Overexpression of dual-specificity phosphatases 4 and 13 attenuates transforming growth factor ß1-induced migration and drug resistance in A549 cells in vitro.

Transforming growth factor-beta (TGFß) proteins induce an epithelial-mesenchymal transition (EMT) programme that is associated with increased invasive and drug-resistant phenotype of carcinoma cells. In addition to the canonical pathway involving SMAD proteins, the mitogen-activated kinase (MAPK) pathway via extracellular signal-regulated kinases ½ (ERK1/2) is also involved in promoting and maintaining a mesenchymal phenotype by tumor cells following TGFß signal activation. As dual-specificity phosphatases (DUSPs) regulate ERK1/2 activity by dephosphorylation, we aimed to examine DUSPs' expression upon TGFß stimulation and whether DUSPs play a role in the EMT and related phenotypes promoted by TGFß1 in A549 cells. We found that TGFß1 stimulation led to marked changes in several DUSP proteins, including significant decreases in DUSP4 and DUSP13 expressions. We then showed that the ectopic co-expression of DUSP4/13 suppresses TGFß1-induced ERK1/2 phosphorylation and protein levels of the EMT transcription factors Snail and Slug proteins. We then demonstrated that DUSP4/13 co-expression partially inhibited TGFß1-promoted migration, invasion, and chemoresistance in A549 cells. Collectively, this report provides data for the involvement of DUSP4/13 in malignant phenotypes regulated by TGFß1 in A549 cells.

SAHA could inhibit TGF-ß1/p38 pathway in MI-induced cardiac fibrosis through DUSP4 overexpression.

Growing evidences have revealed that a histone deacetylase inhibitor (HDACi), suberoylanilide hydroxamic acid (SAHA) has anti-fibrotic effect in different diseases. In this study, we first evaluated whether SAHA could suppress cardiac fibrosis. Mice with MI-induced cardiac fibrosis were treated with SAHA by intraperitoneal injection and their cardiac function was improved after SAHA treatment. Results of western blotting and qRT-PCR in heart tissues suggested that TGFß1/P38 pathway was activated in MI mice, and this effect was reversed by SAHA. Cell proliferation assay suggested that SAHA could suppress TGF-ß1-induced cardiac fibroblasts proliferation. Furthermore, results of western blotting and qRT-PCR in cardiac fibroblasts depicted that SAHA may exert its anti-fibrotic effect through inhibiting TGF-ß1-induced P38 phosphorylation by promoting DUSP4 expression. Our findings may substantiate SAHA as a promising treatment for MI-induced cardiac fibrosis.

DUSP4 inhibits autophagic cell death in PTC by inhibiting JNK-BCL2-Beclin1 signaling.

Dual specificity phosphatase 4 (DUSP4) is a prognostic marker and potential target of papillary thyroid carcinoma (PTC); however, the molecular mechanism underlying DUSP4-regulated PTC carcinogenesis is unknown. DUSP4 is a negative regulator of the autophagy promoter, JNK. This study explored the relationship between DUSP4 and JNK-mediated autophagic cell death in PTC, and the roles of DUSP4 in PTC using gain-of-function and loss-of-function assays. In addition, we further identified the significance of the JNK-BCL2-Beclin1-autophagy signaling pathway on DUSP4-regulated PTC carcinogenesis by combining knockdown of DUSP4 with a JNK-specific inhibitor (SP600125). We found that knockdown of DUSP4 promoted the phosphorylation of JNK and BCL2 in PTC cells, and enhanced the release of Beclin1 from the BCL2-Beclin1 complex. Knockdown of DUSP4 promoted autophagy and the death of PTC cells. The death and autophagy enhanced by knockdown of DUSP4 was reversed by the JNK inhibitor. We further extended the in-vitro experiments by subcutaneously injecting nude mice with K1 cells transfected with DUSP4-silencing vector. In-vivo assays showed that knockdown of DUSP4 not only inhibited tumor growth, but also promoted the phosphorylation of JNK and BCL2 and the expression of LC3II. In conclusion, DUSP4 inhibits BCL2-Beclin1-autophagy signaling by negatively regulating JNK activity, thus inhibiting PTC oncogenesis. The data from this study contribute to the prevention and cure of PTC.

Lysine-Specific Histone Demethylase 1 Promotes Oncogenesis of the Esophageal Squamous Cell Carcinoma by Upregulating DUSP4.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer (EC) and has a poor prognosis due to its aggressive nature. Accordingly, it is necessary to find novel prognostic biomarkers and therapeutic targets for ESCC. Lysine-specific histone demethylase 1 (LSD1) plays a core role in the regulation of ESCC oncogenesis. However, the detailed mechanism of LSD1-regulated ESCC growth has not been elucidated. This study aims to explore molecular mechanism underlying the LSD1-regulated ESCC's oncogenesis. After LSD1 silencing, we detected differentially expressed genes (DEGs) in human ESCC cell line, TE-1, by transcriptome sequencing. Subsequently, we investigated expression pattern of the selected molecules in the ESCC tissues and cell lines by qRT-PCR and Western blotting. Furthermore, we explored the roles of selected molecules in ESCC using gene silencing and overexpression assays. Transcriptome sequencing showed that the expression of dual specificity phosphatase 4 (DUSP4) in TE-1 was significantly attenuated after the LSD1 silencing. In addition, the DUSP4 mRNA expression level was significantly higher in the ESCC tissues, especially in those derived from patients with invasion or metastasis. Moreover, the DUSP4 expression was positively associated with the LSD1 expression in the ESCC tissues. DUSP4 overexpression promoted proliferation, invasion, and migration of the ESCC cells, while DUSP4 silencing had an opposite effect. DUSP4 overexpression also enhanced tumorigenicity of the ESCC cells in vivo, while DUSP4 silencing inhibited tumor growth. Importantly, inhibition of cell proliferation, invasion, and migration by the LSD1 inhibitor (ZY0511) was reversed by DUSP4 overexpression. Conclusively, we found that LSD1 promotes ESCC's oncogenesis by upregulating DUSP4, the potential therapeutic and diagnostic target in ESCC.

Dual-specificity phosphatase 4 is upregulated during skeletal muscle atrophy and modulates extracellular signal-regulated kinase activity.

Skeletal muscle atrophy results from disparate physiological conditions, including denervation, corticosteroid treatment, and aging. The purpose of this study was to describe and characterize the function of dual-specificity phosphatase 4 (Dusp4) in skeletal muscle after it was found to be induced in response to neurogenic atrophy. Quantitative PCR and Western blot analysis revealed that Dusp4 is expressed during myoblast proliferation but rapidly disappears as muscle cells differentiate. The Dusp4 regulatory region was cloned and found to contain a conserved E-box element that negatively regulates Dusp4 reporter gene activity in response to myogenic regulatory factor expression. In addition, the proximal 3'-untranslated region of Dusp4 acts in an inhibitory manner to repress reporter gene activity as muscle cells progress through the differentiation process. To determine potential function, Dusp4 was fused with green fluorescent protein, expressed in C2C12 cells, and found to localize to the nucleus of proliferating myoblasts. Furthermore, Dusp4 overexpression delayed C2C12 muscle cell differentiation and resulted in repression of a MAP kinase signaling pathway reporter gene. Ectopic expression of a Dusp4 dominant negative mutant blocked muscle cell differentiation and attenuated MAP kinase signaling by preferentially targeting the ERK1/2 branch, but not the p38 branch, of the MAP kinase signaling cascade in skeletal muscle cells. The findings presented in this study provide the first description of Dusp4 in skeletal muscle and suggest that Dusp4 may play an important role in the regulation of muscle cell differentiation by regulating MAP kinase signaling.

Sanguinarine inhibits growth and invasion of gastric cancer cells via regulation of the DUSP4/ERK pathway.

Sanguinarine, a bioactive benzophenanthridine alkaloid extracted from plants of the Papaveraceae family, has shown antitumour effects in multiple cancer cells. But the therapeutic effects and regulatory mechanisms of sanguinatine in gastric cancer (GC) remain elusive. This study was aimed to investigate the correlation of dual-specificity phosphatase 4 (DUSP4) expression with clinicopathologic features and overall survival in patients with GC and explore the effects of sanguinarine on tumour growth and invasion in GC cells (SGC-7901 and HGC-27) and underlying molecular mechanisms. Immunohistochemical analysis showed that decreased DUSP4 expression was associated with the sex, tumour size, depth of invasion and distant metastasis in patients with GC. Functional experiments including CCK-8, Transwell and flow cytometry analysis indicated that sanguinarine or DUSP4 overexpression inhibited GC cell viability and invasive potential, and induced cell apoptosis and cycle arrest in S phase, but DUSP4 knockdown attenuated the antitumour activity of sanguinarine. Further observation demonstrated that sanguinarine up-regulated the expression of DUSP4 and Bcl-2-associated X protein (Bax), but down-regulated phosphorylated extracellular signal-regulated kinase (p-ERK), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and B-cell lymphoma 2 (Bcl-2) expression. Taken together, our findings indicate that sanguinarine inhibits growth and invasion of GC cells through regulation of the DUSP4/ERK pathway, suggesting that sanguinarine may have potential for use in GC treatment.

Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin.

BACKGROUND: Breast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial. METHODS: We used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms. RESULTS: Knockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin. CONCLUSIONS: DUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer.

ADSC secretome constrains NK cell activity by attenuating IL-2-mediated JAK-STAT and AKT signaling pathway via upregulation of CIS and DUSP4.

BACKGROUND: Mesenchymal stem cells (MSCs) have immunomodulatory properties and therapeutic effects on autoimmune diseases through their secreted factors, referred to as the secretome. However, the specific key factors of the MSC secretome and their mechanisms of action in immune cells have not been fully determined. Most in vitro experiments are being performed using immune cells, but experiments using natural killer (NK) cells have been neglected, and a few studies using NK cells have shown discrepancies in results. NK cells are crucial elements of the immune system, and adjustment of their activity is essential for controlling various pathological conditions. The aim of this study was to elucidate the role of the adipose tissue-derived stem cell (ADSC) secretome on NK cell activity. METHODS: To obtain the ADSC secretome, we cultured ADSCs in medium and concentrated the culture medium using tangential flow filtration (TFF) capsules. We assessed NK cell viability and proliferation using CCK-8 and CFSE assays, respectively. We analyzed the effects of the ADSC secretome on NK cell activity and pathway-related proteins using a combination of flow cytometry, ELISA, cytotoxicity assay, CD107a assay, western blotting, and quantitative real-time PCR. To identify the composition of the ADSC secretome, we performed LC-MS/MS profiling and bioinformatics analysis. To elucidate the molecular mechanisms involved, we used mRNA sequencing to profile the transcriptional expression of human blood NK cells. RESULTS: The ADSC secretome was found to restrict IL-2-mediated effector function of NK cells while maintaining proliferative potency. This effect was achieved through the upregulation of the inhibitory receptor CD96, as well as downregulation of activating receptors and IL-2 receptor subunits IL-2Rα and IL-2Rγ. These changes were associated with attenuated JAK-STAT and AKT pathways in NK cells, which were achieved through the upregulation of cytokine-inducible SH2-containing protein (CIS, encoded by Cish) and dual specificity protein phosphatase 4 (DUSP4). Furthermore, proteomic analysis revealed twelve novel candidates associated with the immunomodulatory effects of MSCs. CONCLUSIONS: Our findings reveal a detailed cellular outcome and regulatory mechanism of NK cell activity by the ADSC secretome and suggest a therapeutic tool for treating NK-mediated inflammatory and autoimmune diseases using the MSC secretome.

Transcriptomic landscapes of effective and failed liver regeneration in humans.

Background & Aims: Although extensive experimental evidence on the process of liver regeneration exists, in humans, validation is largely missing. However, liver regeneration is critically affected by underlying liver disease. Within this project, we aimed to systematically assess early transcriptional changes during liver regeneration in humans and further assess how these processes differ in people with dysfunctional liver regeneration. Methods: Blood samples of 154 patients and intraoperative tissue samples of 46 patients undergoing liver resection were collected and classified with regard to dysfunctional postoperative liver regeneration. Of those, a matched cohort of 21 patients were used for RNA sequencing. Samples were assessed for circulating cytokines, gene expression dynamics, intrahepatic neutrophil accumulation, and spatial transcriptomics. Results: Individuals with dysfunctional liver regeneration demonstrated an aggravated transcriptional inflammatory response with higher intracellular adhesion molecule-1 induction. Increased induction of this critical leukocyte adhesion molecule was associated with increased intrahepatic neutrophil accumulation and activation upon induction of liver regeneration in individuals with dysfunctional liver regeneration. Comparing baseline gene expression profiles in individuals with and without dysfunctional liver regeneration, we found that dual-specificity phosphatase 4 (DUSP4) expression, a known critical regulator of intracellular adhesion molecule-1 expression in endothelial cells, was markedly reduced in patients with dysfunctional liver regeneration. Mimicking clinical risk factors for dysfunctional liver regeneration, we found liver sinusoidal endothelial cells of two liver disease models to have significantly reduced baseline levels of DUSP4. Conclusions: Exploring the landscape of early transcriptional changes of human liver regeneration, we observed that people with dysfunctional regeneration experience overwhelming intrahepatic inflammation. Subclinical liver disease might account for DUSP4 reduction in liver sinusoidal endothelial cells, which ultimately primes the liver for an aggravated inflammatory response. Impact and implications: Using a unique human biorepository, focused on liver regeneration (LR), we explored the landscape of circulating and tissue-level alterations associated with both functional and dysfunctional LR. In contrast to experimental animal models, people with dysfunctional LR demonstrated an aggravated transcriptional inflammatory response, higher intracellular adhesion molecule-1 (ICAM-1) induction, intrahepatic neutrophil accumulation and activation upon induction of LR. Although inflammatory responses appear rapidly after liver resection, people with dysfunctional LR have exaggerated inflammatory responses that appear to be related to decreased levels of LSEC DUSP4, challenging existing concepts of post-resectional LR.

Low expression of DUSP4 expression predicts unfavorable prognosis in gallbladder adenocarcinoma.

Background: Dual specificity phosphatase 4 (DUSP4), which regulates the mitogen activated protein kinases, has emerged as a tumor suppressor gene in several human malignancies. Aims and Objectives: In this study, we investigated the clinicopathologic significance and the prognostic role of DUSP4 in gallbladder adenocarcinoma. Materials and methods: DUSP4 expression was evaluated immunohistochemically in tissue microarray from 110 gallbladder adenocarcinoma samples and scored by H score system. The cut off (H score <170) was determined by ROC curve analysis. Results: Low expression of DUSP4 expression was observed in 57 (51.8%) out of 110 gallbladder adenocarcinoma samples. Low expression of DUSP4 expression was significantly associated with high histologic grade (P = 0.017), high pT stage (P = 0.002) and high AJCC stage (P = 0.007). Kaplan Meier survival curves revealed that patients with low expression of DUSP4 expression had significantly worse cancer specific survival (P = 0.024, log rank test). However, there was no significant association between DUSP4 expression and recurrence free survival. Conclusions: In conclusion, gallbladder adenocarcinoma with low expression of DUSP4 expression was associated with adverse clinicopathologic characteristics and poor patient outcome.patient outcome.

miR­122­5p suppresses the oncogenesis of PTC by inhibiting DUSP4 expression.

MicroRNAs (miRNAs or miRs) play an important role in regulating the occurrence and development of papillary thyroid carcinoma (PTC). miR­122­5p is widely considered a tumour inhibitor, which has not been fully explored in PTC. Bioinformatics analysis identified dual specificity phosphatase 4 (DUSP4), a tumour promoter gene for PTC, as a downstream target of miR­122­5p. The aim of the present study was to investigate the role and molecular mechanism of miR­122­5p in PTC oncogenesis. In this study, the expression pattern of miR­122­5p in PTC cancer tissues and PTC cell lines was investigated via reverse transcription­quantitative PCR. Furthermore, the roles of miR­122­5p in PTC were explored using gain­of­function and loss­of­function assays. The results revealed that the expression of miR­122­5p was significantly lower in PTC cancer tissues, especially in cancer tissues with significant invasion or metastasis. Overexpression of miR­122­5p caused by miR­122­5p mimics inhibited the proliferation, invasion, and migration of the PTC cell line K1, while knockdown of miR­122­5p by miR­122­5p inhibitors exhibited the opposite effect. Furthermore, in vivo assays revealed that miR­122­5p overexpression inhibited tumour growth. In addition, miR­122­5p was negatively correlated with DUSP4 expression in PTC cancer tissues. miR­122­5p overexpression inhibited DUSP4 expression in K1 cells, while miR­122­5p downregulation produced the inverse effect. Specifically, a luciferase reporter assay confirmed the binding sites of miR­122­5p on the 3'­UTR of DUSP4, demonstrating the targeting effect of miR­122­5p on DUSP4. miR­122­5p inhibited the oncogenesis of PTC by targeting DUSP4, revealing the potential application value of miR­122­5p in the diagnosis and treatment of PTC.

Dendritic Cells Actively Limit Interleukin-10 Production Under Inflammatory Conditions via DC-SCRIPT and Dual-Specificity Phosphatase 4.

Dendritic cell (DC)-based immunotherapy makes use of the DC's ability to direct the adaptive immune response toward activation or inhibition. DCs perform this immune orchestration in part by secretion of selected cytokines. The most potent anti-inflammatory cytokine interleukin-10 (IL-10) is under tight regulation, as it needs to be predominantly expressed during the resolution phase of the immune response. Currently it is not clear whether there is active suppression of IL-10 by DCs at the initial pro-inflammatory stage of the immune response. Previously, knockdown of the DC-specific transcription factor DC-SCRIPT has been demonstrated to mediate an extensive increase in IL-10 production upon encounter with pro-inflammatory immune stimuli. Here, we explored how DC-SCRIPT contributes to IL-10 suppression under pro-inflammatory conditions by applying chromatin immunoprecipitation sequencing analysis of DC-SCRIPT and the epigenetic marks H3K4me3 and H3K27ac in human DCs. The data showed binding of DC-SCRIPT to a GA-rich motif at H3K27ac-marked genomic enhancers that associated with genes encoding MAPK dual-specificity phosphatases (DUSPs). Functional studies revealed that upon knockdown of DC-SCRIPT, human DCs express much less DUSP4 and exhibit increased phosphorylation of the three major MAPKs (ERK, JNK, and p38). Enhanced ERK signaling in DC-SCRIPT-knockdown-DCs led to higher production of IL-10, which was reverted by rescuing DUSP4 expression. Finally, DC-SCRIPT-knockdown-DCs induced less IFN-γ and increased IL-10 production in naïve T cells, indicative for a more anti-inflammatory phenotype. In conclusion, we have delineated a new mechanism by which DC-SCRIPT allows DCs to limit IL-10 production under inflammatory conditions and potentiate pro-inflammatory Th1 responses. These insights may be exploited to improve DC-based immunotherapies.

Dual-Specificity Phosphatase 4 Overexpression in Cells Prevents Hypoxia/Reoxygenation-Induced Apoptosis via the Upregulation of eNOS.

Mitogen-activated protein kinases (MAPKs) signaling cascades regulate several cellular functions, including differentiation, proliferation, survival, and apoptosis. The duration and magnitude of phosphorylation of these MAPKs are decisive determinants of their physiological functions. Dual-specificity phosphatases exert kinetic control over these signaling cascades. Previously, we demonstrated that DUSP4-/- hearts sustain a larger infarct and have poor functional recovery, when isolated hearts were subjected to ischemia/reperfusion. Uncontrolled p38 activation and upregulation of Nox4 expression are the main effectors for this functional alteration. Here, dual-specificity phosphatase 4 (DUSP4) overexpression in endothelial cells was used to investigate the role of DUSP4 on the modulation of reactive oxygen species (ROS) generation and vascular function, when cells were subjected to hypoxia/reoxygenation (H/R) insult. Immunostaining with cleaved caspase-3 revealed that DUSP4 overexpression prevents caspase-3 activation and apoptosis after H/R. The beneficial effects occur via modulating p38 activity, increased NO bioavailability, and reduced oxidative stress. More importantly, DUSP4 overexpression upregulates eNOS protein expression (1.62 ± 0.33 versus 0.65 ± 0.16) during H/R-induced stress. NO is a critical small molecule involved in regulating vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF-2 fluorescence demonstrated that DUSP4 overexpression augments NO production and thus improves vascular function. The level of superoxide generated from cells after being subjected to H/R was determined using dihydroethidium-HPLC method. The results suggested that DUSP4 overexpression in cells decreases H/R-induced superoxide generation (1.56 ± 0.14 versus 1.19 ± 0.05) and thus reduces oxidant stress. This also correlates with the reduction in the total protein S-glutathionylation, an indicator of protein oxidation. These results further support our hypothesis that DUSP4 is an antioxidant gene and a key phosphatase in modulating MAPKs, especially p38, during oxidative stress, which regulates ROS generation and eNOS expression and thus protects against oxidant-induced injury or apoptosis. Overall, DUSP4 may serve as an excellent molecular target for the treatment of ischemic heart disease.

Clinicopathological significance of dual-specificity protein phosphatase 4 expression in invasive ductal carcinoma of the breast.

PURPOSE: Dual-specificity protein phosphatase 4 (DUSP4), also known as mitogen-activated protein kinase phosphatase (MKP) 2 is a member of the inducible nuclear MKP group. The role of DUSP4 in cancer development and progression appears to vary with the type of malignancy. The purpose of this study was to investigate DUSP4 expression in a case series of invasive ductal carcinoma of the breast. METHODS: We constructed tissue microarrays consisting of 16, 14, 47, and 266 cases of normal breast tissue, usual ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma, respectively. DUSP4 expression was investigated by immunohistochemistry. RESULTS: Cytoplasmic DUSP4 expression was observed. DUSP4 was more frequently expressed in malignant than in benign cases (p=0.024). The mean DUSP4 expression score was significantly higher in malignant tumors than in benign lesions (p=0.019). DUSP4 expression was significantly correlated with a larger tumor size (>2 cm, p=0.015). There was no significant correlation between overall survival or disease-free survival and DUSP4 expression in all 266 patients. We evaluated the impact of DUSP4 expression on the survival of 120 patients with T1-stage tumors. Interestingly, Kaplan-Meier survival curves revealed that DUSP4 expression had a significant effect on both overall patient survival (p=0.034, log-rank test) and disease-free survival (p=0.045, log-rank test). In early T-stage breast cancer, DUSP4 expression was associated with a worse prognosis. CONCLUSION: DUSP4 is frequently upregulated in breast malignancy, and may play an important role in cancer development and progression. In addition, it may be a marker of adverse prognosis, especially in patients with early T1-stage cancer.