Mitogen-activated Protein Kinase Dephosphorylation Research Articles

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.

Inhibitory Effects of Ehretia tinifolia Extract on the Excessive Oxidative and Inflammatory Responses in Lipopolysaccharide-Stimulated Mouse Kupffer Cells.

Ehretia tinifolia (E. tinifolia) L., an evergreen tree with substantial biological activity, including antioxidant and anti-inflammatory effects, has been used in many herbal and traditional medicines. To elucidate its antioxidant and anti-inflammatory activity and the underlying mechanisms, we applied a methanol extract of E. tinifolia (ETME) to lipopolysaccharide (LPS)-stimulated mouse immortalized Kupffer cells. ETME suppressed the LPS-induced increase in nitric oxide, a mediator for oxidative stress and inflammation, and restored LPS-mediated depletion of total glutathione level by stabilizing antioxidative nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent increase in heme oxygenase-1 levels. Furthermore, ETME inhibited the LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The inhibitory effects of ETME on pro-inflammatory responses were regulated by ETME-mediated dephosphorylation of mitogen-activated protein kinases (MAPKs: p38, p44/p42, and stress-associated protein kinase/c-Jun N-terminal kinase) and inhibition of nuclear localization of nuclear factor kappa B (NF-κB). These results suggest that ETME is a possible candidate for protecting Kupffer cells from LPS-mediated oxidative stress and excessive inflammatory responses by activating antioxidant Nrf2/HO-1 and inhibiting pro-inflammatory NF-κB and MAPKs, respectively.

MKP1 promotes nonalcoholic steatohepatitis by suppressing AMPK activity through LKB1 nuclear retention

Nonalcoholic steatohepatitis (NASH) is triggered by hepatocyte death through activation of caspase 6, as a result of decreased adenosine monophosphate (AMP)-activated protein kinase-alpha (AMPKα) activity. Increased hepatocellular death promotes inflammation which drives hepatic fibrosis. We show that the nuclear-localized mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP1) is upregulated in NASH patients and in NASH diet fed male mice. The focus of this work is to investigate whether and how MKP1 is involved in the development of NASH. Under NASH conditions increased oxidative stress, induces MKP1 expression leading to nuclear p38 MAPK dephosphorylation and decreases liver kinase B1 (LKB1) phosphorylation at a site required to promote LKB1 nuclear exit. Hepatic deletion of MKP1 in NASH diet fed male mice releases nuclear LKB1 into the cytoplasm to activate AMPKα and prevents hepatocellular death, inflammation and NASH. Hence, nuclear-localized MKP1-p38 MAPK-LKB1 signaling is required to suppress AMPKα which triggers hepatocyte death and the development of NASH.

Cod (Gadus) skin collagen peptide powder reduces inflammation, restores mucosal barrier function, and inhibits fibrosis in dextran sodium sulfate-induced colitis in mice

Ethnopharmacological relevanceUlcerative colitis (UC) is a chronic inflammatory bowel disease of unknown etiology. Cod (Gadus), a kind of herb from the Chinese herb. Traditionally, it has used to treat trauma, reduce swelling and relieve pain in order to exert its anti-inflammatory activity. Recent reports based on its hydrolyzed or enzymatic extracts have shown its anti-inflammatory, mucosal barrier protecting properties. However, its mechanism of improvement in ulcerative colitis is not clear. Aim of the studyThis study aimed to explore the preventive and protective effect of cod skin collagen peptide powder (CP) on mice with UC and to explore the underlying mechanism. Materials and methodsMice with dextran sodium sulfate (DSS)-induced UC were treated with CP by gavage, and the anti-inflammatory effects of CP were assessed using general physical, pro-inflammatory cytokine, histopathological, immunohistochemical, macrophage flow cytometry, and inflammatory signaling pathway assays. ResultsCP ameliorates inflammation by upregulating mitogen-activated protein kinase phosphatase-1 (MKP-1) and thereby decreasing the phosphorylation levels of P38 and JNK. It also polarizes macrophages in the colon towards the M2 phenotype, which helps to reduce tissue damage and promotes colon repair. At the same time, CP also inhibits the development of fibrosis, one of the complications of UC, by upregulating ZO-1, Occludin, and downregulating α-SMA, Vimentin, Snail, and Slug. ConclusionIn this study, we found CP reduced inflammation in mice with UC by inducing MKP-1 expression, which caused dephosphorylation of mitogen-activated protein kinase (MAPK). CP also restored mucosal barrier function and inhibited the development of fibrosis complicating UC in these mice. Taken together, these results suggested that CP improved the pathological manifestations of UC in mice, suggesting that it can play a biological role as a nutritional supplement for preventing and treating UC.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targetedtothe mitochondrial outermembrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has asignificant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data fromour mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

Cannabinoid Receptor Type 2 Agonist Reduces Morphine Tolerance via Mitogen Activated Protein Kinase Phosphatase Induction and Mitogen Activated Protein Kinase Dephosphorylation

Morphine is an opioid drug often used in treating moderate to severe pain. However, morphine tolerance in patients limits its used in clinical settings. Our previous study showed that a cannabinoid type 2 (CB2) receptor agonist attenuated morphine tolerance. However, the exact mechanism by which CB2 agonists reduce morphine tolerance remains unclear. In this study, we investigated the effect of mitogen activated protein kinase (MAPK) and mitogen activated protein kinase phosphatases 1 and 3 (MKP-1 and MKP-3) on the regulation of morphine tolerance by CB2 receptor agonist. Chronic morphine treatments for 7 days reduced the protein expression of MKP-1 and MKP-3 in the spinal cord and increased the phosphorylation of p38, ERK1/2 and the level of proinflammatory mediator, such as IL-1β, IL-6 and TNF-α. Coadministration of CB2 receptor agonist AM1241 alleviated the inhibition of MKP-1 and MKP-3 by chronic morphine administration and reduced the expression of phosphorylated MAPK and proinflammatory factors. The effect of the CB2 receptor agonist on morphine-induced downregulation of MKP-1 and MKP-3 was reversed by the MKP-1 and MKP-3 antagonist triptolide. Our findings suggested that CB2 receptor agonist may induce the expression of MKP-1 and MKP-3 to promote MAPK dephosphorylation and reduce the production of downstream cytokine, thereby reducing morphine tolerance. This finding suggested that MKPs may serve as a new target for alleviating morphine tolerance.

Dephosphorylation of LjMPK6 by Phosphatase LjPP2C is Involved in Regulating Nodule Organogenesis in Lotus japonicus.

The mitogen-activated protein kinase (MAPK) LjMPK6 is a phosphorylation target of SIP2, a MAPK kinase that interacts with SymRK (symbiosis receptor-like kinase) for regulation of legume-rhizobia symbiosis. Both LjMPK6 and SIP2 are required for nodulation in Lotus japonicus. However, the dephosphorylation of LjMPK6 and its regulatory components in nodule development remains unexplored. By yeast two-hybrid screening, we identified a type 2C protein phosphatase, LjPP2C, that specifically interacts with and dephosphorylates LjMPK6 in vitro. Physiological and biochemical assays further suggested that LjPP2C phosphatase is required for dephosphorylation of LjMPK6 in vivo and for fine-tuning nodule development after rhizobial inoculation. A non-phosphorylatable mutant variant LjMPK6 (T224A Y226F) could mimic LjPP2C functioning in MAPK dephosphorylation required for nodule development in hairy root transformed plants. Collectively, our study demonstrates that interaction with LjPP2C phosphatase is required for dephosphorylation of LjMPK6 to fine tune nodule development in L. japonicus.

Design of a MAPK signalling cascade balances energetic cost versus accuracy of information transmission

Cellular processes are inherently noisy, and the selection for accurate responses in presence of noise has likely shaped signalling networks. Here, we investigate the trade-off between accuracy of information transmission and its energetic cost for a mitogen-activated protein kinase (MAPK) signalling cascade. Our analysis of the pheromone response pathway of budding yeast suggests that dose-dependent induction of the negative transcriptional feedbacks in this network maximizes the information per unit energetic cost, rather than the information transmission capacity itself. We further demonstrate that futile cycling of MAPK phosphorylation and dephosphorylation has a measurable effect on growth fitness, with energy dissipation within the signalling cascade thus likely being subject to evolutionary selection. Considering optimization of accuracy versus the energetic cost of information processing, a concept well established in physics and engineering, may thus offer a general framework to understand the regulatory design of cellular signalling systems.

Role of Dual-Specificity Phosphatase 1 in Glucocorticoid-Driven Anti-inflammatory Responses.

Glucocorticoids (GCs) potently inhibit pro-inflammatory responses and are widely used for the treatment of inflammatory diseases, such as allergies, autoimmune disorders, and asthma. Dual-specificity phosphatase 1 (DUSP1), also known as mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), exerts its effects by dephosphorylation of MAPKs, i.e., extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Endogenous DUSP1 expression is tightly regulated at multiple levels, involving both transcriptional and post-transcriptional mechanisms. DUSP1 has emerged as a central mediator in the resolution of inflammation, and upregulation of DUSP1 by GCs has been suggested to be a key mechanism of GC actions. In this review, we discuss the impact of DUSP1 on the efficacy of GC-mediated suppression of inflammation and address the underlying mechanisms.

Kinetic and mechanistic studies of p38α MAP kinase phosphorylation by MKK6.

Bistability (coexistence of two stable steady states in a dynamical system) is a key mechanism of cellular decision-making and has been observed in many biochemical reaction networks such as mitogen-activated protein kinase (MAPK) signaling pathways. Theoretical studies have shown that bistability can arise in a single two-site MAPK phosphorylation and dephosphorylation cycle. However, the bistable behavior mostly relies on the kinetic mechanisms and parameters of this two-site modification. In exploring the system-level properties of MAPK regulation, most models to date focus on two limiting reaction regimes, distributive and processive, and are characterized by high levels of parametric uncertainty. Here, we developed a combined kinetic method which applies a continuous spectrophotometric enzyme-coupled assay incorporated with the viscosity approach, to perform detailed kinetic analyses of p38α MAPK dual phosphorylation by MKK6. Almost all kinetic rate constants for the first and second phosphorylation steps in p38α activation have been quantitatively determined, supporting that the phosphorylation occurs randomly in the first step, albeit preferring the tyrosine residue. The release rates of monophosphorylated p38α from MKK6, either as the product in the first modification or as the substrate in the second step, were comparable to the respective adjacent phosphoryl transfer steps. These results indicated that dual phosphorylation of p38α by MKK6 involves a random, partially processive mechanism. Based on the experimentally determined models and parameters, dynamics of the p38α-MKK6-MKP5 system were explored, demonstrating for the first time that bistability can arise with this model at biologically feasible parameter values. ENZYMES: p38α (EC 2.7.11.24); MKK6 (EC 2.7.12.2).

Dual-specificity phosphatases regulate mitogen-activated protein kinase signaling in adipocytes in response to inflammatory stress

Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3 T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.

Parathyroid Hormone-Related Protein Protects Osteoblastic Cells From Oxidative Stress by Activation of MKP1 Phosphatase.

Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen-activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH-related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase-1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS-induced apoptosis by targeting MKP1. In osteoblastic MC3T3-E1 and MG-63 cells, H2 O2 triggered p38, JNK, ERK and p66Shc phosphorylation, and cell apoptosis. Meanwhile, PTHrP (1-37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK, or p66Shc activation. H2 O2 -induced p38 and ERK phosphorylation and apoptosis were both decreased by pre-treatment with specific kinase inhibitors or PTHrP (1-37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1-37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1-37) promptly enhanced MKP1 protein and gene expression and MKP1-dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1-37) adsorbed in an implanted hydroxyapatite-based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro-apoptotic actions of ROS by a mechanism dependent on MKP1-induced dephosphorylation of MAPKs in osteoblasts. J. Cell. Physiol. 232: 785-796, 2017. © 2016 Wiley Periodicals, Inc.

MTOR-Independent autophagy inducer trehalose rescues against insulin resistance-induced myocardial contractile anomalies: Role of p38 MAPK and Foxo1

Insulin resistance is associated with cardiovascular diseases although the precise mechanisms remain elusive. Akt2, a critical member of the Akt family, plays an essential role in insulin signaling. This study was designed to examine the effect of trehalose, an mTOR-independent autophagy inducer, on myocardial function in an Akt2 knockout-induced insulin resistance model. Adult WT and Akt2 knockout (Akt2⿿/⿿) mice were administered trehalose (1mg/g/day, i.p.) for two days and were then given 2% trehalose in drinking water for two more months. Echocardiographic and myocardial mechanics, intracellular Ca2+ properties, glucose tolerance, and autophagy were assessed. Apoptosis and ER stress were evaluated using TUNEL staining, Caspase 3 assay and Western blot. Autophagy and autophagy flux were examined with a focus on p38 mitogen activated protein kinase (MAPK), Forkhead box O (Foxo1) and Akt. Akt2 ablation impaired glucose tolerance, myocardial geometry and function accompanied with pronounced apoptosis, ER stress and dampened autophagy, the effects of which were ameliorated by trehalose treatment. Inhibition of lysosomal activity using bafilomycin A1 negated trehalose⿿induced induction of autophagy (LC3B-II and p62). Moreover, phosphorylation of p38 MAPK and Foxo1 were upregulated in Akt2⿿/⿿ mice, the effect of which was attenuated by trehalose. Phosphorylation of Akt was suppressed in Akt2⿿/⿿ mice and was unaffected by trehalose. In vitro findings revealed that the p38 MAPK activator anisomycin and the Foxo1 inhibitor (through phosphorylation) AS1842856 effectively masked trehalose-offered beneficial cardiomyocyte contractile response against Akt2 ablation. These data suggest that trehalose may rescue against insulin resistance-induced myocardial contractile defect and apoptosis, via autophagy associated with dephosphorylation of p38 MAPK and Foxo1 without affecting phosphorylation of Akt.

Mitogen-Dependent Regulation of DUSP1 Governs ERK and p38 Signaling During Early 3T3-L1 Adipocyte Differentiation

Knowledge concerning mechanisms that control proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and the development of obesity. Evidence has shown that temporal regulation of mitogen-activated protein kinase (MAPK) phosphorylation and dephosphorylation is critical for coupling extracellular stimuli to cellular growth and differentiation. Using differentiating 3T3-L1 preadipocytes as a model of adipocyte hyperplasia, we examined a role for dual-specificity phosphatase 1 (DUSP1) on the timely modulation of MAPK signaling during states of growth arrest, proliferation, and differentiation. Using real-time reverse transcription PCR (qRT-PCR), we report that DUSP1 is induced during early preadipocyte proliferation concomitant with ERK and p38 dephosphorylation. As deactivation of ERK and p38 is essential for the progression of adipocyte differentiation, we further showed that de novo mRNA synthesis was required for ERK and p38 dephosphorylation, suggesting a role for "inducible" phosphatases in regulating MAPK signaling. Pharmacological and genetic inhibition of DUSP1 markedly increased ERK and p38 phosphorylation during early adipocyte differentiation. Based on these findings, we postulated that loss of DUSP1 would block adipocyte hyperplasia. However, genetic loss of DUSP1 was not sufficient to prevent preadipocyte proliferation or differentiation, suggesting a role for other phosphatases in the regulation of adipogenesis. In support of this, qRT-PCR identified several MAPK-specific DUSPs induced during early (DUSP2, -4, -5, & -6), mid (DUSP4 & -16) and late (DUSP9) stages of adipocyte differentiation. Collectively, these data suggest an important role for DUSPs in regulating MAPK dephosphorylation, with an emphasis on DUSP1, during early adipogenesis.

Polyspermy block in jellyfish eggs: Collaborative controls by Ca2+ and MAPK

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca2+ and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm–egg fusion and initiation of an intracellular Ca2+ rise from this site. The elevated Ca2+ levels lasted for several minutes following the sperm–egg fusion. The Ca2+ rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca2+ rise with EGTA promoted polyspermy, and conversely, triggering a Ca2+ rise by inositol 1,4,5-trisphosphate (IP3) or excess K+ immediately abolished the egg’s capacity for sperm–egg fusion. A Ca2+ rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca2+ rise at fertilization or by IP3 injection, and shut down the subsequent sperm–egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca2+ rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca2+ levels, and prevented sperm–egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm–egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm–egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca2+ and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.

Interaction Between DUSP8 and the Polyglutamine Protein Ataxin-1

Polyglutamine (polyQ) diseases are a group of inherited neurodegenerative disorders caused by the expansion of polyQ repeats in the disease proteins. Expansion of polyQ repeats presumably leads to misfolding and aggregation of polyQ proteins. Although details of the molecular mechanisms are still elusive, transcriptional dysfunction and oxidative damage are suggested as underlying events responsible for polyQ pathogenesis. Misfolded polyQ proteins are frequently concentrated into intranuclear inclusions, which increase generation of reactive oxygen species (ROS) and activate stress signaling pathways. In cultured cells expressing expanded polyQ proteins, mitogen-activated protein kinases (MAPK), e.g., p38 and JNK, are activated and the cytotoxicity induced by polyQ proteins, partially through sustained activation of MAP kinases, can be reduced by administration of a p38 inhibitor. MAPK are activated by MAPK kinase (MEK)-mediated phosphorylation of threonine and tyrosine residues located in the signature sequence (TxY) and MEK is in turn activated by MAPK kinase kinase (MEKK). Since MAPK are involved in a wide variety of cellular processes including cell survival and apoptosis, cells must tightly regulate the magnitude and duration of MAPK activation. The negative regulation of MAPK is often achieved by phosphatase-mediated dephosphorylation of MAPK. A group of protein phosphatases referred as type I cysteinebased protein tyrosine phosphatases including DUSPs (dualspecificity phosphatases) dephosphorylate both tyrosine and serine/threonine residues within the same substrate and as a result counteract MAPK. Involvement of protein tyrosine phosphatases (PTPs) in polyQ-induced cell death has been reported in previous studies. Several PTP proteins including MKP-1 are up-regulated by over-expression of cytotoxic polyQ-expanded huntingtin (Htt) fragment. Moreover, polyQ-expanded Htt fragment activates JNK pathway by reducing solubility of JNK phosphatase M3/6 (also known as hVH5 or DUSP8). A recent study demonstrated that a DUSP protein laforin can suppress the cytotoxicity caused by misfolded proteins and hinted that phosphatases can be potential targets for pharmacological intervention of neurodegenerative disorders. The polyQ protein ataxin-1 is a soluble protein of about 816 amino acids, which varies depending on the length of polyQ repeats, and located both in the cytoplasm and nucleus. Although the exact functions are not completely understood, ataxin-1 is apparently involved in transcription regulation through its ability to interact with several transcription factors as well as RNA. While it is generally assumed that expansion of polyQ repeats causes misfolding of ataxin-1 and leads to neuronal death in spinocerebellar ataxia 1 (SCA1), contribution of other domains to pathogenesis also becomes increasingly clear. Besides N-terminal polyQ region, AXH domain (570-689; for interaction with transcription factors and RNA), endogenous phosphorylation site (776) and nuclear localization signal (795-798) are present in the Cterminus of ataxin-1. Phosphorylation of ataxin-1 is particularly important since it is implicated in pathogenicity of SCA1. Phosphorylation at Ser776 is critical for the interaction of ataxin-1 with 14-3-3 proteins that regulate its entry into the nucleus. Previously we demonstrated that ataxin-1, capable of activating JNK by itself, does not directly interact with this MAPK. Such observations led us to suspect that misfolded polyQ proteins, while not directly binding to JNK, could physically associate with PTPs (e.g., DUSP proteins) and thereby regulate JNK activation. To explore this possibility, we first investigated if ataxin-1 actually interacts with MAPK phosphatases in vivo. Upon screening for PTPs that physically interact with normal ataxin-1[30Q], we found out that a number of human PTPs including DUSP2, 8, 13 and 24 strongly associated with ataxin-1[30Q] (Table 1).

The dual‐specificity phosphatase 8 (Dusp8) regulates cardiac hypertrophic response in vitro and in vivo

BackgroundPhosphorylation and dephosphorylation of mitogen‐activated protein kinases (MAPKs) play important roles in regulating cardiac hypertrophy, remodeling, and heart failure. Dual‐specificity phosphatases (Dusps) directly dephosphorylate and inactivate each of the MAPK terminal kinases (ERK, p38, JNK) within 30 to 60 minutes activation. In this study, we generated mice and mouse embryonic fibroblasts (MEFs) lacking DUSP8 gene and determined the role of Dusp8 in regulating c‐Jun N‐terminal kinases (JNK1/2) signaling in neonatal myocyte culture and mouse heart. We hypothesized that knockout of Dusp8 will lead to increased JNK phosphorylation and decreased cardiac hypertrophy.MethodsDUSP8 null mice were generated using traditional gene‐targeting approach to delete exon 5 and 6. Phenylephrine was injected at final concentration of 10 mg/kg using 2‐month‐old mice.Results: Adenovirus mediated Dusp8 overexpression in cultured cardiomyocytes resulted in decreased JNK1/2 phosphorylation but not that of p38 and ERK1/2, indicating Dusp8 is specific for JNK dephosphorylation. In contrast, injection of Dusp8 null mice with phenylephrine led to sustained JNK phosphorylation compared to that of WT mice, which was also true for the JNK signaling in Dusp8 null MEF cells. Dusp8 null mice had reduced left ventricular weight/body weight ratio at both baseline and angiotensin II/phenylephrine stimulation condition. At cellular level, we found that overexpression of Dusp8 led to increased nuclear accumulation of NFAT and expression of hypertrophy gene ANF.ConclusionsThe results of the present study indicate that knockout of Dusp8 is cardioprotective through increasing JNK phosphorylation to prevent NAFT signaling induced cardiomyopathy.

Abstract B34: Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer

Abstract The interaction between cancer cells and the surrounding microenvironment transforms the stroma into an abnormal phenotype, altering normal function, tissue architecture, cellular morphology, and extracellular matrix-cell interactions that directly contribute to formation of neoplasia. About 80% of reactive stroma associated with breast carcinoma is comprised of activated myofibroblasts or cancer-associated fibroblasts which secrete extracellular matrix proteins resulting in the desmoplasia that is associated with advanced breast tumor progression. Cancer-associated fibroblasts, transformed by cytokines such as transforming growth factor-β (TGF-β), secrete tumor promoting growth factors and extracellular matrix components, to promote tumor initiation, growth, and metastases. Inhibition of cancer-associated fibroblast proliferation and function may be an effective therapeutic strategy, since these cells play a vital role in carcinogenesis. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, seven amino acid peptide hormone of the renin-angiotensin system with anti-proliferative and anti-fibrotic properties. We showed that Ang-(1-7) inhibited the growth of MDA-MB-231 human triple negative breast tumors growing in the mammary fat pads of athymic mice with a concomitant 3-fold decrease in interstitial tumor fibrosis (n=9-11, p<0.0001). Cancer-associated fibroblasts were isolated from orthotopic breast tumors, to assess the effect of Ang-(1-7) on fibroblast proliferation. The isolated tumoral fibroblasts stained positive for fibronectin, vimentin, collagen I, and β-smooth muscle actin, confirming that the isolated cells were activated myofibroblasts. Ang-(1-7) significantly reduced the growth of cancer-associated fibroblasts isolated from orthotopic triple negative breast tumors (n=4-5, p<0.05). The decrease in cancer-associated fibroblast proliferation was accompanied by attenuation of the activation of the growth-promoting mitogen-activated protein kinases (MAPK) ERK1 and ERK2 (n=3, p<0.001) as quantified by Western blot hybridization. In contrast, incubation of the heptapeptide hormone resulted in a 2.5-fold increase in the MAPK phosphatase DUSP1 (dual specificity phosphatase 1) in cancer-associated fibroblasts (n=3, p<0.05). These studies suggest that Ang-(1-7) promotes the dephosphorylation of MAPK by up-regulating DUSP1 to reduce this proliferative signaling pathway in cancer-associated fibroblasts and prevent tumor growth. TGF-β is a potent stimulator of fibroblast activation and plays an important role in promoting fibrosis, including activation of the MAP kinase pathway to stimulate fibroblast proliferation. TGF-β activates tumor-promoting cancer-associated fibroblasts by phosphorylation of Smad2/3 which translocates to the nucleus to induce pro-fibrotic gene expression. Stimulation of the Smad signaling pathway by TGF-β results in extracellular matrix protein synthesis and deposition as well as its own production, creating an autocrine cycle of fibroblast activation and extracellular matrix deposition. TGF-β was quantified by Western blot hybridization in protein homogenates from myofibroblasts isolated from orthotopic breast tumors. TGF-β stimulation of cancer-associated fibroblasts significantly increased phospho-Smad2/3 which was reduced approximately 60% by treatment with Ang-(1-7) (n=3, p<0.001), indicating that the heptapeptide inhibits both the growth of stromal fibroblasts as well as the signaling pathways that participate in extracellular matrix protein production. These findings suggest that Ang-(1-7) may serve as a first-in-class chemotherapeutic agent for triple negative breast cancer in part through a reduction in the proliferation and function of cancer-associated fibroblasts in the tumor microenvironment. Citation Format: Alison L. Arter, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B34.

MicroRNA-101, mitogen-activated protein kinases and mitogen-activated protein kinases phosphatase-1 in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is the prototype of human autoimmune disease in which various inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6 and interferon (IFN) play crucial pathogenic roles. The production of these cytokines is responsible for the mitogen-activated protein kinases (MAPKs), which can also generate mitogen-activated protein kinases phosphatases (MKPs). MKP-1, a prototypical member of the MKP family that can influence outcomes of autoimmune diseases and reduce the inflammatory cytokines by dephosphorylation of p38 and JNK MAPK, plays a critical role in the expression of inflammatory mediators at transcriptional and post-transcriptional levels. MicroRNA-101 (miR) is a small non-coding RNA that regulates the MAPK response by targeting MKP-1 mRNA 3'-UTR, and affects the secretion of the downstream inflammatory cytokines. However, the interaction among the above three in the pathogenesis of SLE has not previously been reported. This review discusses the current understanding of the role of the MAPK/MKP/miR-101 axis in regulating immune responses and the pathogenesis of SLE to provide new ideas for clinical treatment of SLE.

Protein Phosphatase 2A Promotes Endothelial Survival via Stabilization of Translational Inhibitor 4E-BP1 Following Exposure to Tumor Necrosis Factor-α

Tumor necrosis factor-α (TNFα) may change from a stimulator of reversible activation of endothelial cells (ECs) to a killer when combined with cycloheximide (CHX). The means by which endothelial cells are destined to either the survival pathway or the apoptotic pathway are not fully understood. We investigated the role of p38 mitogen-activated protein kinase (MAPK) and protein phosphatase 2A (PP2A) activation and their regulation of 4E-BP1 stability in ECs to determine whether this pathway contributes to apoptosis induced by TNFα and CHX. Apoptosis was induced in human umbilical vein ECs (HUVECs) by treating them with a combination of TNFα and CHX (TNFα/CHX). Activation of p38 MAPK was increased in HUVECs undergoing apoptosis, which was associated with degradation of eukaryotic initiation factor 4A regulator 4E-BP1 in a p38 MAPK-dependent manner. CHX attenuated a TNFα-stimulated increase in the expression and activity of PP2A. Silencing PP2A expression with small interfering RNA transfection mimicked CHX sensitization, increasing HUVEC apoptosis with TNFα stimulation and suggesting a protective role for PP2A in the apoptotic process. Our data suggest that (1) TNFα stimulates PP2A and HUVECs elude apoptosis by PP2A-dependent dephosphorylation of p38 MAPK, and (2) CHX-induced inhibition of PP2A leads to maintenance of p38 activity and degradation of 4E-BP1, resulting in enhanced TNFα-induced apoptosis.