DUSP3 modulates IRES-dependent translation of mRNAs through dephosphorylation of the HNRNPC protein in cells under genotoxic stimulus.

BACKGROUND INFORMATION: The dual-specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC-ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme. RESULTS: The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans-acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr-phosphorylated HNRNPC preferentially binds to IRES-containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c-MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated-HNRNPC/RNA interaction reduces HNRNPC-p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA-dependent manner. CONCLUSIONS AND SIGNIFICANCE: Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC-ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM-domain proteins.

Gastric cancer cell-derived exosomal miR-541-5p induces M2 macrophage polarization through DUSP3/JAK2/STAT3 pathway.

PURPOSE: Exosomal microRNAs have been identified as important mediators of communication between tumor cells and macrophages in the microenvironment. miR-541-5p was reported to be involved in hepatocellular carcinoma progression, but its role in gastric cancer (GC) and in GC cell-macrophage crosstalk is unknown. METHODS: Cell proliferation, migration and invasion were respectively assessed by CCK-8 assay, scratch and Transwell assays. RT-qPCR was used to detect the level of miR-541-5p, macrophage markers and DUSP3. The percentage of CD11b+CD206+ cell population was analyzed by flow cytometry. Western blotting was employed to evaluate DUSP3-JAK2/STAT3 pathway proteins and exosome markers. The interaction between miR-541-5p and DUSP3 was verified by luciferase assay. RESULTS: The results showed that miR-541-5p was upregulated in GC tissues and cells, and stimulated GC cell growth, migration and invasion in vitro. GC cells induce M2 macrophage polarization by secreting the exosomal miR-541-5p. Exosomal miR-541-5p maintained JAK2/STAT3 pathway activation in macrophages by targeting negative regulation of DUSP3. Inhibiting miR-541-5p significantly limited tumor growth in vivo. CONCLUSION: In conclusion, miR-541-5p promotes GC cell progression. GC cells may induce macrophage M2 polarization through the exosomal miR-541-5p-mediated DUSP3/JAK2/STAT3 pathway. miR-541-5p may be a potential therapeutic target for GC.

The role of dual-specificity phosphatase 3 in melanocytic oncogenesis.

Dual-specificity phosphatase 3 (DUSP3), also known as Vaccinia H1-related phosphatase, is a protein tyrosine phosphatase that typically performs its major role in the regulation of multiple cellular functions through the dephosphorylation of its diverse and constantly expanding range of substrates. Many of the substrates described so far as well as alterations in the expression or the activity of DUSP3 itself are associated with the development and progression of various types of neoplasms, indicating that DUSP3 may be an important player in oncogenesis and a promising therapeutic target. This review focuses exclusively on DUSP3's contribution to either benign or malignant melanocytic oncogenesis, as many of the established culprit pathways and mechanisms constitute DUSP3's regulatory targets, attempting to synthesize the current knowledge on the matter. The spectrum of the DUSP3 interactions analysed in this review covers substrates implicated in cellular growth, cell cycle, proliferation, survival, apoptosis, genomic stability/repair, adhesion and migration of tumor melanocytes. Furthermore, the speculations raised, based on the evidence to date, may be considered a fundament for potential research regarding the oncogenesis, evolution, management and therapeutics of melanocytic tumors.

DUSP3 regulates phosphorylation-mediated degradation of occludin and is required for maintaining epithelial tight junction.

BACKGROUND: Tight junctions (TJ) are multi-protein complexes that hold epithelial cells together and form structural and functional barriers for maintaining proper biological activities. Dual specificity phosphatase 3 (DUSP3), a suppressor of multiple protein tyrosine (Tyr) kinases, is decreased in lung cancer tissues. Here we demonstrated the role of DUSP3 in regulation of epithelial TJ. METHODS: Barrier functions of TJ were examined in wild-type or DUSP3-deficient lung epithelial cells. Animal and clinical data were analyzed for the association between DUSP3 deficiency and lung cancer progression. Proximity ligation assay, immunoblotting, and phosphatase assay were performed to study the effect of DUSP3 on the TJ protein occludin (OCLN). Mutations of Tyr residues on OCLN showed the role of Tyr phosphorylation in regulating OCLN. RESULTS: Compared to those of the DUSP3-expressing cells, we found the expression and distribution of ZO-1, a TJ-anchoring molecule, were abnormal in DUSP3-deficient cells. OCLN had an increased phosphorylation level in DUSP3-deficient cells. We identified that OCLN is a direct substrate of DUSP3. DUSP3 regulated OCLN ubiquitination and degradation through decreasing OCLN tyrosine phosphorylation directly or through suppressing focal adhesion kinase, the OCLN kinase. CONCLUSION: Our study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases.

Protein tyrosine kinases (PTK), discovered in the 1970s, have been considered master regulators of biological processes with high clinical significance as targets for human diseases. Their actions are countered by protein tyrosine phosphatases (PTP), enzymes yet underrepresented as drug targets because of the high homology of their catalytic domains and high charge of their catalytic pocket. This scenario is still worse for some PTP subclasses, for example, for the atypical dual-specificity phosphatases (ADUSPs), whose biological functions are not even completely known. In this sense, the present work focuses on the dual-specificity phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-promoting enzyme in different types of human cancers. Furthermore, DUSP3 has other biological functions involving immune response mediation, thrombosis, hemostasis, angiogenesis, and genomic stability that occur through either MAPK-dependent or MAPK-independent mechanisms. This broad spectrum of actions is likely due to the large substrate diversity and molecular mechanisms that are still under scrutiny. The growing advances in characterizing new DUSP3 substrates will allow the development of pharmacological inhibitors relevant for possible future clinical trials. This review covers all aspects of DUSP3, since its gene cloning and crystallographic structure resolution, in addition to its classical and novel substrates and the biological processes involved, followed by an update of what is currently known about the DUSP3/VHR-inhibiting compounds that might be considered potential drugs to treat human diseases.

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways.

BACKGROUND: Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown. METHODS: Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed. RESULTS: The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation. CONCLUSION: The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair. GENERAL SIGNIFICANCE: The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.

Aberrantly methylated DNA regions lead to low activation of CD4+ T-cells in IgA nephropathy.

IgAN (IgA nephropathy) is the most common form of primary glomerulonephritis worldwide and has a strong genetic component. In this setting, DNA methylation could also be an important factor influencing this disease. We performed a genome-wide screening for DNA methylation in CD4(+) T-cells from IgAN patients and found three regions aberrantly methylated influencing genes involved in the response and proliferation of CD4(+) T-cells. Two hypomethylated regions codified genes involved in TCR (T-cell receptor) signalling, TRIM27 (tripartite motif-containing 27) and DUSP3 (dual-specificity phosphatase 3), and an hypermethylated region included the VTRNA2-1 (vault RNA 2-1) non-coding RNA, also known as miR-886 precursor. We showed that the aberrant methylation influences the expression of these genes in IgAN patients. Moreover, we demonstrated that the hypermethylation of the miR-886 precursor led to a decreased CD4(+) T-cell proliferation following TCR stimulation and to the overexpression of TGFß (transforming growth factor ß). Finally, we found a Th1/Th2 imbalance in IgAN patients. The IL (interleukin)-2/IL-5 ratio was notably higher in IgAN patients and clearly indicated a Th1 shift. In conclusion, we identified for the first time some specific DNA regions abnormally methylated in IgAN patients that led to the reduced TCR signal strength of the CD4(+) T-cells and to their anomalous response and activation that could explain the T-helper cell imbalance. The present study reveals new molecular mechanisms underlying the abnormal CD4(+) T-cell response in IgAN patients.

Perspective: Tyrosine phosphatases as novel targets for antiplatelet therapy.

Arterial thrombosis is the primary cause of most cases of myocardial infarction and stroke, the leading causes of death in the developed world. Platelets, highly specialized cells of the circulatory system, are key contributors to thrombotic events. Antiplatelet drugs, which prevent platelets from aggregating, have been very effective in reducing the mortality and morbidity of these conditions. However, approved antiplatelet therapies have adverse side effects, most notably the increased risk of bleeding. Moreover, there remains a considerable incidence of arterial thrombosis in a subset of patients receiving currently available drugs. Thus, there is a pressing medical need for novel antiplatelet agents with a more favorable safety profile and less patient resistance. The discovery of novel antiplatelet targets is the matter of intense ongoing research. Recent findings demonstrate the potential of targeting key signaling molecules, including kinases and phosphatases, to prevent platelet activation and aggregation. Here, we offer perspectives to targeting members of the protein tyrosine phosphatase (PTP) superfamily, a major class of enzymes in signal transduction. We give an overview of previously identified PTPs in platelet signaling, and discuss their potential as antiplatelet drug targets. We also introduce VHR (DUSP3), a PTP that we recently identified as a major player in platelet biology and thrombosis. We review our data on genetic deletion as well as pharmacological inhibition of VHR, providing proof-of-principle for a novel and potentially safer VHR-based antiplatelet therapy.

Dehydroandrographolide facilitates M2 macrophage polarization by downregulating DUSP3 to inhibit sepsis-associated acute kidney injury.

BACKGROUND: Sepsis is perceived as lethal tissue damage and significantly increases mortality in combination with acute kidney injury (AKI). M2 macrophages play important roles in the secretion of anti-inflammatory and tissue repair mediators. We aimed to study the role of Dehydroandrographolide (Deh) in sepsis-associated AKI in vitro and in vivo through lipopolysaccharide (LPS)-induced macrophages model and cecal ligation and puncture-induced AKI mice model, and to reveal the mechanism related to M2 macrophage polarization. METHODS: Enzyme-linked immunosorbent assay kits were used to assess the levels of inflammatory factors. Expression of markers related to M1 macrophages and M2 macrophages were analyzed. Additionally, dual specificity phosphatase 3 (DUSP3) expression was tested. Cell apoptosis was evaluated by flow cytometry analysis and terminal-deoxynucleotidyl transferase-mediated nick end labeling staining. Moreover, renal histological assessment was performed by using hematoxylin and eosin staining. RESULTS: Deh reduced inflammation of THP-1-derived macrophages exposed to LPS. Besides, Deh induced the polarization of M1 macrophages to M2 and downregulated DUSP3 expression in THP-1-derived macrophages under LPS conditions. Further, DUSP3 overexpression reversed the impacts of Deh on the inflammation and M2 macrophages polarization of THP-1-derived macrophages stimulated by LPS. Additionally, human proximal tubular epithelial cells (HK-2) in the condition medium from DUSP3-overexpressed THP-1-derived macrophages treated with LPS and Deh displayed decreased viability and increased apoptosis and inflammation. The in vivo results suggested that Deh improved the renal function, ameliorated pathological injury, induced the polarization of M1 macrophages to M2, suppressed inflammation and apoptosis, and downregulated DUSP3 expression in sepsis-induced mice. CONCLUSION: Deh facilitated M2 macrophage polarization by downregulating DUSP3 to inhibit septic AKI.

Dual Specificity Phosphatase 3 (DUSP3) Knockdown Alleviates Acute Myocardial Infarction Damage via Inhibiting Apoptosis and Inflammation.

BACKGROUND: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response and is associated with ischemia/reperfusion (I/R). However, the precise function of DUSP3 in acute myocardial infarction (AMI) remains to be established. METHODS: In this study, the AMI model in vivo was established in mice by permanent left anterior descending coronary artery (LAD) occlusion, and primary neonatal mouse cardiomyocytes were treated with hypoxia for 12 hours to mimic AMI in vitro. Sh-DUSP3 and AAV9-sh-DUSP3 were used to knock down the DUSP3 expression. LVEF%, LVFS%, SOD1, and HO-1 level, and TTC staining were used to test the cardiac function. Flow cytometric analysis, Western blot, and TUNEL staining were used to investigate the effect of DUSP3 knockdown on apoptosis. Moreover, we detect inflammatory factors expression and oxidative stress by ELISA. Besides, we investigate DUSP3 expression by RT-qPCR. RESULTS: Our findings determined the role of DUSP3 in the progression of AMI. And demonstrated that DUSP3 knockdown alleviated oxidative stress, inflammation, and apoptosis. In addition, our results indicated that DUSP3 knockdown could regulate the expression of p-NF-κB, ICAM1, and VCAM1. CONCLUSION: Our results demonstrated that the knockdown of DUSP3 could effectively alleviate AMI symptoms and be mediated through the NF-κB signaling pathway.

The genetic deletion of the Dual Specificity Phosphatase 3 (DUSP3) attenuates kidney damage and inflammation following ischaemia/reperfusion injury in mouse.

AIM: Dual Specificity Phosphatase 3 (DUSP3) regulates the innate immune response, with a putative role in angiogenesis. Modulating inflammation and perfusion contributes to renal conditioning against ischaemia/reperfusion (I/R). We postulate that the functional loss of DUSP3 is associated with kidney resistance to I/R. METHODS: Ten C57BL/6 male WT and Dusp3-/- mice underwent right nephrectomy and left renal I/R (30 min/48 hours). Renal injury was assessed based on serum levels of urea (BUN) and Jablonski score. The expression of CD31 and VEGF vascular markers was quantified by RT-qPCR and immuno-staining. Renal resistivity index (RRI) was measured in vivo by Doppler ultrasound. Comparative phosphoproteomics was conducted using IMAC enrichment of phosphopeptides. Inflammatory markers were quantified at both mRNA and protein levels in ischaemic vs non-ischaemic kidneys in WT vs Dusp3-/- . RESULTS: At baseline, we located DUSP3 in renal glomeruli and endothelial cells. CD31-positive vascular network was significantly larger in Dusp3-/- kidneys compared to WT, with a lower RRI in Dusp3-/- mice. Following I/R, BUN and Jablonski score were significantly lower in Dusp3-/- vs WT mice. Phosphoproteomics highlighted a down-regulation of inflammatory pathways and up-regulation of phospho-sites involved in cell metabolism and VEGF-related angiogenesis in Dusp3-/- vs WT ischaemic kidneys. Dusp3-/- ischaemic kidneys showed decreased mRNA levels of CD11b, TNF-α, KIM-1, IL-6, IL-1ß and caspase-3 compared to controls. The numbers of PCNA-, F4-80- and CD11b-positive cells were reduced in Dusp3-/- vs WT kidneys post-I/R. CONCLUSION: Genetic inactivation of Dusp3 is associated with kidney conditioning against I/R, possibly due to attenuated inflammation and improved perfusion.

UV Radiation-induced Impairment of Cellular Morphology and Motility is Enhanced by DUSP3/VHR Loss and FAK Activation.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.

Nucleophosmin Protein Dephosphorylation by DUSP3 Is a Fine-Tuning Regulator of p53 Signaling to Maintain Genomic Stability.

The dual-specificity phosphatase 3 (DUSP3), an atypical protein tyrosine phosphatase (PTP), regulates cell cycle checkpoints and DNA repair pathways under conditions of genotoxic stress. DUSP3 interacts with the nucleophosmin protein (NPM) in the cell nucleus after UV-radiation, implying a potential role for this interaction in mechanisms of genomic stability. Here, we show a high-affinity binding between DUSP3-NPM and NPM tyrosine phosphorylation after UV stress, which is increased in DUSP3 knockdown cells. Specific antibodies designed to the four phosphorylated NPM's tyrosines revealed that DUSP3 dephosphorylates Y29, Y67, and Y271 after UV-radiation. DUSP3 knockdown causes early nucleolus exit of NPM and ARF proteins allowing them to disrupt the HDM2-p53 interaction in the nucleoplasm after UV-stress. The anticipated p53 release from proteasome degradation increased p53-Ser15 phosphorylation, prolonged p53 half-life, and enhanced p53 transcriptional activity. The regular dephosphorylation of NPM's tyrosines by DUSP3 balances the p53 functioning and favors the repair of UV-promoted DNA lesions needed for the maintenance of genomic stability.

DUSP3 maintains genomic stability and cell proliferation by modulating NER pathway and cell cycle regulatory proteins.

The DUSP3 phosphatase regulates cell cycle, proliferation, apoptosis and senescence of different cell types, lately shown as a mediator of DNA repair processes. This work evaluated the impact of DUSP3 loss of function (lof) on DNA repair-proficient fibroblasts (MRC-5), NER-deficient cell lines (XPA and XPC) and translesion DNA synthesis (TLS)-deficient cells (XPV), after UV-radiation stress. The levels of DNA strand breaks, CPDs and 6-4-PPs have accumulated over time in all cells under DUSP3 lof, with a significant increase in NER-deficient lines. The inefficient repair of these lesions increased sub-G1 population of XPA and XPC cells 24 hours after UV treatment, notably marked by DUSP3 lof, which is associated with a reduced cell population in G1, S and G2/M phases. It was also detected an increase in S and G2/M populations of XPV and MRC-5 cells after UV-radiation exposure, which was slightly attenuated by DUSP3 lof due to a discrete increase in sub-G1 cells. The cell cycle progression was accompanied by changes in the levels of the main Cyclins (A1, B1, D1 or E1), CDKs (1, 2, 4 or 6), and the p21 Cip1 inhibitor, in a DUSP3-dependent manner. DUSP3 lof affected the proliferation of MRC-5 and XPA cells, with marked worsening of the XP phenotype after UV radiation. This work highlights the roles of DUSP3 in DNA repair fitness and in the fine control of regulatory proteins of cell cycle, essential mechanisms to maintenance of genomic stability.

Lung Cancer Therapy Targeting Histone Methylation: Opportunities and Challenges.

Lung cancer is one of the most common malignancies. In spite of the progress made in past decades, further studies to improve current therapy for lung cancer are required. Dynamically controlled by methyltransferases and demethylases, methylation of lysine and arginine residues on histone proteins regulates chromatin organization and thereby gene transcription. Aberrant alterations of histone methylation have been demonstrated to be associated with the progress of multiple cancers including lung cancer. Inhibitors of methyltransferases and demethylases have exhibited anti-tumor activities in lung cancer, and multiple lead candidates are under clinical trials. Here, we summarize how histone methylation functions in lung cancer, highlighting most recent progresses in small molecular inhibitors for lung cancer treatment.

Reduced levels of Dusp3/Vhr phosphatase impair normal spindle bipolarity in an Erk1/2 activity-dependent manner.

Dual specificity phosphatase-3 (Dusp3/Vhr) regulates cell cycle progression by counteracting the effects of mitogen-activated protein kinases (Mapk) Erk1/2 and Jnk. Despite the known upregulation of Dusp3 at M phase in mammalian cells, its mitotic functions are poorly characterized. Here, we report that loss of Dusp3 by RNAi leads to the formation of multipolar spindles in human mitotic cancer cells in an Erk1/2-dependent manner. In the phosphatase-silenced cells, the normal bipolar spindle structure was restored by chemical inhibition of Erk1/2 and ectopic overexpression of Dusp3. We propose that at M phase Dusp3 keeps Erk1/2 activity in check to facilitate normal mitosis.

Functional Analysis of Dual-Specificity Protein Phosphatases in Angiogenesis.

Therapeutic perspectives targeting angiogenesis in cancer stimulated an intense investigation of the mechanisms triggering and governing angiogenic processes. Several publications have highlighted the importance of typical dual-specificity phosphatases (DSPs) or MKPs in endothelial cells and their role in controlling different biological functions implicated in angiogenesis such as migration, proliferation, apoptosis, tubulogenesis, and cell adhesion. However, among atypical DSPs, the only one investigated in angiogenesis was DUSP3. We recently identified this DSP as a new key player in endothelial cells and angiogenesis. In this chapter we provide with detailed protocols and models used to investigate the role of DUSP3 in endothelial cells and angiogenesis. We start the chapter with an overview of the role of several DSPs in angiogenesis. We continue with providing a full description of a highly efficient transfection protocol to deplete DUSP3 using small interfering RNA (siRNA) in the primary human umbilical vein endothelial cells (HUVEC). We next describe the major assays used to investigate different processes involved in angiogenesis such as tube formation assay, proliferation assay and spheroids sprouting assay. We finish the chapter by validating our results in DUSP3-knockout mice using in vivo angiogenesis assays such as Matrigel plug and Lewis lung carcinoma cell subcutaneous xenograft model followed by anti-CD31 immunofluorescence and ex vivo aortic ring assay. All methods described can be adapted to other phosphatases and signaling molecules.

Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets.

VHR/DUSP3 phosphatase: structure, function and regulation.

Vaccinia H1-related (VHR) phosphatase, also known as dual-specificity phosphatase (DUSP) 3, is a small member of the DUSP (also called DSP) family of phosphatases. VHR has a preference for phospho-tyrosine substrates, and has important roles in cellular signaling ranging from cell-cycle regulation and the DNA damage response to MAPK signaling, platelet activation and angiogenesis. VHR/DUSP3 has been implicated in several human cancers, where its tumor-suppressing and -promoting properties have been described. We give a detailed overview of VHR/DUSP3 phosphatase and compare it with its most closely related phosphatases DUSP13B, DUSP26 and DUSP27.

Revisiting the roles of VHR/DUSP3 phosphatase in human diseases

Protein tyrosine phosphatases have long been considered key regulators of biological processes and are therefore implicated in the origins of various human diseases. Heterozygosity, mutations, deletions, and the complete loss of some of these enzymes have been reported to cause neurodegenerative diseases, autoimmune syndromes, genetic disorders, metabolic diseases, cancers, and many other physiological imbalances. Vaccinia H1-related phosphatase, also known as dual-specificity phosphatase 3, is a protein tyrosine phosphatase enzyme that regulates the phosphorylation of the mitogen-activated protein kinase signaling pathway, a central mediator of a diversity of biological responses. It has been suggested that vaccinia H1-related phosphatase can act as a tumor suppressor or tumor-promoting phosphatase in different cancers. Furthermore, emerging evidence suggests that this enzyme has many other biological functions, such as roles in immune responses, thrombosis, hemostasis, angiogenesis, and genomic stability, and this broad spectrum of vaccinia H1-related phosphatase activity is likely the result of its diversity of substrates. Hence, fully identifying and characterizing these substrate-phosphatase interactions will facilitate the identification of pharmacological inhibitors of vaccinia H1-related phosphatase that can be evaluated in clinical trials. In this review, we describe the biological processes mediated by vaccinia H1-related phosphatase, especially those related to genomic stability. We also focus on validated substrates and signaling circuitry with clinical relevance in human diseases, particularly oncogenesis.