Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

DUSP4 promotes the carcinogenesis of CCRCC via negative regulation of autophagic death.

DUSP4 is considered as an oncogenic gene. However, the effect of DUSP4 on the carcinogenesis of clear cell Renal cell carcinoma (CCRCC) is still unclear. In this study, DUSP4 mRNA levels were significantly increased in CCRCC tissues and cell lines. Furthermore, DUSP4 overexpression promotes the proliferation, migration, and tumorigenicity of CCRCC cells while DUSP4 silencing showed the opposite effects. Importantly, both autophagic activity (LC3 conversion rate and LC3 puncta formation) and total death level promoted by DUSP4 silencing were reversed by treatment with 3-MA in CCRCC cells. Moreover, the proliferation and migration of CCRCC cells inhibited by DUSP4 silencing were also recovered by suppression of autophagy with 3-MA. In conclusion, DUSP4 serves as an oncogenic gene in CCRCC carcinogenesis due to its inhibitory effect on autophagic death, indicating the potential value of DUSP4 in the diagnosis and treatment of CCRCC.

The Role of MKP-5 in Adipocyte-Macrophage Interactions during Obesity.

OBJECTIVE: In obese individuals, chronic low-grade inflammation resulting from adipocyte-macrophage interactions is a major cause of adipose tissue dysfunction and metabolic disease. This study investigated the role of MAP kinase phosphatase-5 (MKP-5) in obesity-induced inflammation during macrophage and adipocyte interactions. METHODS: High-fat diet-induced obese mice were used to explore the role of MKP-5 in obesity-induced adipose tissue inflammation. Macrophage polarization was determined by inflammatory cytokine expression in MKP-5-overexpressed or -silenced Raw264.7 cells exposed to palmitate (PA) or M1/M2 macrophage inducers. To uncover the role of MKP-5 during macrophage-adipocyte interactions, a coculture system composed of differentiated 3T3-L1 and Raw264.7 cells was employed. MAPK inhibitors were used to investigate the involvement of MAPK signaling. RESULTS: Increased MKP-5 expression was observed in adipose stromal vascular cells (SVCs) of obese mice. In Raw264.7 cells, MKP-5 promoted the switching of M1 macrophages to an M2 phenotype. Notably, MKP-5 reduced inflammation during the interaction of macrophages and adipocytes. MKP-5 overexpression in primary SVCs attenuated the expression of inflammatory mediators and increased the number of obesity-induced adipose tissue macrophages. MKP-5 suppressed PA-induced inflammation through the inactivation of P38, JNK, and ERK MAPKs. CONCLUSIONS: MKP-5 promotes macrophages to switch from the M1 to the M2 phenotype and is an inflammatory inhibitor involved in obesity-induced adipose tissue inflammation and PA-triggered macrophage inflammation via the P38, JNK, and ERK MAPK pathways. MKP-5 may be developed into a potential therapeutic target for obesity-related diseases, including type 2 diabetes mellitus and insulin resistance.

Role of dual-specificity protein phosphatase DUSP10/MKP-5 in pulmonary fibrosis.

Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP-5) is a member of the dual-specificity family of protein tyrosine phosphatases that negatively regulates p38 MAPK and the JNK. MKP-5-deficient mice exhibit improved muscle repair and reduced fibrosis in an animal model of muscular dystrophy. Here, we asked whether the effects of MKP-5 on muscle fibrosis extend to other tissues. Using a bleomycin-induced model of pulmonary fibrosis, we found that MKP-5-deficient mice were protected from the development of lung fibrosis, expressed reduced levels of hydroxyproline and fibrogenic genes, and displayed marked polarization towards an M1-macrophage phenotype. We showed that the profibrogenic effects of the transforming growth factor-ß1 (TGF-ß1) were inhibited in MKP-5-deficient lung fibroblasts. MKP-5-deficient fibroblasts exhibited enhanced p38 MAPK activity, impaired Smad3 phosphorylation, increased Smad7 levels, and decreased expression of fibrogenic genes. Myofibroblast differentiation was attenuated in MKP-5-deficient fibroblasts. Finally, we found that MKP-5 expression was increased in idiopathic pulmonary fibrosis (IPF)-derived lung fibroblasts but not in whole IPF lungs. These data suggest that MKP-5 plays an essential role in promoting lung fibrosis. Our results couple MKP-5 with the TGF-ß1 signaling machinery and imply that MKP-5 inhibition may serve as a therapeutic target for human lung fibrosis.

GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

Gliomas are the most common primary brain tumors. Their highly invasive character and the heterogeneity of active oncogenic pathways within single tumors complicate the development of curative therapies and cause poor patient prognosis. Glioma cells express the intermediate filament protein glial fibrillary acidic protein (GFAP), and the level of its alternative splice variant GFAP-δ, relative to its canonical splice variant GFAP-α, is higher in grade IV compared with lower-grade and lower malignant glioma. In this study we show that a high GFAP-δ/α ratio induces the expression of the dual-specificity phosphatase 4 (DUSP4) in focal adhesions. By focusing on pathways up- and downstream of DUSP4 that are involved in the cell-extracellular matrix interaction, we show that a high GFAP-δ/α ratio equips glioma cells to better invade the brain. This study supports the hypothesis that glioma cells with a high GFAP-δ/α ratio are highly invasive and more malignant cells, thus making GFAP alternative splicing a potential therapeutic target.-Van Bodegraven, E. J., van Asperen, J. V., Sluijs, J. A., van Deursen, C. B. J., van Strien, M. E., Stassen, O. M. J. A., Robe, P. A. J., Hol, E. M. GFAP alternative splicing regulates glioma cell-ECM interaction in a DUSP4-dependent manner.

The protective role of the MKP-5-JNK/P38 pathway in glucolipotoxicity-induced islet ß-cell dysfunction and apoptosis.

Hyperglycemia and hyperlipidemia (glycolipotoxicity)-triggered islet ß-cell dysfunction is known to drive the progression of obesity-related type 2 diabetes, however the underlying mechanisms have not been clearly elucidated. The current study aimed to investigate the role of mitogen-activated protein kinase phosphatase 5 (MKP-5) in islet cells under glucolipotoxic conditions. Using gene overexpression and knockdown approaches, we demonstrated that MKP-5 could alleviate glucolipotoxicity-induced apoptosis via the endoplasmic reticulum (ER) stress and mitochondrial apoptosis pathways owing to the altered regulation of caspase family members and ER stress-related molecules in MIN6 and primary islet cells. Overexpression of MKP-5 reversed the glucose and palmitic acid (GP)-induced impairment of insulin secretion as well as the abnormal decreases in the expression of islet functional genes, thereby maintaining the normal insulin secretory functionality, whereas the absence of MKP-5 aggravated islet cell dysfunction. In parallel, the production of ROS and increased inflammation-associated genes in response to GP were also reduced upon MKP-5 overexpression. Further, inhibition of JNK or P38 MAPK pathways resisted to glucolipotoxicity observed in MKP-5 knockdown MIN6 cells. These findings indicate that MKP-5 is an important mediator for glucolipotoxicity-induced islet cell dysfunction and apoptosis, with JNK and P38 as the critical downstream pathways.

JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Osteoblasts are versatile cells involved in multiple whole-body processes, including bone formation and immune response. Secretory amounts and patterns of osteoblast-derived proteins such as osteopontin (OPN) and osteocalcin (OCN) modulate osteoblast function. However, the regulatory mechanism of OPN and OCN expression remains unknown. Here, we demonstrate that p54/p46 c-jun N-terminal kinase (JNK) inhibition suppresses matrix mineralization and OCN expression but increases OPN expression in MC3T3-E1 cells and primary osteoblasts treated with differentiation inducers, including ascorbic acid, bone morphogenic protein-2, or fibroblast growth factor 2. Preinhibition of JNK before the onset of differentiation increased the number of osteoblasts that highly express OPN but not OCN (OPN-OBs), indicating that JNK affects OPN secretory phenotype at the early stage of osteogenic differentiation. Additionally, we identified JNK2 isoform as being critically involved in OPN-OB differentiation. Microarray analysis revealed that OPN-OBs express characteristic transcription factors, cell surface markers, and cytokines, including glycoprotein hormone α2 and endothelial cell-specific molecule 1. Moreover, we found that inhibitor of DNA binding 4 is an important regulator of OPN-OB differentiation and that dual-specificity phosphatase 16, a JNK-specific phosphatase, functions as an endogenous regulator of OPN-OB induction. OPN-OB phenotype was also observed following LPS from Porphyromonas gingivalis stimulation during osteogenic differentiation. Collectively, these results suggest that the JNK-Id4 signaling axis is crucial in the control of OPN and OCN expression during osteoblastic differentiation.-Kusuyama, J., Amir, M. S., Albertson, B. G., Bandow, K., Ohnishi, T., Nakamura, T., Noguchi, K., Shima, K., Semba, I., Matsuguchi, T. JNK inactivation suppresses osteogenic differentiation, but robustly induces osteopontin expression in osteoblasts through the induction of inhibitor of DNA binding 4 (Id4).

Dual-specificity MAP kinase phosphatases in health and disease.

It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions.

Dual-Specificity Phosphatase 9 Protects Against Nonalcoholic Fatty Liver Disease in Mice Through ASK1 Suppression.

Nonalcoholic fatty liver disease (NAFLD), ranging from nonalcoholic fatty liver to nonalcoholic steatohepatitis (NASH), is the leading cause of chronic liver diseases. Until now, no medications for NAFLD have been approved by relevant governmental agencies. Dual-specificity phosphatase 9 (Dusp9) is a member of the DUSP protein family. Dusp9 is expressed in insulin-sensitive tissues, and its expression may be modified with the development of insulin resistance (IR). However, the molecular targets and mechanisms of Dusp9 action on NAFLD and NASH remain poorly understood. In this study, using conditional liver-specific Dusp9-knockout (Dusp9-CKO) mice and Dusp9-transgenic mice, we showed that Dusp9 was a key suppressor of high-fat diet-induced hepatic steatosis and inflammatory responses and that Dusp9 deficiency aggravated high-fat high-cholesterol diet-induced liver fibrosis. Dusp9 was shown to exert its effects by blocking apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and the subsequent activation of p38 and c-Jun NH2-terminal kinase signaling. Conclusion: Hepatocyte Dusp9 prevents NAFLD and NASH progression in mice, including lipid accumulation, glucose metabolism disorders, and enhanced inflammation and liver fibrosis, in an ASK1-dependent manner; these findings suggest that Dusp9 may be a promising therapeutic target for the treatment of NAFLD and NASH.

Cyclin-dependent kinase inhibitor 3 (CDKN3) plays a critical role in prostate cancer via regulating cell cycle and DNA replication signaling.

Cyclin-dependent kinase inhibitor 3 (CDKN3) is proved to be associated with the progressing of many cancers. Whereas, its biological effects on prostate cancer (PC) are less understood. To investigate the functional mechanism of CDKN3 in PC, we examined the expression of CDKN3 in PC tissues and analyzed the disease free survival time of patients. We then transfected LNCaP and PC3 cells with siRNA-CDKN3 to silence CDKN3, and transfected 22RV1 and VCaP cells with full length CDKN3 cDNA for CDKN3 over-expression. Cell growth of these transfected cells were analyzed using CCK-8 assay. And transfected LNCaP and PC3 cells were further submitted to cell cycle, apoptosis, invasion and endogenous protein expression assays. We found that CDKN3 was highly expressed in PC and negatively correlated with disease relapse. And CDKN3 positively control the cell proliferation in prostate carcinoma cell lines. Knockdown of CDKN3 significantly promoted G1 phase arrest, elevated apoptosis rates, and suppressed cell invasion in both LNCaP and PC3 cells. Moreover, in vivo data showed that knockdown of CDKN3 expression dramatically inhibited the PC3 tumor growth in nude mouse model. Gene set enrichment analysis (GSEA) showed that cell cycle and DNA replication signaling were related with elevated CDKN3 expression. And results of western blot showed that the depletion of CDKN3 down-regulated the expression levels of cell cycle- and DNA replication-related proteins. In conclusion, our results highlight the importance of CDKN3 in PC and provide new insights into diagnostics and therapeutics of the PC.

Critical Roles of Dual-Specificity Phosphatases in Neuronal Proteostasis and Neurological Diseases.

Protein homeostasis or proteostasis is a fundamental cellular property that encompasses the dynamic balancing of processes in the proteostasis network (PN). Such processes include protein synthesis, folding, and degradation in both non-stressed and stressful conditions. The role of the PN in neurodegenerative disease is well-documented, where it is known to respond to changes in protein folding states or toxic gain-of-function protein aggregation. Dual-specificity phosphatases have recently emerged as important participants in maintaining balance within the PN, acting through modulation of cellular signaling pathways that are involved in neurodegeneration. In this review, we will summarize recent findings describing the roles of dual-specificity phosphatases in neurodegeneration and offer perspectives on future therapeutic directions.

NEAP/DUSP26 suppresses receptor tyrosine kinases and regulates neuronal development in zebrafish.

Expression of neuroendocrine-associated phosphatase (NEAP, also named as dual specificity phosphatase 26, [DUSP26]) is restricted to neuroendocrine tissues. We found that NEAP, but not its phosphatase-defective mutant, suppressed nerve growth factor (NGF) receptor TrkA and fibroblast growth factor receptor 1 (FGFR1) activation in PC12 cells upon NGF stimulation. Conversely, suppressing NEAP expression by RNA interference enhanced TrkA and FGFR1 phosphorylation. NEAP was capable of de-phosphorylating TrkA and FGFR1 directly in vitro. NEAP-orthologous gene existed in zebrafish. Morpholino (MO) suppression of NEAP in zebrafish resulted in hyper-phosphorylation of TrkA and FGFR1 as well as abnormal body postures and small eyes. Differentiation of retina in zebrafishes with NEAP MO treatment was severely defective, so were cranial motor neurons. Taken together, our data indicated that NEAP/DUSP26 have a critical role in regulating TrkA and FGFR1 signaling as well as proper development of retina and neuronal system in zebrafish.

Dual specificity phosphatase 15 regulates Erk activation in Schwann cells.

Schwann cells and oligodendrocytes are the myelinating cells of the peripheral and central nervous system, respectively. Despite having different myelin components and different transcription factors driving their terminal differentiation there are shared molecular mechanisms between the two. Sox10 is one common transcription factor required for several steps in development of myelinating glia. However, other factors are divergent as Schwann cells need the transcription factor early growth response 2/Krox20 and oligodendrocytes require Myrf. Likewise, some signaling pathways, like the Erk1/2 kinases, are necessary in both cell types for proper myelination. Nonetheless, the molecular mechanisms that control this shared signaling pathway in myelinating cells remain only partially characterized. The hypothesis of this study is that signaling pathways that are similarly regulated in both Schwann cells and oligodendrocytes play central roles in coordinating the differentiation of myelinating glia. To address this hypothesis, we have used genome-wide binding data to identify a relatively small set of genes that are similarly regulated by Sox10 in myelinating glia. We chose one such gene encoding Dual specificity phosphatase 15 (Dusp15) for further analysis in Schwann cell signaling. RNA interference and gene deletion by genome editing in cultured RT4 and primary Schwann cells showed Dusp15 is necessary for full activation of Erk1/2 phosphorylation. In addition, we show that Dusp15 represses expression of several myelin genes, including myelin basic protein. The data shown here support a mechanism by which early growth response 2 activates myelin genes, but also induces a negative feedback loop through Dusp15 to limit over-expression of myelin genes.

DUSP8 Regulates Cardiac Ventricular Remodeling by Altering ERK1/2 Signaling.

RATIONALE: Mitogen-activated protein kinase (MAPK) signaling regulates the growth response of the adult myocardium in response to increased cardiac workload or pathological insults. The dual-specificity phosphatases (DUSPs) are critical effectors, which dephosphorylate the MAPKs to control the basal tone, amplitude, and duration of MAPK signaling. OBJECTIVE: To examine DUSP8 as a regulator of MAPK signaling in the heart and its impact on ventricular and cardiac myocyte growth dynamics. METHODS AND RESULTS: Dusp8 gene-deleted mice and transgenic mice with inducible expression of DUSP8 in the heart were used here to investigate how this MAPK-phosphatase might regulate intracellular signaling and cardiac growth dynamics in vivo. Dusp8 gene-deleted mice were mildly hypercontractile at baseline with a cardiac phenotype of concentric ventricular remodeling, which protected them from progressing towards heart failure in 2 surgery-induced disease models. Cardiac-specific overexpression of DUSP8 produced spontaneous eccentric remodeling and ventricular dilation with heart failure. At the cellular level, adult cardiac myocytes from Dusp8 gene-deleted mice were thicker and shorter, whereas DUSP8 overexpression promoted cardiac myocyte lengthening with a loss of thickness. Mechanistically, activation of extracellular signal-regulated kinases 1/2 were selectively increased in Dusp8 gene-deleted hearts at baseline and following acute pathological stress stimulation, whereas p38 MAPK and c-Jun N-terminal kinases were mostly unaffected. CONCLUSIONS: These results indicate that DUSP8 controls basal and acute stress-induced extracellular signal-regulated kinases 1/2 signaling in adult cardiac myocytes that then alters the length-width growth dynamics of individual cardiac myocytes, which further alters contractility, ventricular remodeling, and disease susceptibility.

Integrin-linked Kinase Controls Renal Branching Morphogenesis via Dual Specificity Phosphatase 8.

Integrin-linked kinase (ILK) is an intracellular scaffold protein with critical cell-specific functions in the embryonic and mature mammalian kidney. Previously, we demonstrated a requirement for Ilk during ureteric branching and cell cycle regulation in collecting duct cells in vivo Although in vitro data indicate that ILK controls p38 mitogen-activated protein kinase (p38MAPK) activity, the contribution of ILK-p38MAPK signaling to branching morphogenesis in vivo is not defined. Here, we identified genes that are regulated by Ilk in ureteric cells using a whole-genome expression analysis of whole-kidney mRNA in mice with Ilk deficiency in the ureteric cell lineage. Six genes with expression in ureteric tip cells, including Wnt11, were downregulated, whereas the expression of dual-specificity phosphatase 8 (DUSP8) was upregulated. Phosphorylation of p38MAPK was decreased in kidney tissue with Ilk deficiency, but no significant decrease in the phosphorylation of other intracellular effectors previously shown to control renal morphogenesis was observed. Pharmacologic inhibition of p38MAPK activity in murine inner medullary collecting duct 3 (mIMCD3) cells decreased expression of Wnt11, Krt23, and Slo4c1 DUSP8 overexpression in mIMCD3 cells significantly inhibited p38MAPK activation and the expression of Wnt11 and Slo4c1. Adenovirus-mediated overexpression of DUSP8 in cultured embryonic murine kidneys decreased ureteric branching and p38MAPK activation. Together, these data demonstrate that Ilk controls branching morphogenesis by regulating the expression of DUSP8, which inhibits p38MAPK activity and decreases branching morphogenesis.

The Tinkerbell (Tink) Mutation Identifies the Dual-Specificity MAPK Phosphatase INDOLE-3-BUTYRIC ACID-RESPONSE5 (IBR5) as a Novel Regulator of Organ Size in Arabidopsis.

Mitogen-activated dual-specificity MAPK phosphatases are important negative regulators in the MAPK signalling pathways responsible for many essential processes in plants. In a screen for mutants with reduced organ size we have identified a mutation in the active site of the dual-specificity MAPK phosphatase indole-3-butyric acid-response5 (IBR5) that we named tinkerbell (tink) due to its small size. Analysis of the tink mutant indicates that IBR5 acts as a novel regulator of organ size that changes the rate of growth in petals and leaves. Organ size and shape regulation by IBR5 acts independently of the KLU growth-regulatory pathway. Microarray analysis of tink/ibr5-6 mutants identified a likely role for this phosphatase in male gametophyte development. We show that IBR5 may influence the size and shape of petals through auxin and TCP growth regulatory pathways.

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function.

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5(-/-) mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5(-/-) eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5(-/-) eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival.

NADPH oxidase-generated reactive oxygen species are required for stromal cell-derived factor-1α-stimulated angiogenesis.

OBJECTIVE: Reactive oxygen species (ROS) act as signaling molecules during angiogenesis; however, the mechanisms used for such signaling events remain unclear. Stromal cell-derived factor-1α (SDF-1α) is one of the most potent angiogenic chemokines. Here, we examined the role of ROS in the regulation of SDF-1α-dependent angiogenesis. APPROACH AND RESULTS: Bovine aortic endothelial cells were treated with SDF-1α, and intracellular ROS generation was monitored. SDF-1α treatment induced bovine aortic endothelial cell migration and ROS generation, with the majority of ROS generated by bovine aortic endothelial cells at the leading edge of the migratory cells. Antioxidants and nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitors blocked SDF-1α-induced endothelial migration. Furthermore, knockdown of either NOX5 or p22phox (a requisite subunit for NOX1/2/4 activation) significantly impaired endothelial motility and tube formation, suggesting that multiple NOXs regulate SDF-1α-dependent angiogenesis. Our previous study demonstrated that c-Jun N-terminal kinase 3 activity is essential for SDF-1α-dependent angiogenesis. Here, we identified that NOX5 is the dominant NOX required for SDF-1α-induced c-Jun N-terminal kinase 3 activation and that NOX5 and MAP kinase phosphatase 7 (MKP7; the c-Jun N-terminal kinase 3 phosphatase) associate with one another but decrease this interaction on SDF-1α treatment. Furthermore, MKP7 activity was inhibited by SDF-1α, and this inhibition was relieved by NOX5 knockdown, indicating that NOX5 promotes c-Jun N-terminal kinase 3 activation by blocking MKP7 activity. CONCLUSIONS: We conclude that NOX is required for SDF-1α signaling and that intracellular redox balance is critical for SDF-1α-induced endothelial migration and angiogenesis.

RNA interference against cancer/testis genes identifies dual specificity phosphatase 21 as a potential therapeutic target in human hepatocellular carcinoma.

UNLABELLED: Cancer/testis (CT) antigens have been considered therapeutic targets for treating cancers. However, a central question is whether their expression contributes to tumorigenesis or if they are functionally irrelevant by-products derived from the process of cellular transformation. In any case, these CT antigens are essential for cancer cell survival and may serve as potential therapeutic targets. Recently, the cell-based RNA interference (RNAi) screen has proven to be a powerful approach for identifying potential therapeutic targets. In this study we sought to identify new CT antigens as potential therapeutic targets for human hepatocellular carcinoma (HCC), and 179 potential CT genes on the X chromosome were screened through a bioinformatics analysis of gene expression profiles. Then an RNAi screen against these potential CT genes identified nine that were required for sustaining the survival of Focus and PLC/PRF/5 cells. Among the nine genes, the physiologically testis-restricted dual specificity phosphatase 21 (DUSP21) encoding a dual specificity phosphatase was up-regulated in 39 (33%) of 118 human HCC specimens. Ectopic DUSP21 had no obvious impact on proliferation and colony formation in HCC cells. However, DUSP21 silencing significantly suppressed cell proliferation, colony formation, and in vivo tumorigenicity in HCC cells. The administration of adenovirus-mediated RNAi and an atelocollagen/siRNA mixture against endogenous DUSP21 significantly suppressed xenograft HCC tumors in mice. Further investigations showed that DUSP21 knockdown led to arrest of the cell cycle in G1 phase, cell senescence, and expression changes of some factors with functions in the cell cycle and/or senescence. Furthermore, the antiproliferative role of DUSP21 knockdown is through activation of p38 mitogen-activated protein kinase in HCC. CONCLUSION: DUSP21 plays an important role in sustaining HCC cell proliferation and may thus act as a potential therapeutic target in HCC treatment.

The tumor suppressor CDKN3 controls mitosis.

Mitosis is controlled by a network of kinases and phosphatases. We screened a library of small interfering RNAs against a genome-wide set of phosphatases to comprehensively evaluate the role of human phosphatases in mitosis. We found four candidate spindle checkpoint phosphatases, including the tumor suppressor CDKN3. We show that CDKN3 is essential for normal mitosis and G1/S transition. We demonstrate that subcellular localization of CDKN3 changes throughout the cell cycle. We show that CDKN3 dephosphorylates threonine-161 of CDC2 during mitotic exit and we visualize CDC2(pThr-161) at kinetochores and centrosomes in early mitosis. We performed a phosphokinome-wide mass spectrometry screen to find effectors of the CDKN3-CDC2 signaling axis. We found that one of the identified downstream phosphotargets, CKß phosphorylated at serine 209, localizes to mitotic centrosomes and controls the spindle checkpoint. Finally, we show that CDKN3 protein is down-regulated in brain tumors. Our findings indicate that CDKN3 controls mitosis through the CDC2 signaling axis. These results have implications for targeted anticancer therapeutics.