cCMP and cUMP in Apoptosis: Concepts and Methods.

The cyclic nucleotides cAMP and cGMP are well-characterized second messenger molecules regulating many important intracellular processes, such as differentiation, proliferation, and apoptosis. The latter is a highly regulated process of programmed cell death wherein several regulatory proteins, like those belonging to the Bcl-2 family, are involved. The initiation of apoptosis is regulated by three different pathways: the intrinsic or mitochondrial, the extrinsic, and the ER stress pathway. Recently, it has been published that the pyrimidine cyclic nucleotides cCMP and cUMP also function as second messenger molecules, and additionally have an effect on apoptosis signaling pathways. cCMP induced PKA-independent apoptosis via the intrinsic and ER-stress pathway in S49 mouse lymphoma cells, and cCMP as well as cUMP induced apoptosis in human HEL cells via the intrinsic pathway. However, in human K-562 cells, which are known to be multidrug-resistant, cCMP and cUMP had no effect. Summarized in this chapter are the initiation of apoptosis by cCMP and cUMP regarding the various apoptotic pathways, the enzymes involved in apoptosis, as well as the most relevant methods for the detection and examination of apoptosis and the corresponding signaling pathways.

Regulation of apoptosis by cyclic nucleotides in human erythroleukemia (HEL) cells and human myelogenous leukemia (K-562) cells.

The cyclic pyrimidine nucleotides cCMP and cUMP have been recently identified in numerous mammalian cell lines, in primary cells and in intact organs, but very little is still known about their biological function. A recent study of our group revealed that the membrane-permeable cCMP analog cCMP-acetoxymethylester (cCMP-AM) induces apoptosis in mouse lymphoma cells independent of protein kinase A via an intrinsic and mitochondria-dependent pathway. In our present study, we examined the effects of various cNMP-AMs in human tumor cell lines. In HEL cells, a human erythroleukemia cell line, cCMP-AM effectively reduced the number of viable cells, effectively induced apoptosis by altering the mitochondrial membrane potential and thereby caused changes in the cell cycle. cCMP itself was biologically inactive, indicating that membrane penetration is required to trigger intracellular effects. cCMP-AM did not induce apoptosis in K-562 cells, a human chronic myelogenous leukemia cell line, due to rapid export via multidrug resistance-associated proteins. The biological effects of cCMP-AM differed from those of other cNMP-AMs. In conclusion, cCMP effectively induces apoptosis in HEL cells, cCMP export prevents apoptosis of K-562 cells and cNMPs differentially regulate various aspects of apoptosis, cell growth and mitochondrial function. In a broader perspective, our data support the concept of distinct second messenger roles of cAMP, cGMP, cCMP and cUMP.

cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines.

cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49kin(-)cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channels 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP-AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspase-dependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP-AM in both cell lines. cAMP-AM, cGMP-AM, cUMP-AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP-AM altered gene expression of the apoptotic-relevant gene Gadd45α and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins.

The heteromeric transcription factor GABP activates the ITGAM/CD11b promoter and induces myeloid differentiation.

The heteromeric transcription factor GA-binding protein (GABP) consists of two subunits, the alpha subunit (GABPA) carrying the DNA-binding ETS domain, and the beta subunit (GABPB1) harbouring the transcriptional activation domain. GABP is involved in haematopoietic stem cell maintenance and differentiation of myeloid and lymphoid lineages in mice. To elucidate the molecular function of GABP in human haematopoiesis, the present study addressed effects of ectopic overexpression of GABP focussing on the myeloid compartment. Combined overexpression of GABPA and GABPB1 caused a proliferation block in cell lines and drastically reduced the colony-forming capacity of murine lineage-negative cells. Impaired proliferation resulted from perturbed cellular cycling and induction of myeloid differentiation shown by surface markers and myelomonocytic morphology of U937 cells. Depending on the dosage and functional integrity of GABP, ITGAM expression was induced. ITGAM encodes CD11b, the alpha subunit of integrin Mac-1, whose beta subunit, ITGB2/CD18, was already described to be regulated by GABP. Finally, Shield1-dependent proteotuning, luciferase reporter assays and chromatin immunoprecipitation showed that GABP activates the ITGAM/CD11b promoter via three binding sites close to the translational start site. In conclusion, the present study supports the crucial role of GABP in myeloid cell differentiation and identified ITGAM/CD11b as a novel GABP target gene.

Flow cytometric analysis with a fluorescently labeled formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2 receptor.

The histamine H2 receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models, or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMP-dependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca(2+)]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression, and potencies and efficacies of fMLP-induced increases in [Ca(2+)]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive, and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression.

cCMP and cUMP occur in vivo.

Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.

N4-monobutyryl-cCMP activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo.

There is increasing evidence for a role of cytidine 3',5'-cyclic monophosphate (cCMP) as second messenger. In a recent study, we showed that cCMP activates both purified guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase Iα (PKG Iα) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) isoenzymes with the regulatory subunits RIα and RIIα. Moreover, the membrane-permeant cCMP analog dibutyryl (DB)-cCMP induces effective vasodilation and inhibition of platelet aggregation via PKG Iα, but not via PKA. These data prompted us to conduct a systematic analysis of the effects of cyclic nucleotide (cNMP) analogs on purified PKG Iα and PKA RIα and RIIα We also studied the effect of DB-cCMP on PKA-dependent phosphorylation of the transcription factor cAMP response-binding protein (CREB) in S49 wild-type lymphoma cells and S49 kin(-) cells, devoid of the catalytic subunit of PKA. The major cellular metabolite of the prodrug DB-cCMP, N(4)-monobutyryl (4-MB)-cCMP, was a partial and low-potency activator of purified PKG Iα and a full and moderate-potency activator of PKA RIα and RIIα. Sp-cCMPS and Sp-cAMPS activated PKA RIα and RIIα with much higher potency and efficacy than PKG Iα. Molecular modeling suggested that the cytidine ring interacts with PKG Iα mainly via hydrophobic interactions, while the butyryl group projects away from the kinase. In contrast to DB-cAMP, DB-cCMP did not induce PKA-dependent phosphorylation in intact cells. Taken together, our data show that N(4)-monobutyryl-cCMP (4-MB-cCMP) activates PKA RIα and PKA RIIα more potently and with higher efficacy than PKG Iα in vitro but not in vivo. cNMP phosphorothioates constitute a starting point for the development of PKA activators with high selectivity relative to PKG.

cNMP-AMs mimic and dissect bacterial nucleotidyl cyclase toxin effects.

In addition to the well-known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. The Pseudomonas aeruginosa toxin ExoY massively increases cGMP and cUMP in cells, whereas the Bordetella pertussis toxin CyaA increases cAMP and, to a lesser extent, cCMP. To mimic and dissect toxin effects, we synthesized cNMP-acetoxymethylesters as prodrugs. cNMP-AMs rapidly and effectively released the corresponding cNMP in cells. The combination of cGMP-AM plus cUMP-AM mimicked cytotoxicity of ExoY. cUMP-AM and cGMP-AM differentially activated gene expression. Certain cCMP and cUMP effects were independent of the known cNMP effectors protein kinases A and G and guanine nucleotide exchange factor Epac. In conclusion, cNMP-AMs are useful tools to mimic and dissect bacterial nucleotidyl cyclase toxin effects.

cAMP, cGMP, cCMP and cUMP concentrations across the tree of life: High cCMP and cUMP levels in astrocytes.

Adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) are well-established second messengers, whereas the physiological role of the cyclic pyrimidine nucleotides cytidine 3',5'-cyclic monophosphate (cCMP) and uridine 3',5'-cyclic monophosphate (cUMP) is poorly understood. Certain mammalian nucleotidyl cyclases (NCs) and bacterial NC toxins can generate cCMP and cUMP. Human HEK293 cells and rat B103 neuroblastoma cells are of neuronal origin and possess high basal concentrations of cCMP and cUMP that can be attributed to soluble adenylyl cyclase activity. These data prompted us to conduct a systematic analysis of basal nucleoside 3',5'-cyclic monophosphate (cNMP) concentrations across the tree of life. cCMP and cUMP were identified in many mammalian cell lines and primary cells. cNMP patterns varied broadly among cells, and in several systems, cCMP and cUMP concentrations were quite high. Prokaryotes, fungi, amoeba and invertebrates lacked cCMP and cUMP, whereas cAMP was found across the tree of life. High cCMP and cUMP concentrations were found in astrocytes. The distinct cNMP patterns support specific second messenger roles of cCMP and cUMP, specifically in astrocytes.

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation.

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

Soluble adenylyl cyclase accounts for high basal cCMP and cUMP concentrations in HEK293 and B103 cells.

Intact HEK293 cells and B103 neuroblastoma cells possess high basal concentrations of the established second messengers cAMP and cGMP and of the emerging second messengers cCMP and cUMP. We asked the question which nucleotidyl cyclase accounts for the high basal cNMP concentrations. Activators and inhibitors of soluble guanylyl cyclase had no major effects on cNMPs, and the activator of membranous adenylyl cyclase forskolin increased only cAMP. Addition of bicarbonate to medium increased, whereas removal of bicarbonate decreased levels of all four cNMPs. The inhibitor of soluble adenylyl cyclase, 2-(1H-benzo[d]imidazol-2-ylthio)-N'-(5-bromo-2-hydroxybenzylidene) propanehydrazide (KH7), reduced bicarbonate-stimulated cNMPs. In conclusion, bicarbonate-stimulated soluble adenylyl cyclase plays an important role in the regulation of basal cellular cNMP levels, most notably cCMP and cUMP.

Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose.

The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)ß(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides.

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC(50) of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.

Spatial and temporal profiles of growth factor expression during CNS demyelination reveal the dynamics of repair priming.

Demyelination is the cause of disability in various neurological disorders. It is therefore crucial to understand the molecular regulation of oligodendrocytes, the myelin forming cells in the CNS. Growth factors are known to be essential for the development and maintenance of oligodendrocytes and are involved in the regulation of glial responses in various pathological conditions. We employed the well established murine cuprizone model of toxic demyelination to analyze the expression of 13 growth factors in the CNS during de- and remyelination. The temporal mRNA expression profile during demyelination and the subsequent remyelination were analyzed separately in the corpus callosum and cerebral cortex using laser microdissection and real-time PCR techniques. During demyelination a similar pattern of growth factor mRNA expression was observed in both areas with a strong up-regulation of NRG1 and GDNF and a slight increase of CNTF in the first week of cuprizone treatment. HGF, FGF-2, LIF, IGF-I, and TGF-ß1 were up-regulated mainly during peak demyelination. In contrast, during remyelination there were regional differences in growth factor mRNA expression levels. GDNF, CNTF, HGF, FGF-2, and BDNF were elevated in the corpus callosum but not in the cortex, suggesting tissue differences in the molecular regulation of remyelination in the white and grey matter. To clarify the cellular source we isolated microglia from the cuprizone lesions. GDNF, IGF-1, and FGF mRNA were detected in the microglial fraction with a temporal pattern corresponding to that from whole tissue PCR. In addition, immunohistochemical analysis revealed IGF-1 protein expression also in the reactive astrocytes. CNTF was located in astrocytes. This study identified seven different temporal expression patterns for growth factors in white and grey matter and demonstrated the importance of early tissue priming and exact orchestration of different steps during callosal and cortical de- and remyelination.

c-Jun controls histone modifications, NF-kappaB recruitment, and RNA polymerase II function to activate the ccl2 gene.

Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5' region close to the transcriptional start site and in the far 3' region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-kappaB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-kappaB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-kappaB and HDAC3 at the level of chromatin.

Transcriptional regulation of EGR-1 by the interleukin-1-JNK-MKK7-c-Jun pathway.

The proinflammatory cytokine interleukin (IL)-1 activates several hundred genes within the same cell. This occurs in part by activation of the MKK7-JNK-c-Jun signaling pathway whose precise role in the regulation of individual inflammatory genes is still incompletely understood. To identify the genes that are under specific control of activated JNK, we used a JNK-MKK7 fusion protein. Genome-wide microarray analysis revealed EGR-1 as the transcript that was most strongly induced by JNK-MKK7. IL-1-stimulated EGR-1 mRNA and protein expression were impaired in cells lacking JNK or c-Jun. Transcriptional activation of the EGR-1 promoter by JNK-MKK7 or by IL-1 required a single upstream AP-1 site and three distal serum-response elements (SRE). Reconstitution experiments in c-Jun-deficient cells revealed that c-Jun is required for EGR-1 transcription through both the AP-1 site and the distal SREs. By chromatin immunoprecipitation analysis, we found IL-1-inducible recruitment of c-Jun to the AP-1 site and to the region containing the three distal SREs. These experiments suggest that c-Jun plays a dual role in EGR-1 transcription. It directly binds to the AP-1 element, and at the same time it is essential for promoter activation through the three distal SREs by an indirect unknown mechanism. As predicted by TRANSFAC analysis and verified by ChIP experiments, IL-1-induced EGR-1 protein binds to the promoter regions of inflammatory mediators such as IL-6, IL-8, and CCL2. Furthermore, short interfering RNA-mediated suppression of EGR-1 partially suppresses IL-1-inducible transcription of IL-8, IL-6, and CCL2. In summary, we provide novel evidence for a complex c-Jun-mediated mechanism that is essential for inducible EGR-1 expression. We identify this pathway as a previously unrecognized part of a multistep gene regulatory network that controls cytokine and chemokine expression via the IL-1-MKK7-JNK-c-Jun-EGR-1 pathway.

Small interfering RNAs generated by recombinant dicer induce inflammatory gene expression independent from the TAK1-NFkappaB-MAPK signaling pathways.

Generation of mixtures of small interfering (si) RNAs by recombinant dicer avoids selection of efficient target sites within mRNAs but little is known about off-target effects of this approach. Using recombinant human dicer we generated siRNA mixtures (dsiRNA) directed against the protein kinase TAK1 and its subunit TAB1, important upstream molecules in the pathways activated by IL-1, TNF, and toll-like receptors (TLR). dsiRNA against TAK1 or TAB1 significantly suppressed their target proteins as well as TAK1-mediated activation of NFkappaB, p38 MAPK, and JNK, and of IL-8 transcription. However, microarray analysis of 136 endogenous inflammatory genes revealed that dsiRNA against TAB1 or TAK1 did not suppress IL-1 or TNF-induced genes but rather induced a broader range of 15 inflammatory genes as well as seven known interferon-response genes. The same genes were induced by dsiRNA directed against luciferase but not by a synthetic control siRNA molecule. Hence, our results show that complex mixtures of siRNA induce an inflammatory gene response that is independent from TAK1-mediated signal transduction. In the light of the increasing usage of enzymatically prepared libraries of siRNA these results provide important insight into potential off-target effects of this approach.

Simultaneous blockade of NFkappaB, JNK, and p38 MAPK by a kinase-inactive mutant of the protein kinase TAK1 sensitizes cells to apoptosis and affects a distinct spectrum of tumor necrosis factor [corrected] target genes.

The inflammatory response is characterized by the induction (or repression) of hundreds of genes. The activity of many of these genes is controlled by MAPKs and the IkappaB kinase-NFkappaB pathway. To reveal the effects of blocking these pathways simultaneously, fibroblasts were infected with retroviruses encoding TAK1K63W, an inactive mutant of the protein kinase TAK1. Expression of this protein inhibited tumor necrosis factor (TNF)-induced activation of NFkappaB, JNK, and p38 MAPK and sensitized the cells to TNF-induced apoptosis. 23 different microarray experiments were used to analyze the expression of >7000 genes in these cells. We identified 518 genes that were regulated by TNF in both TAK1K63W-expressing cells and control cells, 37 genes induced by TNF only when TAK1K63W was present, and 48 TNF-induced genes that were suppressed by TAK1K63W. The TNF-inducible genes that were most strongly suppressed by TAK1K63W, ccl2, ccl7, ccl5, cxcl1, cxcl5, cxcl10, saa3, and slpi also had much lower basal levels of expression, indicating that TAK1 also played a role in their normal expression. Chromatin immunoprecipitation studies on four of these genes suggested that inactivation of TAK1 activity led to direct suppression of expression at the transcriptional level because of impaired recruitment of RNA polymerase II to their promoters. ccl2 induction by TNF or interleukin-1 was also suppressed in cells that expressed TAK1 antisense RNA or that were genetically deficient in JNK1/2 or p65 NFkappaB. These data suggest that regulation of the expression of a selected group of inflammation-related genes is funneled through TAK1, making it a potentially useful target for more specific anti-inflammatory drug development.

Inducible expression of a constitutively active mutant of mitogen-activated protein kinase kinase 7 specifically activates c-JUN NH2-terminal protein kinase, alters expression of at least nine genes, and inhibits cell proliferation.

MKK7 is a recently discovered mitogen-activated protein kinase (MAPK) kinase that is unique in that it specifically activates only the c-JUN NH(2)-terminal protein kinase (JNK) family of enzymes. Very little is known about the biological role of MKK7. We generated inducible cell lines from the human embryonal kidney carcinoma cell line, HEK293, by stable transfection with a constitutively active mutant of MKK7, MKK7(3E), fused to green fluorescent protein (GFP), under the control of an ecdysone-inducible promoter. Treatment of cells with the synthetic ecdysone analog ponasterone A induced expression of GFP-MKK7(3E) and resulted in sustained activation of endogenous JNK, but neither of the other endogenous MAPKs, ERK or p38. Red and green fluorescing cDNA copies of mRNA extracted from cells obtained before and after induction of GFP-MKK7(3E) were hybridized to microarrays containing more than 6,000 cDNAs in eight independent experiments. By selection criteria, 23 genes were differentially regulated after 24 h of induction of GFP-MKK7(3E) and 16 after 48 h. The expression of 9 genes was consistently changed after both 24 and 48 h of induction. These changes included down-regulation of three genes, c-myc, angiopoietin-2, and glucose-regulated protein 58, and up-regulation of 6 genes, tissue factor pathway inhibitor-2, GRP78, autotaxin, PPP1R7, the DKFZ cDNA p434D0818, and 1 unknown gene. Consistent with previously described roles of several of the altered genes, MKK7(3E) inhibited cell proliferation. These data implicate active MKK7 in the negative regulation of cell proliferation and provide evidence for a new role for this kinase in the regulation of a distinct, hitherto unrecognized set of genes.