Mitogen-activated Protein Kinase Isoforms Research Articles

Interdependency and differential expression of ERK1 and ERK2 in breast and melanoma cell lines

BackgroundRegulatory mechanism of ERK1 and ERK2, their mechanisms of action, and how they impact on development, growth, and homeostasis of different organisms have been given much emphasis for long. ERK1 and 2 though are isoforms of ERK mitogen-activated protein kinase but are coded by two different genes MAPK3 and MAPK1 respectively and show differential expressions and interdependency in different cancer cell lines. Our previous investigations substantially stated the effect of ERK1 and ERK2 on different extracellular molecules like MMPs and integrins, responsible for cell growth and differentiation. Here, we aim to study individual roles of ERK1 and ERK2 and their interdependency in progression and invasiveness in various cancer cell lines.MethodsDifferent cancer cell lines namely B16F10 (melanoma), MCF7, and MDAMB231 (breast cancer) for studying this particular question were used. Methodologies like gelatin zymography, immunoprecipitation, Western blotting, cell invasion assay, wound healing assay, siRNA transfection, and double transfection procedures were followed for our study.ResultsOur findings suggest compensation for ERK2 deficiency by pERK1, clear ERK2 predominance in MCF7 cell line, ERK1-ERK2 interdependency in MDAMB231 cells with regard to compensating each other, and significant role of both ERK1 and ERK2 in modulation of MMP9.ConclusionIf summarized, our results prove the contribution of ERK2 in compensating ERK1 loss and vice versa and an evident role of ERK1 in cancer cell invasiveness.

Abstract Mo011: Selective silencing of p38δ is cardioprotective in female mice during doxorubicin chemotherapy

Introduction: Dose-dependent cardiotoxicity limits the efficacy of chemotherapeutic agent doxorubicin (DOX). p38δ is one of four p38 mitogen-activated protein kinase isoforms that regulate DOX-induced cardiotoxicity (DIC). p38δ genetic deletion protects female mice from DIC. In the absence of selective pharmacologic inhibitors of p38δ, docosanoic acid (DCA) conjugate-mediated delivery of chemically engineered small interfering RNA (siRNA) provides a novel avenue to achieve selective, efficient, durable, and nontoxic in vivo p38δ silencing in the heart. Hypothesis: The selective silencing of p38δ is cardioprotective from DIC in female mice. Methods: Following the screening of a panel of p38δ-selective siRNA sequences, the lead siRNA si-p38δ-1, with the highest silencing efficacy and potency in vitro, was identified. 15-week-old female mice were subcutaneously injected with 20 mg/kg of DCA-conjugated chemically stabilized si-p38δ-1 (n = 3) or the non-targeting control (NTC) siRNA (n = 3) twice 10 hours apart. p38δ silencing in mouse hearts was measured 14 days later by assessing mRNA and protein expression. To evaluate the effect of p38δ silencing after acute DOX treatment, the mice were administered si-p38δ-1 (n = 5) or NTC (n = 5), followed by DOX treatment (30 mg/kg) six days later. The study concluded 11 days after DOX treatment. Morbidity was assessed by assigning health scores daily. Electrocardiography, echocardiography, and optical mapping were performed to evaluate cardiac function. Results: si-p38δ-1 led to 80% silencing of p38δ in mouse hearts 14 days after administration, without any cardiotoxic effects. si-p38δ-1-treated female mice exhibited diminished DOX-induced morbidity (p = 0.003) and were protected from developing calcium alternans (CAs) 11 days post-DOX treatment, compared to NTC-treated counterparts. Thus, DOX-induced CAs, indicative of calcium mishandling, were observed in 75% (3/4) NTC-treated mice and 0% (0/5) si-p38δ-1-treated mice. Conclusion: Efficient and well-tolerated in vivo p38δ silencing by si-p38δ-1 in mouse hearts mitigated DOX-induced morbidity and calcium mishandling in female mice, highlighting the therapeutic potential of p38δ silencing for alleviating DIC in female cancer patients.

GLUT2 regulation of p38 MAPK isoform protein expression and p38 phosphorylation in male versus female rat hypothalamic primary astrocyte Cultures

Recent studies documented regulation of hypothalamic astrocyte mitogen-activated protein kinase (MAPK) pathways, including p38, by the plasma membrane glucose carrier/sensor glucose transporter-2 (GLUT2). Sex-specific GLUT2 control of p38 phosphorylation was observed, but effects on individual p38 family protein profiles were not investigated. Current research employed an established primary astrocyte culture model, gene knockdown tools, and selective primary antisera against p38-alpha, p38-beta, p38-gamma, and p38-delta isoforms to investigate whether GLUT2 governs expression of one or more of these variants in a glucose-dependent manner. Data show that GLUT2 inhibits baseline expression of each p38 protein in male cultures, yet stimulates p38-delta profiles without affecting other p38 proteins in female. Glucose starvation caused selective up-regulation of p38-delta profiles in male versus p38-alpha and -gamma proteins in female; these positive responses were amplified by GLUT2 siRNA pretreatment. GLUT2 opposes or enhances basal p38 phosphorylation in male versus female, respectively. GLUT2 siRNA pretreatment did not affect glucoprivic patterns of phospho-p38 protein expression in either sex. Outcomes document co-expression of the four principal p38 MAPK family proteins in hypothalamic astrocytes, and implicate GLUT2 in regulation of all (male) versus one (female) variant(s). Glucoprivation up-regulated expression of distinctive p38 isoforms in each sex; these stimulatory responses are evidently blunted by GLUT2. Glucoprivic-associated loss of GLUT2 gene silencing effects on p38 phosphorylation infers either that glucose status determines whether this sensor controls phosphorylation, or that decrements in screened glucose in each instance are of sufficient magnitude to abolish GLUT2 regulation of that function.

Down-regulation of MKP-1 in hippocampus protects against stress-induced depression-like behaviors and neuroinflammation

Chronic stress is the primary environmental risk factor for major depressive disorder (MDD), and there is compelling evidence that neuroinflammation is the major pathomechanism linking chronic stress to MDD. Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a negative regulator of MAPK signaling pathways involved in cellular stress responses, survival, and neuroinflammation. We examined the possible contributions of MKP-1 to stress-induced MDD by comparing depression-like behaviors (anhedonia, motor retardation, behavioral despair), neuroinflammatory marker expression, and MAPK signaling pathways among rats exposed to chronic unpredictable mild stress (CUMS), overexpressing MKP-1 in the hippocampus, and CUMS-exposed rats underexpressing MKP-1 in the hippocampus. Rats exposed to CUMS exhibited MKP-1 overexpression, greater numbers of activated microglia, and enhanced expressions of neuroinflammatory markers (interleukin [IL]-6, [IL]-1β, tumor necrosis factor [TNF]-ɑ, and decreased phosphorylation levels of ERK and p38 in the hippocampus as well as anhedonia in the sucrose preference test, motor retardation in the open field, and greater immobility (despair) in the forced swimming tests. These signs of neuroinflammation and depression-like behaviors and phosphorylation levels of ERK and p38 were also observed in rats overexpressing MKP-1 without CUMS exposure, while CUMS-induced neuroinflammation, microglial activation, phosphorylation levels of ERK and p38, and depression-like behaviors were significantly reversed by MKP-1 knockdown. Moreover, MKP-1 knockdown promoted the activation of the MAPK isoform ERK, implying that the antidepressant-like effects of MKP-1 knockdown may be mediated by the ERK pathway disinhibition. These findings suggested that hippocampal MKP-1 is an essential regulator of stress-induced neuroinflammation and a promising target for antidepressant development.

Neflamapimod inhibits endothelial cell activation, adhesion molecule expression, leukocyte attachment and vascular inflammation by inhibiting p38 MAPKα and NF-κB signaling

Neflamapimod, a selective inhibitor of the alpha isoform of p38 mitogen-activated protein kinase (MAPKα), was investigated for its potential to inhibit lipopolysaccharide (LPS)-induced activation of endothelial cells (ECs), adhesion molecule induction, and subsequent leukocyte attachment to EC monolayers. These events are known to contribute to vascular inflammation and cardiovascular dysfunction. Our results demonstrate that LPS treatment of cultured ECs and rats leads to significant upregulation of adhesion molecules, both in vitro and in vivo, which can be effectively inhibited by neflamapimod treatment. Western blotting data further reveals that neflamapimod inhibits LPS-induced phosphorylation of p38 MAPKα and the activation of NF-κB signaling in ECs. Additionally, leukocyte adhesion assays demonstrate a substantial reduction in leukocyte attachment to cultured ECs and the aorta lumen of rats treated with neflamapimod. Consistent with vascular inflammation, LPS-treated rat arteries exhibit significantly diminished vasodilation response to acetylcholine, however, arteries from rats treated with neflamapimod maintain their vasodilation capacity, demonstrating its ability to limit LPS-induced vascular inflammation. Overall, our data demonstrate that neflamapimod effectively inhibits endothelium activation, adhesion molecule expression, and leukocyte attachment, thereby reducing vascular inflammation.

P38γ modulates the lipid metabolism in non-alcoholic fatty liver disease by regulating the JAK-STAT signaling pathway.

Non-alcoholic fatty liver disease (NAFLD) is a major health problem in Western countries and has become the most common cause of chronic liver disease. Although NAFLD is closely associated with obesity, inflammation, and insulin resistance, its pathogenesis remains unclear. The disease begins with excessive accumulation of triglycerides in the liver, which in turn leads to liver cell damage, steatosis, inflammation, and so on. P38γ is one of the four isoforms of P38 mitogen-activated protein kinases (P38 MAPKs) that contributes to inflammation in different diseases. In this research, we investigated the role of P38γ in NAFLD. In vivo, a NAFLD model was established by feeding C57BL/6J mice with a methionine- and choline-deficient (MCD) diet and adeno-associated virus (AAV9-shRNA-P38γ) was injected into C57BL/6J mice by tail vein for knockdown P38γ. The results indicated that the expression level of P38γ was upregulated in MCD-fed mice. Furthermore, the downregulation of P38γ significantly attenuated liver injury and lipid accumulation in mice. In vitro, mouse hepatocytes AML-12 were treated with free fatty acid (FFA). We found that P38γ was obviously increased in FFA-treated AML-12 cells, whereas knockdown of P38γ significantly suppressed lipid accumulation in FFA-treated AML-12 cells. Furthermore, P38γ regulated the Janus Kinase-Signal transducers and activators of transcription (JAK-STAT) signaling pathway. Inhibition of P38γ can inhibit the JAK-STAT signaling pathway, thereby inhibiting lipid accumulation in FFA-treated AML-12 cells. In conclusion, our results suggest that targeting P38γ contributes to the suppression of lipid accumulation in fatty liver disease.

Defective synaptic plasticity in a model of Coffin-Lowry syndrome is rescued by simultaneously targeting PKA and MAPK pathways.

Empirical and computational methods were combined to examine whether individual or dual-drug treatments can restore the deficit in long-term synaptic facilitation (LTF) of the Aplysia sensorimotor synapse observed in a cellular model of Coffin-Lowry syndrome (CLS). The model was produced by pharmacological inhibition of p90 ribosomal S6 kinase (RSK) activity. In this model, coapplication of an activator of the mitogen-activated protein kinase (MAPK) isoform ERK and an activator of protein kinase A (PKA) resulted in enhanced phosphorylation of RSK and enhanced LTF to a greater extent than either drug alone and also greater than their additive effects, which is termed synergism. The extent of synergism appeared to depend on another MAPK isoform, p38 MAPK. Inhibition of p38 MAPK facilitated serotonin (5-HT)-induced RSK phosphorylation, indicating that p38 MAPK inhibits activation of RSK. Inhibition of p38 MAPK combined with activation of PKA synergistically activated both ERK and RSK. Our results suggest that cellular models of disorders that affect synaptic plasticity and learning, such as CLS, may constitute a useful strategy to identify candidate drug combinations, and that combining computational models with empirical tests of model predictions can help explain synergism of drug combinations.

A p38α-BLIMP1 signalling pathway is essential for plasma cell differentiation

Plasma cells (PC) are antibody-secreting cells and terminal effectors in humoral responses. PCs differentiate directly from activated B cells in response to T cell-independent (TI) antigens or from germinal center B (GCB) cells in T cell-dependent (TD) antigen-induced humoral responses, both of which pathways are essentially regulated by the transcription factor BLIMP1. The p38 mitogen-activated protein kinase isoforms have already been implicated in B cell development, but the precise role of p38α in B cell differentiation is still largely unknown. Here we show that PC differentiation and antibody responses are severely impaired in mice with B cell-specific deletion of p38α, while B cell development and the GCB cell response are spared. By utilizing a Blimp1 reporter mouse model, we show that p38α-deficiency results in decreased BLIMP1 expression. p38α-driven BLIMP1 up-regulation is required for both TI and TD PCs differentiation. By combining CRISPR/Cas9 screening and other approaches, we identify TCF3, TCF4 and IRF4 as downstream effectors of p38α to control PC differentiation via Blimp1 transcription. This study thus identifies an important signalling pathway underpinning PC differentiation upstream of BLIMP1, and points to a highly specialized and non-redundant role for p38α among p38 isoforms.

Structural characterization and expression analysis of novel MAPK1 transcript variants with the development of a multiplexed targeted nanopore sequencing approach.

Mitogen-activated protein kinases (MAPKs) represent a protein family firmly involved in many signaling cascades, regulating a vast spectrum of stimulated cellular processes. Studies have shown that alternatively spliced isoforms of MAPKs play a crucial role in determining the desired cell fate in response to specific stimulations. Although the implication of most MAPKs transcript variants in the MAPK signaling cascades has been clarified, the transcriptional profile of a pivotal member, MAPK1, has not been investigated for the existence of additional isoforms. In the current study we developed and implemented targeted long-read and short-read sequencing approaches to identify novel MAPK1 splice variants. The combination of nanopore sequencing and NGS enabled the implementation of a long-read polishing pipeline using error-rate correction algorithms, which empowered the high accuracy of the results and increased the sequencing efficiency. The utilized multiplexing option in the nanopore sequencing approach allowed not only the identification of novel MAPK1 mRNAs, but also elucidated their expression profile in multiple human malignancies and non-cancerous cell lines. Our study highlights for the first time the existence of ten previously undescribed MAPK1 mRNAs (MAPK1 v.3 - v.12) and evaluates their relative expression levels in comparison to the main MAPK1 v.1. The optimization and employment of qPCR assays revealed that MAPK1 v.3 - v.12 can be quantified in a wide spectrum of human cell lines with notable specificity. Finally, our findings suggest that the novel protein-coding mRNAs are highly expected to participate in the regulation of MAPK pathways, demonstrating differential localizations and functionalities.

Integration of In Silico Strategies for Drug Repositioning towards P38α Mitogen-Activated Protein Kinase (MAPK) at the Allosteric Site.

P38α mitogen-activated protein kinase (p38α MAPK), one of the p38 MAPK isoforms participating in a signaling cascade, has been identified for its pivotal role in the regulation of physiological processes such as cell proliferation, differentiation, survival, and death. Herein, by shedding light on docking- and 100-ns dynamic-based screening from 3210 FDA-approved drugs, we found that lomitapide (a lipid-lowering agent) and nilotinib (a Bcr-Abl fusion protein inhibitor) could alternatively inhibit phosphorylation of p38α MAPK at the allosteric site. All-atom molecular dynamics simulations and free energy calculations including end-point and QM-based ONIOM methods revealed that the binding affinity of the two screened drugs exhibited a comparable level as the known p38α MAPK inhibitor (BIRB796), suggesting the high potential of being a novel p38α MAPK inhibitor. In addition, noncovalent contacts and the number of hydrogen bonds were found to be corresponding with the great binding recognition. Key influential amino acids were mostly hydrophobic residues, while the two charged residues including E71 and D168 were considered crucial ones due to their ability to form very strong H-bonds with the focused drugs. Altogether, our contributions obtained here could be theoretical guidance for further conducting experimental-based preclinical studies necessary for developing therapeutic agents targeting p38α MAPK.

Multiplexing the Quantitation of MAP Kinase Activities Using Differential Sensing.

Protein kinases are therapeutic targets for many human diseases, but the lack of user-friendly quantitative assays limits the ability to follow the activities of numerous kinases at once (multiplexing). To develop such an assay, we report an array of sulfonamido-oxine (SOX)-labeled peptides showing cross-reactivity to different mitogen-activated protein kinases (MAPKs) for use in a differential sensing scheme. We first verified using linear discriminant analysis that the array could differentiate MAPK isoforms. Then, using principal component analysis, the array was optimized based on the discrimination imparted by each SOX-peptide. Next, the activity of individual MAPK families in ternary mixtures was quantified by support vector machine regression. Finally, we multiplexed the quantification of three MAPK families using partial least squares regression in A549 cell lysates, which has possible interference from other kinase classes. Thus, our method simultaneously quantifies the activity of multiple kinases. The technique could be applied to other protein kinase families and the monitoring of diseases.

Alternative Splicing of MAPKs in the Regulation of Signaling Specificity

The mitogen-activated protein kinase (MAPK) cascades transmit signals from extracellular stimuli to a variety of distinct cellular processes. The MAPKKs in each cascade specifically phosphorylate and activate their cognate MAPKs, indicating that this step funnels various signals into a seemingly linear pathway. Still, the effects of these cascades vary significantly, depending on the identity of the extracellular signals, which gives rise to proper outcomes. Therefore, it is clear that the specificity of the signals transmitted through the cascades is tightly regulated in order to secure the desired cell fate. Indeed, many regulatory components or processes that extend the specificity of the cascades have been identified. Here, we focus on a less discussed mechanism, that is, the role of distinct components in each tier of the cascade in extending the signaling specificity. We cover the role of distinct genes, and the alternatively spliced isoforms of MAPKKs and MAPKs, in the signaling specificity. The alternatively spliced MEK1b and ERK1c, which form an independent signaling route, are used as the main example. Unlike MEK1/2 and ERK1/2, this route’s functions are limited, including mainly the regulation of mitotic Golgi fragmentation. The unique roles of the alternatively spliced isoforms indicate that these components play an essential role in determining the proper cell fate in response to distinct stimulations.

Continuous administration of a p38α inhibitor during the subacute phase after transient ischemia-induced stroke in the rat promotes dose-dependent functional recovery accompanied by increase in brain BDNF protein level.

There is unmet need for effective stroke therapies. Numerous neuroprotection attempts for acute cerebral ischemia have failed and as a result there is growing interest in developing therapies to promote functional recovery through increasing synaptic plasticity. For this research study, we hypothesized that in addition to its previously reported role in mediating cell death during the acute phase, the alpha isoform of p38 mitogen-activated protein kinase, p38α, may also contribute to interleukin-1β-mediated impairment of functional recovery during the subacute phase after acute ischemic stroke. Accordingly, an oral, brain-penetrant, small molecule p38α inhibitor, neflamapimod, was evaluated as a subacute phase stroke treatment to promote functional recovery. Neflamapimod administration to rats after transient middle cerebral artery occlusion at two dose levels was initiated outside of the previously characterized therapeutic window for neuroprotection of less than 24 hours for p38α inhibitors. Six-week administration of neflamapimod, starting at 48 hours after reperfusion, significantly improved behavioral outcomes assessed by the modified neurological severity score at Week 4 and at Week 6 post stroke in a dose-dependent manner. Neflamapimod demonstrated beneficial effects on additional measures of sensory and motor function. It also resulted in a dose-related increase in brain-derived neurotrophic factor (BDNF) protein levels, a previously reported potential marker of synaptic plasticity that was measured in brain homogenates at sacrifice. Taken together with literature evidence on the role of p38α-dependent suppression by interleukin-1β of BDNF-mediated synaptic plasticity and BDNF production, our findings support a mechanistic model in which inhibition of p38α promotes functional recovery after ischemic stroke by blocking the deleterious effects of interleukin-1β on synaptic plasticity. The dose-related in vivo efficacy of neflamapimod offers the possibility of having a therapy for stroke that could be initiated outside the short time window for neuroprotection and for improving recovery after a completed stroke.

P38α MAPK Signaling-A Robust Therapeutic Target for Rab5-Mediated Neurodegenerative Disease.

Multifactorial pathologies, involving one or more aggregated protein(s) and neuroinflammation are common in major neurodegenerative diseases, such as Alzheimer’s disease and dementia with Lewy bodies. This complexity of multiple pathogenic drivers is one potential explanation for the lack of success or, at best, the partial therapeutic effects, respectively, with approaches that have targeted one specific driver, e.g., amyloid-beta, in Alzheimer’s disease. Since the endosome-associated protein Rab5 appears to be a convergence point for many, if not all the most prominent pathogenic drivers, it has emerged as a major therapeutic target for neurodegenerative disease. Further, since the alpha isoform of p38 mitogen-activated protein kinase (p38α) is a major regulator of Rab5 activity and its effectors, a biology that is distinct from the classical nuclear targets of p38 signaling, brain-penetrant selective p38α kinase inhibitors provide the opportunity for significant therapeutic advances in neurogenerative disease through normalizing dysregulated Rab5 activity. In this review, we provide a brief summary of the role of Rab5 in the cell and its association with neurodegenerative disease pathogenesis. We then discuss the connection between Rab5 and p38α and summarize the evidence that through modulating Rab5 activity there are therapeutic opportunities in neurodegenerative diseases for p38α kinase inhibitors.

Lancemaside A from Codonopsis lanceolata prevents hypertension by inhibiting NADPH oxidase 2-mediated MAPK signalling and improving NO bioavailability in rats.

This study investigated whether lancemaside A (LMA) can prevent hypertension and assessed the mechanisms of action of LMA in rats. Hypertension was induced by chronic immobilization stress and nicotine administration. Hypertensive vehicle rats were treated with LMA (1, 20, or 40mg/kg) or nifedipine (10mg/kg) as a positive control daily for 3weeks. In hypertensive vehicle rats, LMA dose-dependently reduced systolic blood pressure. LMA doses of 20 and 40mg/kg reduced the aortic expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX)2 (both P<0.01), and 40mg/kg LMA reduced serum malondialdehyde (P<0.01). Serum nitrite levels were significantly higher in LMA treated rats than in hypertensive vehicle rats, with LMA doses of 20 and 40mg/kg reducing the expression of endothelial nitric oxide synthase in rat aortas (P<0.001 and P<0.01, respectively). LMA also reduced the aortic levels of nuclear factor kappa B and the activation of the three isoforms of mitogen-activated protein kinase (MAPK). Lancemaside A prevents hypertension in rats by inhibiting the activation of MAPK signalling and the impairment in nitric oxide bioavailability due to NOX2-mediated oxidative stress. Thus, LMA may act as a preventive agent for hypertension.

Dissecting the Roles of p38β and p38γ in Cutaneous T Cell Lymphoma

Cutaneous T cell lymphoma (CTCL) is a disfiguring, incurable cancer that develops from clonal expansion of T cells and most commonly presents on the skin. For patients with advanced disease, current therapies are inadequate, and outcome is poor. An incomplete understanding of CTCL molecular regulators has limited development of effective targeted therapies. One candidate molecular regulator is p38γ, a mitogen-activated protein kinase (MAPK) crucial for malignant T cell activity and growth in response to T cell receptor (TCR) signaling. We previously reported that p38β (Bliss-Moreau, JID, 2014) and p38γ (Zhang, JID, 2018) are promising therapeutic targets for CTCL. We examined both RNA-seq and microarray public databases for gene expression and found that both targets are elevated in CTCL cell lines and patient samples, compared to normal T cells. This is consistent with literature showing that in several organs of the healthy human immune system, although p38α and p38δ isoforms are highly expressed, p38β and p38γ are typically very low (Wang, JBC, 1997). We demonstrated that knockdown of p38γ, but not p38β, causes cell death in the human CTCL cell line Hut78. In addition, overexpression of either target causes proliferation in CTCL cells.In this study, we dissected the roles of both p38β and p38γ in CTCL and delineated their downstream pathways. DLGH1 is an important scaffolding protein that directs T-cell signaling through the nuclear factor of activated T-cells (NFAT) pathway rather than through the NF-κB pathway (Round, Nat Immunol, 2007; Sabio, EMBO J, 2005). Our recent data indicate that knockdown of p38β reduced expression of DLGH1 at both the mRNA and protein level, but upregulated gene expression of CARMA1, which directs TCR signaling to the NF-kB pathway (Thome and Tschopp, Trends in Immunology, 2003). p38γ directly phosphorylates DLGH1 at ser158 in Hut78 cells (Sabio, EMBO J, 2005; Zhang, JID, 2018). We showed that inhibition of this phosphorylation reduced the role of both NFAT and NF-kB signaling in regulation of the immune response. Thus, our data indicate that DLGH1 is a common target of both p38β and p38γ isoforms. In addition, curcumin treatment causes induction of both p38γ and DLGH1 protein expression, but causes their reduction in p38β-knockdown Hut78 cells. This suggests an important role for p38β in TCR signaling via DLGH1 and its possible crosstalk with p38γ. We performed confocal microscopy of immunofluorescent p-p38 Tyr323 staining in p38β-knockdown Hut78 cells, which revealed a “ring-like” structure formed in the cytosol, in contrast to a polarized structure in wild-type Hut78 cells. This suggests that loss of p38β abolishes initiation of TCR complex formation in the plasma membrane of Hut78 cells. It is reported that TCR signaling can be modulated by the actin cytoskeleton (Yu, J Cell Sci, 2013); we further pursued its potential mechanisms and found that loss of p38β reduced F-actin expression in Hut78 cells. However, that p38β modulates with cytoskeletal structure in malignant T cells via altered actin expression remains further investigation.In summary, DLGH1 is a common target of p38γ and p38β, which is involved in regulation of TCR signaling through either phosphorylation or downregulation of its protein expression by these two MAPK isoforms. Furthermore, DLGH1 provides a potential pathway for modulation of the T cell cytoskeleton by MAPKs and through which the downstream NFAT and NFkB pathways maybe directed. This opens new possibilities for the development of targeted therapies for CTCL. DisclosuresQuerfeld:Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Bioniz: Membership on an entity's Board of Directors or advisory committees; Kyowa: Membership on an entity's Board of Directors or advisory committees; Acelion: Membership on an entity's Board of Directors or advisory committees.

Dual-specificity MAP kinase phosphatases in health and disease

It is well established that a family of dual-specificity MAP kinase phosphatases (MKPs) play key roles in the regulated dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues. MKPs provide a mechanism of spatiotemporal feedback control of these key signalling pathways, but can also mediate crosstalk between distinct MAP kinase cascades and facilitate interactions between MAP kinase pathways and other key signalling modules. As our knowledge of the regulation, substrate specificity and catalytic mechanisms of MKPs has matured, more recent work using genetic models has revealed key physiological functions for MKPs and also uncovered potentially important roles in regulating the pathophysiological outcome of signalling with relevance to human diseases. These include cancer, diabetes, inflammatory and neurodegenerative disorders. It is hoped that this understanding will reveal novel therapeutic targets and biomarkers for disease, thus contributing to more effective diagnosis and treatment for these debilitating and often fatal conditions.

Effects of Anti-Inflammatory Medications in Patients With Coronary Artery Disease: A Focus on Losmapimod.

Inflammation plays an integral role in atherogenesis and the pathogenesis of coronary artery disease (CAD). The question remains as to whether targeted inhibition of specific pathways of inflammation will have any clinical benefits in CAD. In this article, we will review p38 mitogen-activated protein kinase, one of the key sensors of cellular stress that plays an important role in the inflammatory cascade. In addition, we will review losmapimod, a reversible competitive inhibitor of the α and β isoforms of p38 mitogen-activated protein kinase, and its efficacy when added to standard of care in patients hospitalized with myocardial infarction. In the phase III trial, LATITUDE-TIMI 60, the investigators found that treating patients hospitalized with acute myocardial infarction with losmapimod did not reduce the risk of major adverse cardiovascular events. Lastly, we will briefly review trials recently completed and currently underway, investigating other anti-inflammatory medications such as canakinumab, methotrexate, varespladib, darapladib, and colchicine, and their role in CAD.

P38γ and p38δ Are Involved in T Lymphocyte Development.

p38 mitogen-activated protein kinase (MAPK) signal transduction pathways are essential regulators of the immune response. Particularly, p38γ and p38δ regulate many immune cell functions such as cytokine production, migration, or T cell activation; however, their involvement in immune cell development is largely unknown. Here, we analysed the role of p38 MAPK isoforms p38γ and p38δ in T cell differentiation in the thymus and in lymph nodes, using mice deficient in p38γ, p38δ, or in both. We found that the T cell differentiation program in the thymus was affected at different stages in p38γ-, p38δ-, and p38γ/δ-deficient mice, and also peripheral T cell homaeostasis was compromised. Particularly, p38δ deletion affects different stages of early CD4−CD8− double-negative thymocyte development, whereas lack of p38γ favours thymocyte positive selection from CD4+CD8+ double-positive to CD4+ or CD8+ single-positive cells. Our results identify unreported functions for p38γ and p38δ in T cells.

Intrathecal administration of antisense oligonucleotide against p38α but not p38β MAP kinase isoform reduces neuropathic and postoperative pain and TLR4-induced pain in male mice

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38β; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 μg/10 μl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38β MAPK ASO. Intrathecal injection of p38α or p38β ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38β mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38β MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions.