Nitric Oxide Regulation of Gene Transcription via Soluble Guanylate Cyclase and Type I cGMP-dependent Protein Kinase*

Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2′-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Iβ expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fospromoter by a guanylate cyclase- and G-kinase-dependent mechanism.

Nitric oxide (NO) 1 is a pluripotential molecule involved in regulating blood pressure, neurotransmission, and immune function (1). One of its major intracellular targets is the heme group of soluble guanylate cyclase with NO markedly stimulating enzymatic activity and thereby increasing the intracellular cGMP concentration (2,3). Other NO targets are thiolcontaining proteins, iron sulfur proteins and non-heme iron; in addition, NO can react with oxygen to produce peroxynitrite and hydroxyl radical, both of which can have physiological effects (4).
NO regulates the expression of multiple genes including c-fos, junB, heme oxygenase, smooth muscle ␣-actin, vascular endothelial growth factor, vascular cell adhesion molecule-1 (VCAM-1), and mitogen-activated kinase phosphatase-1 (5)(6)(7)(8)(9)(10). In regulation of VCAM-1, NO appears to act independently of cGMP because it's effect is not mimicked by cGMP analogs, while in regulation of c-fos and junB, NO and cGMP analogs induce similar changes (5,6,10). In cases where NO and cGMP analogs function similarly, it is likely that NO works through activation of soluble guanylate cyclase, but this has not been shown definitively. Moreover, it is not clear which of several cGMP target proteins, e.g. cGMP-dependent protein kinases (G-kinase), cGMP-gated ion channels, cGMP-activated phosphodiesterases, or cGMP-inhibited phosphodiesterases, mediates the effects of NO on gene expression (11).
In this study we used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and G-kinase activity, to determine whether NO regulates the fos promoter via a cGMP/G-kinase signal transduction pathway. We found that maximal stimulation of the fos promoter by NO in BHK cells required expression of both guanylate cyclase and Gkinase. In cells transfected with guanylate cyclase alone, NO caused a small activation of the fos promoter, apparently by cGMP activation of cAMP-dependent protein kinase (protein kinase A), either directly by cross-activation or indirectly by inhibition of phosphodiesterase activity (12). In cells containing endogenous guanylate cyclase and G-kinase, NO stimulation of the fos promoter was inhibited by the guanylate cyclase inhibitor ODQ. This, to our knowledge, is the first demonstration that NO regulates gene expression via cGMP activation of G-kinase.

EXPERIMENTAL PROCEDURES
Cell Culture and Transient Transfection Experiments-BHK cells were cultured and transfected as described previously (13). All cells received 50 ng of pFos-CAT, which contains the chloramphenicol acetyltransferase (CAT) gene under control of the human fos promoter, and 50 ng of the luciferase expression vector pRSV luciferase to serve as an internal control for transfection efficiency (5,13). As indicated, cells also received 300 ng of a human G-kinase I␤ expression vector (14) and/or 100 ng each of expression vectors encoding the ␣ 1 and ␤ 1 subunits of rat soluble guanylate cyclase (15); variable amounts of the empty vector pCB6 were used to keep the total amount of DNA constant. Where indicated, N G -monoethyl-L-arginine monoacetate (NMEA, Alexis Corp., San Diego, CA) was added 16 h prior to cell harvest and 8-Br-cGMP (Biolog Life Sciences, Bremen, Federal Republic of Germany), Deta-NONOate (ethanamine-2,2Ј-(hydroxynitrosohydrazone)bis-, Cayman Chemical, Ann Arbor, MI), and Deta, the parent compound of Deta-NONOate that does not release NO, were added 8 h prior to cell harvest.
CS-54 cells are rat pulmonary arterial smooth muscle cells that maintain differentiated properties through multiple subcultures (16). They were obtained from A. Rothman, University of California, San Diego, and were transfected with pFOS-CAT and pRSV luciferase but not with guanylate cyclase or G-kinase expression vectors. Where indicated, ODQ (Calbiochem, La Jolla, CA) and/or Deta-NONOate were added 16 h prior to cell harvest.
Measurement of Guanylate Cyclase, G-kinase, and Protein Kinase A Activity-Guanylate cyclase activity was measured as described previously in the absence and presence of the NO donor S-nitrosylpenicillamine (SNAP) using [␣-32 P]GTP as substrate and separating substrate and product by thin layer chromatography (17,18). The activities of G-kinase, protein kinase A holoenzyme, and the free catalytic subunit of protein kinase A were measured as described previously following [␥-32 P]ATP phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (13,19). G-kinase activity was measured in the presence of 0.01 mg/ml PKI (protein kinase inhibitor from S. Taylor, University of California, San Diego) and 10 M 8-Br-cGMP; protein kinase A holoenzyme activity was measured in the presence of 1 M cAMP. The activity of the free catalytic subunit of protein kinase A was measured as PKI-inhibitable phosphorylation in the absence of cAMP. All enzyme assays were linear with time and protein concentration.
Reporter Gene Assays-CAT activity and luciferase activity were measured as described previously using [ 14 C]chloramphenicol/butyrylcoenzyme A and luciferin/ATP, respectively (5,13). The data are expressed as relative CAT activity, i.e. CAT activity/luciferase activity.
Measurement of cGMP and Nitrite/Nitrate Concentrations-The intracellular cGMP concentration was measured as described previously by radioimmunoassay (5). After culturing cells for 24 h in low serum medium, nitrite and nitrate concentrations in the medium were measured according to the Griess reaction (20).
Data Analysis-Comparison of data groups was done by the twotailed Student t test, with a p value of Ͻ0.05 considered significant.

Guanylate Cyclase and G-kinase Activity in Transfected BHK
Cells-We found very low soluble guanylate cyclase activity in extracts of BHK cells transfected with empty vector with about a 6-fold increase in enzyme activity on treating the extracts with 50 M SNAP (Table I; difference between minus and plus SNAP was significant at p Ͻ 0.05). When the cells were transfected with guanylate cyclase expression vectors, we found a large increase in enzyme activity, which again was stimulated Ͼ6-fold by SNAP (Table I); these latter experiments demonstrate that the transfected enzyme contained a functional heme moiety. G-kinase activity was also very low in extracts of BHK cells transfected with empty vector and enzyme activity increased substantially when the cells were transfected with a G-kinase expression vector (Table I). The guanylate cyclase and G-kinase activities found in the transfected cells are comparable with amounts of these enzymes expressed endogenously in several cell types, e.g. smooth muscle cells, neuronal cells, and platelets (3,21).

Intracellular cGMP in Guanylate
Cyclase-and G-kinasetransfected BHK Cells-In BHK cells transfected with empty vector, intracellular cGMP was low but measurable, consistent with the cells' low guanylate cyclase activity, and Deta-NONOate caused a small increase in cGMP, compatible with the low endogenous guanylate cyclase being stimulated by NO (Table  II). In cells transfected with guanylate cyclase, intracellular cGMP increased about 8-fold and Deta-NONOate increased cGMP ϳ250-fold (Table II; throughout the text, -fold increase is relative to cells transfected with empty vector receiving no drug treatment).
In cells transfected with both guanylate cyclase and G-kinase expression vectors, intracellular cGMP increased ϳ60fold and Deta-NONOate increased cGMP Ͼ600-fold (Table II). The higher cGMP in cells co-transfected with guanylate cyclase and G-kinase compared with cells transfected with guanylate cyclase alone is probably because the cGMP assay measures total intracellular cGMP, including that which is proteinbound: the intracellular G-kinase concentration in cells transfected with this enzyme is 3-6 pmol/mg of protein corresponding to a cGMP binding capacity of 12-24 pmol/mg of protein (13). In addition, as shown below, BHK cells produce relatively high amounts of NO. Thus, high cGMP in the guanylate cyclase-and G-kinase-transfected cells in the absence of Deta-NONOate probably represents the combination of high endogenous NO stimulating the transfected guanylate cyclase with the cGMP produced binding to the transfected G-kinase. These results suggest that in cells co-transfected with guanylate cyclase and G-kinase, G-kinase is likely to be activated, even in the absence of exogenous NO.
Transactivation of the fos Promoter by NO in BHK Cells-We showed previously that NO-releasing agents activate synthetic TRE-containing promoters in REF52 fibroblasts and rat thyroid cells, and we and others (5,6,(22)(23)(24)(25)(26) have shown that NO increases c-fos mRNA in many cell types. We have also shown that cGMP regulates the fos promoter in BHK cells via Gkinase activation and nuclear translocation (13,27).
To test whether NO's effect on the fos promoter in BHK cells was mediated by cGMP activation of G-kinase, we transfected cells with pFos-CAT in the absence or presence of guanylate cyclase and G-kinase expression vectors. We found that the NO-generating agent Deta-NONOate had no effect on CAT expression in cells transfected with empty vector (Fig. 1). When the cells were transfected singly with guanylate cyclase or G-kinase expression vectors, CAT activity increased a minimal 1.3-fold in the absence of drug treatment which was not statistically significant (Fig. 1). Deta-NONOate increased CAT activity a small, but statistically significant, 2.1-fold in the guanylate cyclase-transfected cells but had no effect in the G-kinase-transfected cells (Fig. 1). The small increase in CAT activity by Deta-NONOate in guanylate cyclase-expressing  cells points to a cGMP-mediated mechanism, and as described above, we found that under these conditions Deta-NONOate increased the intracellular cGMP concentration by ϳ250-fold (Table II). This large increase in cGMP could activate the cells low endogenous G-kinase or, as shown later, cross-activate protein kinase A. Deta-NONOate increased CAT activity more than 8-Br-cGMP in the guanylate cyclase-transfected cells (Fig.  1); as discussed later, this difference could be because endogenously produced cGMP is more available than exogenously provided 8-Br-cGMP. As we showed previously (13), 8-Br-cGMP stimulated CAT expression about 4-fold in cells transfected with the G-kinase expression vector and was without effect in cells transfected with empty vector (Fig. 1), indicating that at the concentration used, 8-Br-cGMP did not cross-activate protein kinase A (Fig. 1). When the cells were co-transfected with guanylate cyclase and G-kinase expression vectors, CAT expression increased 5.5-fold in the absence of drug treatment (Fig. 1). This substantial increase in CAT activity can be attributed to the large increase in intracellular cGMP under these conditions (Table  II) activating the transfected G-kinase (discussed above). Deta-NONOate further increased CAT activity to about 7-fold in cells co-transfected with guanylate cyclase and G-kinase expression vectors (Fig. 1) and, as shown, further increased intracellular cGMP (Table II). The increase in CAT activity by Deta-NONOate in the guanylate cyclase-and G-kinase-transfected cells was similar to that observed when these cells were treated with 8-Br-cGMP ( Fig. 1; the further increase in CAT activity by Deta-NONOate and 8-Br-cGMP was not statistically significant compared with the untreated cells). Deta, the parent compound of Deta-NONOate, which does not release NO, had no effect on CAT expression in guanylate cyclaseand/or G-kinase-expressing cells.
To examine whether high endogenous NO production in the guanylate cyclase-and G-kinase-expressing cells could be the cause of increased CAT expression in the absence of Deta-NONOate or 8-Br-cGMP, we treated cells with the competitive NO synthase inhibitor NMEA. We found that NMEA reduced CAT expression by Ͼ40% in cells transfected with guanylate cyclase and G-kinase expression vectors (Fig. 1). To eliminate possible effects of altered culture conditions, all cells were grown in medium containing 400 M arginine; this relatively high arginine concentration may have prevented more complete inhibition of CAT expression by NMEA and other workers have also found that NO synthase inhibitors are only partially effective at high micromolar arginine concentrations (28). Corresponding to the 40% reduction in CAT expression, NMEA reduced intracellular cGMP by ϳ50% in the guanylate cyclaseand G-kinase-transfected cells (Table II). When Deta-NONOate was added to the NMEA-treated cells, CAT expression returned to the levels observed in the absence of NMEA, providing evidence that NMEA was acting by inhibiting endogenous NO synthase (Fig. 1). Consistent with recovery of CAT activity, Deta-NONOate almost fully returned the intracellular cGMP concentration to the level observed in the absence of NMEA (Table II). NO Production by BHK Cells-NO produced under physiological conditions is converted stoichiometrically to nitrite and nitrate (29). We found the sum of nitrite and nitrate in the medium of serum-starved BHK cells to be Ͼ1 M after 24 h of culture corresponding to an NO production rate of 40 pmol/h/ 10 6 cells. This NO production rate is similar to what vascular endothelial cells and glioma cells, which have high NO synthase activity, produce under nonstimulated conditions (30,31).

Protein Kinase A Activation by Deta-NONOate in Guanylate
Cyclase-transfected BHK Cells-At high intracellular concentrations, cGMP can cross-activate protein kinase A and protein kinase A activation can lead to transactivation of the fos promoter (12,13). The small increase in CAT expression on treating guanylate cyclase-transfected cells with Deta-NONOate ( Fig. 1) could, therefore, be secondary to protein kinase A activation by high intracellular cGMP concentrations (Table  II). To address this question, we measured the activity of the free catalytic subunit of protein kinase A in Deta-NONOatetreated, guanylate cyclase-transfected cells and found a 2.7fold increase in enzyme activity (Table III). This increase in free catalytic subunit activity by Deta-NONOate was about half that observed in cells treated with 8-Br-cAMP (Table III; in control experiments we showed that free catalytic subunit release by 8-Br-cAMP was not secondary to drug carryover from the culture medium because adding 8-Br-cAMP immediately prior to cell washing had no effect on free catalytic subunit activity). In cells transfected with empty vector, Deta-NONOate had no effect, while 8-Br-cAMP yielded the same increase in enzyme activity as in cells transfected with guanylate cyclase expression vectors (Table III). Thus, in cells containing soluble guanylate cyclase, NO can increase the cGMP content sufficiently to cross-activate protein kinase A.
Transactivation of the fos Promoter by NO in CS-54 Cells-Having shown that NO activation of the fos promoter in BHK cells required expression of both soluble guanylate cyclase and G-kinase, we assessed NO's effect on the fos promoter in cells expressing both of these enzymes endogenously. We chose CS-54 cells, derived from the smooth muscle of rat pulmonary arteries (16), because primary vascular smooth muscle cells contain both soluble guanylate cyclase and G-kinase (11). We found that CS-54 cells contained 21 Ϯ 3.4 and 335 Ϯ 39 pmol/ min/mg of protein of guanylate cyclase and G-kinase activity, respectively (mean Ϯ S.D. of three independent experiments performed in duplicate; guanylate cyclase activity was simulated 7-fold by S-nitrosylpenicillamine). The activities of both of these enzymes are considerably more than found in untransfected BHK cells, and although less than in BHK cells transfected with guanylate cyclase or G-kinase expression vectors (Table I), they are within the range reported in primary smooth muscle cells and other mammalian cells (3,21).
In CS-54 cells transfected with pFos-CAT, the guanylate cyclase inhibitor ODQ reduced CAT activity by Ͼ60% compared with untreated cells (Fig. 2). This suggests that CAT expression in untreated cells was determined in large part by endogenously produced cGMP stimulating cellular G-kinase; ODQ was clearly acting via guanylate cyclase inhibition because the membrane-permeable cGMP analog 8-para-chlorophenylthio-cGMP reversed ODQ's inhibitory effect (data not shown). When the cells were treated with Deta-NONOate, CAT expression increased more than 2-fold compared with untreated cells and more than 5-fold compared with cells treated with ODQ (Fig. 2). Deta-NONOate added to ODQ-treated cells was without effect indicating that Deta-NONOate was acting via a cGMP-dependent mechanism (Fig. 2). DISCUSSION We have shown that NO activation of the fos promoter is dependent on both guanylate cyclase and G-kinase activity and have, therefore, defined a specific signal transduction pathway which mediates NO's effect on c-fos expression. We showed previously that G-kinase I␤, the G-kinase isoform used in the present studies, activates the fos promoter via translocation to the nucleus and that this effect of G-kinase is mediated by several sequence elements, including the serum response element, the AP-1 binding site, and the cAMP response element (13,27).
In the absence of drugs, fos promoter activity was not significantly affected when BHK cells were transfected singly with either guanylate cyclase or G-kinase expression vectors. However, we found a substantial increase in fos promoter activity when the cells were co-transfected with expression vectors for both enzymes, even in the absence of drug treatment. These data suggest that the transfected guanylate cyclase generated sufficient cGMP to activate the transfected G-kinase, and indeed, we found a 60-fold increase in the intracellular cGMP concentration compared with untransfected cells. For guanylate cyclase to produce such high amounts of cGMP, it seemed likely that the enzyme was stimulated by NO and we found high constitutive NO production by BHK cells. Moreover, NMEA, a NO synthase inhibitor, decreased CAT expression in the guanylate cyclase and G-kinase transfected cells significantly and adding Deta-NONOate to the NMEA-treated cells caused full recovery of CAT expression to maximal levels. Although CAT activity was high in the guanylate cyclase-and G-kinase-transfected cells in the absence of drugs, both Deta-NONOate and 8-Br-cGMP further increased CAT activity, suggesting that the amount of endogenously produced cGMP was not quite sufficient to maximally stimulate G-kinase. Together, these data provide evidence that NO activates the fos promoter through a guanylate cyclase-and G-kinase-dependent mechanism.
In BHK cells transfected with guanylate cyclase, Deta-NONOate increased the activity of the free catalytic subunit of protein kinase A, although less effectively than 8-Br-cAMP. In vascular smooth muscle cells, Cornwell et al. (32) also found that NO donors activate protein kinase A, although they did not compare the effect of the drugs to that of cAMP analogs. Protein kinase A activation by 8-Br-cAMP stimulates fos promoter activity about 6-fold in BHK cells (13). Thus, protein kinase A activation by Deta-NONOate in the guanylate cyclase-transfected cells likely caused the small increase in fos promoter activity under these conditions; the high amounts of cGMP produced by the cells could also have activated the low endogenous G-kinase present in BHK cells.
The increase in CAT activity in cells transfected with guanylate cyclase and G-kinase expression vectors was significantly more than the increase in CAT activity in cells transfected with the G-kinase expression vector only and treated with 8-Br-cGMP. This may be because 8-Br-cGMP, although more membrane-permeable than cGMP, still does not diffuse readily across cell membranes and intracellular concentrations  achieved by the drug are relatively low. 2,3 In addition, it may be that cGMP produced endogenously from guanylate cyclase is a more effective G-kinase activator than exogenous 8-Br-cGMP because of the intracellular localization of the cyclase and kinase. We found similar results in our previous work with REF52 and rat thyroid cells where NO-releasing agents were more potent activators of TRE-containing reporter genes than was 8-Br-cGMP (5). In the guanylate cyclase-and G-kinasetransfected cells there is also likely some degree of protein kinase A activation that we showed previously does not occur in G-kinase-transfected cells treated with 8-Br-cGMP (13). NO plays an important role in regulating pulmonary vascular resistance and growth of pulmonary arterial cells via activation of soluble guanylate cyclase and G-kinase (33). In CS-54 cells, CAT expression from the fos promoter was markedly reduced when the cells were treated with the guanylate cyclase inhibitor ODQ, suggesting that endogenously produced cGMP is sufficient to stimulate promoter activity in these cells and that CAT activity observed in the presence of ODQ may be the more correct "basal" level than that observed in untreated cells. Although we did not define the type of G-kinase expressed in CS-54 cells, vascular smooth muscle cells express predominantly G-kinase I␣, suggesting that the observed results are applicable to cells expressing either G-kinase I␣ or I␤ (34 -36).
The findings in these studies are likely of physiological significance for the following reasons. First, the amounts of guanylate cyclase and G-kinase in the transfected BHK cells are comparable with the amounts of these enzymes expressed endogenously in several cell types (3,21). Second, fos promoter activity was markedly stimulated by endogenously produced NO in BHK cells expressing both guanylate cyclase and Gkinase, and third, NO increased fos promoter activity in CS-54 cells. Thus, we conclude that one mechanism whereby NO regulates gene expression in vivo is via a guanylate cyclase/ cGMP/G-kinase transduction pathway.