Dual-specificity Phosphatase Research Articles

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.

Dual specificity phosphatase 1 attenuates inflammation-induced cardiomyopathy by improving mitophagy and mitochondrial metabolism

ObjectivesDual specificity phosphatase 1 (DUSP1) is regarded as an anti-inflammatory factor in cardiovascular disorders. Mitophagy removes damaged mitochondria and thus promotes mitochondrial regeneration. We investigated whether DUSP1 could attenuate inflammation-induced cardiomyopathy by improving mitophagy. MethodsLipopolysaccharide was used to induce septic cardiomyopathy in wild-type (WT) and DUSP1 transgenic (DUSP1TG) mice. ResultsEchocardiography revealed that lipopolysaccharide impaired heart function by reducing the cardiac systolic and diastolic capacities of WT mice. Freshly isolated single cardiomyocytes from lipopolysaccharide-treated WT mice also exhibited reduced contractile/relaxation parameters. However, DUSP1 overexpression not only maintained the mechanical properties of cardiomyocytes, but also improved heart performance. Lipopolysaccharide upregulated myocardial inflammatory gene transcription and adhesive factor expression, which increased myocardial neutrophil accumulation and cardiomyocyte apoptosis in WT mice. DUSP1 overexpression inhibited the inflammatory response and therefore promoted cardiomyocyte survival. Lipopolysaccharide disrupted mitochondrial respiration and metabolism in WT cardiomyocytes, but DUSP1 overexpression restored mitochondrial metabolism, maintained the mitochondrial membrane potential and inhibited mitochondrial reactive oxygen species production, possibly by increasing FUN14 domain-containing 1 (FUNDC1)-dependent mitophagy. Silencing of FUNDC1 abolished the protective effects of DUSP1 overexpression on cardiomyocytes and their mitochondria following lipopolysaccharide treatment. ConclusionThese results demonstrated that DUSP1 is a novel anti-inflammatory factor that protects against septic cardiomyopathy by improving FUNDC1-induced mitophagy.

DUSP1 inhibition in the treatment of high grade serous ovarian carcinoma (141.5)

<b>Objectives:</b> Dual Specificity Phosphatase (DUSP) proteins regulate signaling cascades involved in tumor progression, but there are limited studies of DUSPs in epithelial ovarian carcinoma, especially in high-grade serous ovarian carcinoma (HGSOC). The study objective was to elucidate <i>DUSP1</i>'s contribution to HGSOC progression and examine a <i>DUSP1/6</i> inhibitor (DUSPi) in therapy-resistant HGSOC. <b>Methods:</b> Bioinformatic analyses were conducted using The Cancer Genome Atlas (TCGA) Ovarian Serous Cystadenocarcinoma Firehose Database and PanCancer Atlas. qPCR was completed to determine the mRNA expression of <i>DUSP1</i>. Fifty percent inhibitory concentration of DUSPi was determined using colony formation assays. Reverse-phase protein array (RPPA) examined the effect of DUSPi on cellular signaling. The antitumoral effect of DUSPi was examined using a patient-derived xenograft (PDX) model. Quantitative data were expressed as mean +/- standard error of the mean. RPPA relative protein levels were determined for each and designated as log2 intensities and subsequently median-centered. Analysis of variance with Tukey correction was used for multiple comparisons. The level of significance was set at 0.05. <b>Results:</b> Of 24 DUSP proteins examined in HGSOC, <i>DUSP1</i> mRNA was significantly upregulated (p<0.001, one-way ANOVA). Progression-free survival (PFS) and overall survival (OS) were significantly higher in patients with low <i>DUSP1</i> expression (PFS: 15.41 vs 22.22 months, Log-rank test, p=0.0010, HR: 1.65, 95% CI: 1.213-2.259; OS: 41.52 vs 52.40 months, Log-rank test, p=0.0183, HR: 1.462, 95% CI: 1.064-2.00). Elevated <i>DUSP1</i> protein expression was noted in our tissue microarray of 137 serous ovarian carcinoma primary and recurrent tumors. Olaparib-resistant HGSOC cells had significantly increased <i>DUSP1</i> expression compared to olaparib-sensitive HGSOC cells (unpaired t-test, p<0.0001). The RPPA analysis revealed 34 proteins with significantly (p<0.05) altered expression after treatment with DUSPi. DUSPi led to the significant downregulation of <i>WEE1</i> and <i>ATR</i>. Phosphor-AMPK (AMPK-a2_pS345), part of the mTORC pathway that can serve both tumor-suppressive and oncogenic roles, was elevated and dual phosphorylated ERK (MAPK_pT202_Y204). RPPA findings were confirmed in TCGA data and by immunoblotting. A therapy-resistant PDX model was used to examine DUSPi <i>in vivo</i> activity. DUSPi led to a decrease in total flux (photons/sec), indicating tumor regression, in the mice treated with DUSPi compared to control. At the end of the study, there was no evidence of ascites or tumor burden in mice treated with DUSPi. <b>Conclusions:</b> <i>DUSP1</i> expression is associated with poor progression-free and overall survival. After treatment with a novel <i>DUSP1/6</i> inhibitor (DUSPi), cell viability was significantly reduced. We defined the differential activation of signaling following DUSPi via RPPA. DUSP inhibition significantly resulted in altering the expression of multiple pathway proteins, including increasing AMPK phosphorylation. Finally, we identified a robust <i>in vivo</i> antitumor response with DUSPi in PDX models.

Downregulated Dual-Specificity Protein Phosphatase 1 in Ovarian Carcinoma: A Comprehensive Study With Multiple Methods.

Introduction: We aimed to explore the abnormal expression of dual-specificity protein phosphatase 1 (DUSP1) and its latent molecular mechanisms in ovarian carcinoma (OVCA). Materials and Methods: Two clinical cohorts collected from two different hospitals were used to evaluate the expression of DUSP1 protein in OVCA tissues. RNA-sequencing and microarray datasets were utilised to verify DUSP1 expression at mRNA levels in both OVCA tissues and in the peripheral blood of OVCA patients. Furthermore, an integrated calculation was performed to pool the standard mean difference (SMD) from each cohort in order to comprehensively assess the expression of DUSP1 in OVCA. Furthermore, we examined the relationship among DUSP1, tumour microenvironment (TME), and chemotherapy resistance in OVCA. Moreover, we used pathway enrichment analysis to explore the underlying mechanisms of DUSP1 in OVCA. Results: A pooled SMD of −1.19 (95% CI [−2.00, −0.38], p = 0.004) with 1,240 samples revealed that DUSP1 was downregulated in OVCA at both mRNA and protein levels. The area under the receiver operating characteristic curve of 0.9235 indicated the downregulated DUSP1 in peripheral blood may have a non-invasive diagnostic value in OVCA. Through six algorithms, we identified that DUSP1 may related to tumour-infiltrating T cells and cancer associated fibroblasts (CAFs) in OVCA. Pathway enrichment demonstrated that DUSP1 might participate in the mitogen-activated protein kinase (MAPK) signalling pathway. Furthermore, DUSP1 may have relations with chemotherapy resistance, and a favourable combining affinity was observed in the paclitaxel-DUSP1 docking model. Conclusion: DUSP1 was downregulated in OVCA, and this decreasing trend may affect the infiltration of CAFs. Finally, DUSP1 may have a targeting relation with paclitaxel and participate in MAPK signaling pathways.

Renal tubular gen e biomarkers identification based on immune infiltrates in focal segmental glomerulosclerosis

Objective The present study identified novel renal tubular biomarkers that may influence the diagnosis and treatment of focal segmental glomerulosclerosis (FSGS) based on immune infiltration. Methods Three FSGS microarray datasets, GSE108112, GSE133288 and GSE121211, were downloaded from the Gene Expression Omnibus (GEO) database. The R statistical software limma package and the combat function of the sva package were applied for preprocessing and to remove the batch effects. Differentially expressed genes (DEGs) between 120 FSGS and 15 control samples were identified with the limma package. Disease Ontology (DO) pathway enrichment analysis was conducted with statistical R software to search for related diseases. Gene set enrichment analysis (GSEA) was used to interpret the gene expression data and it revealed many common biological pathways. A protein-protein interaction (PPI) network was built using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and hub genes were identified by the Cytoscape (version 3.7.2) plug-in CytoHubba. The plug-in Molecular Complex Detection (MCODE) was used to screen hub modules of the PPI network in Cytoscape, while functional analysis of the hub genes and hub nodes involved in the submodule was performed by ClusterProfiler. The least absolute shrinkage and selection operator (LASSO) regression and support vector machine recursive feature elimination (SVM-RFE) analysis were used to screen characteristic genes and build a logistic regression model. Receiver operating characteristic (ROC) curve analyses were used to investigate the logistic regression model and it was then validated by an external dataset GSE125779, which contained 8 FSGS samples and 8 healthy subjects. Cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) was used to calculate the immune infiltration of FSGS samples. Results We acquired 179 DEGs, 79 genes with downregulated expression (44.1%) and 100 genes with upregulated expression (55.9%), in the FSGS samples. The DEGs were significantly associated with arteriosclerosis, kidney disease and arteriosclerotic cardiovascular disease. GSEA revealed that these gene sets were significantly enriched in allograft rejection signaling pathways and activation of immune response in biological processes. Fifteen genes were demonstrated to be hub genes by PPI, and three submodules were screened by MCODE linked with FSGS. Analysis by machine learning methodologies identified nuclear receptor subfamily 4 group A member 1 (NR4A1) and dual specificity phosphatase 1 (DUSP1) as sensitive tubular renal biomarkers in the diagnosis of FSGS, and they were selected as hub genes, as well as hub nodes which were enriched in the MAPK signaling pathway. Immune cell infiltration analysis revealed that the genetic biomarkers were both correlated with activated mast cells, which may amplify FSGS biological processes. Conclusion DUSP1 and NR4A1 were identified as sensitive potential biomarkers in the diagnosis of FSGS. Activated mast cells have a decisive effect on the occurrence and development of FSGS through tubular lesions and tubulointerstitial inflammation, and they are expected to become therapeutic targets in FSGS.

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

BackgroundTight 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.MethodsBarrier 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.ResultsCompared 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.ConclusionOur study revealed that DUSP3 is an important TJ regulatory protein and its decrease may be involved in progression of epithelial cancers.

Abstract 815: Regulation of p53 by INPP4B and high fat diet in mouse prostate

Abstract Obesity is one of the risk factors for prostate neoplasia development, however the underlying molecular mechanisms that link obesity and prostate cancer remain elusive. The roles of AR, p53, EZH2, INPP4B, and PTEN are implicated in prostate cancer etiology. Androgen receptor (AR) signaling interacts functionally with PTEN, INPP4B, EZH2, and p53 signaling in prostate epithelium, and its transcriptional activity is reprogrammed by changes in the levels of these proteins during prostate cancer progression. Phosphatase and tensin homolog (PTEN) and Inositol Polyphosphate 4-Phosphatase Type-II B (INPP4B) are dual specificity phosphatases which are tumor suppressors in prostate cancer and are lost in nearly half of advanced castration resistant prostate cancers (CRPC). p53 is also a tumor suppressor gene that is frequently inactivated in prostate cancers. Enhancer of Zeste 2 (EZH2) is an epigenetic silencer that increases methylation of histone H3 on K27 and K9 residues and acts as an oncogene in CRPC. We have previously shown that INPP4B inhibits the Akt and PKC signaling pathways, which are activated in obesity. Using an Inpp4b-deficient mouse model, we showed that three-month-old Inpp4b-/- males developed prostatic intraepithelial neoplasia (PIN) when fed a high fat diet (HFD). While the AR protein levels were not altered, the transcriptional activity of AR was reduced in the prostates of Inpp4b-/- males. Furthermore, the prostates of HFD-fed Inpp4b-/- males exhibited increased expression of pro-inflammatory cytokines and macrophage infiltration. It was previously reported that indolent nature of the prostate neoplasia, which is caused by prostate specific Pten knockout, is due to the compensatory increase in other tumor suppressor proteins, such as p53, SMAD4, and PML. Concomitant with Pten, knockout of these individual tumor suppressors led to drastic acceleration of prostate neoplasia. Similar to prostates of Pten-/- mice, p53 levels increased in Inpp4b-/- males fed a low fat diet (LFD). Unlike males fed LFD, p53 protein levels decreased in HFD fed Inpp4b-/- mice, consistent with the development of PIN in this group. EZH2 levels as well as the levels of its targets, H3K27me3 and H3K9me2, decreased in ventral prostates of the Inpp4b-/- males and that decrease was exacerbated by the HFD. Similar to our mouse model, we observed significant positive correlation between the INPP4B and EZH2 expression in the prostates of healthy men and prostate cancer patients. In summary, we showed that loss of INPP4B synergizes with the HFD in reprogramming AR activity, reduction of EZH2 levels, and increases inflammation. Importantly, HFD reverses compensatory increase in p53 protein levels in prostates of Inpp4b-/- males. These alterations contribute to the development of PIN in the prostates of HFD-fed Inpp4b-/- males. Citation Format: Yasemin Ceyhan, Manqi Zhang, Irina U. Agoulnik. Regulation of p53 by INPP4B and high fat diet in mouse prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 815.

Abstract 157: DUSP6 modulates migration and glycolysis in PDAC cells

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor and is the seventh cause of death for cancer worldwide, fifth in the US. The highly malignant profile is mainly caused by the constitutive activation of mutant KRAS - found in approximately 90% of PDAC cases. The undruggability of genetic KRAS mutations has led to efforts to find new therapeutical targets that focus on downstream molecules in this pathway. DUSP6 is a dual-specificity phosphatase that regulates ERK1/2 phosphorylation and, therefore, downstream RAS pathway activation. DUSP6 has been demonstrated to be differentially expressed during PDAC tumorigenesis, which we believe is critical for tumor progression and metastasis. In silico analysis on the TCGA dataset revealed that DUSP6 is overexpressed in primary tumor samples compared to normal pancreatic tissue. This data was confirmed in an independent dataset (GSE71729) that further revealed DUSP6 overexpression in metastatic samples compared to primary tumor samples. Additionally, patients with higher DUSP6 expression have worse overall survival than patients with low DUSP6 expression (P = 0.039), reaffirming its clinical relevance. We then assessed DUSP6 expression in tumor sections derived from KC and KPC mice using the RNAscope technology and observed that DUSP6 was strongly overexpressed in tumorigenic lesions, and largely co-localized with KRT19 expression. Using BCI - a pharmacological DUSP6 inhibitor - we observed significantly reduced viability in vitro in all the cell lines (P &amp;lt; 0.0001). DUSP6 inhibition also promoted ERK1/2 activation, as expected. Surprisingly, we observed that BCI-mediated DUSP6 inhibition increased migratory capacity in AsPC-1 (P &amp;lt; 0.0001), but not on BxPC-3 cells. Furthermore, murine AKC cells presented the completely opposite phenotype, with a significant decrease in migratory capacity (P &amp;lt; 0.0001), indicating a possible context dependent response. To further understand the molecular mechanisms behind DUSP6 in PDAC, we performed a gene set enrichment analysis using the TCGA and GSE15471 datasets and observed a strong correlation between DUSP6 and the glycolysis pathway. Therefore, we knocked down DUSP6 in K8484 cells and evaluated their proliferation in response to 2-DG, which was significantly decreased in comparison to treated parental cells (P &amp;lt; 0.001). In accordance with that, DUSP6 knockdown cells were significantly more impacted by 2-DG treatment than the parental cells regarding migratory capacity, bringing it to the basal level. Collectively, these results suggest that DUSP6 appears to modulate the metastatic process, and this phenotype correlates with the altered glycolytic capacity of pancreatic cancer cells. We are currently expanding our studies to additional cell lines and using in vivo models to further investigate if the observed phenotype is context dependent. To assess glycolytic changes in the cells, we are currently performing Seahorse-based assays. Citation Format: Mariana T. Ruckert, Bailey A. Bye, Michael N. VanSaun, Vanessa S. Silveira. DUSP6 modulates migration and glycolysis in PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 157.

Metabolic Impact of MKP-2 Upregulation in Obesity Promotes Insulin Resistance and Fatty Liver Disease.

The mechanisms connecting obesity with type 2 diabetes, insulin resistance, nonalcoholic fatty liver disease, and cardiovascular diseases remain incompletely understood. The function of MAPK phosphatase-2 (MKP-2), a type 1 dual-specific phosphatase (DUSP) in whole-body metabolism, and how this contributes to the development of diet-induced obesity, type 2 diabetes (T2D), and insulin resistance is largely unknown. We investigated the physiological contribution of MKP-2 in whole-body metabolism and whether MKP-2 is altered in obesity and human fatty liver disease using MKP-2 knockout mice models and human liver tissue derived from fatty liver disease patients. We demonstrate that, for the first time, MKP-2 expression was upregulated in liver tissue in humans with obesity and fatty liver disease and in insulin-responsive tissues in mice with obesity. MKP-2-deficient mice have enhanced p38 MAPK, JNK, and ERK activities in insulin-responsive tissues compared with wild-type mice. MKP-2 deficiency in mice protects against diet-induced obesity and hepatic steatosis and was accompanied by improved glucose homeostasis and insulin sensitivity. Mkp-2−/− mice are resistant to diet-induced obesity owing to reduced food intake and associated lower respiratory exchange ratio. This was associated with enhanced circulating insulin-like growth factor-1 (IGF-1) and stromal cell-derived factor 1 (SDF-1) levels in Mkp-2−/− mice. PTEN, a negative regulator of Akt, was downregulated in livers of Mkp-2−/− mice, resulting in enhanced Akt activity consistent with increased insulin sensitivity. These studies identify a novel role for MKP-2 in the regulation of systemic metabolism and pathophysiology of obesity-induced insulin resistance and fatty liver disease.

Abstract 672: Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3)

Abstract Protein Tyrosine Phosphatase 4A3 (PTP4A3 or PRL-3) is an oncogenic dual-specificity phosphatase that drives tumor metastasis, promotes cancer cell survival, and is correlated with poor patient prognosis in a variety of solid tumors and leukemias. The mechanisms that drive PRL-3’s oncogenic functions are not well understood, in part due to a lack of research tools available to study this protein. The development of such tools has proven difficult, as the PRL family is ~80% homologous and the PRL catalytic binding pocket is shallow and hydrophobic. Currently available small molecules do not exhibit binding specificity for PRL-3 over PRL family members, and the only research antibody specific for PRL-3 can only recognize denatured protein. To address the lack of tools available to study PRL-3, we have developed alpaca-derived single domain antibodies, or nanobodies, targeting PRL-3. Nanobodies have emerged as a valuable research tool and show promise as cancer therapeutics as they are ~15kD and lack light chains, allowing them to reach cavities within active sites that conventional antibodies cannot normally reach. Nanobodies also maintain high specificity and affinity for their antigens. We identified seven unique nanobodies that bind to PRL-3 with no activity towards PRL-1 and PRL-2, making our nanobodies one of the first tools to selectively target PRL-3 in its native state. We used biolayer interferometry and found the nanobody binding affinity for PRL-3 to be within a KD of 30 - 300 nM, similar to that of antibodies currently on the market. We identified PRL-3:nanobody interactions with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and showed binding outside the active site. These data were confirmed by analyzing the effects of nanobodies on PRL-3 phosphatase activity and substrate binding. Our anti-PRL-3 nanobodies specifically pulled down PRL-3 over PRL-1/-2 in immunoprecipitation experiments. Finally, we used these nanobodies to analyze PRL-3 localization in fixed immunofluorescence experiments in human cancer cells. We found that a C-terminal tag on PRL-3, such as FLAG or GFP, enhanced PRL-3 localization to the membrane, compared to untagged protein, which may have confounded previous PRL-3 functional studies. We are currently utilizing these nanobodies in two ways to understand PRL-3’s role in cancer. First, we will use the nanobody to stabilize PRL-3 for X-ray crystallography to develop higher resolution structures that could contribute to substrate identification and drug design. Secondly, we will examine PRL-3 function and trafficking during various cancer processes, such as proliferation, invasion, and stress, to determine how PRL-3 localization contributes to cancer progression. Citation Format: Caroline Noel Smith, Kyle Kihn, Zachary A. Williamson, K. Martin Chow, Louis B. Hersh, Konstantin Korotkov, Daniel Deredge, Jessica S. Blackburn. Development and validation of nanobodies specific to the oncogenic phosphatase protein tyrosine phosphatase 4A3 (PTP4A3 or PRL-3) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 672.

Importance of Tyrosine Phosphorylation in Hormone-Regulated Plant Growth and Development.

Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.

Sevoflurane Post-treatment Mitigates Oxygen-glucose Deprivationinduced Pyroptosis of Hippocampal Neurons by Regulating the Mafb/DUSP14 Axis.

Ischemic brain injury often results in irreversible pyroptosis of neurons. Sevoflurane (Sevo) post-treatment exerts an alleviative role in neuroinflammation. This work evaluated the mechanism of Sevo post-treatment in oxygen-glucose deprivation (OGD)-induced pyroptosis of rat hippocampal neurons. Rat hippocampal neuron cell line H19-7 cells were treated with OGD, followed by posttreatment of 2% Sevo. The expression patterns of Mafb ZIP Transcription Factor B (Mafb) and dual- specificity phosphatase 14 (DUSP14) were determined via quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods. H19-7 cell viability and the release of lactate dehydrogenase (LDH) were examined via the cell counting kit-8 and LDH assay kits. Levels of pyroptosis-related proteins and cytokines NOD-like receptor family, pyrin domain containing 3 (NLRP3), N-term cleaved Gasdermin-D (GSDMD-N), cleaved-caspase-1, interleukin (IL)-1β, and IL-18 were also examined. The binding relation between Mafb and the DUSP14 promoter was detected. Besides, the roles of Mafb/DUSP14 in OGD-induced pyroptosis of rat hippocampal neurons were investigated through functional rescue experiments. Mafb and DUSP14 expression levels were decreased in OGD-induced hippocampal neurons. Sevo post-treatment up-regulated Mafb and DUSP14, facilitated H19-7 cell viability, inhibited LDH release, and reduced levels of NLRP3, GSDMD-N, cleaved-caspase-1, IL-1β, and IL-18. Mafb increased DUSP14 expression via binding to the DUSP14 promoter. Repressing Mafb or DUSP14 exacerbated pyroptosis of hippocampal neurons. Sevo post-treatment increased Mafb and DUSP14 expressions, which repressed OGDinduced pyroptosis of hippocampal neurons.

P-293 Increased expression of dual-specificity phosphatase 7 in gastric cancer is associated with poor prognosis and chemoresistance

Dual specificity phosphatases (DUSPs) are pivotal regulators of protein activation through dephosphorylating key molecules such as mitogen activated protein kinases, thereby co-ordinating signal transduction and biological processes. DUSP7 specifically dephosphorylates extracellular signal regulated kinases (ERK) 1 and 2 (pertinent for cellular growth) whilst being associated to the induction of inflammatory and apoptotic pathways involving p38-α and JNKs. Unlike other members of the DUSP family, DUSP7’s physiological function remains largely elusive with further exploration required of its involvement in healthy and cancerous tissue.

Dual-specificity Phosphatase 1 Suppresses Vascular Smooth Muscle Cell Calcification by Optical Atrophy 1-related Pathway

Objective To investigate the effect of dual-specificity phosphatase 1/optical atrophy 1 (DUSP1/OPA1) signaling pathway on vascular smooth muscle cell (VSMC) calcification.Methods An in vitro model of VSMC calcification was induced by exposure to β-glycerophosphate and calcium chloride.VSMC calcification was assessed by Alizarin Red S staining and calcium content by ELISA.Apoptosis was detected by TUNEL.Western blotting was employed to determine the protein levels of DUSP1,OPA1,Runt-related transcription factor 2 (Runx-2),bone morphogenetic protein 2 (BMP-2),and cysteinyl aspartate-specific proteinase-3 (Caspase-3).The effects of DUSP1 overexpression and OPA1 knockdown on cell calcification were investigated.Results Calcium chloride and β-glycerolphosphate induced VSMC calcification and down-regulated the expression levels of DUSP1 (t=11.951,P<0.001) and OPA1 (t=8.487,P<0.001).DUSP1 overexpression promoted OPA1 expression (t=-8.921,P<0.001),attenuated VSMC calcification,reduced calcium content and apoptosis rate,and down-regulated the expression of Runx-2,BMP-2,and active Caspase-3 (all P<0.001).OPA1 knockdown increased calcium content and apoptosis rate,up-regulated the expression of Runx-2,BMP-2,and active Caspase-3,and promoted VSMC calcification (all P<0.001).Conclusion DUSP1 may inhibit the VSMC calcification through the OPA1 signaling pathway.

Arabidopsis PFA-DSP-Type Phosphohydrolases Target Specific Inositol Pyrophosphate Messengers.

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling, and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologues display phosphohydrolase activity with a high specificity for the 5-β-phosphate of inositol pyrophosphates and only minor activity against the β-phosphates of 4-InsP7 and 6-InsP7. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-β-phosphate-specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

Hypoxia-induced HIF1A activates DUSP18-mediated MAPK14 dephosphorylation to promote hepatocellular carcinoma cell migration and invasion

BackgroundHepatocellular Carcinoma (HCC) is recognized as the second leading cause of cancer-associated deaths globally. Hypoxia-inducible factor 1alpha (HIF1A) has been documented to promote HCC cell migration, invasion and cell cycle. Dual specificity phosphatase 18 (DUSP18) has been predicted to be up-regulated in hypoxia and its expression is positively linked to HIF1A expression in HCC cells. However, their function and molecular mechanism have not been investigated in HCC in depth. PurposeThis study aimed to uncover the functional roles of HIF1A and DUSP18, as well as relevant mechanisms underlying their regulation in HCC cells. MethodsRT-qPCR and western blot were performed to examine gene expression. Functional assays were implemented to reveal the regulatory impact of target genes on HCC cells. Mechanism experiments were conducted to analyze gene interaction. ResultsDUSP18 was found to have significantly high expression in hypoxia-induced HCC cells. HIF1A promoted HCC cell migration, invasion and cell cycle by transcriptionally activating DUSP18. DUSP18 mediated MAPK14 dephosphorylation to weaken MAPK14 activity, which further inhibited MAPK14-mediated TP53 phosphorylation, consequently promoting multiple biological behaviors of HCC cells. ConclusionHypoxia-induced HIF1A activates DUSP18 transcription to further promote MAPK14 dephosphorylation, thereby suppressing TP53 phosphorylation and functionally promoting malignant behaviors of HCC cells.

The Toxoplasma glucan phosphatase TgLaforin utilizes a distinct functional mechanism that can be exploited by therapeutic inhibitors

Toxoplasma gondii is an intracellular parasite that generates amylopectin granules (AGs), a polysaccharide associated with bradyzoites that define chronic T. gondii infection. AGs are postulated to act as an essential energy storage molecule that enable bradyzoite persistence, transmission, and reactivation. Importantly, reactivation can result in the life-threatening symptoms of toxoplasmosis. T. gondii encodes glucan dikinase and glucan phosphatase enzymes that are homologous to the plant and animal enzymes involved in reversible glucan phosphorylation and which are required for efficient polysaccharide degradation and utilization. However, the structural determinants that regulate reversible glucan phosphorylation in T. gondii are unclear. Herein, we define key functional aspects of the T. gondii glucan phosphatase TgLaforin (TGME49_205290). We demonstrate that TgLaforin possesses an atypical split carbohydrate-binding-module domain. AlphaFold2 modeling combined with hydrogen–deuterium exchange mass spectrometry and differential scanning fluorimetry also demonstrate the unique structural dynamics of TgLaforin with regard to glucan binding. Moreover, we show that TgLaforin forms a dual specificity phosphatase domain–mediated dimer. Finally, the distinct properties of the glucan phosphatase catalytic domain were exploited to identify a small molecule inhibitor of TgLaforin catalytic activity. Together, these studies define a distinct mechanism of TgLaforin activity, opening up a new avenue of T. gondii bradyzoite biology as a therapeutic target.

Remuscularization with triiodothyronine and β1-blocker therapy reverses post-ischemic left ventricular dysfunction and adverse remodeling

Renewal of the myocardium by preexisting cardiomyocytes is a powerful strategy for restoring the architecture and function of hearts injured by myocardial infarction. To advance this strategy, we show that combining two clinically approved drugs, but neither alone, muscularizes the heart through cardiomyocyte proliferation. Specifically, in adult murine cardiomyocytes, metoprolol, a cardioselective β1-adrenergic receptor blocker, when given with triiodothyronine (T3, a thyroid hormone) accentuates the ability of T3 to stimulate ERK1/2 phosphorylation and proliferative signaling by inhibiting expression of the nuclear phospho-ERK1/2-specific phosphatase, dual-specificity phosphatase-5. While short-duration metoprolol plus T3 therapy generates new heart muscle in healthy mice, in mice with myocardial infarction-induced left ventricular dysfunction and pathological remodeling, it remuscularizes the heart, restores contractile function and reverses chamber dilatation; outcomes that are enduring. If the beneficial effects of metoprolol plus T3 are replicated in humans, this therapeutic strategy has the potential to definitively address ischemic heart failure.

Long noncoding RNA GK-IT1 promotes esophageal squamous cell carcinoma by regulating MAPK1 phosphorylation.

Long noncoding RNAs (lncRNAs) are implicated in the oncogenesis and metastasis of multiple human cancers. Nonetheless, the precise molecular mechanisms underlying the oncogenic role of lncRNA in esophageal squamous cell carcinoma (ESCC) remains to be clarified. The expression of GK intronic transcript 1 (GK-IT1) was analyzed using ESCC RNA-seq data from The Cancer Genome Atlas database. Quantitative real-time PCR was used to measure the expression of GK-IT1 in ESCC clinical samples and cells. The correlation between GK-IT1 expression and clinicopathological variables was examined using chi-squared tests. Kaplan-Meier survival and Cox regression analyses were employed to generate the survival curve and assess the prognostic value of GK-IT1. Functional experiments were utilized to explore the role of GK-IT1 in promoting cell migration, invasion, proliferation, and suppressing apoptosis and autophagy in ESCC. To understand the mechanism, an RNA pulldown assay, RNA immunoprecipitation, agarose gel electrophoresis, immunofluorescence, and co-immunoprecipitation assays were used. In this study we identified an unreported lncRNA, termed GK-IT1 that was aberrantly overexpressed in ESCC tissues and cells. GK-IT1 was closely associated with advanced clinical stage, and it was an independent prognostic indicator of ESCC. Functional assays verified that GK-IT1 significantly promoted ESCC proliferation, invasion, and migration, and suppressed ESCC apoptosis and autophagy. Furthermore, tumorigenesis experiments in nude mice indicated that GK-IT1 promoted ESCC tumor growth and metastasis. Mechanistically, GK-IT1 competitively bound to mitogen-activated protein kinase 1 (MAPK1) to prevent the interaction between dual specificity phosphatase 6 (DUSP6) and MAPK1, thereby controlling the phosphorylation of MAPK1 and promoting ESCC progression. Our study revealed that GK-IT1 competed with DUSP6 to attenuate the interaction between DUSP6 and MAPK1, leading to activation of the ERK/MAPK pathway, thereby promoting progression of ESCC. Our research indicated that GK-IT1 served as a novel potential target for the diagnosis and treatment of ESCC.

Dual Specific Phosphatase 7 Exacerbates Dilated Cardiomyopathy, Heart Failure, and Cardiac Death by Inactivating the ERK1/2 Signaling Pathway.

Heart failure is one of the most common but complicated end-stage syndromes in clinical practice. Dilated cardiomyopathy is a myocardial structural abnormality that is associated with heart failure. Dual-specificity phosphatases (DUSPs) are a group of protein phosphatases that regulate signaling pathways in numerous diseases; however, their physiological and pathological impact on cardiovascular disease remains unknown. In the present study, we generated two transgenic mouse models, a DUSP7 knockout and a cardiac-specific DUSP7 overexpressor. Mice overexpressing DUSP7 showed an exacerbated disease phenotype, including severe dilated cardiomyopathy, heart failure, and cardiac death. We further demonstrated that high levels of DUSP7 inhibited ERK1/2 phosphorylation and influenced downstream c-MYC, c-FOS, and c-JUN gene expression but did not affect upstream activators. Taken together, our study reveals a novel molecular mechanism for DUSP7 and provides a new therapeutic target and clinical path to alleviate dilated cardiomyopathy and improve cardiac function.