Dual-specificity phosphatases: therapeutic targets in cancer therapy resistance.

PURPOSE: Therapy resistance is the principal obstacle to achieving cures in cancer patients and its successful tackling requires a deep understanding of the resistance mediators. Increasing evidence indicates that tumor phosphatases are novel and druggable targets in translational oncology and their modulation may hinder tumor growth and motility and potentiate therapeutic sensitivity in various neoplasms via regulation of various signal transduction pathways. Dual-specificity phosphatases (DUSPs) are key players of cell growth, survival and death and have essential roles in tumor initiation, malignant progression and therapy resistance through regulation of the MAPK signaling pathway. In this review, different aspects of DUSPs are discussed. METHODS: A comprehensive literature review was performed using various websites including PubMed. RESULTS: We provide mechanistic insights into the roles of well-known DUSPs in resistance to a wide range of cancer therapeutic approaches including chemotherapy, radiation and molecular targeted therapy in human malignancies. Moreover, we discuss the development of DUSP modulators, with a focus on DUSP1 and 6 inhibitors. Ultimately, the preclinical investigations of small molecule inhibitors of DUSP1 and 6 are outlined. CONCLUSION: Emerging evidence indicates that the DUSP family is aberrantly expressed in human malignancies and plays critical roles in determining sensitivity to a wide range of cancer therapeutic strategies through regulation of the MAPK signaling pathways. Consequently, targeting DUSPs and their downstream molecules can pave the way for more effective cancer therapies.

Reciprocal regulation of endothelial-mesenchymal transition by MAPK7 and EZH2 in intimal hyperplasia and coronary artery disease.

Endothelial-mesenchymal transition (EndMT) is a form of endothelial dysfunction wherein endothelial cells acquire a mesenchymal phenotype and lose endothelial functions, which contributes to the pathogenesis of intimal hyperplasia and atherosclerosis. The mitogen activated protein kinase 7 (MAPK7) inhibits EndMT and decreases the expression of the histone methyltransferase Enhancer-of-Zeste homologue 2 (EZH2), thereby maintaining endothelial quiescence. EZH2 is the catalytic subunit of the Polycomb Repressive Complex 2 that methylates lysine 27 on histone 3 (H3K27me3). It is elusive how the crosstalk between MAPK7 and EZH2 is regulated in the endothelium and if the balance between MAPK7 and EZH2 is disturbed in vascular disease. In human coronary artery disease, we assessed the expression levels of MAPK7 and EZH2 and found that with increasing intima/media thickness ratio, MAPK7 expression decreased, whereas EZH2 expression increased. In vitro, MAPK7 activation decreased EZH2 expression, whereas endothelial cells deficient of EZH2 had increased MAPK7 activity. MAPK7 activation results in increased expression of microRNA (miR)-101, a repressor of EZH2. This loss of EZH2 in turn results in the increased expression of the miR-200 family, culminating in decreased expression of the dual-specificity phosphatases 1 and 6 who may repress MAPK7 activity. Transfection of endothelial cells with miR-200 family members decreased the endothelial sensitivity to TGFß1-induced EndMT. In endothelial cells there is reciprocity between MAPK7 signaling and EZH2 expression and disturbances in this reciprocal signaling associate with the induction of EndMT and severity of human coronary artery disease.

p38-MAP Kinase Negatively Regulates the Slow Force Response to Stretch in Rat Myocardium through the Up-Regulation of Dual Specificity Phosphatase 6 (DUSP6).

BACKGROUND/AIMS: Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. METHODS: Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. RESULTS: The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. CONCLUSION: Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.

Up-regulation of DUSP5 and DUSP6 by gonadotropin-releasing hormone in cultured hypothalamic neurons, GT1-7 cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates anterior pituitary gonadotrophs to synthesize and secrete gonadotropins. In addition to gonadotrophs, GnRH neurons also express GnRH receptors, and the autocrine action of GnRH is reportedly involved in the regulation of functions of GnRH neurons. There is accumulating evidence that extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases (MAPKs), is activated by GnRH and involved in various effects of GnRH in GnRH neurons. In the present study, we performed microarray analysis to examine the types of genes whose expression was regulated by GnRH in immortalized mouse GnRH neurons (GT1-7 cells). We found that 257 genes among 55,681 genes examined were up-regulated after 30-min treatment of GT1-7 cells with GnRH. These up-regulated genes included four dual-specificity MAPK phosphatases (DUSPs), DUSP1, DUSP2, DUSP5, and DUSP6. Reverse transcription-polymerase chain reaction analysis confirmed that the mRNA levels of DUSP5 and DUSP6 were robustly increased within 30 min. U0126, an inhibitor of ERK activation, completely inhibited the increases in the mRNA levels of DUSP5 and DUSP6. Immunoblotting analysis revealed that ERK activation peaked at 5 min and declined steeply at 60 min, whereas DUSP5 and DUSP6 proteins were increased from 60 min. It was notable that down-regulation of DUSP6 augmented GnRH-induced ERK activation approximately 1.7-fold at 60 min. These results suggested that the up-regulation of DUSP6 regulates the duration of ERK activation at least in part.

Translational control of ERK signaling through miRNA/4EHP-directed silencing.

MicroRNAs (miRNAs) exert a broad influence over gene expression by directing effector activities that impinge on translation and stability of mRNAs. We recently discovered that the cap-binding protein 4EHP is a key component of the mammalian miRNA-Induced Silencing Complex (miRISC), which mediates gene silencing. However, little is known about the mRNA repertoire that is controlled by the 4EHP/miRNA mechanism or its biological importance. Here, using ribosome profiling, we identify a subset of mRNAs that are translationally controlled by 4EHP. We show that the Dusp6 mRNA, which encodes an ERK1/2 phosphatase, is translationally repressed by 4EHP and a specific miRNA, miR-145. This promotes ERK1/2 phosphorylation, resulting in augmented cell growth and reduced apoptosis. Our findings thus empirically define the integral role of translational repression in miRNA-induced gene silencing and reveal a critical function for this process in the control of the ERK signaling cascade in mammalian cells.

Overexpression of Mitogen-activated protein kinase phosphatase-3 (MKP-3) reduces FoxO1 phosphorylation in mice hypothalamus.

The mitogen-activated kinase phosphatase-3 (MKP-3) has gained great importance in the scientific community by acting as a regulator of the cell cycle through dephosphorylation of FoxO1, an important transcription factor involved in the insulin intracellular signaling cascade. When dephosphorylated and translocated to the nuclei, FoxO1 can promote the transcription of orexigenic neuropeptides (NPY/AgRP) in the hypothalamus, whereas insulin signaling is responsible for the disruption of this process. However, it is not understood if the hypothalamic activation of MKP-3 affects FoxO1 phosphorylation, and we hypothesized that MKP-3 overexpression reduces the capacity of the insulin signal to phosphorylate FoxO1. In the present study, we overexpressed the DUSP6 gene through an injection of adenovirus directly into the hypothalamic third ventricle of Swiss mice. The colocalization of the adenovirus was confirmed by the immunofluorescence assay. Then, MKP-3 overexpression resulted in a significant reduction of hypothalamic FoxO1 phosphorylation after insulin stimulation. This effect was independent of changes in Akt phosphorylation. Thus, the role of MKP-3 in the hypothalamus is closely associated with FoxO1 dephosphorylation and may provide a potential therapeutic target against hypothalamic disorders related to obesity and unbalanced food intake control.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma.

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies.We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression.

A Screen for Key Genes and Pathways Involved in High-Quality Brush Hair in the Yangtze River Delta White Goat.

The Yangtze River Delta White Goat is the only goat breed that produces high-quality brush hair, or type III hair, which is specialized for use in top-grade writing brushes. There has been little research, especially molecular research, on the traits that result in high-quality brush hair in the Yangtze River Delta White Goat. To explore the molecular mechanisms of the formation of high-quality brush hair, High-throughput RNA-Seq technology was used to compare skin samples from Yangtze River Delta White Goats that produce high-quality hair and non high-quality hair for identification of the important genes and related pathways that might influence the hair quality traits. The results showed that 295 genes were expressed differentially between the goats with higher and lower hair quality, respectively. Of those genes, 132 were up-regulated, 62 were down-regulated, and 101 were expressed exclusively in the goats with high-quality brush hair. Gene Ontology and Metabolic Pathway Significant Enrichment analyses of the differentially expressed genes indicated that the MAP3K1, DUSP1, DUSP6 and the MAPK signaling pathway might play important roles in the traits important for high-quality brush hair.

Docosahexaenoic acid up-regulates both PI3K/AKT-dependent FABP7-PPARγ interaction and MKP3 that enhance GFAP in developing rat brain astrocytes.

The astrocyte marker, glial fibrillary acidic protein (GFAP), has essential functions in the brain, but may trigger astroglial scarring when expressed in excess. Docosahexaenoic acid (DHA) is an n-3 fatty acid that is protective during brain development. However, the effect of DHA on GFAP levels of developing brain remains unexplored. Here, we detected that treating developing rats with DHA-enriched fish-oil caused dose-dependent GFAP augmentation. We investigated the mechanism promoting GFAP, hypothesizing the participation of fatty acid-binding protein-7 (FABP7), known to bind DHA. We identified that DHA stimulated FABP7 expression in astrocytes, and FABP7-silencing suppressed DHA-induced GFAP, indicating FABP7-mediated GFAP increase. Further investigation proved FABP7 expression to be phosphatidylinositide 3-kinases (PI3K)/AKT and nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ)-dependent. We found that PI3K/AKT activated PPARγ that triggered FABP7 expression via PPARγ-responsive elements within its gene. Towards identifying FABP7-downstream pathways, we considered our previous report that demonstrated cyclin-dependent kinase-5 (CDK5)-PPARγ-protein-protein complex to suppress GFAP. We found that the DHA-induced FABP7 underwent protein-protein interaction with PPARγ, which impeded CDK5-PPARγ formation. Hence, it appeared that enhanced FABP7-PPARγ in lieu of CDK5-PPARγ resulted in increased GFAP. PI3K/AKT not only stimulated formation of FABP7-PPARγ protein-protein complex, but also up-regulated a FABP7-independent MAP-kinase-phosphatase-3 pathway that inactivated CDK5 and hence attenuated CDK5-PPARγ. Overall, our data reveal that via the proximal PI3K/AKT, DHA induces FABP7-PPARγ, through genomic and non-genomic mechanisms, and MAP-kinase-phosphatase-3 that converged at attenuated CDK5-PPARγ and therefore, enhanced GFAP. Accordingly, our study demonstrates a DHA-mediated astroglial hyperactivation, pointing toward a probable injurious role of DHA in brain development.

Prognostic value of dual-specificity phosphatase 6 expression in non-small cell lung cancer.

Dual-specificity phosphatase 6 (DUSP6/MKP-3) is a mitogen-activated protein kinase phosphatase that regulates extracellular signal-regulated kinases (ERKs) activity via feedback mechanisms, with an increasingly recognized role in tumour biology. The aim of this study was to explore the role of DUSP6 expression in the prognosis of human non-small cell lung cancer (NSCLC). DUSP6 expression levels were evaluated by real-time quantitative reverse transcription polymerase chain reaction (PCR) in 60 NSCLC samples from patients who underwent pulmonary resection at 12 de Octubre University Hospital. We performed a statistical analysis to investigate the correlation of DUSP6 expression and the clinical outcomes. We found that 66.7% of the tumour samples show the downregulation of DUSP6 at the messenger RNA (mRNA) levels compared to benign epithelial lung tissues and 55% of them show at least twofold downregulation of DUSP6 gene expression. Patients were classified into three groups according to their DUSP6 expression levels and those with very low levels (at least twofold downregulation) had the worst outcomes. Using the value of twice below the mean value in benign epithelial lung tissue as a cutoff, the overall survival of patients with very low DUSP6 levels was significantly lower than that in the rest of patients (31.9 ± 18.8 months vs. not reached, P = 0.049). This was most pronounced in adenocarcinoma histology and high-stage tumour samples. Our results suggest that DUSP6 gene expression in tumour samples may be a prognostic marker in NSCLC.

p53 protein-mediated up-regulation of MAP kinase phosphatase 3 (MKP-3) contributes to the establishment of the cellular senescent phenotype through dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2).

Growth arrest is one of the essential features of cellular senescence. At present, the precise mechanisms responsible for the establishment of the senescence-associated arrested phenotype are still incompletely understood. Given that ERK1/2 is one of the major kinases controlling cell growth and proliferation, we examined the possible implication of ERK1/2. Exposure of normal rat epithelial cells to etoposide caused cellular senescence, as manifested by enlarged cell size, a flattened cell body, reduced cell proliferation, enhanced ß-galactosidase activity, and elevated p53 and p21. Senescent cells displayed a blunted response to growth factor-induced cell proliferation, which was preceded by impaired ERK1/2 activation. Further analysis revealed that senescent cells expressed a significantly higher level of mitogen-activated protein phosphatase 3 (MKP-3, a cytosolic ERK1/2-targeted phosphatase), which was suppressed by blocking the transcriptional activity of the tumor suppressor p53 with pifithrin-α. Inhibition of MKP-3 activity with a specific inhibitor or siRNA enhanced basal ERK1/2 phosphorylation and promoted cell proliferation. Apart from its role in growth arrest, impairment of ERK1/2 also contributed to the resistance of senescent cells to oxidant-elicited cell injury. These results therefore indicate that p53-mediated up-regulation of MKP-3 contributes to the establishment of the senescent cellular phenotype through dephosphorylating ERK1/2. Impairment of ERK1/2 activation could be an important mechanism by which p53 controls cellular senescence.

Hepatitis C virus-induced reduction in miR-181a impairs CD4(+) T-cell responses through overexpression of DUSP6.

UNLABELLED: T cells play a crucial role in viral clearance or persistence; however, the precise mechanisms that control their responses during viral infection remain incompletely understood. MicroRNA (miR) has been implicated as a key regulator controlling diverse biological processes through posttranscriptional repression. Here, we demonstrate that hepatitis C virus (HCV)-mediated decline of miR-181a expression impairs CD4(+) T-cell responses through overexpression of dual specific phosphatase 6 (DUSP6). Specifically, a significant decline of miR-181a expression along with overexpression of DUSP6 was observed in CD4(+) T cells from chronically HCV-infected individuals compared to healthy subjects, and the levels of miR-181a loss were found to be negatively associated with the levels of DUSP6 overexpression in these cells. Importantly, reconstitution of miR-181a or blockade of DUSP6 expression in CD4(+) T cells led to improved T-cell responses including enhanced CD25 and CD69 expression, increased interleukin-2 expression, and improved proliferation of CD4(+) T cells derived from chronically HCV-infected individuals. CONCLUSION: Since a decline of miR-181a concomitant with DUSP6 overexpression is the signature marker for age-associated T-cell senescence, these findings provide novel mechanistic insights into HCV-mediated premature T-cell aging through miR-181a-regulated DUSP6 signaling and reveal new targets for therapeutic rejuvenation of impaired T-cell responses during chronic viral infection.

Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers.

Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER-) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2+ and ERBB2- in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2+ subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.

c-Myb negatively regulates Ras signaling through induction of dual phosphatase MKP-3 in NIH3T3 cells.

Mitogen-activated protein kinase phosphatase-3 (MKP-3) negatively regulates ERK1/2 MAPK in a feedback loop. However, little is known about the molecular mechanism by which Ras signaling induces MKP-3 expression. In the present study, we demonstrate that exogenous expression of constitutively active H-Ras increases the level of MKP-3 mRNA. A transfection study using a series of MKP-3 promoter deletion constructs revealed that the c-Myb binding site is required for Ras-induced transcriptional activation of the MKP-3 gene promoter. Furthermore, we show that c-Myb directly binds to the MKP-3 promoter, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation. Knock-down of c-Myb expression using siRNA abrogated Ras-induced MKP-3 promoter activity. These findings propose a novel mechanism through which Ras signaling activates c-Myb-dependent transcriptional activation of the MKP-3 gene.

Distinct populations within Isl1 lineages contribute to appendicular and facial skeletogenesis through the ß-catenin pathway.

Isl1 expression marks progenitor populations in developing embryos. In this study, we investigated the contribution of Isl1-expressing cells that utilize the ß-catenin pathway to skeletal development. Inactivation of ß-catenin in Isl1-expressing cells caused agenesis of the hindlimb skeleton and absence of the lower jaw (agnathia). In the hindlimb, Isl1-lineages broadly contributed to the mesenchyme; however, deletion of ß-catenin in the Isl1-lineage caused cell death only in a discrete posterior domain of nascent hindlimb bud mesenchyme. We found that the loss of posterior mesenchyme, which gives rise to Shh-expressing posterior organizer tissue, caused loss of posterior gene expression and failure to expand chondrogenic precursor cells, leading to severe truncation of the hindlimb. In facial tissues, Isl1-expressing cells broadly contributed to facial epithelium. We found reduced nuclear ß-catenin accumulation and loss of Fgf8 expression in mandibular epithelium of Isl1(-/-) embryos. Inactivating ß-catenin in Isl1-expressing epithelium caused both loss of epithelial Fgf8 expression and death of mesenchymal cells in the mandibular arch without affecting epithelial proliferation and survival. These results suggest a Isl1→ß-catenin→Fgf8 pathway that regulates mesenchymal survival and development of the lower jaw in the mandibular epithelium. By contrast, activating ß-catenin signaling in Isl1-lineages caused activation of Fgf8 broadly in facial epithelium. Our results provide evidence that, despite its broad contribution to hindlimb mesenchyme and facial epithelium, the Isl1-ß-catenin pathway regulates skeletal development of the hindlimb and lower jaw through discrete populations of cells that give rise to Shh-expressing posterior hindlimb mesenchyme and Fgf8-expressing mandibular epithelium.

MAP kinase phosphatase-3 (MKP-3) is transcriptionally and post-translationally up-regulated by hCG and modulates cAMP-induced p21 expression in MA-10 Leydig cells.

Luteinizing hormone (LH) activates ERK1/2, MAP kinases (MAPKs) necessary for its action on steroidogenesis and cell proliferation, and also induces MAPK phosphatase-1 (MKP-1), which rapidly dephosphorylates nuclear ERK1/2. MKP-3 is a cytoplasmic ERK-phosphatase up-regulated by proliferative stimuli. MKP-3 also dephosphorylates transcription factor FOXO1, promoting its transport to the nucleus. Here we analyzed MKP-3 expression in MA-10 Leydig cells and demonstrated that LH receptor (LHR) activation with human gonadotropin hormone (hCG) and an analog of its second messenger, 8Br-cAMP, up-regulates MKP-3 by transcriptional and post-translational mechanisms. It is known that FOXO1 drives the expression of the cell cycle inhibitor p21. Since the activation of this transcription factor by MKP-3 has been reported, we assessed the effect of shRNA against MKP-3 on p21mRNA levels. 8Br-cAMP increased these levels (2-fold at 2h) and MKP-3 down-regulation reduced this effect. Our work demonstrates that LH/hCG tightly up-regulates MKP-3 which in turn, dephosphorylates ERK1/2 and drives p21 expression. These events could contribute to counteract hormonal action on cell proliferation.

Mapping MKP-3/FOXO1 interaction and evaluating the effect on gluconeogenesis.

BACKGROUND: MAP kinase phosphatase 3 (MKP-3) is known to attenuate the ERK signaling pathway. It has been recently demonstrated that MKP-3 is also a player in promoting hepatic glucose output in obese state by interacting and activating FOXO1. Reduction of hepatic MKP-3 expression is sufficient to reduce blood glucose levels in both diet-induced and genetically obese mice. METHODOLOGY/PRINCIPAL FINDINGS: In current study, the mechanism of MKP-3/FOXO1 interaction and the effects on transcription of gluconeogenic gene and glucose output was investigated in Fao hepatoma cells by using mutated MKP-3 and FOXO1 adenoviral constructs. The results indicate that MKP-3 phosphatase activity is not required for MKP-3/FOXO1 interaction but is essential for FOXO1 nuclear translocation and MKP-3 promoted gluconeogenesis. Compared to GFP control (1±0.38), MKP-3 increased G6Pase gene expression by 242% (3.42±0.62) while inactive MKP-3 does not change G6Pase expression (0.98±0.17). The residues 200-260 of MKP-3 and the residues 360-456 of FOXO1 are essential for mediating MKP-3/FOXO1 interaction. Interestingly, ERK phosphorylation deficient but not Akt phosphorylation deficient FOXO1 mutant lost interaction with MKP-3. Furthermore, in vivo experiments showed that Akt phosphorylation resistant FOXO1 3A mutant is sufficient to rescue the hypoglycemia caused by MKP-3 knock down in the liver of lean mice (from 141±6.78 to 209±14.64 mg/dL). CONCLUSIONS/SIGNIFICANCE: 1) Critical residues mediating MKP-3/FOXO1 interaction have been identified; 2) ERK phosphorylation deficient FOXO1 mutant is as potent as Akt phosphorylation deficient FOXO1 mutant in activating transcription of gluconeogenic genes; 3) Constitutively active FOXO1 can rescue the hypoglycemic effect caused by reduced hepatic MKP-3 expression in vivo.

Ischemia/reperfusion-induced MKP-3 impairs endothelial NO formation via inactivation of ERK1/2 pathway.

Mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases. Endothelial cells express multiple MKP family members, such as MKP-3. However, the effects of MKP-3 on endothelial biological processes have not yet been fully elucidated. Here, we address the association between MKP-3 and endothelial Nitric oxide (NO) formation under ischemia/reperfusion (IS/RP) condition. Human umbilical vein endothelial cells (HUVECs) were subjected to IS/RP treatment. The MKP-3 expression and NO formation were examined. IS/RP induced endothelial MKP-3 expression and inhibited eNOS expression and NO formation, accompanied by an increase of endothelial apoptosis. The siRNA experiments showed that MKP-3 was an important mediator in impairing eNOS expression and NO production in endothelial cells. Transfection of HUVECs with constitutively active ERK plasmids suggested that the above mentioned effect of MKP-3 was via inactivation of ERK1/2 pathway. Furthermore, impairment of eNOS expression was restored by treatment of histone deacetylase (HDAC) inhibitor and related to histone deacetylation and recruitment of HDAC1 to the eNOS promoter. Finally, Salvianolic acid A (SalA) markedly attenuated induction of MKP-3 and inhibition of eNOS expression and NO formation under endothelial IS/RP condition. Overall, these results for the first time demonstrated that IS/RP inhibited eNOS expression by inactivation of ERK1/2 and recruitment of HDAC1 to the gene promoter, leading to decreased NO formation through a MKP-3-dependent mechanism in endothelial cells, and SalA has therapeutic significance in protecting endothelial cells from impaired NO formation in response to IS/RP.

Dual-specific phosphatase-6 (Dusp6) and ERK mediate AMPA receptor-induced oligodendrocyte death.

Oligodendrocytes, the myelinating cells of the CNS, are highly vulnerable to glutamate excitotoxicity, a mechanism involved in tissue damage in multiple sclerosis. Thus, understanding oligodendrocyte death at the molecular level is important to develop new therapeutic approaches to treat the disease. Here, using microarray analysis and quantitative PCR, we observed that dual-specific phosphatase-6 (Dusp6), an extracellular regulated kinase-specific phosphatase, is up-regulated in oligodendrocyte cultures as well as in optic nerves after AMPA receptor activation. In turn, Dusp6 is overexpressed in optic nerves from multiple sclerosis patients before the appearance of evident damage in this structure. We further analyzed the role of Dusp6 and ERK signaling in excitotoxic oligodendrocyte death and observed that AMPA receptor activation induces a rapid increase in ERK1/2 phosphorylation. Blocking Dusp6 expression, which enhances ERK1/2 phosphorylation, significantly diminished AMPA receptor-induced oligodendrocyte death. In contrast, MAPK/ERK pathway inhibition with UO126 significantly potentiates excitotoxic oligodendrocyte death and increases cytochrome c release, mitochondrial depolarization, and mitochondrial calcium overload produced by AMPA receptor stimulation. Upstream analysis demonstrated that MAPK/ERK signaling alters AMPA receptor properties. Indeed, Dusp6 overexpression as well as incubation with UO126 produced an increase in AMPA receptor-induced inward currents and cytosolic calcium overload. Together, these data suggest that levels of phosphorylated ERK, controlled by Dusp6 phosphatase, regulate glutamate receptor permeability and oligodendroglial excitotoxicity. Therefore, targeting Dusp6 may be a useful strategy to prevent oligodendrocyte death in multiple sclerosis and other diseases involving CNS white matter.

Infrequent methylation of the DUSP6 phosphatase in endometrial cancer.

OBJECTIVE: Dual-specificity phosphatase six (DUSP6, MKP3, or PYST1) dephosphorylates phosphotyrosine and phosphothreonine residues on ERK-2 (MAPK1) to inactivate the ERK-2 kinase. DUSP6 is a critical regulator of the ERK signaling cascade and has been implicated as a tumor suppressor. DNA methylation in the first intron of DUSP6 abrogates expression in a subset of pancreatic cancers. We sought to determine whether DUSP6 was similarly silenced by methylation in endometrial cancer, a tumor type in which there is frequent activation of the ERK pathway. METHODS: One hundred and nine endometrial cancers were analyzed for DUSP6 methylation using combined bisulfite restriction analysis (COBRA). The cohort included 70 primary endometrioid endometrial cancers, 21 primary endometrial tumors of adverse histological types, and 18 endometrial cancer cell lines. Primary tumors, cell lines, and normal endometrial tissues were analyzed for DUSP6 mRNA levels using quantitative RT-PCR and pERK levels by Western blots and/or immunohistochemistry. RESULTS: Methylation of the first intron of the DUSP6 gene was seen in 1/91 primary endometrial cancers investigated. The methylated tumor was also methylated at the more 5' regulatory region of DUSP6. Q-RT-PCR revealed that DUSP6 transcript levels varied widely in primary endometrial tumors. DUSP6 mRNA levels did not correlate with pERK status in primary tumors, consistent with the existence of negative feedback loops activated by pERK that result in transcription of DUSP6. CONCLUSION: DUSP6 methylation is a rare event in endometrial cancer. Silencing of the DUSP6 phosphatase is unlikely to contribute to constitutive activation of the ERK kinase cascade in endometrial cancer.