Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

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.

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.

Acute Endoplasmic Reticulum Stress Suppresses Hepatic Gluconeogenesis by Stimulating MAPK Phosphatase 3 Degradation.

Drug-induced liver injury (DILI) is a widespread and harmful disease, and is closely linked to acute endoplasmic reticulum (ER) stress. Previous reports have shown that acute ER stress can suppress hepatic gluconeogenesis and even leads to hypoglycemia. However, the mechanism is still unclear. MAPK phosphatase 3 (MKP-3) is a positive regulator for gluconeogenesis. Thus, this study was conducted to investigate the role of MKP-3 in the suppression of gluconeogenesis by acute ER stress, as well as the regulatory role of acute ER stress on the expression of MKP-3. Results showed that acute ER stress induced by tunicamycin significantly suppressed gluconeogenesis in both hepatocytes and mouse liver, reduced glucose production level in hepatocytes, and decreased fasting blood glucose level in mice. Additionally, the protein level of MKP-3 was reduced by acute ER stress in both hepatocytes and mouse liver. Mkp-3 deficiency eliminated the inhibitory effect of acute ER stress on gluconeogenesis in hepatocytes. Moreover, the reduction effect of acute ER stress on blood glucose level and hepatic glucose 6-phosphatase (G6pc) expression was not observed in the liver-specific Mkp-3 knockout mice. Furthermore, activation of protein kinase R-like ER kinase (PERK) decreased the MKP-3 protein level, while inactivation of PERK abolished the reduction effect of acute ER stress on the MKP-3 protein level in hepatocytes. Taken together, our study suggested that acute ER stress could suppress hepatic gluconeogenesis by stimulating MKP-3 degradation via PERK, at least partially. Thus, MKP-3 might be a therapeutic target for DILI-related hypoglycemia.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

Introduction: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. Methods: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. Results: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. Discussion: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

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.

A novel site on dual-specificity phosphatase MKP7/DUSP16 is required for catalysis and MAPK binding.

The dual-specificity phosphatases responsible for the inactivation of the mitogen-activated protein kinases (MAPKs) are designated as the MAPK phosphatases (MKPs). We demonstrated previously that MKP5 is regulated through a novel allosteric site suggesting additional regulatory mechanisms of catalysis exist amongst the MKPs. Here, we sought to determine whether the equivalent site within the phosphatase domain of a highly similar MKP family member, MKP7, is also important for phosphatase function. We found that mutation of tyrosine 271 (Y271) in MKP7, which represents the comparable Y435 within the MKP5 allosteric pocket, inhibited MKP7 catalytic activity. Consistent with this, when MKP7 Y271 mutants were overexpressed in cells, the substrates of MKP7, p38 MAPK or JNK, failed to undergo dephosphorylation. The binding efficiency of MKP7 to p38 MAPK and JNK1/2 was also reduced when MKP7 Y271 is mutated. Consistent with reduced MAPK binding, we observed a greater accumulation of nuclear p38 MAPK and JNK when the MKP7 Y271 mutants are expressed in cells as compared with WT MKP7, which sequesters p38 MAPK/JNK in the cytoplasm. Therefore, we propose that Y271 is critical for effective MAPK dephosphorylation through a mechanism whereby binding to this residue precedes engagement of the catalytic site and upon overexpression, MKP7 allosteric site mutants potentiate MAPK signaling. These results provide insight into the regulatory mechanisms of MKP7 catalysis and interactions with the MAPKs. Furthermore, these data support the generality of the MKP allosteric site and provide a basis for small molecule targeting of MKP7.

Manipulation of pantothenate kinase in Anopheles stephensi suppresses pantothenate levels with minimal impacts on mosquito fitness.

Pantothenate (Pan) is an essential nutrient required by both the mosquito vector and malaria parasite. We previously demonstrated that increasing pantothenate kinase (PanK) activity and co-enzyme A (CoA) biosynthesis led to significantly decreased parasite infection prevalence and intensity in the malaria mosquito Anopheles stephensi. In this study, we demonstrate that Pan stores in A. stephensi are a limited resource and that manipulation of PanK levels or activity, via small molecule modulators of PanK or transgenic mosquitoes, leads to the conversion of Pan to CoA and an overall reduction in Pan levels with minimal to no effects on mosquito fitness. Transgenic A. stephensi lines with repressed insulin signaling due to PTEN overexpression or repressed c-Jun N-terminal kinase (JNK) signaling due to MAPK phosphatase 4 (MKP4) overexpression exhibited enhanced PanK levels and significant reductions in Pan relative to non-transgenic controls, with the PTEN line also exhibiting significantly increased CoA levels. Provisioning of the PTEN line with the small molecule PanK modulator PZ-2891 increased CoA levels while provisioning Compound 7 decreased CoA levels, affirming chemical manipulation of mosquito PanK. We assessed effects of these small molecules on A. stephensi lifespan, reproduction and metabolism under optimized laboratory conditions. PZ-2891 and Compound 7 had no impact on A. stephensi survival when delivered via bloodmeal throughout mosquito lifespan. Further, PZ-2891 provisioning had no impact on egg production over the first two reproductive cycles. Finally, PanK manipulation with small molecules was associated with minimal impacts on nutritional stores in A. stephensi mosquitoes under optimized rearing conditions. Together with our previous data demonstrating that PanK activation was associated with significantly increased A. stephensi resistance to Plasmodium falciparum infection, the studies herein demonstrate a lack of fitness costs of mosquito Pan depletion as a basis for a feasible, novel strategy to control parasite infection of anopheline mosquitoes.

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.

Measurement of synovium and serum dual specificity phosphatase 22 level: Their inter-correlation and potency as biomarkers in rheumatoid arthritis.

BACKGROUND: Dual specificity phosphatase 22 (DUSP22), also named as Jun N-terminal kinase pathway associated phosphatase recently, is reported to be closely engaged in immune and inflammation regulation. This study aimed to investigate the interaction between synovium DUSP22 and serum DUSP22 levels and to explore their correlation with rheumatoid arthritis (RA) risk, inflammation, and disease activity. METHODS: Synovium and serum samples from 42 RA patients with knee involvement underwent arthroscopy, and 20 knee trauma patients were collected. Besides, serum samples from 40 healthy controls were also obtained. Synovium DUSP22 expression was detected by reverse transcription quantitative polymerase chain reaction, while serum DUSP22 level was detected by enzyme-linked immunosorbent assay. RESULTS: Synovium DUSP22 level was greatly decreased in RA patients compared to trauma controls (p < 0.001), and it was negatively correlated with tender joint count (TJC) (r = -0.318, p = 0.040), C-reactive protein (CRP) (r = -0.330, p = 0.033), and Lysholm score (r = -0.423, p = 0.005) in RA patients. Serum DUSP22 level was lowest in RA patients, followed by trauma controls, then highest in healthy controls (p < 0.001). Serum DUSP22 level was negatively associated with TJC (r = -0.438, p = 0.004), swollen joint count (SJC) (r = -0.372, p = 0.015), CRP (r = -0.391, p = 0.011), and disease activity score in 28 joints (DAS28ESR ) score (r = -0.406, p = 0.008), and it increased after treatment (p = 0.001) in RA patients. In addition, serum DUSP22 level positively related to synovium DUSP22 level in RA patients (r = 0.394, p = 0.010). CONCLUSION: Synovium and serum DUSP22 are intercorrelated and insufficiently expressed in RA patients; meanwhile, their deficiency correlates with increased systemic inflammation, disease activity, and joint dysfunction.