Dual-specificity Protein Phosphatase Research Articles

DUSP5 deficiency suppresses the progression of acute kidney injury by enhancing autophagy through AMPK/ULK1 pathway

Acute kidney injury (AKI) represents a critical clinical disease characterized by the rapid decline in renal function, carrying a substantial burden of morbidity and mortality. The treatment of AKI is frequently limited by its variable clinical presentations and intricate pathophysiology, highlighting the urgent need for a deeper understanding of its pathogenesis and potential therapeutic targets. Dual-specific protein phosphatase 5 (DUSP5), a member of the serine-threonine phosphatase family, possesses the capability to dephosphorylate extracellular regulated protein kinases (ERK). DUSP5 has emerged as a pivotal player in modulating metabolic signals, inflammatory responses, and cancer progression, while also being closely associated with various kidney diseases. This study systematically scrutinized the function and mechanism of DUSP5 in AKI for the first time, unveiling a substantial increase in DUSP5 expression during AKI. Moreover, DUSP5 knockdown was observed to attenuate the production of inflammatory factors and apoptotic cells in renal tubular epithelial cells by enhancing AMPK/ULK1-mediated autophagy, thus improving renal function. In a word, DUSP5 knockdown in AKI effectively impede disease progression by activating autophagy. This finding holds promise for introducing fresh perspectives and targets for AKI treatment.

The Impact of Calcium Depletion on Proliferation of Chlorella sorokiniana Strain DSCG150.

This study analyzed the effects of Ca2+ metal ions among culture medium components on the Chlorella sorokiniana strain DSCG150 strain cell growth. The C. sorokiniana strain DSCG150 grew based on a multiple fission cell cycle and growth became stagnant in the absence of metal ions in the medium, particularly Ca2+. Flow cytometry and confocal microscopic image analysis results showed that in the absence of Ca2+, cell growth became stagnant as the cells accumulated into four autospores and could not transform into daughter cells. Genetic analysis showed that the absence of Ca2+ caused upregulation of calmodulin (calA) and cell division control protein 2 (CDC2_1) genes, and downregulation of origin of replication complex subunit 6 (ORC6) and dual specificity protein phosphatase CDC14A (CDC14A) genes. Analysis of gene expression patterns by qRT-PCR showed that the absence of Ca2+ did not affect cell cycle progression up to 4n autospore, but it inhibited Chlorella cell fission (liberation of autospores). The addition of Ca2+ to cells cultivated in the absence of Ca2+ resulted in an increase in n cell population, leading to the resumption of C. sorokiniana growth. These findings suggest that Ca2+ plays a crucial role in the fission process in Chlorella.

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.

Early growth response 1 regulates dual‑specificity protein phosphatase 1 and inhibits cell migration and invasion of tongue squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the head and neck, and among the OSCCs, tongue squamous cell carcinoma (TSCC) is one of the most common types. Although therapy strategies have recently advanced, the prognosis of TSCC has not substantially improved. Metastasis is one of the main causes of patient mortality in TSCC; therefore, it is necessary to elucidate the mechanism by which TSCC metastasis is regulated. In the present study, the early growth response 1 (Egr-1) expression in TSCC was analyzed based on GEO datasets and the effect of Egr-1 in TSCC tumor cell migration and invasion was measured using Transwell assay. By overexpressing dual-specificity protein phosphatase 1 (DUSP1) in cells with Egr-1 knockdown using lentivirus infection, the role of DUSP1 in Egr-1-regulated TSCC cell migration and invasion was determined. By using luciferase and ChIP assays, the mechanism behind how DUSP1 is regulated by Egr-1 was detected. In the present study, it was demonstrated that Egr-1 was downregulated in TSCC and the knockdown of Egr-1 increased TSCC cell migration and invasion. The expression of Egr-1 was also correlated with DUSP1. The overexpression of DUSP1 in Egr-1 knockdown cells, reduced the level of cell migration and invasion. Furthermore, it was demonstrated that knockdown of Egr-1 inhibited the promoter activity of DUSP1 and the site through which Egr-1 regulates DUSP1 transcription was identified. In conclusion, the present study demonstrated that Egr-1 regulates TSCC cell migration and invasion through modulating DUSP1, suggesting the potential of Egr-1 and DUSP1 as therapy targets for TSCC.

DUSP22 Ameliorates Endothelial-to-Mesenchymal Transition in HUVECs through Smad2/3 and MAPK Signaling Pathways.

Endothelial-to-mesenchymal transition (EndMT) is the process by which endothelial cells lose their endothelial properties and acquire mesenchymal characteristics. Dual-specific protein phosphatase 22 (DUSP22) inactivates various protein kinases and transcription factors by dephosphorylating serine/threonine residues: hence, it plays a key role in many diseases. The aim of this study was to explore the functional role of DUSP22 in EndMT. In the transforming growth factor-β-induced EndMT model in human umbilical vein endothelial cells (HUVECs), we observed a downregulation of DUSP22 expression. This DUSP22 deficiency could aggravate EndMT. Conversely, the overexpression of DUSP22 could ameliorate EndMT. We used signaling pathway inhibitors to verify our results and found that DUSP22 could regulate EndMT through the smad2/3 and the mitogen-activated protein kinase (MAPK) signaling pathways. In summary, DUSP22 ameliorates EndMT in HUVECs in vitro through the smad2/3 and MAPK signaling pathways.

Enhancing protein dynamics analysis with hydrophilic polyethylene glycol cross-linkers.

Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.

Abstract C081: The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma

Abstract Deregulation of proteins involved in chromatin regulation is common in pancreatic ductal adenocarcinoma (PDAC), occurring via both genomic and non-genomic mechanisms. Lysine demethylase 4C (KDM4C) is one of the chromatin-modifying proteins frequently overexpressed across multiple solid cancers. We observed upregulation of Kdm4c protein in a panel of human and murine PDAC cell lines, as well as in PDAC patient samples compared to non-neoplastic controls. CRISPR/Cas9-mediated deletion of KDM4C in human and murine PDAC cells resulted in reduced proliferation and clonogenicity, and increased survival of orthotopically implanted murine PDAC allografts. Notably, in multiple KDM4C-null PDAC clones analyzed by reverse phase protein arrays (RPPA), we observed downregulation of activated mitogen-activated protein kinase (MAP kinase) pathway components (including phospho-MEK and phospho-ERK), which are key effector pathways downstream of mutant KRAS. In vitro propagation of KDM4C-null PDAC lines eventually led to adaptation and restitution of MAP kinase signaling via compensatory upregulation of other KDM4 family members (KDM4A and KDM4B). In order to bypass genetic compensation by KDM4 family members, we utilized a preclinical grade pan-KDM4 inhibitor (TACH107, Tachyon Therapeutics) in PDAC cell lines, which confirmed profound reduction in proliferation and colony formation; in vivo xenograft and allograft studies with TACH107 are now ongoing. To characterize signaling pathways perturbed by KDM4C deregulation, we investigated the transcriptional changes in PDAC cells with and without KDM4C deletion by RNA-seq and integrated these with alterations in H3K9me3 and H3K36me3 marks using histone ChIP-Seq. Regulatory network analysis identified ATF4 as one of the top downregulated transcription factor networks, validating a previously published observation linking KDM4C to amino acid biosynthesis and cell proliferation. Gene set enrichment analysis showed downregulation of RAS signatures, consistent with the RPPA data on attenuation of MAP Kinase pathway. DUSP2, a member of the dual specificity protein phosphatase subfamily that inactivates ERK, is transcriptionally upregulated and has increased H3K36me3 marks near the promoter region in KDM4C-depleted cells, nominating it as a candidate of KDM4C-mediated epigenetic regulation. To further delineate the essential binding partners of KDM4C in chromatin regulation, we have created HA-tagged catalytic domain and reader domain deletion constructs that will be used for immunoprecipitation followed by mass spectrometry analysis. Overall, our data suggests that KDM4C - and other KDM4 family members – regulate MAPK signaling in KRAS-mutant cells, at least partially via attenuation of transcripts encoding specific protein phosphatases. To the best of our knowledge, this is the first demonstration linking the requirement of sustained activity of the KDM4 family of lysine demethylases to MAPK signaling in cancer and presents an opportunity to leverage this oncogenic pathway for therapeutic purposes. Citation Format: Menna-t-Allah Shaheen, Laila Barkoudeh, Sarah Dhebat, Kimal I. Rajapakshe, Bidyut Ghosh, Anirban Maitra. The lysine demethylase KDM4C is an oncogenic driver in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C081.

Impact of knockout of dual-specificity protein phosphatase 5 on structural and mechanical properties of rat middle cerebral arteries: implications for vascular aging.

Vascular aging influences hemodynamics, elevating risks for vascular diseases and dementia. We recently demonstrated that knockout (KO) of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This improvement correlates with elevated pPKC and pERK1/2 levels in the brain and kidneys. Additionally, we observed that Dusp5 KO modulates the passive mechanical properties of cerebral and renal arterioles, associated with increased myogenic tone at low pressure, enhanced distensibility, greater compliance, and reduced stiffness. The present study evaluates the structural and mechanical properties of the middle cerebral artery (MCA) in Dusp5 KO rats. We found that vascular smooth muscle cell layers and the collagen content in the MCA wall are comparable between Dusp5 KO and control rats. The internal elastic lamina in the MCA of Dusp5 KO rats exhibits increased thickness, higher autofluorescence intensity, smaller fenestrae areas, and fewer fenestrations. Despite an enhanced myogenic response and tone of the MCA in Dusp5 KO rats, other passive mechanical properties, such as wall thickness, cross-sectional area, wall-to-lumen ratio, distensibility, incremental elasticity, circumferential wall stress, and elastic modulus, do not significantly differ between strains. These findings suggest that while Dusp5 KO has a limited impact on altering the structural and mechanical properties of MCA, its primary role in ameliorating hemodynamics and cognitive functions is likely attributable to its enzymatic activity on cerebral arterioles. Further research is needed to elucidate the specific enzymatic mechanisms and explore potential clinical applications in the context of vascular aging.

Dual-specificity protein phosphatase 6 (DUSP6) overexpression reduces amyloid load and improves memory deficits in male 5xFAD mice.

Dual specificity protein phosphatase 6 (DUSP6) was recently identified as a key hub gene in a causal VGF gene network that regulates late-onset Alzheimer's disease (AD). Importantly, decreased DUSP6 levels are correlated with an increased clinical dementia rating (CDR) in human subjects, and DUSP6 levels are additionally decreased in the 5xFAD amyloidopathy mouse model. To investigate the role of DUSP6 in AD, we stereotactically injected AAV5-DUSP6 or AAV5-GFP (control) into the dorsal hippocampus (dHc) of both female and male 5xFAD or wild type mice, to induce overexpression of DUSP6 or GFP. Barnes maze testing indicated that DUSP6 overexpression in the dHc of 5xFAD mice improved memory deficits and was associated with reduced amyloid plaque load, Aß1-40 and Aß1-42 levels, and amyloid precursor protein processing enzyme BACE1, in male but not in female mice. Microglial activation, which was increased in 5xFAD mice, was significantly reduced by dHc DUSP6 overexpression in both males and females, as was the number of "microglial clusters," which correlated with reduced amyloid plaque size. Transcriptomic profiling of female 5xFAD hippocampus revealed upregulation of inflammatory and extracellular signal-regulated kinase pathways, while dHc DUSP6 overexpression in female 5xFAD mice downregulated a subset of genes in these pathways. Gene ontology analysis of DEGs (p < 0.05) identified a greater number of synaptic pathways that were regulated by DUSP6 overexpression in male compared to female 5xFAD. In summary, DUSP6 overexpression in dHc reduced amyloid deposition and memory deficits in male but not female 5xFAD mice, whereas reduced neuroinflammation and microglial activation were observed in both males and females, suggesting that DUSP6-induced reduction of microglial activation did not contribute to sex-dependent improvement in memory deficits. The sex-dependent regulation of synaptic pathways by DUSP6 overexpression, however, correlated with the improvement of spatial memory deficits in male but not female 5xFAD.

MID-LIFE PLASMA PROTEINS ASSOCIATE WITH PREFRAILTY AND FRAILTY: A PROTEOMIC ANALYSIS

Abstract Physical frailty is a syndrome that typically manifests in later life, although the pathogenic process causing physical frailty likely begins decades earlier. To date, few studies have examined the biological signatures in mid-life associated with frailty in late life. We evaluated 4955 plasma proteins (log 2-transformed and standardized) measured using the SomaScan platform among 3810 participants (57.8+/-5.0 years, 58.2% women) at Visit 3 of the Atherosclerosis Risk in Community (ARIC) study, and their frailty status across up to 3 study visits in late-life (median follow-up 19 years). We used multinomial logistic regression models to examine the associations between the protein levels and the frailty status, adjusting for demographics, health indicators, comorbidities, and follow-up time. Two sets of 2 proteins were identified to be associated with prefrailty and frailty, respectively, after Bonferroni correction (p&amp;lt; 1.01×10-5). Higher concentration of hydroxymethylglutaryl-CoA synthase (HMGCS1; OR=1.22), a regulator of cholesterol synthesis, and lower concentration of dual specificity protein phosphatase 13 isoform A (DUSP13A; OR=0.82), a protein involved in cell apoptosis, were associated with higher odds of prefrailty. Higher concentration of growth differentiation factor 15 (GDF15; OR=1.39), a protein related to inflammation and cell senescence, and lower concentration of contactin-1 (CNTN1; OR=0.72), a protein involved in nervous system development and signalling, were associated with higher odds of frailty. The pathway and upstream regulator analyses of the physical frailty- or prefrailty-associated proteins (p&amp;lt; 0.01) revealed the dysregulation of apoptosis, inflammation, and neuromuscular communication in mid-life. Our findings provides new insights into frailty etiology earlier in the life course.

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

BackgroundMesenchymal 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.MethodsTo 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.ResultsThe 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.ConclusionsOur 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.

Abstract 16749: Influence of Dual-Specificity Protein Phosphatase 5 on Structural and Mechanical Properties of Rat Middle Cerebral Artery

Dual-specificity protein phosphatase 5 (DUSP5) is a multifunctional phosphatase that modulates signaling cascades by catalyzing the dephosphorylation of both threonine and tyrosine residues. We recently reported that KO of Dusp5 enhances cerebral and renal hemodynamics and cognitive function. This effect is associated with increased pPKC and pERK1/2 levels in arteries and arterioles of the brain and kidneys. Further, we found that KO of Dusp5 contributes to the regulation of arteriolar passive mechanical properties with higher myogenic tones, better distensibility, greater compliance, and less stiffness. The present study focuses on the structural and mechanical properties of rat middle cerebral artery (MCA) to evaluate whether it contributes to enhanced vascular and cognitive function in Dusp5 KO rats. Vascular smooth muscle cell layer numbers and collagen content in the wall of the MCA are similar in Dusp5 KO and control rats. While external elastic lamina was absent in the MCA in both strains, internal elastic lamina was thicker in the MCA of Dusp5 KO vs. control rats (2.33 ± 0.02 vs. 1.85 ± 0.03 μm) and associated with higher autofluorescence intensity (953.76 ± 10.03 vs. 737.31 ± 64.88 au), smaller fenestrae areas (51.31 ± 3.62 vs. 70.46 ± 2.24), and fewer number of fenestrations (138.19 ± 8.86 vs. 374.73 ± 17.02 μm 2 ). Even though the myogenic tone is enhanced in Dusp5 KO vs. control rats (52.37 ± 1.65 vs. 44.87 ± 2.00%), other passive mechanical properties (wall thickness, cross-sectional area, wall-to-lumen ratio, wall tension, distensibility, incremental distensibility, circumferential wall strain, circumferential wall stress, and elastin modulus) of the MCA of Dusp5 KO rats do not exhibit significant changes. The findings suggest that while Dusp5 KO has a limited impact on changing the structural and mechanical properties of the MCA, its role in enhancing hemodynamics and cognitive function is primarily attributed to its enzymatic function.

Role of N-formyl peptide receptor 2 in germinal matrix hemorrhage: an intrinsic review of a hematoma resolving pathway.

Germinal matrix hemorrhage is one of the leading causes of morbidity, mortality, and acquired infantile hydrocephalus in preterm infants in the United States, with little progress made in its clinical management. Blood clots have been shown to elicit secondary brain injury after germinal matrix hemorrhage, by disrupting normal cerebrospinal fluid circulation and absorption after germinal matrix hemorrhage causing post-hemorrhagic hydrocephalus development. Current evidence suggests that rapid hematoma resolution is necessary to improve neurological outcomes after hemorrhagic stroke. Various articles have demonstrated the beneficial effects of stimulating the polarization of microglia cells into the M2 phenotype, as it has been suggested that they play an essential role in the rapid phagocytosis of the blood clot after hemorrhagic models of stroke. N-formyl peptide receptor 2 (FPR2), a G-protein-coupled receptor, has been shown to be neuroprotective after stroke. FPR2 activation has been associated with the upregulation of phagocytic macrophage clearance, yet its mechanism has not been fully explored. Recent literature suggests that FPR2 may play a role in the stimulation of scavenger receptor CD36. Scavenger receptor CD36 plays a vital role in microglia phagocytic blood clot clearance after germinal matrix hemorrhage. FPR2 has been shown to phosphorylate extracellular-signal-regulated kinase 1/2 (ERK1/2), which then promotes the transcription of the dual-specificity protein phosphatase 1 (DUSP1) gene. In this review, we present an intrinsic outline of the main components involved in FPR2 stimulation and hematoma resolution after germinal matrix hemorrhage.

MicroRNA-375 restrains the progression of lung squamous cell carcinoma by modulating the ERK pathway via UBE3A-mediated DUSP1 degradation

MiRNA-375 has been reported to play critical roles in a variety of cancers. To unravel its biological roles, especially its specific mechanisms of action in lung squamous cell carcinoma (LUSC), LUSC tissue microarrays and miRNAscope were performed to identify the miR-375 expression. Associations with clinicopathologic features, survival, and the prognostic value of miR-375 in LUSC were clarified in a retrospective study of 90 pairs of LUSC tissues. In vitro and in vivo gain- and loss-of-function assays were conducted to validate the effects and mechanism of miR-375 in LUSC. The mechanism responsible for interactions was verified by dual-luciferase reporter gene assay, immunoprecipitation (IP) analysis, immunofluorescence (IF) assay and ubiquitination assay. We found that miR-375 had higher expression in noncancerous adjacent tissues than in LUSC tissues. Clinicopathologic analyses showed that miR-375 expression was correlated with pathologic stage and was an independent predictor of overall survival (OS) for LUSC. MiR-375, as a tumor inhibitor, inhibited proliferation and metastasis while promoting apoptosis of LUSC cells. Mechanistic research indicated that miR-375 targeted ubiquitin-protein ligase E3A (UBE3A), which in turn promoted the activity of the ERK signaling pathway via ubiquitin-mediated dual-specificity protein phosphatase 1 (DUSP1) degradation. Collectively, we propose a novel mechanism of tumorigenesis and metastasis of LUSC via the miR-375/UBE3A/DUSP1/ERK axis, which could potentially facilitate new strategies for the treatment of LUSC.

Knockdown of phosphatases of regenerating liver-1 prolongs the lifespan of Caenorhabditis elegans via activating DAF-16/FOXO.

Phosphatases of regenerating liver (PRLs) are dual-specificity protein phosphatases. The aberrant expression of PRLs threatens human health, but their biological functions and pathogenic mechanisms are unclear yet. Herein, the structure and biological functions of PRLs were investigated using the Caenorhabditis elegans (C. elegans). Structurally, this phosphatase in C. elegans, named PRL-1, consisted of a conserved signature sequence WPD loop and a single C(X)5 R domain. Besides, by Western blot, immunohistochemistry and immunofluorescence staining, PRL-1 was proved to mainly express in larval stages and express in intestinal tissues. Afterward, by feeding-based RNA-interference method, knockdown of prl-1 prolonged the lifespan of C. elegans but also improved their healthspan, such as locomotion, pharyngeal pumping frequency, and defecation interval time. Furthermore, the above effects of prl-1 appeared to be taken without acting on germline signaling, diet restriction pathway, insulin/insulin-like growth factor 1 signaling pathway, and SIR-2.1 but through a DAF-16-dependent pathway. Moreover, knockdown of prl-1 induced the nuclear translocation of DAF-16, and upregulated the expression of daf-16, sod-3, mtl-1, and ctl-2. Finally, suppression of prl-1 also reduced the ROS. In conclusion, suppression of prl-1 enhanced the lifespan and survival quality of C. elegans, which provides a theoretical basis for the pathogenesis of PRLs in related human diseases.

Enhanced Cerebral Hemodynamics and Cognitive Function Via Knockout of Dual-Specificity Protein Phosphatase 5.

Alzheimer's Disease (AD) and Alzheimer's Disease-Related Dementias (ADRD) are neurodegenerative disorders. Recent studies suggest that cerebral hypoperfusion is an early symptom of AD/ADRD. Dual-specificity protein phosphatase 5 (DUSP5) has been implicated in several pathological conditions, including pulmonary hypertension and cancer, but its role in AD/ADRD remains unclear. The present study builds on our previous findings, demonstrating that inhibition of ERK and PKC leads to a dose-dependent dilation of the middle cerebral artery and penetrating arteriole, with a more pronounced effect in Dusp5 KO rats. Both ERK and PKC inhibitors resulted in a significant reduction of myogenic tone in vessels from Dusp5 KO rats. Dusp5 KO rats exhibited stronger autoregulation of the surface but not deep cortical cerebral blood flow. Inhibition of ERK and PKC significantly enhanced the contractile capacity of vascular smooth muscle cells from both strains. Finally, a significant improvement in learning and memory was observed in Dusp5 KO rats 24 hours after initial training. Our results suggest that altered vascular reactivity in Dusp5 KO rats may involve distinct mechanisms for different vascular beds, and DUSP5 deletion could be a potential therapeutic target for AD/ADRD. Further investigations are necessary to determine the effects of DUSP5 inhibition on capillary stalling, blood-brain barrier permeability, and neurodegeneration in aging and disease models.

Phosphatase Ssu72 Is Essential for Homeostatic Balance Between CD4+ T Cell Lineages.

Ssu72, a dual-specificity protein phosphatase, not only participates in transcription biogenesis, but also affects pathophysiological functions in a tissue-specific manner. Recently, it has been shown that Ssu72 is required for T cell differentiation and function by controlling multiple immune receptor-mediated signals, including TCR and several cytokine receptor signaling pathways. Ssu72 deficiency in T cells is associated with impaired fine-tuning of receptor-mediated signaling and a defect in CD4+ T cell homeostasis, resulting in immune-mediated diseases. However, the mechanism by which Ssu72 in T cells integrates the pathophysiology of multiple immune-mediated diseases is still poorly elucidated. In this review, we will focus on the immunoregulatory mechanism of Ssu72 phosphatase in CD4+ T cell differentiation, activation, and phenotypic function. We will also discuss the current understanding of the correlation between Ssu72 in T cells and pathological functions which suggests that Ssu72 might be a therapeutic target in autoimmune disorders and other diseases.

Dual-Specificity Protein Phosphatase 4 (DUSP4) Overexpression Improves Learning Behavior Selectively in Female 5xFAD Mice, and Reduces β-Amyloid Load in Males and Females.

Recent multiscale network analyses of banked brains from subjects who died of late-onset sporadic Alzheimer's disease converged on VGF (non-acronymic) as a key hub or driver. Within this computational VGF network, we identified the dual-specificity protein phosphatase 4 (DUSP4) [also known as mitogen-activated protein kinase (MAPK) phosphatase 2] as an important node. Importantly, DUSP4 gene expression, like that of VGF, is downregulated in postmortem Alzheimer's disease (AD) brains. We investigated the roles that this VGF/DUSP4 network plays in the development of learning behavior impairment and neuropathology in the 5xFAD amyloidopathy mouse model. We found reductions in DUSP4 expression in the hippocampi of male AD subjects, correlating with increased CDR scores, and in 4-month-old female and 12-18-month-old male 5xFAD hippocampi. Adeno-associated virus (AAV5)-mediated overexpression of DUSP4 in 5xFAD mouse dorsal hippocampi (dHc) rescued impaired Barnes maze performance in females but not in males, while amyloid loads were reduced in both females and males. Bulk RNA sequencing of the dHc from 5-month-old mice overexpressing DUSP4, and Ingenuity Pathway and Enrichr analyses of differentially expressed genes (DEGs), revealed that DUSP4 reduced gene expression in female 5xFAD mice in neuroinflammatory, interferon-gamma (IFNγ), programmed cell death protein-ligand 1/programmed cell death protein 1 (PD-L1/PD-1), and extracellular signal-regulated kinase (ERK)/MAPK pathways, via which DUSP4 may modulate AD phenotype with gender-specificity.

Negative Feedback Regulation of MAPK Signaling Is an Important Driver of CLL Progression

Chronic lymphocytic leukemia (CLL) represents approximately 25% of all new cases of leukemia in the USA. Insight into the pathogenesis of CLL has led to the development of highly effective treatments in the past decade. Nevertheless, CLL patients with activating mutations in the mitogen-activated protein kinase (MAPK) pathway, which enables cell proliferation and differentiation, comprise a distinct subgroup with poor clinical prognosis such as shorter treatment-free survival and higher drug resistance. Dual specificity protein phosphatases (DUSPs) negatively regulate MAPK signaling and are required to limit and terminate the activating signal. Whether DUSP signaling is crucial for the development and progression of MAPK driven CLL is unknown. Thus, in this study we investigated the expression and functional relevance of negative MAPK regulators DUSP1 and DUSP6 in CLL. Analyzing mRNA data sets of CLL patients, we discovered that DUSP1 and DUSP6 were broadly expressed in CLL. High expression levels of DUSP6, though not DUSP1, were found in cases with activating mutations within the MAPK pathway and correlated with a poor clinical outcome, indicated by shorter time-to-treatment and overall survival. To investigate the functional relevance of DUSP1- and DUSP6-mediated negative MAPK regulation in CLL, we tested the effects of the dual-specific DUSP1/6 inhibitor BCI on CLL cells. DUSP1/6 inhibition in vitro resulted in a dose-dependent induction of specific cell death of CLL cells, whereas B cells from healthy donors remained unaffected. Treatment of mice with an adoptively transferred murine CLL with an in vivo applicable derivate of BCI also significantly decreased CLL levels in the spleen and peritoneal cavity, compared to a vehicle treated control group. Further, CRISPR/Cas9-mediated knockout of DUSP1 or DUSP6 in the CLL cell line MEC-1 reduced cell proliferation and fitness; however, DUSP1/6 double knockouts lead to syngeneic lethality. Thus, DUSP1 and DUSP6 are crucial for negative MAPK pathway regulation to enable cell survival. To assess changes in intracellular signaling pathways after DUSP1/6 inhibition, we performed a phospho-proteome analysis of BCI treated patient-derived CLL cells and compared them to vehicle treated cells. As expected, a significant deregulation of B-cell receptor and MAPK signaling was detected after inhibition, with an increase in the activating phosphorylation on ERK1 and ERK2, which are targeted by DUSP1/6, confirming on-target specificity of BCI using an unbiased approach. To gain insight into the mechanism driving cell death, we conducted a BCI time course analysis followed by a phospho-proteome screen and observed that the DNA damage response (DDR) and the apoptosis pathway were among the strongest regulated pathways upon DUSP1/6 inhibition in MEC-1 cells. To validate these findings, we used immunoblotting and detected phosphorylation of DDR key molecules, such as ATF2 and c-JUN, in human CLL samples after DUSP1/6 inhibition. We also used flow cytometry to confirm acute activation of the MAPK pathway after BCI treatment by detecting increased phospho-γH2AX (DNA damage) and phosphatidylserine (apoptosis) levels. Furthermore, we showed that the apoptotic cell death is promoted by CHK1/2 kinases and depends on MAPK activity. Chemotherapeutic drug resistances are often detected in patients with deletion of p53 or ATM, central regulators of the DDR. Hence, we tested the effectiveness of DUSP1/6 inhibition on patient derived CLLs carrying a loss of function variation of p53 or ATM. We did not detect a significant difference in cytotoxicity, indicating DUSP1/6 inhibition effectively decreases CLL viability, even in cases without functional p53/ATM-mediated DDR. Finally, we generated a MEC-1 cell line resistant to the chemotherapeutic agent idelalisib, as a model for drug resistant CLLs. As the sensitivity to idelalisib decreased, the cells became more sensitive to BCI. When comparing the effects of BCI on CLL cells from treatment naïve patients compared to ibrutinib-refractory patients, we found no differences in sensitivity to DUSP1/6 inhibition. In conclusion, we show that MAPK deregulation by DUSP1/6 inhibition is highly effective to induce cytotoxic cell death in CLL cells, even without functional p53/ATM-mediated DDR; hence, we suggest DUSP1/6 inhibition is a promising approach to treat drug resistant and refractory CLLs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK/STAT3 Signaling Activation Related to Distinct Clinicopathologic Features in Systemic ALK - Anaplastic Large Cell Lymphomas : New Insights into Their Heterogeneity.

Systemic anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) is a group of heterogenous CD30 + T-cell non-Hodgkin lymphomas. Previous studies have highlighted the importance of JAK/STAT3 signaling activation in the molecular pathogenesis of ALK - ALCLs. In the present study, we aimed to establish a potential relationship between JAK/STAT3 signaling activation and clinicopathologic features in ALK - ALCLs, and further recognize the heterogenous nature of these neoplasms. Immunohistochemistry staining of the phosphorylated-STAT3 (p-STAT3) and dual-specificity protein phosphatase 22 ( DUSP22 ) gene rearrangement analysis were performed. Forty-five cases of ALK - ALCL were divided into 3 groups, including 9 DUSP22 -rearranged ALCLs, 21 p-STAT3 + double-negative (DN) ALCLs (both ALK and DUSP22 rearrangement negative), and 15 p-STAT3 - DN-ALCLs. Morphologically, p-STAT3 + DN-ALCLs exhibited sheet-like neoplastic cells and sometimes showed large pleomorphic cells scattered in a lymphocyte-rich background more frequently than those in other ALK - ALCLs subtypes. Phenotypically, the p-STAT3 + DN-ALCLs frequently expressed cytotoxic molecules, epithelial membrane antigen, and programmed death-ligand 1, whereas CD3 and CD5 expression was not observed. Clinically, patients with p-STAT3 + DN-ALCLs had a better prognosis than those with p-STAT3 - DN-ALCLs. These observations suggest that p-STAT3 + DN-ALCLs represent a distinct subtype of ALK - ALCLs. Identifying ALK - ALCL subtypes by using p-STAT3 staining and DUSP22 rearrangement is a promising approach that may contribute to risk stratification and better treatment decisions in the future clinical practice.