The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling.

DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.

DUSP22 suppresses tumor progression by directly dephosphorylating AKT in non-small cell lung cancer.

Protein kinase B (AKT) plays a pivotal in regulating cell migration, proliferation, apoptosis, and survival, making it a prominent target for anticancer therapy. While the kinase activity of AKT has been extensively explored, its dephosphorylation have largely remained uncharted. Herein, we aimed to unravel the molecular mechanisms governing AKT dephosphorylation, with a specific emphasis on dual-specificity phosphatases DUSP22. Our investigation sought to shed light on the potential of DUSP22 as a potential therapeutic target for non-small cell lung cancer (NSCLC). To determine the expression level of DUSP22 in NSCLC cell lines, the gene expression profiling interactive analysis (GEPIA) and Oncomine database were searched. Additionally, the effect of DUSP22 on patient survival was analyzed with Kaplan-Meier database. Antitumor effects of DUSP22 were tested in A549 and H1299 cell lines. Experiments are based on: (1) cell viability determined by the cell counting kit-8 assay and colony-formation assay; (2) cell migratory ability assessed through the scratch assay and the transwell migration assay; (3) the mechanism behind the antitumor effects of DUSP22 dissected with co-immunoprecipitation (Co-IP) and in vitro kinase assays. Our study revealed a significant downregulation of DUSP22 in both NSCLC cell lines and tissues. Meanwhile, survival rate analysis results demonstrated that reduced DUSP22 expression was correlated with poorer overall survival in lung cancer patients. Moreover, DUSP22 exhibited an inhibitory effect on the cell viability and migratory capacity of A549 and H1299 cells. This inhibition was accompanied by the decrease in the phosphorylation of AKT and p38. Mechanistically, the phosphatase domain of DUSP22 interacted with AKT, resulting in the inhibition of AKT phosphorylation. This inhibitory effect was contingent upon the phosphatase activity of DUSP22. These findings provide compelling evidence that DUSP22 directly interacted with AKT, leading to the dephosphorylation of AKT at S473 and T308 residues, ultimately curbing the proliferation and migration of lung cancer cells. Additionally, our results also highlight a preclinical rationale for utilizing DUSP22 as a prognostic marker in NSCLC.

DOCK1 insufficiency disrupts trophoblast function and pregnancy outcomes via DUSP4-ERK pathway.

Abnormal trophoblast function is associated with diseases such as recurrent spontaneous abortion, pre-eclampsia, and preterm birth, and endangers maternal and fetal health. However, the underlying regulatory mechanisms remain unclear. In this study, we found DOCK1 expression is decreased in the placental villi of patients with recurrent spontaneous abortion, and that its expression determined the invasive properties of extravillous trophoblasts (EVTs), highlighting a previously unknown role of DOCK1 in regulating EVT function. Furthermore, DOCK1 deficiency disturbed the ubiquitinated degradation of DUSP4, leading to its accumulation. This caused inactivation of the ERK signaling pathway, resulting in inadequate EVT migration and invasion. DOCK1 was implicated in regulating the ubiquitin levels of DUSP4, possibly by modulating the E3 ligase enzyme HUWE1. The results of our in vivo experiments confirmed that the DOCK1 inhibitor TBOPP caused miscarriage in mice by inactivating the DUSP4/ERK pathway. Collectively, our results revealed the crucial role of DOCK1 in the regulation of EVT function via the DUSP4-ERK pathway and a basis for the development of novel treatments for adverse pregnancy outcomes caused by trophoblast dysfunction.

ARID1A loss activates MAPK signaling via DUSP4 downregulation.

BACKGROUND: ARID1A, a tumor suppressor gene encoding BAF250, a protein participating in chromatin remodeling, is frequently mutated in endometrium-related malignancies, including ovarian or uterine clear cell carcinoma (CCC) and endometrioid carcinoma (EMCA). However, how ARID1A mutations alter downstream signaling to promote tumor development is yet to be established. METHODS: We used RNA-sequencing (RNA-seq) to explore transcriptomic changes in isogenic human endometrial epithelial cells after deleting ARID1A. Chromatin immunoprecipitation sequencing (ChIP-seq) was employed to assess the active or repressive histone marks on DUSP4 promoter and regulatory regions. We validated our findings using genetically engineered murine endometroid carcinoma models, human endometroid carcinoma tissues, and in silico approaches. RESULTS: RNA-seq revealed the downregulation of the MAPK phosphatase dual-specificity phosphatase 4 (DUSP4) in ARID1A-deficient cells. ChIP-seq demonstrated decreased histone acetylation marks (H3K27Ac, H3K9Ac) on DUSP4 regulatory regions as one of the causes for DUSP4 downregulation in ARID1A-deficient cells. Ectopic DUSP4 expression decreased cell proliferation, and pharmacologically inhibiting the MAPK pathway significantly mitigated tumor formation in vivo. CONCLUSIONS: Our findings suggest that ARID1A protein transcriptionally modulates DUSP4 expression by remodeling chromatin, subsequently inactivating the MAPK pathway, leading to tumor suppression. The ARID1A-DUSP4-MAPK axis may be further considered for developing targeted therapies against ARID1A-mutated cancers.

Placenta microRNA profile of patient with Obstetric Antiphospholipid Syndrome.

The onset of obstetric antiphospholipid syndrome (APS) occurs when antiphospholipid antibodies act upon the placenta. During pregnancy, APS exhibits traits such as vascular thrombosis, inflammation, and hindered trophoblast implantation. The involvement of microRNA expression has been proposed as a genetic factor contributing to the syndrome's development. MicroRNAs play a role in regulating gene expression in various cellular processes, including the formation of placental tissue. Therefore, additional research is needed to explore the control of placental miRNA in APS. In this study, we aimed to profile miRNA expressions from placenta tissue of patients with APS. Differentially expressed miRNAs were determined for its targeted genes and pathways. Agilent microarray platform was used to measure placental microRNA expressions between normal placental tissue and those obtained from patients with APS. Differentially expressed miRNAs were detected using GeneSpring GX software 14.2 and sequences were mapped using TargetScan software to generate the predicted target genes. Pathway analysis for the genes was then performed on PANTHER and REACTOME software. Selected miRNAs and their associated genes of interest were validated using qPCR. Microarray findings revealed, 9 downregulated and 21 upregulated miRNAs expressed in placenta of patients with APS. Quantitative expressions of 3 selected miRNAs were in agreement with the microarray findings, however only miR-525-5p expression was statistically significant. Pathway analysis revealed that the targeted genes of differentially expressed miRNAs were involved in several hypothesised signalling pathways such as the vascular endothelial (VE) growth factor (VEGF) and inflammatory pathways. VE-cadherin, ras homolog member A (RHOA) and tyrosine kinase receptor (KIT) showed significant downregulation while Retinoblastoma gene (RET), Dual specificity protein phosphatase 10 (DUSP10) and B-lymphocyte kinase (BLK) genes were significantly upregulated. These preliminary findings suggest the involvement of miRNAs and identified novel associated genes involvement in the mechanism of obstetric APS, particularly through the alteration of vascular-associated regulators and the inflammatory signalling cascade.

Expression of dual-specificity phosphatases in TGFß1-induced EMT in SKOV3 cells.

BACKGROUND: The study aims to profile the dual-specificity phosphatases (DUSP) expression in response to Transforming growth factor ß1 (TGFß1)-induced epithelial- mesenchymal transition (EMT) in ovarian adenocarcinoma cells. METHODS: The ovarian adenocarcinoma cell line SKOV3 was used as a TGFß1-induced EMT model. Cells were incubated with 5 ng/mL TGFß1 to induce EMT. EMT was confirmed with real-time qPCR, western blot, and immunofluorescence analyses of various EMT markers. Western blot was used to analyze phospho- and total MAPK protein levels. Typical and atypical DUSPs mRNA expression profile was determined by real-time qPCR. RESULTS: The epithelial marker E-cadherin expressions were decreased and mesenchymal EMT markers Snail and Slug expression levelswere increased after TGFß1 induction. Phosphorylation of ERK1/2 and p38 MAPK were enhanced in response to TGFß1 treatment. The expression of DUSP2, DUSP6, DUSP8, DUSP10, and DUSP13 were decreased while DUSP7, DUSP16, DUSP18, DUSP21, and DUSP27 were increased by TGFß1. DISCUSSION: TGFß1 induced EMT which was accompanied by increased activity of MAPKs, and led to marked changes in expressions of several DUSPs in SKOV3 cells.

DUSP4 promotes esophageal squamous cell carcinoma progression by dephosphorylating HSP90ß.

The molecular and pathogenic mechanisms of esophageal squamous cell carcinoma (ESCC) development are still unclear, which hinders the development of effective treatments. In this study, we report that DUSP4 is highly expressed in human ESCC and is negatively correlated with patient prognosis. Knockdown of DUSP4 suppresses cell proliferation and patient-derived xenograft (PDX)-derived organoid (PDXO) growth and inhibits cell-derived xenograft (CDX) development. Mechanistically, DUSP4 directly binds to heat shock protein isoform ß (HSP90ß) and promotes the ATPase activity of HSP90ß by dephosphorylating HSP90ß on T214 and Y216. These dephosphorylation sites are critical for the stability of JAK1/2-STAT3 signaling and p-STAT3 (Y705) nucleus translocation. In vivo, Dusp4 knockout in mice significantly inhibits 4-nitrochinoline-oxide-induced esophageal tumorigenesis. Moreover, DUSP4 lentivirus or treatment with HSP90ß inhibitor (NVP-BEP800) significantly impedes PDX tumor growth and inactivates the JAK1/2-STAT3 signaling pathway. These data provide insight into the role of the DUSP4-HSP90ß-JAK1/2-STAT3 axis in ESCC progression and describe a strategy for ESCC treatment.

Identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using tandem affinity purification and mass spectrometry.

BACKGROUND: According to the World Health Organization Report, depressive disorders affect about 10% of the population. The molecular mechanism of the pathogenesis of depression is still not well understood. The new findings point to phosphatases as potential targets for effective depression therapy. The aim of the present work was the development of a method that would enable the identification of mitogen-activated protein kinase phosphatase-1 (MKP-1) protein partners using a proteomic approach. METHODS: The research was carried out using the PC12 cell line, often used as a model for neurobiological research. The use of the procedure for efficient purification of protein complexes-tandem affinity purification (TAP) will facilitate the identification of proteins interacting with MKP-1, a potential goal of effective antidepressant therapy. RESULTS: Identified proteins belong to various groups: cytoskeletal, ribosomal, nucleic acid binding, chaperones, and enzymes and may potentially be involved in the molecular mechanism of depression. CONCLUSIONS: The presented protocol for the purification of protein complexes is universal and can be successfully used in different mammalian cell lines. Proteins identified in the present work have been reported in the literature concerning studies on depressive disorders, which speaks in favour of their role in depression.

Investigating the Role of DUSP4 in Uveal Melanoma.

Purpose: Dual-specificity phosphatase 4 (DUSP4) inactivates factors in the mitogen-activated protein kinase (MAPK) signaling cascade, activated in uveal melanoma (UM) by mutations in upstream G-protein α subunits GNAQ/11 in >90% cases. This study examined whether DUSP4 (1) protein expression in primary UM (pUM) was a biomarker of metastatic risk and (2) knockdown sensitized UM cells to therapeutic agents, selumetinib or doxorubicin. Methods: DUSP4 mRNA data from The Cancer Genome Atlas and DUSP4 protein expression examined using immunohistochemistry in 28 cases of pUM were evaluated for association with clinical, genetic, and histological features. In vitro cytotoxic drug assays tested the efficacy of selumetinib and doxorubicin in UM cell lines with/without small interfering RNA DUSP4 gene silencing. Results: DUSP4 protein expression was observed in 93% of cases, with strong nuclear positivity in 79%. Despite higher DUSP4 messenger RNA levels in disomy 3/wild-type BAP1 UM, there was no significant association of nDUSP4 protein with these metastatic risk predictors or outcome. DUSP4 expression in UM cell lines varied. DUSP4 silencing in Mel202, MP46, and MP41 cells did not affect ERK1/2 or phospho-ERK levels. Despite increased phospho-ERK levels in Mel285, no cell line showed enhanced sensitivity to selumetinib/doxorubicin. Conclusions: DUSP4 protein expression is not a biomarker of UM metastatic risk. DUSP4 plays a complex role in oncogenesis, as reported in other cancers, and further work is required to fully understand its functional role in the MAPK pathway. Translational Relevance: Understanding the role of phosphatases, such as DUSP4, in the control of intracellular signaling cascades will facilitate our ability to identify successful treatment options.

DUSP4 Silencing Enhances the Sensitivity of Breast Cancer Cells to Doxorubicin through the Activation of the JNK/c-Jun Signalling Pathway.

Doxorubicin (Dox) has limited efficiency in breast cancer (BC) due to drug-acquired resistance. The epithelial-mesenchymal transition (EMT) plays a major role in the survival and drug resistance of cancer cells. It was suggested that the JNK pathway was implicated in the response to Dox by regulating EMT. DUSP4/or MKP-2 is a well-known regulator of the JNK pathway and was found to be highly expressed in BC. However, its functional significance is not yet fully understood. In the present study, the possible involvement of MKP-2 in Dox-induced EMT was investigated in breast cancer cells. Immunohistochemistry for tissues obtained from BC patients (n = 108) revealed 71.3% of tissues stained positively for MKP-2 while only 28.7% stained negatively. However, MKP-2 protein expression exhibited no significant relationship between BC prognostic factors, such as histological grade, histological type, hormonal status, and Ki-67 marker, its expression was significantly correlated with age 40 or below. In MDA-MB-231 cells, Dox-induced phosphorylation of JNK was sufficiently enhanced in MKP-2 silenced cells. This resulted in the attenuation of Dox-induced EMT, cell cycle arrest, and ultimately accelerated apoptosis. It was confirmed that the acquisition of Dox sensitivity by MKP-2 silencing largely depends on the stimulation of the JNK pathway. Indeed, results showed that overexpressing MKP-2 in non-tumorigenic MCF-12A cells dramatically inhibited Dox-induced JNK activation and, subsequently, cell death. The present study, to our knowledge, is the first to provide evidence for the potential role of MKP-2 in chemoresistance to Dox through modulating the JNK pathway and enhancing EMT.

Hepatocyte phosphatase DUSP22 mitigates NASH-HCC progression by targeting FAK.

Nonalcoholic steatohepatitis (NASH), a common clinical disease, is becoming a leading cause of hepatocellular carcinoma (HCC). Dual specificity phosphatase 22 (DUSP22, also known as JKAP or JSP-1) expressed in numerous tissues plays essential biological functions in immune responses and tumor growth. However, the effects of DUSP22 on NASH still remain unknown. Here, we find a significant decrease of DUSP22 expression in human and murine fatty liver, which is mediated by reactive oxygen species (ROS) generation. Hepatic-specific DUSP22 deletion particularly exacerbates lipid deposition, inflammatory response and fibrosis in liver, facilitating NASH and non-alcoholic fatty liver disease (NAFLD)-associated HCC progression. In contrast, transgenic over-expression, lentivirus or adeno-associated virus (AAV)-mediated DUSP22 gene therapy substantially inhibit NASH-related phenotypes and HCC development in mice. We provide mechanistic evidence that DUSP22 directly interacts with focal adhesion kinase (FAK) and restrains its phosphorylation at Tyr397 (Y397) and Y576 + Y577 residues, subsequently prohibiting downstream activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and nuclear factor-κB (NF-κB) cascades. The binding of DUSP22 to FAK and the dephosphorylation of FAK are indispensable for DUSP22-meliorated NASH progression. Collectively, our findings identify DUSP22 as a key suppressor of NASH-HCC, and underscore the DUSP22-FAK axis as a promising therapeutic target for treatment of the disease.

Combined Dusp4 and p53 loss with Dbf4 amplification drives tumorigenesis via cell cycle restriction and replication stress escape in breast cancer.

AIM: Deregulated signaling pathways are a hallmark feature of oncogenesis and driver of tumor progression. Dual specificity protein phosphatase 4 (DUSP4) is a critical negative regulator of the mitogen-activated protein kinase (MAPK) pathway and is often deleted or epigenetically silenced in tumors. DUSP4 alterations lead to hyperactivation of MAPK signaling in many cancers, including breast cancer, which often harbor mutations in cell cycle checkpoint genes, particularly in TP53. METHODS: Using a genetically engineered mouse model, we generated mammary-specific Dusp4-deleted primary epithelial cells to investigate the necessary conditions in which DUSP4 loss may drive breast cancer oncogenesis. RESULTS: We found that Dusp4 loss alone is insufficient in mediating tumorigenesis, but alternatively converges with loss in Trp53 and MYC amplification to induce tumorigenesis primarily through chromosome 5 amplification, which specifically upregulates Dbf4, a cell cycle gene that promotes cellular replication by mediating cell cycle checkpoint escape. CONCLUSIONS: This study identifies a novel mechanism for breast tumorigenesis implicating Dusp4 loss and p53 mutations in cellular acquisition of Dbf4 upregulation as a driver of cellular replication and cell cycle checkpoint escape.

Falnidamol and cisplatin combinational treatment inhibits non-small cell lung cancer (NSCLC) by targeting DUSP26-mediated signal pathways.

Non-small-cell lung cancer (NSCLC) is one of the most commonly diagnosed cancers worldwide with limited effective therapies. Cisplatin (DDP), as the first-line treatment, is always served as a mainstay of chemotherapeutic agents in combination with other drugs for NSCLC treatment. Nevertheless, DDP-based therapy is limited due to the frequent development of chemoresistance and adverse effects. Herein, it is necessary to find a more effective therapeutic approach with less toxicity. Falnidamol (FLD) is a pyrimido-pyrimidine compound and exerts anti-cancer activity. In the present study, we found that FLD could strongly promote the cytotoxicity of DDP and markedly reduce the IC50 values to restrain the proliferation of NSCLC cells. Furthermore, combination of FLD and DDP remarkably induced G2/M cell cycle arrest, DNA damage and mitochondrial apoptosis, which was largely through the induction of reactive oxygen species (ROS). Additionally, FLD/DDP in combination greatly triggered ferroptosis, along with free iron accumulation and enhanced lipid peroxidation. Epithelial to mesenchymal transition (EMT) and epidermal growth factor receptor (EGFR) phosphorylation were also considerably restrained in NSCLC cells co-treated with FLD/DDP. Mechanistically, the combinative treatment significantly reduced DUSP26 expression in NSCLC cells. More studies showed that DUSP26 was strongly up-regulated in human NSCLC samples compared with the paired normal tissues, and high DUSP26 predicted poor overall survival rate among patients. Importantly, we found that DUSP26 suppression intensively reduced the proliferation, EMT process and pEGFR expression in NSCLC cells, whereas facilitated ROS production, DNA damage and cell death; however, opposite phenotype was observed in NSCLC cells over-expressing DUSP26. More importantly, DUSP26 over-expression completely abolished the anti-cancer function of FLD/DDP in NSCLC cells. Animal studies finally confirmed that FLD/DDP in combination efficiently reduced tumor growth and lung metastasis in mice with ameliorated side effects. In conclusion, all these data illustrated that FLD and DDP combinational treatment effectively restrained NSCLC progression, and thus can be served as a promising therapeutic strategy.

DUSP4 Inactivation Leads to Reduced Extracellular Signal‒Regulated Kinase Activity through Upregulation of DUSP6 in Melanoma Cells.

A subset of dual-specificity phosphatases is a major negative regulator of MAPKs, and their involvement in tumorigenesis remains controversial. Among them, DUSP4 is reported to preferentially dephosphorylate extracellular signal‒regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase over p38. In this study, we aimed to identify a possible role of DUSP4 in melanoma genesis. An examination of large-scale public data on gene expression and dependency revealed a considerably high DUSP4 expression and dependency of the melanoma cell lines compared with those of other tumor cell lines, which was not apparent for the other 24 dual-specificity phosphatases genes encoded in the human genome. Using two melanoma lines, we confirmed that DUSP4 depletion impaired cell growth without notably inducing apoptosis. Interestingly, immunoblotting and kinase translocation reporter data revealed that DUSP4 depletion induces a decrease in ERK1/2 phosphorylation but barely affects c-Jun N-terminal kinase phosphorylation, suggesting that neither ERK nor c-Jun N-terminal kinase is a direct target of DUSP4 in our experimental setting. Notably, DUSP4 depletion led to an increase in DUSP6 level, possibly through a post-transcriptional process, and DUSP6 knockout almost eliminated the DUSP4-depletion effect on cell growth and ERK activity. Our findings suggest that DUSP4 plays a role in maintaining a high ERK1/2 activity by negatively regulating DUSP6 and thus contributes to the survival and growth of melanoma cells.

Characterization of Type 1 Angiotensin II Receptor Activation Induced Dual-Specificity MAPK Phosphatase Gene Expression Changes in Rat Vascular Smooth Muscle Cells.

Activation of the type I angiotensin receptor (AT1-R) in vascular smooth muscle cells (VSMCs) plays a crucial role in the regulation of blood pressure; however, it is also responsible for the development of pathological conditions such as vascular remodeling, hypertension and atherosclerosis. Stimulation of the VSMC by angiotensin II (AngII) promotes a broad variety of biological effects, including gene expression changes. In this paper, we have taken an integrated approach in which an analysis of AngII-induced gene expression changes has been combined with the use of small-molecule inhibitors and lentiviral-based gene silencing, to characterize the mechanism of signal transduction in response to AngII stimulation in primary rat VSMCs. We carried out Affymetrix GeneChip experiments to analyze the effects of AngII stimulation on gene expression; several genes, including DUSP5, DUSP6, and DUSP10, were identified as upregulated genes in response to stimulation. Since various dual-specificity MAPK phosphatase (DUSP) enzymes are important in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways, these genes have been selected for further analysis. We investigated the kinetics of gene-expression changes and the possible signal transduction processes that lead to altered expression changes after AngII stimulation. Our data shows that the upregulated genes can be stimulated through multiple and synergistic signal transduction pathways. We have also found in our gene-silencing experiments that epidermal growth factor receptor (EGFR) transactivation is not critical in the AngII-induced expression changes of the investigated genes. Our data can help us understand the details of AngII-induced long-term effects and the pathophysiology of AT1-R. Moreover, it can help to develop potential interventions for those symptoms that are induced by the over-functioning of this receptor, such as vascular remodeling, cardiac hypertrophy or atherosclerosis.

DUSP9, a Dual-Specificity Phosphatase with a Key Role in Cell Biology and Human Diseases.

Mitogen-activated protein kinases (MAPKs) are essential for proper cell functioning as they regulate many molecular effectors. Careful regulation of MAPKs is therefore required to avoid MAPK pathway dysfunctions and pathologies. The mammalian genome encodes about 200 phosphatases, many of which dephosphorylate the MAPKs and bring them back to an inactive state. In this review, we focus on the normal and pathological functions of dual-specificity phosphatase 9 (DUSP9)/MAP kinase phosphatases-4 (MKP-4). This cytoplasmic phosphatase, which belongs to the threonine/tyrosine dual-specific phosphatase family and was first described in 1997, is known to dephosphorylate ERK1/2, p38, JNK and ASK1, and thereby to control various MAPK pathway cascades. As a consequence, DUSP9 plays a major role in human pathologies and more specifically in cardiac dysfunction, liver metabolic syndromes, diabetes, obesity and cancer including drug response and cell stemness. Here, we recapitulate the mechanism of action of DUSP9 in the cell, its levels of regulation and its roles in the most frequent human diseases, and discuss its potential as a therapeutic target.

Enhancement of DUSP14 (dual specificity phosphatase 14) limits osteoarthritis progression by alleviating chondrocyte injury, inflammation and metabolic homeostasis.

Osteoarthritis (OA) is a proverbial inflammatory degenerative joint disease associated with the acceleration of the aging process and is characterized by chondrocyte injury and articular cartilage degradation. Dual-specificity phosphatase 14 (Dusp14), a common member of the DUSP family, has been implicated in multiple inflammatory diseases and bone loss. Nevertheless, the function of DUSP14 in OA remains unclear. In the present study, down-regulation of DUSP14 was corroborated in anterior cruciate ligament transection (ACLT)-induced OA rats and interleukin-1ß (IL-1ß)-stimulated chondrocytes. Additionally, the gain of DUSP14 reversed IL-1ß-induced inhibition of chondrocyte viability but attenuated cell apoptosis. Concomitantly, DUSP14 overexpression muted IL-1ß-induced release of pro-inflammatory mediators NO and prostaglandin E2 (PGE2), as well as pro-inflammatory cytokine levels (IL-6 and TNF-α). Furthermore, up-regulation of DUSP14 overturned the effects of IL-1ß on the inhibition of collagen II and aggrecan expression, and enhancement of A Disintegrin and Metalloproteinase with Thrombospondin Motifs 5 (ADAMTS5) and matrix metalloproteinases (MMPs; MMP3 and MMP-13). Mechanistically, DUSP14 elevation increased the p-Adenosine 5'-monophosphate-activated protein activated protein kinase(AMPK), inhibitor of NF-κB (IκB) expression and decreased p-p65 NF-κB expression, indicating that DUSP14 might restore the AMPK-IκB pathway to restrain NF-κB signaling under IL-1ß exposure. Notably, blockage of AMPK signaling muted the protective efficacy of DUSP14 elevation against IL-1ß-induced inflammatory injury and metabolism disturbance in chondrocytes. Interestingly, histological evaluation substantiated that DUSP14 injection alleviated cartilage degradation in OA rats. Together, DUSP14 may ameliorate OA progression by affecting chondrocyte injury, inflammatory response and cartilage metabolism homeostasis, implying a promising therapeutic strategy against OA.

Biphasic Regulation of Mitogen-Activated Protein Kinase Phosphatase 3 in Hypoxic Colon Cancer Cells.

Hypoxia, or low oxygen tension, is a hallmark of the tumor microenvironment. The hypoxia-inducible factor-1α (HIF-1α) subunit plays a critical role in the adaptive cellular response of hypoxic tumor cells to low oxygen tension by activating gene-expression programs that control cancer cell metabolism, angiogenesis, and therapy resistance. Phosphorylation is involved in the stabilization and regulation of HIF-1α transcriptional activity. HIF-1α is activated by several factors, including the mitogen-activated protein kinase (MAPK) superfamily. MAPK phosphatase 3 (MKP-3) is a cytoplasmic dual-specificity phosphatase specific for extracellular signal-regulated kinase 1/2 (Erk1/2). Recent evidence indicates that hypoxia increases the endogenous levels of both MKP-3 mRNA and protein. However, its role in the response of cells to hypoxia is poorly understood. Herein, we demonstrated that small-interfering RNA (siRNA)-mediated knockdown of MKP-3 enhanced HIF-1α (not HIF-2α) levels. Conversely, MKP-3 overexpression suppressed HIF-1α (not HIF-2α) levels, as well as the expression levels of hypoxia-responsive genes (LDHA, CA9, GLUT-1, and VEGF), in hypoxic colon cancer cells. These findings indicated that MKP-3, induced by HIF-1α in hypoxia, negatively regulates HIF-1α protein levels and hypoxia-responsive genes. However, we also found that long-term hypoxia (>12 h) induced proteasomal degradation of MKP-3 in a lactic acid-dependent manner. Taken together, MKP-3 expression is modulated by the hypoxic conditions prevailing in colon cancer, and plays a role in cellular adaptation to tumor hypoxia and tumor progression. Thus, MKP-3 may serve as a potential therapeutic target for colon cancer treatment.

Transcriptional regulation of human DUSP4 gene by cancer-related transcription factors.

Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is responsible for the dephosphorylation and inactivation of ERK, JNK and p38, which are mitogen-activated protein kinases involved in cell proliferation, differentiation and apoptosis, but also in inflammation processes. Given its importance for cellular signalling, DUSP4 is subjected to a tight regulation and there is growing evidence that its expression is dysregulated in several tumours. However, the mechanisms underlying DUSP4 transcriptional regulation remain poorly understood. Here, we analysed the regulation of the human DUSP4 promoters 1 and 2, located upstream of exons 1 and 2, respectively, by the cancer-related transcription factors (TFs) STAT3, FOXA1, CTCF and YY1. The presence of binding sites for these TFs was predicted in both promoters through the in silico analysis of DUSP4, and their functionality was assessed through luciferase activity assays. Regulatory activity of the TFs tested was found to be promoter-specific. While CTCF stimulated the activity of promoter 2 that controls the transcription of variants 2 and X1, STAT3 stimulated the activity of promoter 1 that controls the transcription of variant 1. YY1 positively regulated both promoters, although to different extents. Through site-directed mutagenesis, the functionality of YY1 binding sites present in promoter 2 was confirmed. This study provides novel insights into the transcriptional regulation of DUSP4, contributing to a better comprehension of the mechanisms of its dysregulation observed in several types of cancer.