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The strength and duration of mitogen-activated protein kinase signaling is regulated through phosphorylation and dephosphorylation by dedicated dual-specificity kinases and phosphatases, respectively. Here we investigated the physiological role that extracellular signal-regulated kinases 1/2 (ERK1/2) dephosphorylation plays in vivo through targeted disruption of the gene encoding dual-specificity phosphatase 6 (Dusp6) in the mouse. Dusp6(-/-) mice, which were viable, fertile, and otherwise overtly normal, showed an increase in basal ERK1/2 phosphorylation in the heart, spleen, kidney, brain, and fibroblasts, but no change in ERK5, p38, or c-Jun N-terminal kinases activation. However, loss of Dusp6 did not increase or prolong ERK1/2 activation after stimulation, suggesting that its function is more dedicated to basal ERK1/2 signaling tone. In-depth analysis of the physiological effect associated with increased baseline ERK1/2 signaling was performed in cultured mouse embryonic fibroblasts (MEFs) and the heart. Interestingly, mice lacking Dusp6 had larger hearts at every age examined, which was associated with greater rates of myocyte proliferation during embryonic development and in the early postnatal period, resulting in cardiac hypercellularity. This increase in myocyte content in the heart was protective against decompensation and hypertrophic cardiomyopathy following long term pressure overload and myocardial infarction injury in adult mice. Dusp6(-/-) MEFs also showed reduced apoptosis rates compared with wild-type MEFs. These results demonstrate that ERK1/2 signaling is physiologically restrained by DUSP6 in coordinating cellular development and survival characteristics, directly impacting disease-responsiveness in adulthood. Show less

The zebrafish adult brain contains numerous neural progenitors and is a good model to approach the general mechanisms of adult neural stem cell maintenance and neurogenesis. Here we use this model to test for a correlation between Fgf signaling and cell proliferation in adult progenitor zones. We report expression of Fgf signals (fgf3,4,8a,8b,17b), receptors (fgfr1-4), and targets (erm, pea3, dusp6, spry1,2,4, and P-ERK) and document that genes of the embryonic fgf8 synexpression group acquire strikingly divergent patterns in the adult brain. We further document the specific expression of fgf3, fgfr1-3, dusp6, and P-ERK in ventricular zones, which contain neural progenitors. In these locations, however, a comparison at the single-cell level of fgfr/P-ERK expression with bromo-deoxy-uridine (BrdU) incorporation and the proliferation marker MCM5 indicates that Fgf signaling is not specifically associated with proliferating progenitors. Rather, it correlates with the ventricular radial glia state, some of which only are progenitors. Together these results stress the importance of Fgf signaling in the adult brain and establish the basis to study its function in zebrafish, in particular in relation to adult neurogenesis. Show less

The mechanism(s) underlying neurodegeneration-associated activation of ERK1/2 remain poorly understood. We report that in cultured rat cortical neurons, whose basal ERK1/2 phosphorylation required NMDA receptors (NMDAR), the neurotoxic DNA intercalating drug cisplatin increased ERK1/2 phosphorylation via NMDAR despite reducing their activity. The rate of ERK1/2 dephosphorylation was lowered by cisplatin. Cisplatin-treated neurons showed general transcription inhibition likely accounting for the reduced expression of the ERK1/2-selective phosphatases including the dual specificity phosphatase-6 (DUSP6) and the DUSP3 activator vaccinia-related kinase-3 (VRK3). Hence, cisplatin effects on ERK1/2 may be due to the deficient ERK1/2 inhibition by the transcription-regulated phosphatases. Indeed, the transcription inhibitor actinomycin D reduced expression of DUSP6 and VRK3 while inducing the NMDAR-dependent activation of ERK1/2 and the impairment of ERK1/2 dephosphorylation. Thus, cisplatin-mediated transcriptional inhibition of ERK1/2 phosphatases contributed to delayed and long lasting accumulation of phospho-ERK1/2 that was driven by the basal NMDAR activity. Our results provide the first direct evidence for transcriptionally-regulated inactivation of neuronal ERK1/2. Its disruption likely contributes to neurodegeneration-associated activation of ERK1/2. Show less

The RAS-RAF-MEK-extracellular signal-regulated kinase (ERK) pathway plays a pivotal role in various cellular responses, including cellular growth, differentiation, survival and motility. Constitutive activation of the ERK pathway has been linked to the development and progression of human cancers. Here, we reported that mitogen-activated protein kinase phosphatase (MKP)-3, a negative regulator of ERK1/2, lost its expression particularly in the protein level, was significantly correlated with high ERK1/2 activity in primary human ovarian cancer cells using quantitative reverse transcription-polymerase chain reaction and western blot analyses. Intriguingly, the loss of MKP3 protein was associated with ubiquitination/proteosome degradation mediated by high intracellular reactive oxygen species (ROS) accumulation such as hydrogen peroxide in ovarian cancer cells. Functionally, short hairpin RNA knock down of endogenous MKP3 resulted in increased ERK1/2 activity, cell proliferation rate, anchorage-independent growth ability and resistance to cisplatin in ovarian cancer cells. Conversely, enforced expression of MKP3 in MKP3-deficient ovarian cancer cells significantly reduced ERK1/2 activity and inhibited cell proliferation, anchorage-independent growth ability and tumor development in nude mice. Furthermore, the enforced expression of MKP3 succeeded to sensitize ovarian cancer cells to cisplatin-induced apoptosis in vitro and in vivo. These results suggest a molecular mechanism by which the accumulation of ROS during ovarian cancer progression may cause the degradation of MKP3, which in turn leads to aberrant ERK1/2 activation and contributes to tumorigenicity and chemoresistance of human ovarian cancer cells. Show less

The antidepressant and cocaine sensitive plasma membrane monoamine transporters are the primary mechanism for clearance of their respective neurotransmitters and serve a pivotal role in limiting monoamine neurotransmission. To identify molecules in pathways that regulate dopamine transporter (DAT) internalization, we used a genetic complementation screen in Xenopus oocytes to identify a mitogen-activated protein (MAP) kinase phosphatase, MKP3/Pyst1/DUSP6, as a molecule that inhibits protein kinase C-induced (PKC) internalization of transporters, resulting in enhanced DAT activity. The involvement of MKP3 in DAT internalization was verified using both overexpression and shRNA knockdown strategies in mammalian cell models including a dopaminergic cell line. Although the isolation of MKP3 implies a role for MAP kinases in DAT internalization, MAP kinase inhibitors have no effect on internalization. Moreover, PKC-dependent down-regulation of DAT does not correlate with the phosphorylation state of several well-studied MAP kinases (ERK1/2, p38, and SAPK/JNK). We also show that MKP3 does not regulate PKC-induced ubiquitylation of DAT but acts at a more downstream step to stabilize DAT at the cell surface by blocking dynamin-dependent internalization and delaying the targeting of DAT for degradation. These results indicate that MKP3 can act to enhance DAT function and identifies MKP3 as a phosphatase involved in regulating dynamin-dependent endocytosis. Show less

PYST1/MKP3 is a negative feedback modulator of fibroblast growth factor 8 (Fgf8) signaling in the mammalian isthmic organizer of the brain, which affects the development of the midbrain and the hindbrain. The Dusp6 (Mkp3) gene is also highly expressed in the mouse embryonic forebrain. However, its role in forebrain development and function remains largely unknown. In this study, association analyses were preformed in silico between the variation I62M (rs13480726) of the Dusp6 (Mkp3) gene and the mouse forebrain weight/structure in 385 mice of 20 strains. It was found that I62M (allele A) was associated with reduced mouse forebrain structure in both sexes and lower brain weight in males. Show less

There are ten mitogen-activated protein kinase (MAPK) phosphatases (MKPs) that act as negative regulators of MAPK activity in mammalian cells and these can be subdivided into three groups. The first comprises DUSP1/MKP-1, DUSP2/PAC1, DUSP4/MKP-2 and DUSP5/hVH-3, which are inducible nuclear phosphatases. With the exception of DUSP5, these MKPs display a rather broad specificity for inactivation of the ERK, p38 and JNK MAP kinases. The second group contains three closely related ERK-specific and cytoplasmic MKPs encoded by DUSP6/MKP-3, DUSP7/MKP-X and DUSP9/MKP-4. The final group consists of three MKPs DUSP8/hVH-5, DUSP10/MKP-5 and DUSP16/MKP-7 all of which preferentially inactivate the stress-activated p38 and JNK MAP kinases. Abnormal MAPK signalling will have important consequences for processes critical to the development and progression of human cancer. In addition, MAPK signalling also plays a key role in determining the response of tumour cells to conventional cancer therapies. The emerging roles of the dual-specificity MKPs in the regulation of MAPK activities in normal tissues has highlighted the possible pathophysiological consequences of either loss (or gain) of function of these enzymes as part of the oncogenic process. This review summarises the current evidence implicating the dual-specificity MKPs in the initiation and development of cancer and also on the outcome of treatment. Show less

DUSP6 (dual-specificity phosphatase 6), also known as MKP-3 [MAPK (mitogen-activated protein kinase) phosphatase-3] specifically inactivates ERK1/2 (extracellular-signal-regulated kinase 1/2) in vitro and in vivo. DUSP6/MKP-3 is inducible by FGF (fibroblast growth factor) signalling and acts as a negative regulator of ERK activity in key and discrete signalling centres that direct outgrowth and patterning in early vertebrate embryos. However, the molecular mechanism by which FGFs induce DUSP6/MKP-3 expression and hence help to set ERK1/2 signalling levels is unknown. In the present study, we demonstrate, using pharmacological inhibitors and analysis of the murine DUSP6/MKP-3 gene promoter, that the ERK pathway is critical for FGF-induced DUSP6/MKP-3 transcription. Furthermore, we show that this response is mediated by a conserved binding site for the Ets (E twenty-six) family of transcriptional regulators and that the Ets2 protein, a known target of ERK signalling, binds to the endogenous DUSP6/MKP-3 promoter. Finally, the murine DUSP6/MKP-3 promoter coupled to EGFP (enhanced green fluorescent protein) recapitulates the specific pattern of endogenous DUSP6/MKP-3 mRNA expression in the chicken neural plate, where its activity depends on FGFR (FGF receptor) and MAPK signalling and an intact Ets-binding site. These findings identify a conserved Ets-factor-dependent mechanism by which ERK signalling activates DUSP6/MKP-3 transcription to deliver ERK1/2-specific negative-feedback control of FGF signalling. Show less

The formation of somites in the course of vertebrate segmentation is governed by an oscillator known as the segmentation clock, which is characterized by a period ranging from 30 min to a few hours depending on the organism. This oscillator permits the synchronized activation of segmentation genes in successive cohorts of cells in the presomitic mesoderm in response to a periodic signal emitted by the segmentation clock, thereby defining the future segments. Recent microarray experiments [Dequeant, M.L., Glynn, E., Gaudenz, K., Wahl, M., Chen, J., Mushegian, A., Pourquie, O., 2006. A complex oscillating network of signaling genes underlies the mouse segmentation clock. Science 314, 1595-1598] indicate that the Notch, Wnt and Fibroblast Growth Factor (FGF) signaling pathways are involved in the mechanism of the segmentation clock. By means of computational modeling, we investigate the conditions in which sustained oscillations occur in these three signaling pathways. First we show that negative feedback mediated by the Lunatic Fringe protein on intracellular Notch activation can give rise to periodic behavior in the Notch pathway. We then show that negative feedback exerted by Axin2 on the degradation of beta-catenin through formation of the Axin2 destruction complex can produce oscillations in the Wnt pathway. Likewise, negative feedback on FGF signaling mediated by the phosphatase product of the gene MKP3/Dusp6 can produce oscillatory gene expression in the FGF pathway. Coupling the Wnt, Notch and FGF oscillators through common intermediates can lead to synchronized oscillations in the three signaling pathways or to complex periodic behavior, depending on the relative periods of oscillations in the three pathways. The phase relationships between cycling genes in the three pathways depend on the nature of the coupling between the pathways and on their relative autonomous periods. The model provides a framework for analyzing the dynamics of the segmentation clock in terms of a network of oscillating modules involving the Wnt, Notch and FGF signaling pathways. Show less

Extracellular signal-regulated kinase-1 and -2 (ERK1/2) are activated by dual threonine and tyrosine phosphorylation of a TEY motif. The highly related kinase ERK5 is also activated by phosphorylation at a TEY motif. Inactivation of ERK1/2 is achieved by distinct members of the dual-specificity protein phosphatase (DUSP) family, which are responsible for the specific, regulated de-phosphorylation of the TEY motif. These include both nuclear (DUSP5) and cytoplasmic (DUSP6) enzymes. DUSP6, a candidate tumour suppressor gene, is thought to be highly specific for inactivation of ERK1/2 but several reports have suggested that it may also inactivate ERK5. Here we have compared the ability of DUSP6 to regulate the ERK1/2 and ERK5 protein kinases. We find that DUSP6 binds to ERK1/2 in both yeast and human cells but fails to bind to ERK5. Recombinant ERK2 can induce catalytic activation of DUSP6 whereas ERK5 cannot. Ectopic expression of DUSP6 can de-phosphorylate a co-expressed ERK2 construct but does not de-phosphorylate ERK5. Finally, expression of DUSP6 blocks the MEK1-driven activation of GAL4-ELK1, an ERK1/2-regulated transcription factor, but fails to block the MEK5-driven activation of GAL4-MEF2D, an ERK5-regulated transcription factor. These results demonstrate that even upon over-expression DUSP6 fails to inactivate ERK5, confirming that it is indeed an ERK1/2-specific DUSP. Show less

Previous studies showed that expression of the novel candidate tumor suppressor gene, DEC1 (Deleted in Esophageal Cancer 1), is reduced in esophageal carcinoma and suppresses cancer cell growth in vitro and tumor growth in vivo in nude mice. This study shows that DEC1 gene expression was downregulated in 100% of 16 esophageal squamous cell carcinoma (ESCC) cell lines and 52 and 45%, respectively, of esophageal tumor specimens from Hong Kong and a high-risk ESCC region of Henan, China. Using epitope tagging, the DEC1 protein was localized to both the cytoplasm and nucleus of the cell. In 3D Matrigel culture, no significant difference in colony numbers formed was observed for DEC1 stable transfectants, as compared to vector-alone transfectant controls. However, significantly smaller colony sizes were observed for the DEC1 transfectants. In in vitro cell migration, invasion and soft agar assays of DEC1 transfectants, only the soft agar assay showed statistically significant differences in colony numbers with the vector-alone controls, indicating that DEC1 may be involved in anchorage-independent cell growth. In addition, the global gene expression affected by DEC1 in tumor-suppressive stable transfectants was investigated using cDNA oligonucleotide microarray hybridization. Three candidate genes, TFPI-2, GDF15 and DUSP6, were identified through this approach; they are downregulated in tumor segregants of DEC1 stable transfectants, ESCC cell lines and esophageal tumors and have a potential role in tumor growth and progression. These studies show that DEC1 is involved in esophageal cancer development and help elucidate its functional role in tumor development. Show less

Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression. Show less

MAP kinases phosphatases (MKPs) belong to the dual-specificity phosphatase family (DUSP) and dephosphorylate phosphothreonine and phosphotyrosine within MAP kinases. We had previously shown that DUSP6/MKP-3 was phosphorylated and degraded upon growth factor stimulation, in a MEK-dependent manner. Here we show that another pathway involved in growth factor signaling, the PI3K/mTOR signaling pathway, accounts for a part of the phosphorylation and degradation of DUSP6 induced by serum growth factors, as evidenced by experiments using pharmacological inhibitors of PI3 kinase and mammalian target of rapamycin (mTOR). Moreover, specific agonists of the mTOR pathway, such as amino acids or insulin/IGF-1, which do not activate extracellular signal regulated kinases (ERKs) in our cellular model, were also able to induce the phosphorylation and degradation of DUSP6. However, a basal activity of MEK was required for the mTOR pathway-mediated phosphorylation to occur. Mutagenesis studies identified serine 159 within DUSP6 as the target of the mTOR pathway. The ERK phosphatase DUSP6 may thus constitute a novel branch-point of the crosstalk between two major signaling pathways induced by growth factors, the MEK/ERK pathway and the PI3K/mTOR pathway. Show less

Epstein-Barr virus (EBV) infection plays a key role in the pathogenesis of nasopharyngeal carcinoma (NPC). This study was to explore the effects of the recurrent infection by EBV reactivation on the genomic expression profile of NPC. The microarray expression data from different cell lines subjected to primary infection of EBV+ vs. EBV- targets in NPC and recurrent EBV reactivation were collected from public data depository. Cross comparison, t-test analysis as well as filtering by flag, expression level and fold change were used to analyze the data and identify differential genes. Moreover, a set of web-based applications, such as DAVID (database for annotation, visualization and integrated discovery), pSTIING (protein, signaling, transcriptional interactions and inflammation networks gateway), GATHER (gene annotation tool to help explain relationships) and TELiS (transcription element listening system), were used to analyze and predict the probable expression profile of the differential genes. As compared with the genes expressed during primary infection of EBV, 25 genes, including DUSP1, TOP1, HOXA9, DEK, PABPC1 and IMPDH2, were differentially expressed during EBV reactivation. Many of them were oncogenic. The differential genes together with related transcriptional factors were interacted mainly through 2 mechanisms: one mainly included TOP1, DUSP1, DUSP6, and RPS28; the other one was a circuit of PITX1, CD9, HOXA9 and IMPDH2. The differential genes might participate in EBV reactivation by changing their expression level through two mechanisms, which contributes to the final development of NPC. Show less

Philadelphia (Ph) chromosome-positive leukemia is characterized by the BCR/ABL1 fusion protein that affects a wide range of signal transduction pathways. The knowledge about its downstream target genes is, however, still quite limited. To identify novel BCR/ABL1-regulated genes we used global gene expression profiling of several Ph-positive and Ph-negative cell lines treated with imatinib. Following imatinib treatment, the Ph-positive cells showed decreased growth, viability, and reduced phosphorylation of BCR/ABL1 and STAT5. In total, 142 genes were identified as being dependent on BCR/ABL1-mediated signaling, mainly including genes involved in signal transduction, e.g. the JAK/STAT, MAPK, TGFB, and insulin signaling pathways, and in regulation of metabolism. Interestingly, BCR/ABL1 was found to activate several genes involved in negative feedback regulation (CISH, SOCS2, SOCS3, PIM1, DUSP6, and TNFAIP3), which may act to indirectly suppress the tumor promoting effects exerted by BCR/ABL1. In addition, several genes identified as deregulated upon BCR/ABL1 expression could be assigned to the TGFB and NFkB signaling pathways, as well as to reflect the metabolic adjustments needed for rapidly growing cells. Apart from providing important pathogenetic insights into BCR/ABL1-mediated leukemogenesis, the present study also provides a number of pathways/individual genes that may provide attractive targets for future development of targeted therapies. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. Show less

The use of embryonic stem cells is currently the most promising approach to assess developmental toxicity in vitro. In addition, the possibility of using human embryonic stem (hES) cells will increase safety of consumers and patients as false classification of substances due to inter-species variations can be avoided. One validated test based on murine embryonic stem cells, the embryonic stem cell test (EST), consists of following endpoints: IC(50) values of fibroblasts and embryonic stem cells as well as the inhibition of differentiation of mES cells into cardiomyocytes. As a follow up of its successful validation study we established a cytotoxicity assay based on hES cells and human fibroblasts employing two developmental toxicants: 5-fluorouracil (5-FU) and all-trans retinoic acid (RA). The results were compared to historical data from the EST. For 5-FU, no significant differences were obtained between the different cell lines. However, for RA, both test systems produced higher IC(50) values for the fibroblasts than for the stem cells, which is a well-known effect of developmental toxicants. Moreover, the reliability and relevance of several marker genes as possible toxicological endpoints were tested. During early differentiation Oct-4, hTert and Dusp6 showed the most reliable results. Brachyury and GATA-4 were found to be best suited to monitor cardiac differentiation. The late cardiac marker gene TNNT2 demonstrated significant results until day 18. Therefore, these marker genes have the highest potential to serve as endpoints for a developmental toxicity test. Show less

Fibroblast Growth Factors (FGFs) represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis. Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3) is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP) in transgenic embryos (Tg(Dusp6:d2EGFP)). Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf) within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB), pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR) inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFP)y1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF) signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs). From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway. The generation of transgenic reporter zebrafish lines has allowed direct visualization of FGF signalling within the developing embryo. These FGF reporter transgenic lines provide a tool to screen for specific compounds that can distinguish between two conserved members of the RTK family. Show less

Tienilic acid (TA) was withdrawn due to idiosyncratic hepatotoxicity. Two hypotheses for the mechanisms of idiosyncratic reactions are the hapten and danger hypotheses, which are not mutually exclusive. Both human CYP 2C9 and rat CYP 2C11 metabolize TA to a reactive metabolite that was reported to bind exclusively to these enzymes. TA-Induced liver toxicity is associated with antibodies against CYP 2C9, thus TA appears to act as a hapten. However, if the binding were limited to CYP 2C, it is unlikely that this would lead to significant cell stress. If TA does not cause cell stress it would suggest that acting as a hapten is sufficient to induce an idiosyncratic reaction. To test whether TA can cause cell stress rats were dosed with TA and hepatic gene expression was profiled at 6 and 24 hr after drug administration. TA induced changes in genes involved in oxidative stress (aldo-keto reductase, glutathione-S-transferase, thioredoxin reductase, epoxide hydrolase), inflammation (IL-1beta, interferon regulatory factor 1, macrophage stimulating protein 1), cytotoxicity (caspase-12), and liver regeneration (p27(Kip1), DUSP6, serine dehyratase, spectrin beta II, inhibin beta(A)). These data support the hypothesis that danger signals in the form of cell-stress may be involved in initiating the immune response observed in TA-induced toxicity. In separate experiments, we examined the changes in gene expression induced in mice by sulfamethoxazole, which also causes idiosyncratic reactions. Sulfamethoxazole is an aromatic amine, and aromatic amines in general are associated with idiosyncratic drug reactions. They form reactive metabolites that both act as electrophiles and can redox cycle; therefore, it was assumed that sulfamethoxazole would cause some type of cell stress, the only question was what changes in mRNA expression would occur. In contrast to expectations, no changes induced by sulfamethoxazole could easily be interpreted as a danger signal. These data are presented together because they are the opposite of the expected results and convey a complex story. Show less

Intraductal papillary-mucinous neoplasms of the pancreas show characteristic clinicopathological and molecular pathobiological features which are distinct from those of conventional ductal adenocarcinomas. Alterations of KRAS, AKT/PKB, CDKN2A, TP53, SMAD4, STK11/LKB1, and DUSP6, and other molecular alterations, including global expression studies as well as their clinical implications, are discussed. Show less

Interictal spikes are hallmarks of epileptic neocortex that are used commonly in both EEG and electrocorticography (ECoG) to localize epileptic brain regions. Despite their prevalence, the exact relationship between interictal spiking and the molecular pathways that drive the production and propagation of seizures is not known. We have recently identified a common group of genes induced in human epileptic foci, including EGR1, EGR2, c-fos, and MKP-3. We found that the expression levels of these genes correlate precisely with the frequency of interictal activity and can thus serve as markers of epileptic activity. Here, we explore this further by comparing the expression of these genes within human epileptic neocortex to both ictal and specific electrical parameters of interictal spiking from subdural recordings prior to surgical resection in order to determine the electrical properties of the human neocortex that correlate best to the expression of these genes. Seizure frequency as well as quantitative electrophysiological parameters of interictal spikes including frequency, amplitude, duration, and area were calculated at each electrode channel and compared to quantitative real-time RT-PCR measurements of four activity-dependent genes (c-fos, EGR1, EGR2, and MKP-3) in the underlying neocortical tissue. Local neocortical regions of seizure onset had consistently higher spike firing frequencies and higher spike amplitudes compared to nearby "control" cortex. In contrast, spike duration was not significantly different between these two areas. There was no relationship observed between seizure frequency and the expression levels of activity-dependent genes for the patients examined in this study. However, within each patient, there were highly significant correlations between the expression of three of these genes (c-fos, EGR1, EGR2) and the frequency, amplitude, and total area of the interictal spikes at individual electrodes. We conclude that interictal spiking is closely associated with the expression of a group of activity-dependent transcription factors in neocortical human epilepsy. Since there was little correlation between gene expression and seizure frequency, our results suggest that interictal spiking is a stronger driving force behind these activity-dependent gene changes and may thus participate in the development and maintenance of the abnormal neuronal hyperactivity seen in human epileptic neocortex. Show less

Phthalates are chemical plasticizers used in a variety of consumer products; in rodents, they alter testicular development, leading to decreased testosterone synthesis and maldevelopment of the reproductive tract. Here, our goals were to discover a set of biomarker genes that respond early after relatively low-dose-level dibutyl phthalate (DBP) exposure and map the responding testicular cell types. To identify testicular phthalate biomarker genes, 34 candidate genes were examined by quantitative PCR at 1, 2, 3, or 6 h after exposure of Gestational Day 19 rats to DBP dose levels ranging from 0.1 to 500 mg/kg body weight. Twelve genes (Ctgf, Cxcl10, Dusp6, Edn1, Egr1, Fos, Ier3, Junb, Nr4a1, Stc1, Thbs1, and Tnfrsf12a) were identified with increased expression by 1-3 h at 100 or 500 mg/kg DBP, and 7 of these 12 genes had increased expression by 6 h at 10 mg/kg DBP. Using in situ hybridization of fetal testis cryosections from DBP-exposed rats, the temporal cellular expression of 10 biomarker genes was determined. Genes with a robust response at 1 h (Dusp6, Egr1, Fos, and Thbs1) were induced in peritubular myoid cells. For Egr1 and Fos, the interstitial compartment also showed increased expression at 1 h. Cxcl10 and Nr4a1 were induced by 1-3 h in both sparsely located interstitial cells and peritubular myoid cells. By 3 h, Stc1 was induced in Leydig cells, and Edn1, Ier3, and Tnfrsf12a were increased in Sertoli cells. These data reveal a complex early cascade of phthalate-induced cellular responses in the fetal testis, and for the first time suggest that peritubular myoid cells are an important proximal phthalate target cell. Show less

MAPK signaling pathways function as critical regulators of cellular differentiation, proliferation, stress responsiveness, and apoptosis. One branch of the MAPK signaling pathway that culminates in ERK1/2 activation is hypothesized to regulate the growth and adaptation of the heart to both physiologic and pathologic stimuli, given its known activation in response to virtually every stress- and agonist-induced hypertrophic stimulus examined to date. Here we investigated the requirement of ERK1/2 signaling in mediating the cardiac hypertrophic growth response in Erk1(-/-) and Erk2(+/-) mice, as well as in transgenic mice with inducible expression of an ERK1/2-inactivating phosphatase in the heart, dual-specificity phosphatase 6. Although inducible expression of dual-specificity phosphatase 6 in the heart eliminated ERK1/2 phosphorylation at baseline and after stimulation without affecting any other MAPK, it did not diminish the hypertrophic response to pressure overload stimulation, neuroendocrine agonist infusion, or exercise. Similarly, Erk1(-/-) and Erk2(+/-) mice showed no reduction in pathologic or physiologic stimulus-induced cardiac growth in vivo. However, blockade or deletion of cardiac ERK1/2 did predispose the heart to decompensation and failure after long-term pressure overload in conjunction with an increase in myocyte TUNEL. Thus, ERK1/2 signaling is not required for mediating physiologic or pathologic cardiac hypertrophy in vivo, although it does play a protective role in response to pathologic stimuli. Show less

To investigate the association between the configurational and compositional changes of nuclear matrix and the differentiation of carcinoma cells. Cells cultured with or without 5 x 10(-3) mmol/L of hexamethylene bisacetamide (HMBA) on Nickel grids were treated by selective extraction and prepared for whole mount observation under electron microscopy. The samples were examined under transmission electron microscope. Nuclear matrix proteins were selectively extracted and subjected to subcellular proteomics study. The protein expression patterns were analyzed by PDQuest software. Spots of differentially expressed nuclear matrix proteins were excised and subjected to in situ digestion with trypsin. The peptides were analyzed by matrix-assisted laser-desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Data were submitted for database searching using Mascot tool (www.matrixscience.com). The nuclear matrix (NM) and intermediate filament (IF) in SMMC-7721 hepatocarcinoma cells were found relatively sparse and arranged irregularly. The nuclear lamina was non-uniform, and two kinds of filaments were not tightly connected. After induction for differentiation by HMBA, the NM-IF filaments were concentrated and distributed uniformly. The heterogeneous population of filaments, including highly branched ultrathin filaments could also be seen in the regular meshwork. The connection between the two kinds of filaments and the relatively thin, condensed and sharply demarcated lamina composed of intermediate-sized filaments was relatively fastened. Meanwhile, 21 NM proteins changed remarkably during SMMC-7721 cell differentiation. Four proteins, i.e. mutant Pyst1, hypothetical protein, nucleophosmin 1, and LBP were downregulated, whereas four other proteins, eIF6, p44 subunit, beta-tubulin, and SIN3B were upregulated with the last one, SR2/ASF found only in the differentiated SMMC-7721 cells. The induced differentiation of SMMC-7721 cells by HMBA is accompanied by the configurational changes of nuclear matrix-intermediate filament (NM-IF) system and the compositional changes of nuclear matrix protein expression. These changes may be important morphological or functional indications of the cancer cell reversion. Show less

Small molecule inhibitors of protein tyrosine kinases have become both powerful chemical probes of biological processes and clinically effective therapeutics. In contrast, few small molecule inhibitors of protein tyrosine phosphatases have been identified and none are currently approved for clinical use. New cell-based high-content methods have been developed that should enable investigators to probe for selective inhibitors of diseases-relevant protein phosphatases. Details of these methods are described herein. Show less

The regulated dephosphorylation of mitogen-activated protein kinases (MAPKs) plays a key role in determining the magnitude and duration of kinase activation and hence the physiological outcome of signalling. In mammalian cells, an important component of this control is mediated by the differential expression and activities of a family of 10 dual-specificity (Thr/Tyr) MAPK phosphatases (MKPs). These enzymes share a common structure in which MAPK substrate recognition is determined by sequences within an amino-terminal non-catalytic domain whereas MAPK binding often leads to a conformational change within the C-terminal catalytic domain resulting in increased enzyme activity. MKPs can either recognize and inactivate a single class of MAP kinase, as in the specific inactivation of extracellular signal regulated kinase (ERK) by the cytoplasmic phosphatase DUSP6/MKP-3 or can regulate more than one MAPK pathway as illustrated by the ability of DUSP1/MKP-1 to dephosphorylate ERK, c-Jun amino-terminal kinase and p38 in the cell nucleus. These properties, coupled with transcriptional regulation of MKP expression in response to stimuli that activate MAPK signalling, suggest a complex negative regulatory network in which individual MAPK activities can be subject to negative feedback control, but also raise the possibility that signalling through multiple MAPK pathways may be integrated at the level of regulation by MKPs. Show less

MAP kinase phosphatase 3 (MKP3, also known as DUSP6 and PYST1) is involved in extracellular signal receptor kinase (ERK) regulation and functions as a specific phosphatase to the activated (phosphorylated) forms of ERK1 and ERK2. MKP3 displays allosteric activation, which aids in tightly regulating its function to ERK substrates, but not other related MAPKs. Due to MKP3's specificity for the ERK signaling pathway, the development of specific activators or inhibitors to the enzyme have been suggested in order to expressly influence the ERK1 and ERK2 pathways. To produce the high yields of MKP3 protein necessary for physico-chemical characterization of MKP3 and for high throughput screening of its small-molecule activators and inhibitors, we have cloned, purified and, and identified refolding conditions suitable for producing full-length, human MKP3 from Escherichia coli inclusion bodies. Furthermore, we demonstrate the use of a 96-well plate format refolding assay in which the ERK-induced activity of MKP3 is simulated by 33% DMSO. The assay allowed for rapid detection of MKP3's function following a refolding screen in the absence of ERK and thus provides quick and inexpensive testing of MKP3 activity. Following screening, the refolded product was confirmed to be correctly folded by steady-state kinetic analysis and by the CD spectroscopy-determined secondary structure content. CD data were consistent with 36% helix and 14% sheet, which compared to an expected 32.9% helix and 12.4% sheet. These data indicated that MKP3 was properly folded, making it a suitable protein for use in functional studies. Show less

Activation of extracellular signal-regulated kinase (ERK) is known to be regulated by cell adhesion, namely "anchorage dependence". Most studies on the anchorage-dependent regulation have focused on the upstream activating components. We previously reported that the focal adhesion protein vinexin beta can induce the anchorage-independent activation of ERK2. We show here that vinexin beta-induced anchorage-independent activation of ERK2 involves prevention of the dephosphorylation of ERK2, but not the promotion of MEK1 or Raf1 activity. Furthermore, knockdown of vinexin beta resulted in a faster dephosphorylation of ERK2 in A549 cells. Moreover, the coexpression of MKP3/rVH6, an ERK2 specific phosphatase, suppressed the anchorage-independent activation of ERK2 induced by vinexin beta. These results suggest that vinexin beta can prevent the dephosphorylation of ERK2 stimulated by cell detachment, leading to the anchorage-independent activation of ERK2. Furthermore, we found that phosphatase activity directed against activated ERK2 was higher in suspended cells than in adherent cells. In addition, orthovanadate efficiently induces anchorage-independent activation of ERK2 without marked activation of MEK1 in NIH3T3 cells. These observations suggest that the anchorage dependence of ERK1/2 activation is regulated not only by upstream kinases, Raf1 and MEK, but also by phosphatases acting against ERK1/2 and that vinexin beta can induce anchorage-independent activation of ERK by preventing the inactivation of ERK1/2. Show less

Famine and viral infection, as well as interferon therapy have been reported to increase the risk of developing bipolar disorder. In addition, almost 100 polymorphic genes have been associated with this disease. Several form most of the components of a phosphatidyl-inositol signalling/AKT1 survival pathway (PIK3C3, PIP5K2A, PLCG1, SYNJ1, IMPA2, AKT1, GSK3B, TCF4) which is activated by growth factors (BDNF, NRG1) and also by NMDA receptors (GRIN1, GRIN2A, GRIN2B). Various other protein products of genes associated with bipolar disorder either bind to or are affected by phosphatidyl-inositol phosphate products of this pathway (ADBRK2, HIP1R, KCNQ2, RGS4, WFS1), are associated with its constituent elements (BCR, DUSP6, FAT, GNAZ) or are downstream targets of this signalling cascade (DPYSL2, DRD3, GAD1, G6PD, GCH1, KCNQ2, NOS3, SLC6A3, SLC6A4, SST, TH, TIMELESS). A further pathway relates to endoplasmic reticulum-stress (HSPA5, XBP1), caused by problems in protein glycosylation (ALG9), growth factor receptor sorting (PIK3C3, HIP1R, SYBL1), or aberrant calcium homoeostasis (WFS1). Key processes relating to these pathways appear to be under circadian control (ARNTL, CLOCK, PER3, TIMELESS). DISC1 can also be linked to many of these pathways. The growth factor pathway promotes protein synthesis, while the endoplasmic reticulum stress pathway, and other stress pathways activated by viruses and cytokines (IL1B, TNF, Interferons), oxidative stress or starvation, all factors associated with bipolar disorder risk, shuts down protein synthesis via control of the EIF2 alpha and beta translation initiation complex. For unknown reasons, oligodendrocytes appear to be particularly prone to defects in the translation initiation complex (EIF2B) and the convergence of these environmental and genomic signalling pathways on this area might well explain their vulnerability in bipolar disorder. Show less

Current staging methods are inadequate for predicting the outcome of treatment of non-small-cell lung cancer (NSCLC). We developed a five-gene signature that is closely associated with survival of patients with NSCLC. We used computer-generated random numbers to assign 185 frozen specimens for microarray analysis, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis, or both. We studied gene expression in frozen specimens of lung-cancer tissue from 125 randomly selected patients who had undergone surgical resection of NSCLC and evaluated the association between the level of expression and survival. We used risk scores and decision-tree analysis to develop a gene-expression model for the prediction of the outcome of treatment of NSCLC. For validation, we used randomly assigned specimens from 60 other patients. Sixteen genes that correlated with survival among patients with NSCLC were identified by analyzing microarray data and risk scores. We selected five genes (DUSP6, MMD, STAT1, ERBB3, and LCK) for RT-PCR and decision-tree analysis. The five-gene signature was an independent predictor of relapse-free and overall survival. We validated the model with data from an independent cohort of 60 patients with NSCLC and with a set of published microarray data from 86 patients with NSCLC. Our five-gene signature is closely associated with relapse-free and overall survival among patients with NSCLC. Show less

Mitogen-activated protein kinase (MAPK) pathways are major mediators of extracellular signals that are transduced to the nucleus. MAPK signaling is attenuated at several levels, and one class of dual-specificity phosphatases, the MAPK phosphatases (MKPs), inhibit MAPK signaling by dephosphorylating activated MAPKs. Several of the MKPs are themselves induced by the signaling pathways they regulate, forming negative feedback loops that attenuate the signals. We show here that in mouse embryos, Fibroblast growth factor receptors (FGFRs) are required for transcription of Dusp6, which encodes MKP3, an extracellular signal-regulated kinase (ERK)-specific MKP. Targeted inactivation of Dusp6 increases levels of phosphorylated ERK, as well as the pERK target, Erm, and transcripts initiated from the Dusp6 promoter itself. Finally, the Dusp6 mutant allele causes variably penetrant, dominant postnatal lethality, skeletal dwarfism, coronal craniosynostosis and hearing loss; phenotypes that are also characteristic of mutations that activate FGFRs inappropriately. Taken together, these results show that DUSP6 serves in vivo as a negative feedback regulator of FGFR signaling and suggest that mutations in DUSP6 or related genes are candidates for causing or modifying unexplained cases of FGFR-like syndromes. Show less