test whether microarray results accurately predicted changes in protein expression .
three induced genes, e2f transcription factor  <dig> , p21/waf1/cip <dig> , and cell division cycle  <dig>  were examined.
e2f <dig> and p <dig> are well-characterized master regulatory proteins that control the cell cycle.
four repressed genes, cyclin a <dig> , cyclin b <dig> , polo-like kinase  and cyclin f  were also measured by western blotting.
in all cases, directional changes in protein expression were consistent with the differential effect of no• on corresponding transcripts as determined by microarray analysis.
no•-regulation of cell cycle genes independent of camp
no• induces tnfα by decreasing intracellular camp; dibutyryl-camp , a cell permeable camp analog, blocks this effect  <cit> .
moreover, camp is an omnipresent second messenger that affects cell proliferation and the cell cycle in a variety of contexts  <cit> .
therefore, bt2camp was added to some conditions to test for the camp-dependence of no•-mediated effects.
of  <dig> bt2camp-responsive genes,  <dig> of  <dig>  compared to  <dig> of  <dig> on the microarray , as annotated in the go biological process database, were cell cycle related, but this effect was not statistically significant .
only one additional camp-responsive gene was subsequently annotated to the cell cycle by searching pubmed .
however, like no•, camp-regulated genes did contain are more frequently than expected .
this finding was consistent with the known ability of camp to stabilize transcripts that contain are  <cit> .
to further compare the effects of no• and camp, a hierarchical cluster analysis was performed using the  <dig> cell cycle genes regulated by no• .
for each of the  <dig> cell cycle genes significantly affected by both no• and bt2camp , the direction of regulation was the same.
further, for cell cycle genes regulated by no•, camp analog either had no effect on or produced expression changes that were similar to and additive with those observed for no• .
these results suggest that no• effects on cell cycle genes are independent of its interference with camp signaling, since camp analog  was not antagonistic to the actions of no•.
analysis of no• effects on the cell cycle
no• causes arrest in either the g <dig> or g2/m phase of the cell cycle  <cit> .
however, the mechanisms underlying this effect are not well characterized.
annotation of no•-regulated genes to their respective phase of the cell cycle revealed that expression changes were not random .
most no• up-regulated genes  were g1/s associated, while down-regulated genes were strikingly g <dig> and g2/m phase associated .
the latter included topoisomerase ii alpha , cyclin b <dig>  plk, and cdc25b, genes that are necessary factors for mitosis.
these results show that no• suppresses the cell cycle in early g2/m by triggering a highly integrated program of gene regulation that does not require soluble guanylate cyclase or cgmp.
a fold change comparing glutathione  to s-nitrosoglutathione -treated cells, expressed as the mean ± se
b represented by more than one probe set on the microarray that reached statistical significance; each result is shown
to further test this hypothesis, cell cycle analysis was performed on u <dig> cells using flow cytometry.
pma-differentiation significantly increased the portion of cells in g0/g <dig> , while decreasing cells in s  and g2/m  compared to a naïve, undifferentiated cell population .
differentiated cells were then treated with glutathione  or gsno in the absence or presence of bt2camp.
consistent with no•-induced changes in mrna expression at  <dig> h, cell cycle analysis at  <dig> h demonstrated that no• increased the portion of cells in g2/m , and in combination with bt2camp, no• synergistically increased g2/m phase cells .
no• induction of p <dig>  a master cell cycle regulator; dependence on p <dig> mapk and role of mrna stabilization
the cdk inhibitor, p <dig> is known to induce cell cycle arrest in late g <dig> or early g2/m,  <cit>  effects similar to those of no•.
no• increased both p <dig> mrna and protein expression in the current experiments.
we have previously shown that no• activates p <dig> mapk in u <dig> and thp- <dig> cells  <cit> .
activation of p <dig> mapk induces p <dig> in other cell types,  <cit>  and like no• here, can trigger g2/m cell arrest  <cit> .
we therefore reasoned that no• might up-regulate p <dig> by activating p <dig> mapk in the present system.
gsno was first confirmed in pma-differentiated u <dig> cells to dose-dependently increase p <dig> mapk activation.
this effect reached significance at the lowest  gsno concentration tested .
next, three chemically distinct no• donors were tested for their ability to up-regulate p <dig> protein in pma-differentiated u <dig> cells.
gsno, s-nitroso-n-acetylpenicillamine , and deta-nonoate similarly increased p <dig> expression in these cells compared to degraded controls .
thus, independent of cgmp and type of donor molecule, no• consistently increased the expression of p <dig> protein in u <dig> cells.
a specific p <dig> mapk inhibitor  was used to determine whether blocking this pathway could prevent no• induction of p <dig>  as shown in fig.
6b, sb <dig> dose-dependently reduced no•-induced p <dig> protein expression .
collectively, these results suggest that no• induces p <dig> through p <dig> mapk activation.
finally, we investigated the effects of no• and p <dig> mapk inhibition on the stability of p <dig> mrna, which harbors are in its 3' utr  <cit> .
after  <dig> h of pma exposure, p <dig> expression increased almost  <dig> fold compared to naïve u <dig> cells .
no• stabilized p <dig> mrna in the absence of sb <dig> , but had no effect in the presence of sb <dig> .
no• regulation of the cell cycle through e2f <dig> and p21
the e2f family of transcription factors and p <dig> act as master regulatory switches that control the cell cycle.
e2f <dig> regulates target genes through e2f-binding sites and thereby plays an essential role in dna synthesis and the g1/s transition  <cit> .
some p <dig> effects are mediated by inhibition of e2f factor binding, while other downstream targets contain cell cycle dependent element/cell cycle gene homology region  repressor sites within their promoters  <cit> .
protein binding to cde/chr sites, triggered by p <dig> expression, causes repression of a diverse group of cell cycle genes and subsequent late g <dig> or early g2/m phase arrest, responses that are highly similar to no• effects shown here.
the ability of no• to increase the expression of e2f <dig> and p <dig> may explain much of its broad control over the cell cycle that ultimately involves dozens of gene products.
we therefore identified no•-regulated genes that contain e2f or cde/chr promoter sites by searching transfac  <cit>  and pubmed <cit> .
sixteen no•-regulated genes contain apparent e2f sites .
seven of these are annotated to the g1/s phase of the cell cycle, six of which have reported e2f <dig> responses that are concordant with no• effects in the current experiment.
notably, of  <dig> no• down-regulated transcripts with possible e2f-binding sites, only c-myb is a g1/s phase gene.
further,  <dig> of these genes are known to also contain a cde/chr binding site that appears to be functionally dominant .
moreover, of the  <dig> cell cycle genes down-regulated by no•,  <dig> are known targets of p <dig> repression , including  <dig> genes with putative cde/chr sites  and two others, lamin b <dig>  and centromere protein f   <cit> .
a as reported in the literature; citations shown in parentheses
b these genes also contain cde/chr sites as shown in table 3b
a as reported in the literature  <cit>
b these genes also contain e2f sites as shown in table 3a
next, electrophoretic mobility shift assays  were performed to test whether no• altered protein binding to e2f and cde/chr consensus sequences.
pma-differentiated u <dig> cells were treated with pbs, gsh, or gsno followed by preparation of nuclear extract.
no• increased binding to both e2f  and cde/chr probes .
site-directed mutagenesis of each consensus sequence abolished competition  and e2f1-directed antibody blocked complex formation with labeled e2f probe .
summary
no•, independent of cgmp, regulated a diverse subset of genes involved in inflammation, metabolism, apoptosis, the cell cycle, proliferation, signal transduction, and transport.
notably, genes associated with the cell cycle and proliferation, including the master cell cycle regulatory genes e2f <dig> and p <dig>  were over-represented.
further, no•-regulated transcripts had are  in their 3' utr and were p <dig> mapk responsive more frequently than expected.
e2f <dig> induction by no• was associated with up-regulation of several genes involved in g1/s transition that contain e2f-binding sites.
no• also induced p <dig>  an are-containing gene, through p <dig> mapk activation and mrna stabilization.
this was associated with the down-regulation of g2/m phase genes, at least in part, through changes in protein binding to cde/chr promoter sites.
collectively, these results demonstrate that no•, independent of cgmp and camp, triggers a specific and highly coordinated genetic program that alters the g1/s transition and induces arrest in early g2/m .
mapk pathways and mrna stability are major mechanisms by which no• regulates the transcriptome.
discussion
no• has potent anti-tumor and anti-atherosclerotic effects that are closely associated with its ability to block cell proliferation  <cit> .
this activity of no• has been ascribed to both cgmp-dependent and -independent mechanisms.
experiments in rodents have found, with a few notable exceptions  <cit> , that no• controls the cell cycle through cgmp.
these studies have focused on the importance of a no•-cgmp-pka-erk 1/ <dig> signal transduction pathway  <cit> .
accordingly, camp itself has also been reported to inhibit cell proliferation through activation of pka and/or erk 1/ <dig> with the up-regulation of p <dig> or p <dig> in a cell-specific manner  <cit> .
in contrast, the anti-proliferation effects of no• in human cells have been frequently associated with cgmp-independent signaling  <cit> .
here, a transcriptome-wide approach revealed that no• exerts broad control over the cell cycle through p <dig> mapk activation and mrna stabilization.
in a previous study, we found that no• up-regulates tnfα by decreasing camp, an effect antagonized by camp analogs.
therefore bt2camp was used in this investigation to explore whether some effects of no• on the transcriptome could be attributed to its interaction with camp signaling.
however, our results indicate that no•-camp signaling appears to be a minor pathway, regulating less than  <dig> of the affected transcripts in u <dig> cells .
these included tnfα, as well as pim- <dig> oncogene , tnfα-induced protein  <dig> , and glutathione reductase .
importantly, for cell cycle genes, no• and bt2camp consistently had the same directional effect on transcripts, although no• regulated more genes overall.
thus, decreases in intracellular camp appear unrelated to no• effects on the cell cycle.
furthermore, treatment with both no• and bt2camp synergistically provoked cell cycle arrest in g2/m, suggesting that no•-induced decreases in camp may attenuate some of its effects on the cell cycle.
although this experiment also provides useful intormation on gene regulation by camp in u <dig> cells, the reader should keep in mind that bt2camp was the only analog studied and some effects may have been caused by its butyryl component.
u <dig> cells were pma-differentiated in the current experiments to render them capable of producing tnfα and il- <dig>  two cytokines previously identified as no•-responsive  <cit> .
further, this treatment also reduced cell proliferation and forced >80% of the cells into the g0/g <dig> phase of the cell cycle, allowing for a more coherent analysis of cell cycle regulation .
however, pma itself had large effects on no•-regulated genes such as p <dig>  and the findings here cannot be extrapolated directly to naïve u <dig> cells.
fortunately, turpev and colleagues have recently reported selected microarray results from no• exposure of undifferentiated u <dig> and mono mac  <dig> cells  <cit> .
of interest, a number of key genes identified by this group were also found to be similarly regulated by no• in pma-differentiated cells including hmox <dig>  il- <dig>  activating transcription factor  <dig> , bcl2/adenovirus e1b  <dig> kd-interacting protein  <dig> , and importantly p21/waf1/cip <dig>
the no• donor gsno was found to activate p <dig> mapk in u <dig> cells, which was consistent with our previous result using snap, another no• donor  <cit> .
furthermore, three different no• donors were shown here to consistently increase p <dig> protein expression, indicating that this effect is no•-specific and donor independent.
importantly, very low concentrations of the p <dig> mapk inhibitor sb <dig> were found to block the induction of p <dig> protein by no•, further establishing the role of p <dig> mapk as an intermediary signal transduction event.
finally, p <dig> mrna was measured serially by rt-pcr after transcriptional blockade in the absence or presence of sb <dig> showing that this transcript is stabilized by no• through a p <dig> mapk-dependent mechanism.
others have found that no• increases p <dig> mrna and protein expression in human vascular smooth muscle independent of cgmp  <cit> .
in addition, p <dig> mapk activation has been shown to increase p <dig> expression by both transcriptional activation and protein stabilization  <cit> .
e2f <dig> is also known to induce p <dig> transcription  <cit>  providing another mechanism by which no• may have increased p <dig> expression in the current experiment.
conversely, as already discussed, no• decreases camp, reducing the ability of sp <dig> to bind to gc box elements and thereby repressing the transcription of sp1-dependent genes such as enos  <cit> .
interestingly, p <dig> is highly dependent on sp <dig> for transcription  <cit>  and is induced by camp  <cit> , findings consistent with the possibility that p <dig> may be transcriptionally repressed by no•-camp-sp <dig> signal transduction.
nonetheless, no• induction of p <dig> demonstrates that other mechanisms dominate over any negative effects of no• on sp <dig> binding to the p <dig> promoter.
here, we focused on mrna stabilization, because of the strong indication in our microarray data that this is a major mechanism of gene regulation by no•.
however, other mechanisms such as changes in transcription, translation, or protein stability may have contributed substantially to the net effects of no• on p <dig> expression.
the e2f family of transcription factors play important roles in g1/s phase transition.
e2f <dig> up-regulates many g1/s phase genes including itself, cyclin e <dig> , cdc <dig>  uracil-dna glycosylase , jun, p <dig> and c-myb  <cit> .
except for c-myb, all were up-regulated by no• in the present study, suggesting that no• may drive differentiated u <dig> cells through the g1/s transition by inducing e2f <dig> expression.
this conclusion is further supported by emsa showing that no• increases e2f <dig> binding to e2f consensus sequence.
however, increased e2f <dig> expression may not be the only mechanism contributing to these observed changes in dna binding.
no• activates p <dig> mapk in u <dig> cells and p <dig> mapk has been shown to increase e2f <dig> binding to e2f sites  <cit> .
further, cyclin a <dig> was down-regulated by no• and has been shown to turn off e2f <dig> target genes by decreasing e2f <dig> dna binding  <cit> .
notably, c-myb and a number of g <dig> or g2/m phase genes that contain e2f sites were down-regulated by no•.
e2f sites can function as repressors in some genes and their disruption by mutation leads to promoter activation  <cit> .
further, no•-responsive genes with both e2f elements and cde/chr repressor sites were uniformly down-regulated.
promoters with cde/chr motifs are repressed by p <dig>  <cit> , which was also induced by no•.
therefore, even for promoters activated by e2f <dig>  repression through cde/chr sites appears to be the dominant action of no• in this cellular context.
moreover, e2f and chr sites may cooperate as co-repressors  <cit> .
although cdc25b lacks an identifiable cde/chr site, it does have a proximal repressor and its regulation is similar to cde/chr-containing genes  <cit> .
cell cycle arrest induced by p <dig> occurs in late g1/s  <cit>  or early g2/m  <cit>  and is mediated, at least in part, by the repression of target genes with cde/chr sites  <cit> .
cde/chr sites are present in the promoters of cyclin a <dig>  <cit> , cdc <dig>  <cit> , cyclin b <dig>  <cit> , and top2a  <cit> , centromere protein a   <cit>  and plk  <cit> .
all of these genes are g <dig> or g2/m related and are down-regulated by p <dig>  results consistent with the effects of no• observed here.
emsa confirmed the hypothesis that no• regulates protein binding to cde/chr sites.
collectively, these findings suggest that no• regulates many g2/m phase cell cycle genes through p <dig>  however, no• may also regulate some of these downstream p <dig> targets through additional mechanisms.
for example, c-myb, cyclin a <dig>  cyclin b <dig>  and cenpa have are in their 3' utr, indicating that no• might alter the stability of these transcripts.
notably, cyclin a <dig> and cyclin b <dig> are down-regulated by p <dig> mapk, a signal transduction pathway that was activated by no• in the current experiments.
importantly, p <dig> mapk has been shown to induce cell cycle arrest at the g <dig> checkpoint through mechanisms that were only partially dependent on p <dig>  <cit> .
are in 3' utr have been implicated in the control of transcript stability and have an important post-transcriptional impact on transcriptome content  <cit> .
we previously demonstrated that independent of cgmp, no• up-regulates il- <dig>  but not tnfα post-transcriptionally through p <dig> mapk activation  <cit> .
in the current investigation, are- containing genes including il- <dig> and p <dig> were over-represented among no•-regulated genes.
nearly half of these are genes have been reported to be regulated by p <dig> mapk .
notably, no• responses were all in the same direction as those reported for p <dig> mapk activation.
previous microarray experiments that globally tested mrna stability found that 10% of transcripts were associated with p <dig> mapk-dependent regulation  <cit> .
the over-representation of p <dig> mapk-regulated genes in our experiments indicates that this stress kinase is an important target of no•.
CONCLUSIONS
the present investigation was focused on understanding cgmp-independent gene regulation by no•.
major themes within the identified gene list were the predominance of cell cycle-related genes and are-containing transcripts.
no• was found to trigger a specific and coordinated cell cycle arrest independent of both cgmp and camp.
e2f <dig> induction up-regulated target genes involved in g1/s transition through e2f sites.
no• stabilization of p <dig> mrna was p <dig> mapk dependent and led to increased protein binding to cde/chr promoter sites and the down-regulation of g2/m phase genes.
the cell cycle is a major target of no•-mediated gene regulation.
importantly, p <dig> mapk and mrna stability are major intermediary mechanisms through which no• affects the human transcriptome.

 nitric oxide plays a pivotal role in vascular biology through both cgmp-dependent and -independent mechanisms. in health, no• regulates vascular tone by activating soluble guanylate cyclase. however, other important effects of no• in the vasculature such as cytoprotection and anti-adhesion appear to occur independent of cgmp signaling. likewise, no• regulation of inflammation has frequently been associated with signal transduction events that do not involve cgmp. no• induces tnfα in human cells by decreasing intracellular levels of camp, thereby removing camp-mediated repression of the tnfα promoter through a proximal sp element. analogs of camp and sp site mutation both block, while antagonists of camp-dependent protein kinase simulate the effect of no• on tnfα.. in contrast to tnfα, no• induces interleukin- through a distinct post-transcriptional mechanism that is both cgmp- and camp-independent. il- mrna is stabilized by no• activation of p mapk, increasing its half-life and translation. these and other reports. suggest that cgmp-independent gene regulation by no• occurs through multiple pathways. similar to the regulation of blood pressure and inflammatory responses, no• regulation of cell proliferation is of central importance to circulatory health. failure of this regulatory pathway has been linked to atherosclerosis and other forms of vascular dysfunction. despite extensive investigation, the relative contribution of cgmp-independent no• signaling in the regulation of cell cycle genes remains controversial. in rats, no• has been shown to activate transcription through cgmp-dependent effects on ap- promoter sites. also in rodents, a no•-cgmp-pka-erk1/ signal transduction pathway has been described that inhibits cell proliferation and increases expression of p21/waf1/cip. a master regulatory gene, p directly inhibits cdk complexes and represses the transcription of many cell cycle genes through cde/chr promoter elements. in contrast to rodents, no• regulation of cell cycle genes in humans, including regulation of p appears to occur, at least in part, independent of cgmp. however, a global examination of cgmp-independent no• effects on the transcriptome in general or on cell cycle genes specifically has not been undertaken in either rodents or humans. here, oligonucleotide microarrays and human u cells that lack soluble guanylate cyclase were used to globally characterize the cgmp-independent effects of no• on gene expression. differentiation with pma was employed to render cells capable of cytokine production. this treatment also forced >80% of cells into the g0/g phase of the cell cycle, which facilitated the analysis of cell cycle gene regulation. since no• lowers camp levels in u cells and camp is known to affect cell proliferation, no• effects were also tested in the absence and presence of a cell permeable camp analog. for genes affected by no•-induced decreases in camp, camp analog would be expected to produce an apposite effect. hypotheses generated from microarray results were further investigated by examining downstream changes in protein expression and signal transduction pathways.  functional distribution of no•-regulated genes and hypothesis generation of no•-responsive genes, were induced, and were repressed; the majority were not previously known to be no•-responsive. both naïve and differentiated u cells lack no•-sensitive soluble guanylate cyclase, and therefore gene regulation by no• in these cells can be attributed to cgmp-independent mechanisms. genes were annotated into functional categories. no• had broad biological effects independent of cgmp. heme oxygenase, a known no•-responsive gene, had the second largest fold change among up-regulated genes. tnfα and il- cytokines previously associated with specific cgmp-independent mechanisms of no• regulation, were also detected as differentially regulated by the microarray analysis. no•-regulated genes annotated disproportionately to the cell cycle biological process database; p = ]. in particular, a large majority of no• down-regulated genes annotated specifically to the cell cycle. additional annotation using pubmed identified of genes as cell cycle or cell proliferation related. of down-regulated transcripts, were ultimately annotated specifically to the cell cycle. previously, we demonstrated that no• activates p mapk and thereby stabilizes il- mrna through effects on au-rich elements in 3' untranslated regions. therefore, the are database was used to identify are-containing genes among those regulated by no•. twenty-two of genes contained are compared to are genes of on the microarray. an additional are-containing genes were identified in pubmed for a total of. nearly half of these genes have been reported to be p mapk regulated. importantly, for these genes, p mapk activation produces responses that are in the same direction as those observed here for no•. the broad influence of no• on cell cycle-related genes and are-containing transcripts independent of cgmp was unexpected, as was the strong association of these effects with p mapk. therefore, further experiments were performed to confirm these results and to define underlying regulatory mechanisms that might link no• effects on the cell cycle with post-transcriptional gene regulation through are sites. a as reported in the literature; citations shown in parentheses validation of no•-regulated genes to determine whether no• and camp effects on mrna, as measured by microarray, produced downstream effects on secreted protein, tnfα, il- and il-1β were measured in supernatants collected from parallel cell cultures incubated for h. s-nitrosoglutathione significantly increased tnfα, il- and il-1β protein. in contrast, camp decreased tnfα, increased il-1β, and had no significant effect on il- no•-induced changes in transcript abundance as determined by microarray were consistent with these results was used to validate no•-mediated changes in mrna levels. of selected genes, were no• up-regulated, and were down-regulated. fold changes from microarray experiments strongly correlated with results from rt-pcr. western blotting of key cell cycle genes regulated by no• was performed to test whether microarray results accurately predicted changes in protein expression. three induced genes, e2f transcription factor, p21/waf1/cip, and cell division cycle were examined. e2f and p are well-characterized master regulatory proteins that control the cell cycle. four repressed genes, cyclin a, cyclin b, polo-like kinase and cyclin f were also measured by western blotting. in all cases, directional changes in protein expression were consistent with the differential effect of no• on corresponding transcripts as determined by microarray analysis. no•-regulation of cell cycle genes independent of camp no• induces tnfα by decreasing intracellular camp; dibutyryl-camp, a cell permeable camp analog, blocks this effect. moreover, camp is an omnipresent second messenger that affects cell proliferation and the cell cycle in a variety of contexts. therefore, bt2camp was added to some conditions to test for the camp-dependence of no•-mediated effects. of bt2camp-responsive genes, of compared to of on the microarray, as annotated in the go biological process database, were cell cycle related, but this effect was not statistically significant. only one additional camp-responsive gene was subsequently annotated to the cell cycle by searching pubmed. however, like no•, camp-regulated genes did contain are more frequently than expected. this finding was consistent with the known ability of camp to stabilize transcripts that contain are. to further compare the effects of no• and camp, a hierarchical cluster analysis was performed using the cell cycle genes regulated by no•. for each of the cell cycle genes significantly affected by both no• and bt2camp, the direction of regulation was the same. further, for cell cycle genes regulated by no•, camp analog either had no effect on or produced expression changes that were similar to and additive with those observed for no•. these results suggest that no• effects on cell cycle genes are independent of its interference with camp signaling, since camp analog was not antagonistic to the actions of no•. analysis of no• effects on the cell cycle no• causes arrest in either the g or g2/m phase of the cell cycle. however, the mechanisms underlying this effect are not well characterized. annotation of no•-regulated genes to their respective phase of the cell cycle revealed that expression changes were not random. most no• up-regulated genes were g1/s associated, while down-regulated genes were strikingly g and g2/m phase associated. the latter included topoisomerase ii alpha, cyclin b plk, and cdc25b, genes that are necessary factors for mitosis. these results show that no• suppresses the cell cycle in early g2/m by triggering a highly integrated program of gene regulation that does not require soluble guanylate cyclase or cgmp. a fold change comparing glutathione to s-nitrosoglutathione -treated cells, expressed as the mean ± se b represented by more than one probe set on the microarray that reached statistical significance; each result is shown to further test this hypothesis, cell cycle analysis was performed on u cells using flow cytometry. pma-differentiation significantly increased the portion of cells in g0/g, while decreasing cells in s and g2/m compared to a naïve, undifferentiated cell population. differentiated cells were then treated with glutathione or gsno in the absence or presence of bt2camp. consistent with no•-induced changes in mrna expression at h, cell cycle analysis at h demonstrated that no• increased the portion of cells in g2/m, and in combination with bt2camp, no• synergistically increased g2/m phase cells. no• induction of p a master cell cycle regulator; dependence on p mapk and role of mrna stabilization the cdk inhibitor, p is known to induce cell cycle arrest in late g or early g2/m, effects similar to those of no•. no• increased both p mrna and protein expression in the current experiments. we have previously shown that no• activates p mapk in u and thp- cells. activation of p mapk induces p in other cell types, and like no• here, can trigger g2/m cell arrest. we therefore reasoned that no• might up-regulate p by activating p mapk in the present system. gsno was first confirmed in pma-differentiated u cells to dose-dependently increase p mapk activation. this effect reached significance at the lowest gsno concentration tested. next, three chemically distinct no• donors were tested for their ability to up-regulate p protein in pma-differentiated u cells. gsno, s-nitroso-n-acetylpenicillamine, and deta-nonoate similarly increased p expression in these cells compared to degraded controls. thus, independent of cgmp and type of donor molecule, no• consistently increased the expression of p protein in u cells. a specific p mapk inhibitor was used to determine whether blocking this pathway could prevent no• induction of p as shown in fig. 6b, sb dose-dependently reduced no•-induced p protein expression. collectively, these results suggest that no• induces p through p mapk activation. finally, we investigated the effects of no• and p mapk inhibition on the stability of p mrna, which harbors are in its 3' utr. after h of pma exposure, p expression increased almost fold compared to naïve u cells. no• stabilized p mrna in the absence of sb, but had no effect in the presence of sb. no• regulation of the cell cycle through e2f and p21 the e2f family of transcription factors and p act as master regulatory switches that control the cell cycle. e2f regulates target genes through e2f-binding sites and thereby plays an essential role in dna synthesis and the g1/s transition. some p effects are mediated by inhibition of e2f factor binding, while other downstream targets contain cell cycle dependent element/cell cycle gene homology region repressor sites within their promoters. protein binding to cde/chr sites, triggered by p expression, causes repression of a diverse group of cell cycle genes and subsequent late g or early g2/m phase arrest, responses that are highly similar to no• effects shown here. the ability of no• to increase the expression of e2f and p may explain much of its broad control over the cell cycle that ultimately involves dozens of gene products. we therefore identified no•-regulated genes that contain e2f or cde/chr promoter sites by searching transfac and pubmed. sixteen no•-regulated genes contain apparent e2f sites. seven of these are annotated to the g1/s phase of the cell cycle, six of which have reported e2f responses that are concordant with no• effects in the current experiment. notably, of no• down-regulated transcripts with possible e2f-binding sites, only c-myb is a g1/s phase gene. further, of these genes are known to also contain a cde/chr binding site that appears to be functionally dominant. moreover, of the cell cycle genes down-regulated by no•, are known targets of p repression, including genes with putative cde/chr sites and two others, lamin b and centromere protein f. a as reported in the literature; citations shown in parentheses b these genes also contain cde/chr sites as shown in table 3b a as reported in the literature b these genes also contain e2f sites as shown in table 3a next, electrophoretic mobility shift assays were performed to test whether no• altered protein binding to e2f and cde/chr consensus sequences. pma-differentiated u cells were treated with pbs, gsh, or gsno followed by preparation of nuclear extract. no• increased binding to both e2f and cde/chr probes. site-directed mutagenesis of each consensus sequence abolished competition and e2f1-directed antibody blocked complex formation with labeled e2f probe. summary no•, independent of cgmp, regulated a diverse subset of genes involved in inflammation, metabolism, apoptosis, the cell cycle, proliferation, signal transduction, and transport. notably, genes associated with the cell cycle and proliferation, including the master cell cycle regulatory genes e2f and p were over-represented. further, no•-regulated transcripts had are in their 3' utr and were p mapk responsive more frequently than expected. e2f induction by no• was associated with up-regulation of several genes involved in g1/s transition that contain e2f-binding sites. no• also induced p an are-containing gene, through p mapk activation and mrna stabilization. this was associated with the down-regulation of g2/m phase genes, at least in part, through changes in protein binding to cde/chr promoter sites. collectively, these results demonstrate that no•, independent of cgmp and camp, triggers a specific and highly coordinated genetic program that alters the g1/s transition and induces arrest in early g2/m. mapk pathways and mrna stability are major mechanisms by which no• regulates the transcriptome. discussion no• has potent anti-tumor and anti-atherosclerotic effects that are closely associated with its ability to block cell proliferation. this activity of no• has been ascribed to both cgmp-dependent and -independent mechanisms. experiments in rodents have found, with a few notable exceptions, that no• controls the cell cycle through cgmp. these studies have focused on the importance of a no•-cgmp-pka-erk 1/ signal transduction pathway. accordingly, camp itself has also been reported to inhibit cell proliferation through activation of pka and/or erk 1/ with the up-regulation of p or p in a cell-specific manner. in contrast, the anti-proliferation effects of no• in human cells have been frequently associated with cgmp-independent signaling. here, a transcriptome-wide approach revealed that no• exerts broad control over the cell cycle through p mapk activation and mrna stabilization. in a previous study, we found that no• up-regulates tnfα by decreasing camp, an effect antagonized by camp analogs. therefore bt2camp was used in this investigation to explore whether some effects of no• on the transcriptome could be attributed to its interaction with camp signaling. however, our results indicate that no•-camp signaling appears to be a minor pathway, regulating less than of the affected transcripts in u cells. these included tnfα, as well as pim- oncogene, tnfα-induced protein, and glutathione reductase. importantly, for cell cycle genes, no• and bt2camp consistently had the same directional effect on transcripts, although no• regulated more genes overall. thus, decreases in intracellular camp appear unrelated to no• effects on the cell cycle. furthermore, treatment with both no• and bt2camp synergistically provoked cell cycle arrest in g2/m, suggesting that no•-induced decreases in camp may attenuate some of its effects on the cell cycle. although this experiment also provides useful intormation on gene regulation by camp in u cells, the reader should keep in mind that bt2camp was the only analog studied and some effects may have been caused by its butyryl component. u cells were pma-differentiated in the current experiments to render them capable of producing tnfα and il- two cytokines previously identified as no•-responsive. further, this treatment also reduced cell proliferation and forced >80% of the cells into the g0/g phase of the cell cycle, allowing for a more coherent analysis of cell cycle regulation. however, pma itself had large effects on no•-regulated genes such as p and the findings here cannot be extrapolated directly to naïve u cells. fortunately, turpev and colleagues have recently reported selected microarray results from no• exposure of undifferentiated u and mono mac cells. of interest, a number of key genes identified by this group were also found to be similarly regulated by no• in pma-differentiated cells including hmox il- activating transcription factor, bcl2/adenovirus e1b kd-interacting protein, and importantly p21/waf1/cip the no• donor gsno was found to activate p mapk in u cells, which was consistent with our previous result using snap, another no• donor. furthermore, three different no• donors were shown here to consistently increase p protein expression, indicating that this effect is no•-specific and donor independent. importantly, very low concentrations of the p mapk inhibitor sb were found to block the induction of p protein by no•, further establishing the role of p mapk as an intermediary signal transduction event. finally, p mrna was measured serially by rt-pcr after transcriptional blockade in the absence or presence of sb showing that this transcript is stabilized by no• through a p mapk-dependent mechanism. others have found that no• increases p mrna and protein expression in human vascular smooth muscle independent of cgmp. in addition, p mapk activation has been shown to increase p expression by both transcriptional activation and protein stabilization. e2f is also known to induce p transcription providing another mechanism by which no• may have increased p expression in the current experiment. conversely, as already discussed, no• decreases camp, reducing the ability of sp to bind to gc box elements and thereby repressing the transcription of sp1-dependent genes such as enos. interestingly, p is highly dependent on sp for transcription and is induced by camp, findings consistent with the possibility that p may be transcriptionally repressed by no•-camp-sp signal transduction. nonetheless, no• induction of p demonstrates that other mechanisms dominate over any negative effects of no• on sp binding to the p promoter. here, we focused on mrna stabilization, because of the strong indication in our microarray data that this is a major mechanism of gene regulation by no•. however, other mechanisms such as changes in transcription, translation, or protein stability may have contributed substantially to the net effects of no• on p expression. the e2f family of transcription factors play important roles in g1/s phase transition. e2f up-regulates many g1/s phase genes including itself, cyclin e, cdc uracil-dna glycosylase, jun, p and c-myb. except for c-myb, all were up-regulated by no• in the present study, suggesting that no• may drive differentiated u cells through the g1/s transition by inducing e2f expression. this conclusion is further supported by emsa showing that no• increases e2f binding to e2f consensus sequence. however, increased e2f expression may not be the only mechanism contributing to these observed changes in dna binding. no• activates p mapk in u cells and p mapk has been shown to increase e2f binding to e2f sites. further, cyclin a was down-regulated by no• and has been shown to turn off e2f target genes by decreasing e2f dna binding. notably, c-myb and a number of g or g2/m phase genes that contain e2f sites were down-regulated by no•. e2f sites can function as repressors in some genes and their disruption by mutation leads to promoter activation. further, no•-responsive genes with both e2f elements and cde/chr repressor sites were uniformly down-regulated. promoters with cde/chr motifs are repressed by p, which was also induced by no•. therefore, even for promoters activated by e2f repression through cde/chr sites appears to be the dominant action of no• in this cellular context. moreover, e2f and chr sites may cooperate as co-repressors. although cdc25b lacks an identifiable cde/chr site, it does have a proximal repressor and its regulation is similar to cde/chr-containing genes. cell cycle arrest induced by p occurs in late g1/s or early g2/m and is mediated, at least in part, by the repression of target genes with cde/chr sites. cde/chr sites are present in the promoters of cyclin a, cdc, cyclin b, and top2a, centromere protein a and plk. all of these genes are g or g2/m related and are down-regulated by p results consistent with the effects of no• observed here. emsa confirmed the hypothesis that no• regulates protein binding to cde/chr sites. collectively, these findings suggest that no• regulates many g2/m phase cell cycle genes through p however, no• may also regulate some of these downstream p targets through additional mechanisms. for example, c-myb, cyclin a cyclin b and cenpa have are in their 3' utr, indicating that no• might alter the stability of these transcripts. notably, cyclin a and cyclin b are down-regulated by p mapk, a signal transduction pathway that was activated by no• in the current experiments. importantly, p mapk has been shown to induce cell cycle arrest at the g checkpoint through mechanisms that were only partially dependent on p. are in 3' utr have been implicated in the control of transcript stability and have an important post-transcriptional impact on transcriptome content. we previously demonstrated that independent of cgmp, no• up-regulates il- but not tnfα post-transcriptionally through p mapk activation. in the current investigation, are- containing genes including il- and p were over-represented among no•-regulated genes. nearly half of these are genes have been reported to be regulated by p mapk. notably, no• responses were all in the same direction as those reported for p mapk activation. previous microarray experiments that globally tested mrna stability found that 10% of transcripts were associated with p mapk-dependent regulation. the over-representation of p mapk-regulated genes in our experiments indicates that this stress kinase is an important target of no•.  the present investigation was focused on understanding cgmp-independent gene regulation by no•. major themes within the identified gene list were the predominance of cell cycle-related genes and are-containing transcripts. no• was found to trigger a specific and coordinated cell cycle arrest independent of both cgmp and camp. e2f induction up-regulated target genes involved in g1/s transition through e2f sites. no• stabilization of p mrna was p mapk dependent and led to increased protein binding to cde/chr promoter sites and the down-regulation of g2/m phase genes. the cell cycle is a major target of no•-mediated gene regulation. importantly, p mapk and mrna stability are major intermediary mechanisms through which no• affects the human transcriptome. 
no• coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cgmp. differentiated u cells were exposed to s-nitrosoglutathione, a no• donor, or glutathione alone for h without or with dibutyryl-camp, and then harvested to extract total rna for microarray analysis. no• activated p mapk, stabilizing p mrna and increasing p protein; this increased protein binding to cde/chr promoter sites of p target genes, repressing key g2/m phase genes, and increasing the proportion of cells in g2/m. stress kinase signaling and alterations in mrna stability appear to be major pathways by which no• regulates the transcriptome. no• regulated transcripts that annotated disproportionately to the cell cycle and cell proliferation and more frequently than expected contained au-rich, post-transcriptional regulatory elements. cell cycle genes induced by no• annotated to g1/s and included e2f and p21/waf1/cip1; of these were e2f target genes involved in g1/s transition. here, cgmp-independent effects of no• on gene expression were globally examined in u cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. 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. bt2camp regulated genes; cell cycle gene enrichment did not reach significance. regulatory functions of nitric oxide that bypass the second messenger cgmp are incompletely understood. in humans, antiproliferative effects of no• may rely substantially on cgmp-independent mechanisms.
 regulatory functions of nitric oxide that bypass the second messenger cgmp are incompletely understood. here, cgmp-independent effects of no• on gene expression were globally examined in u cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. differentiated u cells were exposed to s-nitrosoglutathione, a no• donor, or glutathione alone for h without or with dibutyryl-camp, and then harvested to extract total rna for microarray analysis. bt2camp was used to block signaling attributable to no•-induced decreases in camp.  no• regulated transcripts that annotated disproportionately to the cell cycle and cell proliferation and more frequently than expected contained au-rich, post-transcriptional regulatory elements. bt2camp regulated 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 and included e2f and p21/waf1/cip1; of these were e2f target genes involved in g1/s transition. repressed genes were g2/m associated; of were known targets of p e2f mrna and protein were increased by no•, as was e2f binding to e2f promoter elements. no• activated p mapk, stabilizing p mrna and increasing p protein; this increased protein binding to cde/chr promoter sites of p target genes, repressing key g2/m phase genes, and increasing the proportion of cells in g2/m.  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. 