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.

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.

Identification of Vaccinia-H1 Related Phosphatase as an Anticancer Target for 1,2,3,4,6-O-Pentagalloylglucose.

Protein tyrosine phosphatases are involved in diverse human diseases, including cancer, diabetes and inflammatory disorders. Loss of Vaccinia-H1 related phosphatase (VHR) has been shown to arrest at the G1-S and G2-M transitions of the cell cycle, and to increases cell death of prostate cancer cells through JNK activation, suggesting that VHR can be considered as an anticancer target. In this study, 658 natural products were screened through in vitro enzyme assay to identify VHR inhibitor. Among the VHR-inhibitory compounds, 1,2,3,4,6-O-pentagalloylglucose (PGG) was selected for further study as it has been reported to show antitumor effects against tumor model mice, but its direct target has not been identified. PGG inhibited the catalytic activity of VHR (Ki =53 nm) in vitro. Furthermore, the incubation of HeLa cervical cancer cells with PGG dramatically decreased cell viability and markedly increased the protein levels of the cleaved PARP, a hallmark of apoptosis. In addition, treatment of HeLa cells with PGG significantly reduced the protein levels of cyclin D1, Bcl-2 and STAT3 phosphorylation. Taken together, these results suggest that PGG could be a potential therapeutic candidate for the treatment of cervical cancer through VHR inhibition.

Dusp3 and Psme3 are associated with murine susceptibility to Staphylococcus aureus infection and human sepsis.

Using A/J mice, which are susceptible to Staphylococcus aureus, we sought to identify genetic determinants of susceptibility to S. aureus, and evaluate their function with regard to S. aureus infection. One QTL region on chromosome 11 containing 422 genes was found to be significantly associated with susceptibility to S. aureus infection. Of these 422 genes, whole genome transcription profiling identified five genes (Dcaf7, Dusp3, Fam134c, Psme3, and Slc4a1) that were significantly differentially expressed in a) S. aureus -infected susceptible (A/J) vs. resistant (C57BL/6J) mice and b) humans with S. aureus blood stream infection vs. healthy subjects. Three of these genes (Dcaf7, Dusp3, and Psme3) were down-regulated in susceptible vs. resistant mice at both pre- and post-infection time points by qPCR. siRNA-mediated knockdown of Dusp3 and Psme3 induced significant increases of cytokine production in S. aureus-challenged RAW264.7 macrophages and bone marrow derived macrophages (BMDMs) through enhancing NF-κB signaling activity. Similar increases in cytokine production and NF-κB activity were also seen in BMDMs from CSS11 (C57BL/6J background with chromosome 11 from A/J), but not C57BL/6J. These findings suggest that Dusp3 and Psme3 contribute to S. aureus infection susceptibility in A/J mice and play a role in human S. aureus infection.

Specificity profiling of dual specificity phosphatase vaccinia VH1-related (VHR) reveals two distinct substrate binding modes.

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/ϕ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/ϕ)(T/S)(D/E)pY(G/A/S/Q) (where ϕ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1-6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp(164), which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.

DUSP3/VHR is a pro-angiogenic atypical dual-specificity phosphatase.

BACKGROUND: DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies showed that DUSP3 is a negative regulator of ERK and JNK pathways in several cell lines. On the other hand, DUSP3 is implicated in human cancer. It has been alternatively described as having tumor suppressive and oncogenic properties. Thus, the available data suggest that DUSP3 plays complex and contradictory roles in tumorigenesis that could be cell type-dependent. Since most of these studies were performed using recombinant proteins or in cell-transfection based assays, the physiological function of DUSP3 has remained elusive. RESULTS: Using immunohistochemistry on human cervical sections, we observed a strong expression of DUSP3 in endothelial cells (EC) suggesting a contribution for this phosphatase to EC functions. DUSP3 downregulation, using RNA interference, in human EC reduced significantly in vitro tube formation on Matrigel and spheroid angiogenic sprouting. However, this defect was not associated with an altered phosphorylation of the documented in vitro DUSP3 substrates, ERK1/2, JNK1/2 and EGFR but was associated with an increased PKC phosphorylation. To investigate the physiological function of DUSP3, we generated Dusp3-deficient mice by homologous recombination. The obtained DUSP3-/- mice were healthy, fertile, with no spontaneous phenotype and no vascular defect. However, DUSP3 deficiency prevented neo-vascularization of transplanted b-FGF containing Matrigel and LLC xenograft tumors as evidenced by hemoglobin (Hb) and FITC-dextran quantifications. Furthermore, we found that DUSP3 is required for b-FGF-induced microvessel outgrowth in the aortic ring assay. CONCLUSIONS: All together, our data identify DUSP3 as a new important player in angiogenesis.

Proteomic, cellular, and network analyses reveal new DUSP3 interactions with nucleolar proteins in HeLa cells.

DUSP3 (or Vaccinia virus phosphatase VH1-related; VHR) is a small dual-specificity phosphatase known to dephosphorylate c-Jun N-terminal kinases and extracellular signal-regulated kinases. In human cervical cancer cells, DUSP3 is overexpressed, localizes preferentially to the nucleus, and plays a key role in cellular proliferation and senescence triggering. Other DUSP3 functions are still unknown, as illustrated by recent and unpublished results from our group showing that this enzyme mediates DNA damage response or repair processes. In this study, we sought to identify new interactions between DUSP3 and proteins directly or indirectly involved in or correlated with its biological roles in HeLa cells exposed to gamma or UV radiation. By using GST-DUSP as bait, we pulled down interacting proteins and identified them by LC-MS/MS. Of the 46 proteins obtained, six hits were extensively validated by immune techniques; the proteins Nucleophosmin, HnRNP C1/C2, and Nucleolin were the most promising targets found to directly interact with DUSP3. We then analyzed the DUSP3 interactomes using physical protein-protein interaction networks using our hits as the seed list. The validated hits as well as unvalidated hits fluctuated on the DUSP3 interactomes of HeLa cells, independent of the time post radiation, which confirmed our proteomic and experimental data and clearly showed the proximity of DUSP3 to proteins involved in processes intimately related to DNA repair and senescence, such as Ku70 and Tert, via interactions with nucleolar proteins, which were identified in this study, that regulate DNA/RNA structure and functions.

Vaccinia H1-related phosphatase is a phosphatase of ErbB receptors and is down-regulated in non-small cell lung cancer.

Vaccinia H1-related phosphatase (VHR) is classified as a dual specificity phosphatase. Unlike typical dual specificity phosphatases, VHR lacks the MAPK-binding domain and shows poor activity against MAPKs. We found that EGF receptor (EGFR) was a direct substrate of VHR and that overexpression of VHR down-regulated EGFR phosphorylation, particularly at Tyr-992 residue. Expression of VHR inhibited the activation of phospholipase Cγ and protein kinase C, both downstream effectors of Tyr-992 phosphorylation of EGFR. Decreasing VHR expression by RNA interference caused higher EGFR phosphorylation at Tyr-992. In addition to EGFR, VHR also directly dephosphorylated ErbB2. Consistent with these results, suppression of VHR augmented the foci formation ability of H1299 non-small cell lung cancer (NSCLC) cells, whereas overexpression of VHR suppressed cell growth in both two- and three-dimensional cultures. Expression of VHR also suppressed tumor formation in a mouse xenograft model. Furthermore, VHR expression was significantly lower in NSCLC tissues in comparison to that in normal lung tissues. Collectively, this study shows that down-regulation of VHR expression enhances the signaling of ErbB receptors and may be involved in NSCLC pathogenesis.

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.

Dual-specificity phosphatase 3 deletion promotes obesity, non-alcoholic steatohepatitis and hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic hepatic pathology in Western countries. It encompasses a spectrum of conditions ranging from simple steatosis to more severe and progressive non-alcoholic steatohepatitis (NASH) that can lead to hepatocellular carcinoma (HCC). Obesity and related metabolic syndrome are important risk factors for the development of NAFLD, NASH and HCC. DUSP3 is a small dual-specificity protein phosphatase with a poorly known physiological function. We investigated its role in metabolic syndrome manifestations and in HCC using a mouse knockout (KO) model. While aging, DUSP3-KO mice became obese, exhibited insulin resistance, NAFLD and associated liver damage. These phenotypes were exacerbated under high fat diet (HFD). In addition, DEN administration combined to HFD led to rapid HCC development in DUSP3-KO compared to wild type (WT) mice. DUSP3-KO mice had more serum triglycerides, cholesterol, AST and ALT compared to control WT mice under both regular chow diet (CD) and HFD. The level of fasting insulin was higher compared to WT mice, though, fasting glucose as well as glucose tolerance were normal. At the molecular level, HFD led to decreased expression of DUSP3 in WT mice. DUSP3 deletion was associated with increased and consistent phosphorylation of the insulin receptor (IR) and with higher activation of the downstream signaling pathway. In conclusion, our results support a new role for DUSP3 in obesity, insulin resistance, NAFLD and liver damage.

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.

Identification of serum miR-1915-3p and miR-455-3p as biomarkers for breast cancer.

Breast cancer is one of the most malignant diseases in women worldwide. Serum microRNAs (miRNAs), with the characteristics of high sensitivity and specificity, have recently attracted more attentions to serve as potential biomarkers for tumor diseases. In this study, 194 breast cancer patients' serum samples were collected before surgery and enrolled into different groups based on their diagnostic information. To search for breast cancer diagnostic biomarkers, serum miRNAs were screened by microarray in pooled samples of healthy volunteers and breast cancer patients in different clinical stages. The miRNAs were further verified in each individual patient's serum samples in diagnostic and predictive sets. The serum level of miR-1915-3p was upregulated and miR-455-3p was downregulated significantly in breast cancer patients compared with healthy volunteers. Furthermore, the patients with infiltrating carcinoma or lymph node metastasis had a higher serum level of miR-1915-3p and lower serum level of miR-455-3p than patients with the carcinoma in situ or patients without lymph node metastasis. ROC analysis suggested that miR-1915-3p and miR-455-3p had the potential as a promising serum diagnostic and predictive biomarkers of breast cancer. miR-1915-3p was over-expressed in certain human breast cancer cells. Functional experiments in vitro showed that miR-1915-3p enhanced cell proliferative and migrational abilities. Overexpression of miR-1915-3p repressed target gene DUSP3 and activated ERK1/2. Collectively, this study provided a new insight that miR-1915-3p might play a role in the development of breast cancer and that serum miR-1915-3p and miR-455-3p could serve as diagnostic and predictive biomarkers for breast cancer.

Identification of a potent inhibitor of human dual-specific phosphatase, VHR, from computer-aided and NMR-based screening to cellular effects.

Human vaccinia H1-related phosphatase (VHR) is a dual-specific phosphatase (DSPs) that plays an important role in the mitogen-activated protein (MAP) kinase cascade regulation. It is also a potential drug target for diseases that are related to immune response. By combining a virtual and NMR-based ligand-screening strategy, we successfully identified four VHR inhibitors, of which GATPT ((glucosamine-aminoethoxy)triphenyltin) can bind to VHR with a K(i) value of 2.54 muM. The putative binding mode of GATPT was constructed by a molecular docking simulation to provide structural insights into the ligand-binding mechanism. Furthermore, we found that this compound can significantly inhibit the dephosphorylation of the extracellular regulated kinases (ERKs), and c-Jun N-terminal kinases (JNKs) and block the G(1)-S phase transition in the cell cycle. Therefore, GATPT is a promising lead structure for designing more effective inhibitors of VHR.

Leveraging Reciprocity to Identify and Characterize Unknown Allosteric Sites in Protein Tyrosine Phosphatases.

Drug-like molecules targeting allosteric sites in proteins are of great therapeutic interest; however, identification of potential sites is not trivial. A straightforward approach to identify hidden allosteric sites is demonstrated in protein tyrosine phosphatases (PTP) by creation of single alanine mutations in the catalytic acid loop of PTP1B and VHR. This approach relies on the reciprocal interactions between an allosteric site and its coupled orthosteric site. The resulting NMR chemical shift perturbations (CSPs) of each mutant reveal clusters of distal residues affected by acid loop mutation. In PTP1B and VHR, two new allosteric clusters were identified in each enzyme. Mutations in these allosteric clusters altered phosphatase activity with changes in kcat/KM ranging from 30% to nearly 100-fold. This work outlines a simple method for identification of new allosteric sites in PTP, and given the basis of this method in thermodynamics, it is expected to be generally useful in other systems.

Diagnostic accuracy of a selected signature gene set that discriminates active pulmonary tuberculosis and other pulmonary diseases.

OBJECTIVE: We validated the accuracy of host selected signature gene set using unstimulated whole blood (WB), and peripheral blood mononuclear cells (PBMC) in the diagnosis of tuberculosis (TB). METHODS: The unstimulated WB and PBMC from 1417 individuals with active pulmonary TB patients, other lung diseases and healthy participants were analyzed using real time polymerase chain reaction (RT-PCR). RESULTS: The WB cohort test demonstrates that the combination of GBP5 and KLF2 can differentiate active TB versus HC with sensitivity and specificity of 77.8% and 87.1%, respectively; but most importantly active TB versus OD with sensitivity and specificity of 96.1% and 85.2%, respectively. Again during treatment course, the TB score of GBP5 and KLF2, analytes secretion and clinical parameters were found to be associated in disease progression. In the PBMC cohort test, we found that the only and best discriminatory combination was GBP5, DUSP3 and KLF2 inthe active TB versus HC with a sensitivity and specificity of 76.4% and 85.9%, respectively. CONCLUSIONS: Our study reveals that GBP5 and KLF2 may be useful as a diagnostic tool for active TB, also the two-gene set may serve as surrogate biomarkers for monitoring TB therapy.

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.

Discrimination of Dysplastic Nevi from Common Melanocytic Nevi by Cellular and Molecular Criteria.

Dysplastic nevi (DNs), also known as Clark's nevi or atypical moles, are distinguished from common melanocytic nevi by variegation in pigmentation and clinical appearance, as well as differences in tissue patterning. However, cellular and molecular differences between DNs and common melanocytic nevi are not completely understood. Using cDNA microarray, quantitative RT-PCR, and immunohistochemistry, we molecularly characterized DNs and analyzed the difference between DNs and common melanocytic nevi. A total of 111 probesets (91 annotated genes, fold change > 2.0 and false discovery rate < 0.25) were differentially expressed between the two lesions. An unexpected finding in DNs was altered differentiation and activation of epidermal keratinocytes with increased expression of hair follicle-related molecules (keratin 25, trichohyalin, ribonuclease, RNase A family, 7) and inflammation-related molecules (S100A7, S100A8) at both genomic and protein levels. The immune microenvironment of DNs was characterized by an increase of T helper type 1 (IFNγ) and T helper type 2 (IL13) cytokines as well as an upregulation of oncostatin M and CXCL1. DUSP3, which regulates cellular senescence, was identified as one of the disease discriminative genes between DNs and common melanocytic nevi by three independent statistical approaches and its altered expression was confirmed by immunohistochemistry. The molecular and cellular changes in which the epidermal-melanin unit undergoes follicular differentiation as well as upregulation of defined cytokines could drive complex immune, epidermal, and pigmentary alterations.

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.