Mechanistic Understanding of Dexamethasone-Mediated Protection against Remdesivir-Induced Hepatotoxicity.

Remdesivir (RDV), a broad-spectrum antiviral agent, is often used together with dexamethasone (DEX) for hospitalized COVID-19 patients requiring respiratory support. Potential hepatic adverse drug reaction is a safety concern associated with the use of RDV. We previously reported that DEX cotreatment effectively mitigates RDV-induced hepatotoxicity and reduces elevated serum alanine aminotransferase and aspartate aminotransferase levels in cultured human primary hepatocytes (HPH) and hospitalized COVID-19 patients, respectively. Yet, the precise mechanism behind this protective drug-drug interaction remains largely unknown. Here, we show that through the activation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases 1 and 2 (ERK1/2) signaling, RDV induces apoptosis (cleavage of caspases 8, 9, and 3), autophagy (increased autophagosome and LC3-II), and mitochondrial damages (decreased membrane potential, respiration, ATP levels, and increased expression of Bax and the released cytosolic cytochrome C) in HPH. Importantly, cotreatment with DEX partially reversed RDV-induced apoptosis, autophagy, and cell death. Mechanistically, DEX deactivates/dephosphorylates p38, JNK, and ERK1/2 signaling by enhancing the expression of dual specificity protein phosphatase 1 (DUSP1), a mitogen-activated protein kinase (MAPK) phosphatase, in a glucocorticoid receptor (GR)-dependent manner. Knockdown of GR in HPH attenuates DEX-mediated DUSP1 induction, MAPK dephosphorylation, as well as protection against RDV-induced hepatotoxicity. Collectively, our findings suggest a molecular mechanism by which DEX modulates the GR-DUSP1-MAPK regulatory axis to alleviate the adverse actions of RDV in the liver. SIGNIFICANCE STATEMENT: The research uncovers the molecular mechanisms by which dexamethasone safeguards against remdesivir-associated liver damage in the context of COVID-19 treatment.

p38 MAPK and MKP-1 control the glycolytic program via the bifunctional glycolysis regulator PFKFB3 during sepsis.

Hyperlactatemia often occurs in critically ill patients during severe sepsis/septic shock and is a powerful predictor of mortality. Lactate is the end product of glycolysis. While hypoxia due to inadequate oxygen delivery may result in anaerobic glycolysis, sepsis also enhances glycolysis under hyperdynamic circulation with adequate oxygen delivery. However, the molecular mechanisms involved are not fully understood. Mitogen-activated protein kinase (MAPK) families regulate many aspects of the immune response during microbial infections. MAPK phosphatase (MKP)-1 serves as a feedback control mechanism for p38 and JNK MAPK activities via dephosphorylation. Here, we found that mice deficient in Mkp-1 exhibited substantially enhanced expression and phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) 3, a key enzyme that regulates glycolysis following systemic Escherichia coli infection. Enhanced PFKFB3 expression was observed in a variety of tissues and cell types, including hepatocytes, macrophages, and epithelial cells. In bone marrow-derived macrophages, Pfkfb3 was robustly induced by both E. coli and lipopolysaccharide, and Mkp-1 deficiency enhanced PFKFB3 expression with no effect on Pfkfb3 mRNA stability. PFKFB3 induction was correlated with lactate production in both WT and Mkp-1-/- bone marrow-derived macrophage following lipopolysaccharide stimulation. Furthermore, we determined that a PFKFB3 inhibitor markedly attenuated lactate production, highlighting the critical role of PFKFB3 in the glycolysis program. Finally, pharmacological inhibition of p38 MAPK, but not JNK, substantially attenuated PFKFB3 expression and lactate production. Taken together, our studies suggest a critical role of p38 MAPK and MKP-1 in the regulation of glycolysis during sepsis.

Upregulation of hsa-miR-141-3p promotes uterine cervical carcinoma progression via targeting dual-specificity protein phosphatase 1.

We aimed to explore the aberrant expression status of hsa-miR-141-3p and dual-specificity protein phosphatase 1 (DUSP1) and their relative mechanisms in uterine cervical carcinoma (UCC).Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was conducted to detect the expression of hsa-miR-141-3p. Immunohistochemical (IHC) staining was performed to examine the expression of DUSP1 in UCC. Gene chips and RNA-seq datasets were also obtained to assess the expression level. Integrated standardized mean difference (SMD) was calculated to evaluate the expression status of hsa-miR-141-3p in UCC tissues comprehensively. DUSP1-overexpression and hsa-miR-141-3p-inhibition HeLa cells were established, and CCK-8, transwell, wound healing, cell cycle, and apoptosis assays were implemented. The targets of hsa-miR-141-3p were obtained with online tools, and the combination of hsa-miR-141-3p and DUSP1 was validated via dual-luciferase reporter assay. Single-cell RNA-seq data were analyzed to explore hsa-miR-141-3p and DUSP1 in different cells. An integrated SMD of 1.41 (95% CI[0.45, 2.38], p = 0.0041) with 558 samples revealed the overexpression of hsa-miR-141-3p in UCC tissues. And the pooled SMD of -1.06 (95% CI[-1.45, -0.66], p < 0.0001) with 1,268 samples indicated the downregulation of DUSP1. Inhibition of hsa-miR-141-3p could upregulate DUSP1 expression and suppress invasiveness and metastasis of HeLa cells. Overexpression of DUSP1 could hamper proliferation, invasion, and migration and boost apoptosis and distribution of G1 phase. The dual-luciferase reporter assay validated the combination of hsa-miR-141-3p and DUSP1. Moreover, the targets of hsa-miR-141-3p were mainly enriched in the MAPK signaling pathway and activated in fibroblasts and endothelial cells. The current study illustrated the upregulation of hsa-miR-141-3p and the downregulation of DUSP1 in UCC tissues. Hsa-miR-141-3p could promote UCC progression by targeting DUSP1.

CRISPR-targeted mutagenesis of mitogen-activated protein kinase phosphatase 1 improves both immunity and yield in wheat.

Plants have evolved a sophisticated immunity system for specific detection of pathogens and rapid induction of measured defences. Over- or constitutive activation of defences would negatively affect plant growth and development. Hence, the plant immune system is under tight positive and negative regulation. MAP kinase phosphatase1 (MKP1) has been identified as a negative regulator of plant immunity in model plant Arabidopsis. However, the molecular mechanisms by which MKP1 regulates immune signalling in wheat (Triticum aestivum) are poorly understood. In this study, we investigated the role of TaMKP1 in wheat defence against two devastating fungal pathogens and determined its subcellular localization. We demonstrated that knock-down of TaMKP1 by CRISPR/Cas9 in wheat resulted in enhanced resistance to rust caused by Puccinia striiformis f. sp. tritici (Pst) and powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt), indicating that TaMKP1 negatively regulates disease resistance in wheat. Unexpectedly, while Tamkp1 mutant plants showed increased resistance to the two tested fungal pathogens they also had higher yield compared with wild-type control plants without infection. Our results suggested that TaMKP1 interacts directly with dephosphorylated and activated TaMPK3/4/6, and TaMPK4 interacts directly with TaPAL. Taken together, we demonstrated TaMKP1 exert negative modulating roles in the activation of TaMPK3/4/6, which are required for MAPK-mediated defence signalling. This facilitates our understanding of the important roles of MAP kinase phosphatases and MAPK cascades in plant immunity and production, and provides germplasm resources for breeding for high resistance and high yield.

Dual-specificity phosphatase 1 interacts with prohibitin 2 to improve mitochondrial quality control against type-3 cardiorenal syndrome.

Type-3 cardiorenal syndrome (CRS-3) is acute kidney injury followed by cardiac injury/dysfunction. Mitochondrial injury may impair myocardial function during CRS-3. Since dual-specificity phosphatase 1 (DUSP1) and prohibitin 2 (PHB2) both promote cardiac mitochondrial quality control, we assessed whether these proteins were dysregulated during CRS-3-related cardiac depression. We found that DUSP1 was downregulated in heart tissues from a mouse model of CRS-3. DUSP1 transgenic (DUSP1Tg) mice were protected from CRS-3-induced myocardial damage, as evidenced by their improved heart function and myocardial structure. CRS-3 induced the inflammatory response, oxidative stress and mitochondrial dysfunction in wild-type hearts, but not in DUSP1Tg hearts. DUSP1 overexpression normalized cardiac mitochondrial quality control during CRS-3 by suppressing mitochondrial fission, restoring mitochondrial fusion, re-activating mitophagy and augmenting mitochondrial biogenesis. We found that DUSP1 sustained cardiac mitochondrial quality control by binding directly to PHB2 and maintaining PHB2 phosphorylation, while CRS-3 disrupted this physiological interaction. Transgenic knock-in mice carrying the Phb2S91D variant were less susceptible to cardiac depression upon CRS-3, due to a reduced inflammatory response, suppressed oxidative stress and improved mitochondrial quality control in their heart tissues. Thus, CRS-3-induced myocardial dysfunction can be attributed to reduced DUSP1 expression and disrupted DUSP1/PHB2 binding, leading to defective cardiac mitochondrial quality control.

DUSP1 interacts with and dephosphorylates VCP to improve mitochondrial quality control against endotoxemia-induced myocardial dysfunction.

Dual specificity phosphatase 1 (DUSP1) and valosin-containing protein (VCP) have both been reported to regulate mitochondrial homeostasis. However, their impact on mitochondrial quality control (MQC) and myocardial function during LPS-induced endotoxemia remains unclear. We addressed this issue by modeling LPS-induced endotoxemia in DUSP1 transgenic (DUSP1TG) mice and in cultured DUSP1-overexpressing HL-1 cardiomyocytes. Accompanying characteristic structural and functional deficits, cardiac DUSP1 expression was significantly downregulated following endotoxemia induction in wild type mice. In contrast, markedly reduced myocardial inflammation, cardiomyocyte apoptosis, cardiac structural disorder, cardiac injury marker levels, and normalized systolic/diastolic function were observed in DUSP1TG mice. Furthermore, DUSP1 overexpression in HL-1 cells significantly attenuated LPS-mediated mitochondrial dysfunction by preserving MQC, as indicated by normalized mitochondrial dynamics, improved mitophagy, enhanced biogenesis, and attenuated mitochondrial unfolded protein response. Molecular assays showed that VCP was a substrate of DUSP1 and the interaction between DUSP1 and VCP primarily occurred on the mitochondria. Mechanistically, DUSP1 phosphatase domain promoted the physiological DUSP1/VCP interaction which prevented LPS-mediated VCP Ser784 phosphorylation. Accordingly, transfection with a phosphomimetic VCP mutant abolished the protective actions of DUSP1 on MQC and aggravated inflammation, apoptosis, and contractility/relaxation capacity in HL-1 cardiomyocytes. These findings support the involvement of the novel DUSP1/VCP/MQC pathway in the pathogenesis of endotoxemia-caused myocardial dysfunction.

Mucosal acidosis elicits a unique molecular signature in epithelia and intestinal tissue mediated by GPR31-induced CREB phosphorylation.

Metabolic changes associated with tissue inflammation result in significant extracellular acidosis (EA). Within mucosal tissues, intestinal epithelial cells (IEC) have evolved adaptive strategies to cope with EA through the up-regulation of SLC26A3 to promote pH homeostasis. We hypothesized that EA significantly alters IEC gene expression as an adaptive mechanism to counteract inflammation. Using an unbiased RNA sequencing approach, we defined the impact of EA on IEC gene expression to define molecular mechanisms by which IEC respond to EA. This approach identified a unique gene signature enriched in cyclic AMP response element-binding protein (CREB)-regulated gene targets. Utilizing loss- and gain-of-function approaches in cultured epithelia and murine colonoids, we demonstrate that EA elicits prominent CREB phosphorylation through cyclic AMP-independent mechanisms that requires elements of the mitogen-activated protein kinase signaling pathway. Further analysis revealed that EA signals through the G protein-coupled receptor GPR31 to promote induction of FosB, NR4A1, and DUSP1. These studies were extended to an in vivo murine model in conjunction with colonization of a pH reporter Escherichia coli strain that demonstrated significant mucosal acidification in the TNFΔARE model of murine ileitis. Herein, we observed a strong correlation between the expression of acidosis-associated genes with bacterial reporter sfGFP intensity in the distal ileum. Finally, the expression of this unique EA-associated gene signature was increased during active inflammation in patients with Crohn's disease but not in the patient control samples. These findings establish a mechanism for EA-induced signals during inflammation-associated acidosis in both murine and human ileitis.

Calcitriol alleviates noise-induced hearing loss by regulating the ATF3/DUSP1 signalling pathway.

BACKGROUND: Calcitriol (Cal) is the most active metabolite of vitamin D and has antioxidant and anti-inflammatory properties. The aim of this study was to investigate the role of Cal in noise-induced hearing loss (NIHL) to further elucidate the mechanism of noise-induced oxidative stress in the mouse cochlea. METHODS: C57BL/6 J mice were given six intraperitoneal injections of Cal (500 ng/kg/d). After 14 days of noise exposure, auditory brainstem response (ABR) thresholds, and the cochlear outer hair cell loss rate were analysed to evaluate auditory function. Real-time fluorescence quantitative PCR, immunofluorescence and western blotting were performed in vitro after the treatment of cochlear explants with 100 µM tert-butyl hydroperoxide (TBHP) for 2.5 h and HEI-OC1 cells with 250 µM TBHP for 1.5 h. RESULTS: In vivo experiments confirmed that Cal pretreatment mitigated NIHL and outer hair cell death. The in vitro results demonstrated that Cal significantly reduced TBHP-induced cochlear auditory nerve fibre degradation and spiral ganglion neuron damage. Moreover, treatment with Cal inhibited the expression of oxidative stress-related factors (3-NT and 4-HNE) and DNA damage-related factors (γ-H2A.X) and attenuated TBHP-induced apoptosis in cochlear explants and HEI-OC1 cells. A total of 1479 upregulated genes and 1443 downregulated genes were screened in cochlear tissue 1 h after noise exposure. The level of transcription factor 3 (ATF3) was significantly elevated in HEI-OC1 cells after TBHP stimulation. Gene Transcription Regulation Database （GTRD）and Cistrome database analyses revealed that the downstream target gene of ATF3 is dual specificity phosphatase 1 (DUSP1). Cistrome DB Toolkit database results showed that the transcription factor of DUSP1 was ATF3. In addition, the ChIP-PCR results indicated that ATF3 might be a direct transcription factor of DUSP1. CONCLUSION: The results of our study suggest that Cal attenuates NIHL and inhibits noise-induced apoptosis by regulating the ATF3/DUSP1 signalling pathway.

Mitogen-activated protein kinase phosphatase-1 controls PD-L1 expression by regulating type I interferon during systemic Escherichia coli infection.

Mitogen-activated protein kinase phosphatase 1 (Mkp-1) KO mice produce elevated cytokines and exhibit increased mortality and bacterial burden following systemic Escherichia coli infection. To understand how Mkp-1 affects immune defense, we analyzed the RNA-Seq datasets previously generated from control and E. coli-infected Mkp-1+/+ and Mkp-1-/- mice. We found that E. coli infection markedly induced programmed death-ligand 1 (PD-L1) expression and that Mkp-1 deficiency further amplified PD-L1 expression. Administration of a PD-L1-neutralizing monoclonal antibody (mAb) to Mkp-1-/- mice increased the mortality of the animals following E. coli infection, although bacterial burden was decreased. In addition, the PD-L1-neutralizing mAb increased serum interferon (IFN)-γ and tumor necrosis factor alpha, as well as lung- and liver-inducible nitric oxide synthase levels, suggesting an enhanced inflammatory response. Interestingly, neutralization of IFN-α/ß receptor 1 blocked PD-L1 induction in Mkp-1-/- mice following E. coli infection. PD-L1 was potently induced in macrophages by E. coli and lipopolysaccharide in vitro, and Mkp-1 deficiency exacerbated PD-L1 induction with little effect on the half-life of PD-L1 mRNA. In contrast, inhibitors of Janus kinase 1/2 and tyrosine kinase 2, as well as the IFN-α/ß receptor 1-neutralizing mAb, markedly attenuated PD-L1 induction. These results suggest that the beneficial effect of type I IFNs in E. coli-infected Mkp-1-/- mice is, at least in part, mediated by Janus kinase/signal transducer and activator of transcription-driven PD-L1 induction. Our studies also support the notion that enhanced PD-L1 expression contributes to the bactericidal defect of Mkp-1-/- mice.

Transient deSUMOylation of IRF2BP proteins controls early transcription in EGFR signaling.

Molecular switches are essential modules in signaling networks and transcriptional reprogramming. Here, we describe a role for small ubiquitin-related modifier SUMO as a molecular switch in epidermal growth factor receptor (EGFR) signaling. Using quantitative mass spectrometry, we compare the endogenous SUMO proteomes of HeLa cells before and after EGF stimulation. Thereby, we identify a small group of transcriptional coregulators including IRF2BP1, IRF2BP2, and IRF2BPL as novel players in EGFR signaling. Comparison of cells expressing wild type or SUMOylation-deficient IRF2BP1 indicates that transient deSUMOylation of IRF2BP proteins is important for appropriate expression of immediate early genes including dual specificity phosphatase 1 (DUSP1, MKP-1) and the transcription factor ATF3. We find that IRF2BP1 is a repressor, whose transient deSUMOylation on the DUSP1 promoter allows-and whose timely reSUMOylation restricts-DUSP1 transcription. Our work thus provides a paradigm how comparative SUMO proteome analyses serve to reveal novel regulators in signal transduction and transcription.

MiR-337-3p confers protective effect on facet joint osteoarthritis by targeting SKP2 to inhibit DUSP1 ubiquitination and inactivate MAPK pathway.

OBJECTIVE: To probe the performance of miR-337-3p on the facet joint osteoarthritis (FJOA) and its underlying mechanism. METHODS: qRT-PCR and Western blot were utilized to analyze the levels of miR-337-3p and DUSP1 in the synovial tissues from 36 FJOA patients and 10 healthy controls. The human synovial fibroblasts of FJOA were isolated and cultured followed by cell transfection. Then, cells were exposed to 10 ng/mL of IL-1ß to induce inflammatory response of synovial fibroblasts. The alternation on cell biological function in cell models was determined. The binding of miR-337-3p and SKP2 was predicted by StarBase, TargetScan, DIANA-microT and miRmap, and further verified by RIP assay and dual-luciferase reporter assay. Co-IP experiment and ubiquitination assay were used to display the binding of SKP2 and DUSP1 as well as the ubiquitination and degradation of DUSP1. After that, the FJOA rat model was established and miR-337-3p mimic or negative control was given to rats by tail vein injection. The pathological changes of synovial tissues, synovitis score, and inflammation level in rats were assessed. RESULTS: The low expressions of miR-337-3p and DUSP1 were noticed in the synovial tissues of FJOA patients and in IL-1ß-induced synovial fibroblasts, and highly expressed p-p38 MAPK was noticed. Upregulation of miR-337-3p/DUSP1 or downregulation of SKP2 inhibited IL-1ß-induced proliferation and inflammatory response of synovial fibroblasts. SKP2 was the target gene of miR-337-3p, and SKP2 induced the ubiquitination and degradation of DUSP1. MiR-337-3p exerted a protective effect on FJOA rats by alleviating damage of rat synovial tissues, promoting cell apoptosis and repressing inflammatory response. CONCLUSION: MiR-337-3p plays a protective role in FJOA by negatively targeting SKP2 to suppress DUSP1 ubiquitination and inactivate the p38 MAPK pathway.

Knockout of MAPK Phosphatase-1 Exaggerates Type I IFN Response during Systemic Escherichia coli Infection.

We have previously shown that Mkp-1-deficient mice produce elevated TNF-α, IL-6, and IL-10 following systemic Escherichia coli infection, and they exhibited increased mortality, elevated bacterial burden, and profound metabolic alterations. To understand the function of Mkp-1 during bacterial infection, we performed RNA-sequencing analysis to compare the global gene expression between E. coli-infected wild-type and Mkp-1 -/- mice. A large number of IFN-stimulated genes were more robustly expressed in E. coli-infected Mkp-1 -/- mice than in wild-type mice. Multiplex analysis of the serum cytokine levels revealed profound increases in IFN-ß, IFN-γ, TNF-α, IL-1α and ß, IL-6, IL-10, IL-17A, IL-27, and GMSF levels in E. coli-infected Mkp-1 -/- mice relative to wild-type mice. Administration of a neutralizing Ab against the receptor for type I IFN to Mkp-1 -/- mice prior to E. coli infection augmented mortality and disease severity. Mkp-1 -/- bone marrow-derived macrophages (BMDM) produced higher levels of IFN-ß mRNA and protein than did wild-type BMDM upon treatment with LPS, E. coli, polyinosinic:polycytidylic acid, and herring sperm DNA. Augmented IFN-ß induction in Mkp-1 -/- BMDM was blocked by a p38 inhibitor but not by an JNK inhibitor. Enhanced Mkp-1 expression abolished IFN-ß induction by both LPS and E. coli but had little effect on the IFN-ß promoter activity in LPS-stimulated RAW264.7 cells. Mkp-1 deficiency did not have an overt effect on IRF3/7 phosphorylation or IKK activation but modestly enhanced IFN-ß mRNA stability in LPS-stimulated BMDM. Our results suggest that Mkp-1 regulates IFN-ß production primarily through a p38-mediated mechanism and that IFN-ß plays a beneficial role in E. coli-induced sepsis.

DUSP1 regulates the JAK2/STAT3 signaling pathway through targeting miR-21 in cervical cancer cells.

This study was to investigate the effect of DUSP1 on cervical cancer (CC) cells by targeting the miR-21 regulatory JAK2/STAT3 signaling pathway. For this purpose, fifteen CC patients treated at our hospital from January 2021 to February 2023 were selected. CC tissues and para-cancerous (PC) tissues were collected from the patients, and DUSP1 protein and mRNA expression levels were detected by Western blot and qPCR. The C33a control group (COG) and DUSP1 overexpression group (OVG) were set up: human cervical squamous carcinoma cells (CSCC) in the C33a COG were cultured without any treatment, while the DUSP1 OVG was cultured using DUSP1 gene overexpression lentivirus infection progeny. The proliferation ability of the three groups of cells was measured by CCK8, protein and mRNA expression by Western blot and qPCR, and cell migration and invasion ability by Transwell. It was found that DUSP1 protein and mRNA in CC tissues were reduced compared with those in PC tissues (P<0.05). The miR-21 in the DUSP1 OVG was reduced than those in the C33a COG (P<0.05). The expression of JAK2, STAT3 mRNA and protein in the DUSP1 OVG were reduced compared with those in the C33a COG (P<0.05). In conclusion, overexpression of DUSP1 can target and reduce the expression of miR-21, block the JAK2/STAT3 signaling pathway, reduce the viability of CC cells, inhibit the proliferation and migration and invasion ability of CC cells, and induce apoptosis of CC cells, thus providing a theoretical basis for the targeted treatment of clinical CC.

Loss of FOXM1 in macrophages promotes pulmonary fibrosis by activating p38 MAPK signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease with high mortality and is refractory to treatment. Pulmonary macrophages can both promote and repress fibrosis, however molecular mechanisms regulating macrophage functions during fibrosis remain poorly understood. FOXM1 is a transcription factor and is not expressed in quiescent lungs. Herein, we show that FOXM1 is highly expressed in pulmonary macrophages within fibrotic lungs of IPF patients and mouse fibrotic lungs. Macrophage-specific deletion of Foxm1 in mice (myFoxm1-/-) exacerbated pulmonary fibrosis. Inactivation of FOXM1 in vivo and in vitro increased p38 MAPK signaling in macrophages and decreased DUSP1, a negative regulator of p38 MAPK pathway. FOXM1 directly activated Dusp1 promoter. Overexpression of DUSP1 in FOXM1-deficient macrophages prevented activation of p38 MAPK pathway. Adoptive transfer of wild-type monocytes to myFoxm1-/- mice alleviated bleomycin-induced fibrosis. Altogether, contrary to known pro-fibrotic activities in lung epithelium and fibroblasts, FOXM1 has anti-fibrotic function in macrophages by regulating p38 MAPK.

Aloe-emodin targets multiple signaling pathways by blocking ubiquitin-mediated degradation of DUSP1 in nasopharyngeal carcinoma cells.

Aloe-emodin (AE) has been shown to inhibit the proliferation of several cancer cell lines, including human nasopharyngeal carcinoma (NPC) cell lines. In this study, we confirmed that AE inhibited malignant biological behaviors, including cell viability, abnormal proliferation, apoptosis, and migration of NPC cells. Western blotting analysis revealed that AE upregulated the expression of DUSP1, an endogenous inhibitor of multiple cancer-associated signaling pathways, resulting in blockage of the extracellular signal-regulated kinase (ERK)-1/2, protein kinase B (AKT), and p38-mitogen activated protein kinase（p38-MAPK） signaling pathways in NPC cell lines. Moreover, the selective inhibitor of DUSP1, BCI-hydrochloride, partially reversed the AE-induced cytotoxicity and blocked the aforementioned signaling pathways in NPC cells. In addition, the binding between AE and DUSP1 was predicted via molecular docking analysis using AutoDock-Vina software and further verified via a microscale thermophoresis assay. The binding amino acid residues were adjacent to the predicted ubiquitination site (Lys192) of DUSP1. Immunoprecipitation with the ubiquitin antibody, ubiquitinated DUSP1 was shown to be upregulated by AE. Our findings revealed that AE can stabilize DUSP1 by blocking its ubiquitin-proteasome-mediated degradation and proposed an underlying mechanism by which AE-upregulated DUSP1 may potentially target multiple pathways in NPC cells.

LINC00702-mediated DUSP1 transcription in the prevention of bladder cancer progression: Implications in cancer cell proliferation and tumor inflammatory microenvironment.

BACKGROUND: Long noncoding RNAs (lncRNAs) can mediate the biological processes during tumorigenesis which may be affected by tumor associated macrophages (TAMs). Hence, we aim to identify the functionality of LINC00702 in regulation of bladder cancer cells and M2-TAMs. METHODS: After induction of M2-TAMs from THP-1 monocyte, we evaluated effects of LINC00702 on bladder cancer cells and M2-TAMs, which were validated in a xenograft tumor mouse model. RESULTS: Low LINC00702 expression was determined in bladder cancer tissues. LINC00702 could promote DUSP1 transcription by recruiting JUND to its promoter. Ectopic LINC00702 expression suppressed the bladder cancer cell proliferation and secretion of inflammatory cytokines by M2-TAMs through up-regulation of DUSP1. The anti-tumor activity of LINC00702 was ultimately validated in vivo. CONCLUSION: LINC00702 promoted DUSP1 by recruiting JUND to inhibit the proliferation of bladder cancer cells and the secretion of inflammatory factors, thus modulating bladder cancer inflammatory microenvironment.

Genkwanin suppresses mitochondrial dysfunction to alleviate IL-1ß-elicited inflammation, apoptosis, and degradation of extracellular matrix in chondrocytes through upregulating DUSP1.

Osteoarthritis (OA) is a form of chronic degenerative disease contributing to elevated disability rate among the elderly. Genkwanin is an active component extracted from Daphne genkwa possessing pharmacologic effects. Here, this study is designed to expound the specific role of genkwanin in OA and elaborate the probable downstream mechanism. First, the viability of chondrocytes in the presence or absence of interleukin-1 beta (IL-1ß) treatment was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay was used to assess cell apoptosis. Inflammatory response was estimated through enzyme-linked immunosorbent assay and Western blot. In addition, immunofluorescence staining and Western blot were utilized to measure the expression of extracellular matrix (ECM)-associated proteins. Dual-specificity protein phosphatase-1 (DUSP1) expression was tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot. Following DUSP1 elevation in genkwanin-treated chondrocytes exposed to IL-1ß, inflammatory response and ECM-associated factors were evaluated as forementioned. In addition, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining was to assess the mitochondrial membrane potential. Adenosine triphosphate (ATP) level was examined with ATP assay kit, and RT-qPCR was used to test mitochondrial DNA expression. Results indicated that genkwanin administration enhanced the viability while ameliorated the apoptosis, inflammatory response, and ECM degradation in IL-1ß-induced chondrocytes. Besides, genkwanin treatment fortified DUSP1 expression in IL-1ß-exposed chondrocytes. DUSP1 interference further offsets the impacts of genkwanin on the inflammation, ECM degradation, and mitochondrial dysfunction in IL-1ß-challenged chondrocytes. In short, genkwanin enhanced DUSP1 expression to mitigate mitochondrial dysfunction, thus ameliorating IL-1ß-elicited inflammation, apoptosis, and degradation of ECM in chondrocytes.

MKP-1 Deficiency Exacerbates Skin Fibrosis in a Mouse Model of Scleroderma.

Scleroderma is a chronic fibrotic disease, where proinflammatory and profibrotic events precede collagen accumulation. MKP-1 [mitogen-activated protein kinase (MAPK) phosphatase-1] downregulates inflammatory MAPK pathways suppressing inflammation. MKP-1 also supports Th1 polarization, which could shift Th1/Th2 balance away from profibrotic Th2 profile prevalent in scleroderma. In the present study, we investigated the potential protective role of MKP-1 in scleroderma. We utilized bleomycin-induced dermal fibrosis model as a well-characterized experimental model of scleroderma. Dermal fibrosis and collagen deposition as well as the expression of inflammatory and profibrotic mediators were analyzed in the skin samples. Bleomycin-induced dermal thickness and lipodystrophy were increased in MKP-1-deficient mice. MKP-1 deficiency enhanced collagen accumulation and increased expression of collagens, 1A1 and 3A1, in the dermis. Bleomycin-treated skin from MKP-1-deficient mice also showed enhanced expression of inflammatory and profibrotic factors IL-6, TGF-ß1, fibronectin-1 and YKL-40, and chemokines MCP-1, MIP-1α and MIP-2, as compared to wild-type mice. The results show, for the first time, that MKP-1 protects from bleomycin-induced dermal fibrosis, suggesting that MKP-1 favorably modifies inflammation and fibrotic processes that drive the pathogenesis of scleroderma. Compounds enhancing the expression or activity of MKP-1 could thus prevent fibrotic processes in scleroderma and possess potential as a novel immunomodulative drug.

Biomarkers in the Rat Hippocampus and Peripheral Blood for an Early Stage of Mental Disorders Induced by Water Immersion Stress.

It is difficult to evaluate the pre-symptomatic state of mental disorders and prevent its onset. Since stress could be a trigger of mental disorders, it may be helpful to identify stress-responsive biomarkers (stress markers) for the evaluation of stress levels. We have so far performed omics analyses of the rat brain and peripheral blood after various kinds of stress and have found numerous factors that respond to stress. In this study, we investigated the effects of relatively moderate stress on these factors in the rat to identify stress marker candidates. Adult male Wistar rats underwent water immersion stress for 12 h, 24 h, or 48 h. Stress caused weight loss and elevated serum corticosterone levels, and alterations regarded as anxiety and/or fear-like behaviors. Reverse-transcription PCR and Western blot analyses revealed significant alterations in the expressions of hippocampal genes and proteins by the stress for no longer than 24 h, such as mitogen-activated protein kinase phosphatase 1 (MKP-1), CCAAT/enhancer-binding protein delta (CEBPD), small ubiquitin-like modifier proteins 1/sentrin-specific peptidase 5 (SENP5), matrix metalloproteinase-8 (MMP-8), kinase suppressor of Ras 1 (KSR1), and MKP-1, MMP-8, nerve growth factor receptor (NGFR). Similar alterations were observed in three genes (MKP-1, CEBPD, MMP-8) in the peripheral blood. The present results strongly suggest that these factors may serve as stress markers. The correlation of these factors in the blood and brain may enable the evaluation of stress-induced changes in the brain by blood analysis, which will contribute to preventing the onset of mental disorders.

Dual-Specificity Phosphatases in Regulation of Tumor-Associated Macrophage Activity.

The regulation of protein kinases by dephosphorylation is a key mechanism that defines the activity of immune cells. A balanced process of the phosphorylation/dephosphorylation of key protein kinases by dual-specificity phosphatases is required for the realization of the antitumor immune response. The family of dual-specificity phosphatases is represented by several isoforms found in both resting and activated macrophages. The main substrate of dual-specificity phosphatases are three components of mitogen-activated kinase signaling cascades: the extracellular signal-regulated kinase ERK1/2, p38, and Janus kinase family. The results of the study of model tumor-associated macrophages supported the assumption of the crucial role of dual-specificity phosphatases in the formation and determination of the outcome of the immune response against tumor cells through the selective suppression of mitogen-activated kinase signaling cascades. Since mitogen-activated kinases mostly activate the production of pro-inflammatory mediators and the antitumor function of macrophages, the excess activity of dual-specificity phosphatases suppresses the ability of tumor-associated macrophages to activate the antitumor immune response. Nowadays, the fundamental research in tumor immunology is focused on the search for novel molecular targets to activate the antitumor immune response. However, to date, dual-specificity phosphatases received limited discussion as key targets of the immune system to activate the antitumor immune response. This review discusses the importance of dual-specificity phosphatases as key regulators of the tumor-associated macrophage function.