Members Of Mitogen-activated Protein Kinase Family Research Articles

CaMAPK1 Plays a Vital Role in the Regulation of Resistance to Ralstonia solanacearum Infection and Tolerance to Heat Stress.

As an important member of mitogen-activated protein kinase (MAPK) cascades, MAPKs play an important role in plant defense response against biotic and abiotic stresses; however, the involvement of the majority of the MAPK family members against Ralstonia solanacearum and heat stress (HS) remains poorly understood. In the present study, CaMAPK1 was identified from the genome of pepper and its function against R. solanacearum and HS was analyzed. The transcript accumulations of CaMAPK1 and the activities of its native promoter were both significantly induced by R. solanacearum inoculation, HS, and the application of exogenous hormones, including SA, MeJA, and ABA. Transient expression of CaMAPK1 showed that CaMAPK1 can be targeted throughout the whole cells in Nicotiana benthamiana and triggered chlorosis and hypersensitive response-like cell death in pepper leaves, accompanied by the accumulation of H2O2, and the up-regulations of hormones- and H2O2-associated marker genes. The knock-down of CaMAPK1 enhanced the susceptibility to R. solanacearum partially by down-regulating the expression of hormones- and H2O2-related genes and impairing the thermotolerance of pepper probably by attenuating CaHSFA2 and CaHSP70-1 transcripts. Taken together, our results revealed that CaMAPK1 is regulated by SA, JA, and ABA signaling and coordinates responses to R. solanacearum infection and HS in pepper.

Chronic binge alcohol-induced hippocampal plasticity in SIV-infected female rhesus macaques

Human immunodeficiency virus (HIV) infection is detrimental to central nervous system (CNS) function and may result in HIV-associated neurocognitive disorder (HAND) over time. HIV-associated cognitive deficits are further exacerbated by chronic alcohol use. With the rising prevalence of alcohol use disorder (AUD) in females and people living with HIV (PLWH), understanding the neurobiological impact of chronic alcohol exposure in these populations is increasingly important. As the hippocampus plays a prominent role in cognition, examining neuroadaptations in this brain region in the context of comorbid HIV infection and alcohol exposure is critical for understanding the mechanisms of neurocognitive impairment. We hypothesized that simian immunodeficiency virus (SIV)-infected female rhesus macaques with a history of chronic binge alcohol (CBA) administration (n=7-8 per group) would exhibit increased phosphorylation of mitogen-activated protein kinase (MAPK) family members, indicative of increased hippocampal activity and potential hyperexcitability. We also measured cognitive performance in macaques via the novel object recognition (NOR) task and further hypothesized a manifestation of cognitive deficits in the CBA-treated group. Finally, we incorporated additional groups of ovariectomized (OVX) females in the design as we hypothesized that the loss of ovarian hormones would reduce cognitive performance. Data were analyzed via two-way ANOVA with CBA treatment and OVX as factors. Western blot analyses demonstrated that CBA significantly increased phosphorylation of MAPK-family members extracellular signal-regulated kinase (ERK; F=8.278, p=0.0093) and p38 mitogen-activated protein kinase (p38 MAPK; F=4.414, p=0.0485). Furthermore, CBA significantly increased both phosphorylated and total levels of Jun N-terminal kinase (JNK; F=5.989, p=0.0233; F=7.158, p=0.0145). While analysis of the NOR task revealed a significant reduction in latency to approach the novel versus familiar object on test day across all groups (F = 11.97, p=0.0013), no significant difference was seen between CBA and control groups. Interestingly, no main effects of OVX or interactive CBA/OVX effects were observed for either molecular or behavioral outcomes. We are currently considering alternative cognitive tasks that may better reveal CBA-associated cognitive deficits, as well as neuroadaptations in nuclear signaling downstream of MAPK activity. Together, these data suggest that chronic alcohol use results in profound changes in hippocampal plasticity, potentially facilitating cognitive decline and other CNS symptoms observed in PLWH. This research was supported by grant # T32AA007577 and #P60AA009803 from the NIAAA at the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain cysteine residues that are predicted to be oxidized by H2O2 that lie within or in close proximity to the substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following treatment with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pretreatment, both ERK2 and p38α exhibited an increase in the relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcatfor both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). Likewise, fluorescence polarization assays suggest that oxidation of ERK2 increases its affinity for some ligands while decreasing its affinity for others. Interestingly, the upstream MAPK kinase, MEK1, also recognizes ERK1/2 via interactions with the DRS, suggesting that ERK1/2 activation dynamics may be affected by signal‐generated H2O2 within the cellular environment. Indeed, cell‐based assays suggested that pre‐treatment of NIH‐3T3 cells with the cell permeable H2O2 scavenger, PEG‐catalase, alters ERK1/2 phosphorylation following platelet‐derived growth factor (PDGF) treatment. Together, these findings suggest that redox modification of the cysteines located within the MAPK DRS alters the activation dynamics and may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.

Functional Roles of JNK and p38 MAPK Signaling in Nasopharyngeal Carcinoma.

c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) family members integrate signals that affect proliferation, differentiation, survival, and migration in a cell context- and cell type-specific way. JNK and p38 MAPK activities are found upregulated in nasopharyngeal carcinoma (NPC). Studies have shown that activation of JNK and p38 MAPK signaling can promote NPC oncogenesis by mechanisms within the cancer cells and interactions with the tumor microenvironment. They regulate multiple transcription activities and contribute to tumor-promoting processes, ranging from cell proliferation to apoptosis, inflammation, metastasis, and angiogenesis. Current literature suggests that JNK and p38 MAPK activation may exert pro-tumorigenic functions in NPC, though the underlying mechanisms are not well documented and have yet to be fully explored. Here, we aim to provide a narrative review of JNK and p38 MAPK pathways in human cancers with a primary focus on NPC. We also discuss the potential therapeutic agents that could be used to target JNK and p38 MAPK signaling in NPC, along with perspectives for future works. We aim to inspire future studies further delineating JNK and p38 MAPK signaling in NPC oncogenesis which might offer important insights for better strategies in diagnosis, prognosis, and treatment decision-making in NPC patients.

(E)-9-Octadecenoic Acid Ethyl Ester Derived from Lotus Seedpod Ameliorates Inflammatory Responses by Regulating MAPKs and NF-κB Signalling Pathways in LPS-Induced RAW264.7 Macrophages.

Inflammation is generally considered a key risk factor in the progress of several chronic diseases, such as arthritis, gastritis, and hepatitis. Natural products with anti-inflammatory ability have played a great role in the process of overcoming these inflammatory diseases. In this study, we evaluated the anti-inflammatory activities of ten natural compounds derived from lotus seedpod and discovered (E)-9-octadecenoic acid ethyl ester (E9OAEE) inhibited the production of nitric oxide (NO) optimally in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. Furthermore, we explored the effects of E9OAEE on inflammatory responses and the underlying mechanisms in LPS-induced RAW264.7 macrophages. The results indicated that E9OAEE significantly suppressed the production of NO, prostaglandin E2 (PGE2), and tumour necrosis factor-α (TNFα) in a dose-dependent manner. The protein expression and mRNA levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were inhibited by pretreatment of E9OAEE. Furthermore, E9OAEE restrained the phosphorylation of mitogen-activated protein kinase (MAPKs) family members, ERK, P38, and JNK stimulated by LPS-treated for 30 min and prevented the nuclear translocation of nuclear factor-kappa B (NF-κB) prompted by LPS-treated for 6 h in RAW264.7 macrophages. Taken together, we discovered an anti-inflammatory component from lotus seedpod and identified E9OAEE attenuated the inflammatory response in LPS-induced RAW264.7 macrophages probably by regulating the activation of MAPKs and NF-κB signalling pathways, which would provide some base for the development of new anti-inflammatory drugs.

Understanding Abnormal c-JNK/p38MAPK Signaling in Amyotrophic Lateral Sclerosis: Potential Drug Targets and Influences on Neurological Disorders.

c-JNK (c-Jun N-terminal kinase) and p38 mitogen-activated protein kinase (MAPK) family members work in a cell-specific manner to regulate neuronal signals. The abnormal activation of these cellular signals can cause glutamate excitotoxicity, disrupted protein homeostasis, defective axonal transport, and synaptic dysfunction. Various pre-clinical and clinical findings indicate that the up-regulation of c-JNK and p38MAPK signaling is associated with neurological disorders. Exceptionally, a significant amount of experimental data has recently shown that dysregulated c-JNK and p38MAPK are implicated in the damage to the central nervous system, including amyotrophic lateral sclerosis. Furthermore, currently available information has shown that c- JNK/p38MAPK signaling inhibitors may be a promising therapeutic alternative for improving histopathological, functional, and demyelination defects related to motor neuron disabilities. Understanding the abnormal activation of c-JNK/p38MAPK signaling and the prediction of motor neuron loss may help identify important therapeutic interventions that could prevent neurocomplications. Based on the involvement of c-JNK/p38MAPK signaling in the brain, we have assumed that the downregulation of the c-JNK/p38MAPK signaling pathway could trigger neuroprotection and neurotrophic effects towards clinicopathological presentations of ALS and other brain diseases. Thus, this research-based review also outlines the inhibition of c-JNK and p38MAPK signal downregulation in the pursuit of disease-modifying therapies for ALS.

ROS generation is involved in titanium dioxide nanoparticle-induced AP-1 activation through p38 MAPK and ERK pathways in JB6 cells.

Titanium dioxide (TiO2 ) is generally regarded as a nontoxic and nongenotoxic white mineral, which is mainly applied in the manufacture of paper, paint, plastic, sunscreen lotion and other products. Recently, TiO2 nanoparticles (TiO2 NPs) have been demonstrated to cause chronic inflammation and lung tumor formation in rats, which may be associated with the particle size of TiO2 . Considering the important role of activator protein-1 (AP-1) in regulating multiple genes involved in the cell proliferation and inflammation and the induction of neoplastic transformation, we aimed to evaluate the potency of TiO2 NPs (≤ 20 nm) on the activation of AP-1 signaling pathway and the generation of reactive oxygen species (ROS) in a mouse epidermal cell line, JB6 cells. MTT, electron spin resonance (ESR), AP-1 luciferase activity assay in vitro and in vivo, and Western blotting assay were used to clarify this problem. Our results indicated that TiO2 NPs dose-dependently caused the hydroxyl radical (·OH) generation and sequentially increased the AP-1 activity in JB6 cells. Using AP-1-luciferase reporter transgenic mice models, an obvious increased AP-1 activity was detected in dermal tissue after exposure to TiO2 NPs for 24 h. Interestingly, TiO2 NPs increased the AP-1 activity via stimulating the expression of mitogen-activated protein kinases (MAPKs) family members, including extracellular signal-regulated protein kinases (ERKs), p38 kinase, and C-Jun N-terminal kinases (JNKs). Of note, the AP-1 activation induced by TiO2 NPs could be blocked by specific inhibitors (SB203580, PD98059, and SP 600125, respectively) that inhibit ERKs and p38 kinase but not JNKs. These findings indicate that ROS generation is involved in TiO2 NPs-induced AP-1 activation mediated by MAPKs signal pathway.

The active component of ginseng, ginsenoside Rb1, improves erythropoiesis in models of Diamond–Blackfan anemia by targeting Nemo-like kinase

Nemo-like kinase (NLK) is a member of the mitogen-activated protein kinase family of kinases and shares a highly conserved kinase domain with other mitogen-activated protein kinase family members. The activation of NLK contributes to the pathogenesis of Diamond–Blackfan anemia (DBA), reducing c-myb expression and mechanistic target of rapamycin activity, and is therefore a potential therapeutic target. Unlike other anemias, the hematopoietic effects of DBA are largely restricted to the erythroid lineage. Mutations in ribosomal genes induce ribosomal insufficiency and reduced protein translation, dramatically impacting early erythropoiesis in the bone marrow of patients with DBA. We sought to identify compounds that suppress NLK and increases erythropoiesis in ribosomal insufficiency. We report that the active component of ginseng, ginsenoside Rb1, suppresses NLK expression and improves erythropoiesis in in vitro models of DBA. Ginsenoside Rb1–mediated suppression of NLK occurs through the upregulation of miR-208, which binds to the 3′-UTR of NLK mRNA and targets it for degradation. We also compare ginsenoside Rb1–mediated upregulation of miR-208 with metformin-mediated upregulation of miR-26. We conclude that targeting NLK expression through miRNA binding of the unique 3′-UTR is a viable alternative to the challenges of developing small-molecule inhibitors to target the highly conserved kinase domain of this specific kinase.

Molecular characterization of a novel p38 MAPK cDNA from Cyclina sinensis and its potential immune-related function under the threat of Vibrio anguillarum

The p38 mitogen-activated protein kinase (MAPK) is one important member of MAPK family and reported to serve a predominant function in regulating innate immunity after the occurrence of certain infection. In the present study, one novel p38 MAPK gene was acquired from Cyclina sinensis based on the RNA-seq analysis and designated as Csp38 MAPK. This novel gene contained a full length of 1781bp, 1104bp of which was deemed as open reading frames and gave rise to a peptide of 367 amino acids with a predicted molecular weight of 42.31 KDa. A conserved serine/threonine protein kinase (S_Tkc) region along with a Thr-Gly-Tyr motif was discovered in the deduced sequence. According to the phylogenetic analysis, there was a close relationship between this kinase and Meretrix petechialis p38 MAPK. As for the expression pattern, this newly-identified Csp38 MAPK was ubiquitously distributed in several tissues throughout the body but with varied abundance. After the challenge of Vibrio anguillarum, both the transcription and phosphorylation level of Csp38 MAPK in hemolymph were coordinately altered with a time-dependent manner. Besides, with the application of double strand RNA homologous to myeloid differentiation factor 88 (MyD88) of C. sinensis, the activation of Csp38 MAPK was found to obviously decrease in hemolymph after the pathogen stimulation. Hence, our experimental data presented evidence for the potential involvement of p38 MAPK in response to bacterial invaders in C. sinensis, possibly facilitating the understanding for pathogen-induced innate immunity in clams.

Salt-inducible kinases are required for the IL-33–dependent secretion of cytokines and chemokines in mast cells

Cytokines and chemokines are important regulators of airway hyper-responsiveness, immune cell infiltration, and inflammation and are produced when mast cells are stimulated with interleukin-33 (IL-33). Here, we establish that the salt-inducible kinases (SIKs) are required for the IL-33-stimulated transcription of il13, gm-csf and tnf and hence the production of these cytokines. The IL-33–stimulated secretion of IL-13, granulocyte-macrophage colony stimulating factor, and tumor necrosis factor was strongly reduced in fetal liver–derived mast cells from mice expressing a kinase-inactive mutant of SIK3 and abolished in cells expressing kinase-inactive mutants of SIK2 and SIK3. The IL-33–dependent secretion of these cytokines and several chemokines was also abolished in SIK2/3 double knock-out bone marrow–derived mast cells (BMMC), reduced in SIK3 KO cells but little affected in BMMC expressing kinase-inactive mutants of SIK1 and SIK2 or lacking SIK2 expression. In SIK2 knock-out BMMC, the expression of SIK3 was greatly increased. Our studies identify essential roles for SIK2 and SIK3 in producing inflammatory mediators that trigger airway inflammation. The effects of SIKs were independent of IκB kinase β, IκB kinase β-mediated NF-κB-dependent gene transcription, and activation of the mitogen-activated protein kinase family members p38α and c-jun N-terminal kinases. Our results suggest that dual inhibitors of SIK2 and SIK3 may have therapeutic potential for the treatment of mast cell–driven diseases.

Differential Expression of Mitogen Activated Protein Kinase (MAPK) and Stress-Related Genes in Rice Overexpressing MPK3 and MPK6 under Abiotic Stress

Plants being sessile have ability to adapt themselves in constantly changing and challenging environment by perceiving external/internal cues and transducing them down to the nucleus for appropriate cellular reorganization. Activation of mitogen-activated protein kinases (MAPKs) is a common reaction of plant cells towards such cues in development-related signal transduction pathways. The downstream events after the activation of MAPKs are largely unknown in plants. In the present study, we examined the effect of arsenic, salt and drought stress on growth parameters and transcript profiling of MAPKs, silicon transporters andstress responsivegenes in transgenic rice overexpressing OsMPK3 and OsMPK6 under the control of a dexamethasone (DEX)-inducible promoter, and possible role MAPKs in stress tolerance. Our results revealed that OsMPK3 and OsMPK6 overexpressing lines under arsenic stress and drought stress enhanced the transcript level of stress responsive genes, encoding enzymes superoxide dismutase, ascorbate peroxidase, glutamine synthetase and aldehyde oxidase. Upregulation in transcripts profiling of three silicon transporter genes OsLsi1, OsLsi2 and OsLsi6 showed improved stress tolerance ability of OsMPK3 and OsMPK6 overexpressing lines by effective absorption of silicon. The fundamental knowledge about the function of MPK3 and MPK6, important member of MAPKs family may also lead to an engineered plant tolerant to various abiotic and biotic stresses.

Epimedin C modulates the balance between Th9 cells and Treg cells through negative regulation of noncanonical NF-κB pathway and MAPKs activation to inhibit airway inflammation in the ovalbumin–induced murine asthma model

Allergic asthma is a common airway inflammatory disease and mainly caused by abnormal immune responses to allergens and viruses. The precise mechanisms of airway inflammation and airway hyper-responsiveness (AHR) are still not completely understood. CD4+ helper T cells (Th cells) serve as critical regulators of allergic immunity. The imbalance between T helper 9 (Th9) cells and forkhead box protein 3 (Foxp3)+ regulatory T (Treg) cells may contribute to airway inflammation in asthma. Epimedin C, a dominant compound isolated from Herba Epimedii, has shown anti-inflammatory effects and the immunoregulatory activity, such as increase of lymphocyte proliferation. However, the protective role of epimedin C in an experimental model of ovalbumin (OVA)-induced allergic airway inflammation and the underlying mechanism remain unknown. Female BALB/c mice were sensitized by intraperitoneal injection (i.p.) of OVA plus aluminum hydroxide (Alum) and subsequently challenged with an aerosol of 3% OVA in saline. Mice were treated with different concentrations of epimedin C (20 mg/kg/d, 40 mg/kg/d, 80 mg/kg/d) for 4 weeks. Experimental endpoints were evaluated via the analysis of AHR to acetyl-β-methacholine (Mch), differential inflammatory cell counts, concentrations of cytokines interleukin-9 (IL-9), IL-4 and IL-10 in bronchoalveolar lavage fluid (BALF), serum OVA-specific IgE level, as well as airway inflammation, mucus secretion and collagen deposition in mice. Mechanistically, we investigated the percentages of Th9 cells and Treg cells, as well as mRNA levels of IL-9 and transcription factor Foxp3 in lungs. Furthermore, the proteins expression of nuclear factor-κB (NF-κB) family members p105/p50, RelA, p100/p52 and RelB, as well as mitogen-activated protein kinase (MAPK) family members extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK was detected. Epimedin C dose-dependently attenuated AHR, airway inflammation, mucus hypersecretion and collagen deposition in OVA-induced murine asthma model. The expression levels of IL-9, IL-4 and OVA-specific IgE were significantly decreased while IL-10 was increased by epimedin C. We further confirmed that epimedin C decreased the percentage of lung Th9 cells with lower mRNA expression of IL-9 and increased the percentage of lung Treg cells with higher mRNA expression of Foxp3. In addition, epimedin C dose-dependently decreased the protein levels of p52, RelB, phosphorylation of ERK1/2 and p38 MAPK which are pivotal to the development of Th9 cells and Treg cells. Collectively, epimedin C could inhibit pathophysiological features of asthma by reconstruction of the balance between Th9 cells and Treg cells through regulation of the noncanonical NF-κB p52/RelB pathway and MAPKs activation. These findings suggest epimedin C as a potential remedy for inflammatory airway diseases.

Impact of Conventional and Atypical MAPKs on the Development of Metabolic Diseases.

The family of mitogen-activated protein kinases (MAPKs) consists of fourteen members and has been implicated in regulation of virtually all cellular processes. MAPKs are divided into two groups, conventional and atypical MAPKs. Conventional MAPKs are further classified into four sub-families: extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK1, 2 and 3), p38 (α, β, γ, δ), and extracellular signal-regulated kinase 5 (ERK5). Four kinases, extracellular signal-regulated kinase 3, 4, and 7 (ERK3, 4 and 7) as well as Nemo-like kinase (NLK) build a group of atypical MAPKs, which are activated by different upstream mechanisms than conventional MAPKs. Early studies identified JNK1/2 and ERK1/2 as well as p38α as a central mediators of inflammation-evoked insulin resistance. These kinases have been also implicated in the development of obesity and diabetes. Recently, other members of conventional MAPKs emerged as important mediators of liver, skeletal muscle, adipose tissue, and pancreatic β-cell metabolism. Moreover, latest studies indicate that atypical members of MAPK family play a central role in the regulation of adipose tissue function. In this review, we summarize early studies on conventional MAPKs as well as recent findings implicating previously ignored members of the MAPK family. Finally, we discuss the therapeutic potential of drugs targeting specific members of the MAPK family.

ERK5 Is Required for Tumor Growth and Maintenance Through Regulation of the Extracellular Matrix in Triple Negative Breast Cancer.

Conventional mitogen-activated protein kinase (MAPK) family members regulate diverse cellular processes involved in tumor initiation and progression, yet the role of ERK5 in cancer biology is not fully understood. Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. ERK5 signaling contributes to drug resistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). More recently a role for ERK5 in regulation of the extracellular matrix (ECM) has been proposed, and here we investigated the necessity of ERK5 in TNBC tumor formation. Depletion of ERK5 expression using the CRISPR/Cas9 system in MDA-MB-231 and Hs-578T cells resulted in loss of mesenchymal features, as observed through gene expression profile and cell morphology, and suppressed TNBC cell migration. In vivo xenograft experiments revealed ERK5 knockout disrupted tumor growth kinetics, which was restored using high concentration Matrigel™ and ERK5-ko reduced expression of the angiogenesis marker CD31. These findings implicated a role for ERK5 in the extracellular matrix (ECM) and matrix integrity. RNA-sequencing analyses demonstrated downregulation of matrix-associated genes, integrins, and pro-angiogenic factors in ERK5-ko cells. Tissue decellularization combined with cryo-SEM and interrogation of biomechanical properties revealed that ERK5-ko resulted in loss of key ECM fiber alignment and mechanosensing capabilities in breast cancer xenografts compared to parental wild-type cells. In this study, we identified a novel role for ERK5 in tumor growth kinetics through modulation of the ECM and angiogenesis axis in breast cancer.

Mapping p38α mitogen-activated protein kinase signaling by proximity-dependent labeling.

Mitogen-activated protein (MAP) kinase signaling is central to multiple cellular responses and processes. MAP kinase p38α is the best characterized member of the p38 MAP kinase family. Upstream factors and downstream targets of p38α have been identified in the past by conventional methods such as coimmunoprecipitation. However, a complete picture of its interaction partners and substrates in cells is lacking. Here, we employ a proximity-dependent labeling approach using biotinylation tagging to map the interactome of p38α in cultured 293T cells. Fusing the advanced biotin ligase BioID2 to the N-terminus of p38α, we used mass spectrometry to identify 37 biotin-labeled proteins that putatively interact with p38α. Gene ontology analysis confirms known upstream and downstream factors in the p38 MAP kinase cascade (e.g., MKK3, MAPKAPK2, TAB2, and c-jun). We furthermore identify a cluster of zinc finger (ZnF) domain-containing proteins that is significantly enriched among proximity-labeled interactors and is involved in gene transcription and DNA damage response. Fluorescence imaging and coimmunoprecipitation with overexpressed p38α in cells supports an interaction of p38α with ZnF protein XPA, a key factor in the DNA damage response, that is promoted by UV irradiation. These results define an extensive network of interactions of p38α in cells and new direct molecular targets of MAP kinase p38α in gene regulation and the DNA damage response.

Impact of Redox Modification on MAPK Global Substrate Selection

Mitogen activated protein kinases (MAPKs) are activated by various stimuli, ranging from growth factors to environmental stress factors. However, given this diverse set of stimuli, it is not currently clear how specificity is achieved in MAPK signaling pathways. We are exploring the hypothesis that, by modulating the substrate selectivity of MAPK family members, cells are able to control which “arm” of a branched pathway is activated in response to a given signal. Specifically, we are investigating the impact of redox modification on the global substrate selection of the canonical MAPKs, extracellular regulated kinase 2 (ERK2) and p38α, which play important roles in regulating a variety of cellular outcomes, including cellular migration, differentiation, apoptosis, proliferation and survival. MAPK family members recognize the majority of their substrates via one of two binding surfaces, termed the D‐recognition site (DRS) and the F‐recognition site (FRS). Interestingly, MAPKs contain redox‐sensitive cysteine residues within or in close proximity to their substrate recognition sites. Therefore, to explore the effects of oxidation on MAPK substrate selection, we first investigated the ability of ERK2 and p38α to phosphorylate model DRS and FRS peptide substrates following pre‐treatment of the kinases with various concentrations of H2O2. While their activity toward FRS substrates was not changed by H2O2 pre‐treatment, both ERK2 and p38α exhibited an increase in their relative activity toward the DRS substrate, Sub‐D, at H2O2 concentrations that were stoichiometric with the kinase. Kinetic analysis demonstrated that treatment with H2O2 leads to substantial changes in Km and, to a lesser extent, kcat for both kinases. Together, these data suggest that redox modification of p38α and ERK2 may alter their ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter the activity of p38α and ERK2 toward some, but not all, of their downstream substrates. To explore this possibility further, we investigated the impact of redox modification on the global substrate selectivity of these MAPKs using functional protein microarrays. Consistent with our hypothesis, phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2‐dependent changes in their phosphorylation status (both increases and decreases depending on the substrate). These findings suggest that redox modification of the cysteines located within the MAPK DRS may help to modulate downstream substrate selection of MAPK family members under physiological and pathological states.Support or Funding InformationThis research was supported by the NIH National Institute of General Medical Sciences through grants SC2GM113784 and SC1GM130545 (to RHN) and R01GM119227 (to LBP). Likewise, LM is supported through a Title III HBGI grant from the U.S. Department of Education.

Protective role of endothelial calpain knockout in lipopolysaccharide-induced acute kidney injury via attenuation of the p38-iNOS pathway and NO/ROS production

To explore the role of calpain and its signaling pathway in lipopolysaccharide (LPS)-induced acute kidney injury (AKI), animal models of endotoxemia were established by administration of LPS to mice with endothelial-specific Capn4 knockout (TEK/Capn4−/−), mice with calpastatin (an endogenous calpain inhibitor) overexpression (Tg-CAST) and mice with myeloid-specific Capn4 knockout (LYZ/Capn4−/−). Mouse pulmonary microvascular endothelial cells (PMECs) were used as a model of the microvascular endothelium and were stimulated with LPS. Renal function, renal inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expression, cellular apoptosis, plasma and renal levels of NO and reactive oxygen species (ROS), and phosphorylation of mitogen-activated protein kinase (MAPK) family members (p38, ERK1/2, and JNK1/2) were examined. Moreover, a calpain inhibitor, calpastatin overexpression adenoviruses and MAPK inhibitors were used. Significant renal dysfunction was induced by LPS stimulation, and recovery was observed in TEK/Capn4−/− and Tg-CAST mice but not in LYZ/Capn4−/− mice. Endothelial Capn4 knockout also abrogated the LPS-induced increases in renal iNOS expression, caspase-3 activity and apoptosis and plasma and renal NO and ROS levels but did not obviously affect renal eNOS expression. Moreover, LPS increased both calpain and caspase-3 activity, and only the expression of iNOS in PMECs was accompanied by increased phosphorylation of p38 and JNK. Inhibiting calpain activity or p38 phosphorylation alleviated the increased iNOS expression, NO/ROS production, and cellular apoptosis induced by LPS. These results suggest that endothelial calpain plays a protective role in LPS-induced AKI by inhibiting p38 phosphorylation, thus attenuating iNOS expression and further decreasing NO and ROS overproduction-induced endothelial apoptosis.

Mitogen-Activated Protein Kinase Inhibitors and T-Cell-Dependent Immunotherapy in Cancer.

Mitogen-activated protein kinase (MAPK) signaling networks serve to regulate a wide range of physiologic and cancer-associated cell processes. For instance, a variety of oncogenic mutations often lead to hyperactivation of MAPK signaling, thereby enhancing tumor cell proliferation and disease progression. As such, several components of the MAPK signaling network have been proposed as viable targets for cancer therapy. However, the contributions of MAPK signaling extend well beyond the tumor cells, and several MAPK effectors have been identified as key mediators of the tumor microenvironment (TME), particularly with respect to the local immune infiltrate. In fact, a blockade of various MAPK signals has been suggested to fundamentally alter the interaction between tumor cells and T lymphocytes and have been suggested a potential adjuvant to immune checkpoint inhibition in the clinic. Therefore, in this review article, we discuss the various mechanisms through which MAPK family members contribute to T-cell biology, as well as circumstances in which MAPK inhibition may potentiate or limit cancer immunotherapy.

Adenosine N1-Oxide Exerts Anti-inflammatory Effects through the PI3K/Akt/GSK-3β Signaling Pathway and Promotes Osteogenic and Adipocyte Differentiation.

Previously, we reported that adenosine N1-oxide (ANO), which is found in royal jelly, inhibited the secretion of inflammatory mediators by activated macrophages and reduced lethality in lipopolysaccharide (LPS)-induced endotoxin shock. Here, we examined the regulatory mechanisms of ANO on the release of pro-inflammatory cytokines, with a focus on the signaling pathways activated by toll-like receptor (TLR)4 in response to LPS. ANO inhibited both tumor necrosis factor (TNF)-α and interleukin (IL)-6 secretion from LPS-stimulated RAW264.7 cells without affecting cell proliferation. In this response, phosphorylation of mitogen-activated protein kinase (MAPK) family members (extracellular signal-regulated kinase (ERK)1/2, p38 and SAPK/c-Jun N-terminal kinase (JNK)) and nuclear factor-κB (NF-κB) p65 was not affected by treatment with ANO. In contrast, phosphorylation of Akt (Ser473) and its downstream molecule glycogen synthase kinase-3β (GSK-3β) (Ser9) was up-regulated by ANO, suggesting that ANO stimulated GSK-3β phosphorylation via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The phosphorylation of GSK-3β on Ser9 has been shown to negatively regulate the LPS-induced inflammatory response. Activation of PI3K/Akt signaling pathway has also been implicated in differentiation of mesenchymal stem cells into osteoblasts and adipocytes. As expected, ANO induced alkaline phosphatase activity and promoted calcium deposition in a mouse pre-osteoblastic MC3T3-E1 cell line. The ANO-induced differentiation into osteoblasts was abrogated by coincubation with Wortmannin. Furthermore, ANO promoted insulin/dexamethasone-induced differentiation of mouse 3T3-L1 preadipocytes into adipocytes at much lower concentrations than adenosine. The protective roles of PI3K/Akt/GSK-3β signaling pathway in inflammatory disorders have been well documented. Our data suggest that ANO may serve as a potential candidate for the treatment of inflammatory disorders. Promotion of osteogenic and adipocyte differentiation further suggests its application for regenerative medicine.

Selective activation of tumor-suppressive MAPKP signaling pathway by triptonide effectively inhibits pancreatic cancer cell tumorigenicity and tumor growth

The mitogen-activated protein kinase (MAPK, 1K) family members ERK, JNK, and p38 play a divergent role in either promoting tumorigenesis or tumor-suppression. Activation of ERK and JNK promotes tumorigenesis; whereas, escalation of p38 inhibits carcinogenesis. As these three MAPK members are controlled by the common up-stream MAPK signaling proteins which consist of MAPK kinases (2K) and MAPK kinase kinases (3K), how to selectively actuate tumor-suppressive p38, not concurrently stimulate tumorigenic ERK and JNK, in cancer cells is a challenge for cancer researchers, and a new opportunity for novel anti-cancer drug discovery. Using human pancreatic cancer cells and xenograft mice as models, we found that a small molecule triptonide first discerningly activated the up-stream MAPK kinase kinase MEKK4, not the other two 3K members ASK1 and GADD45; and then selectively actuated the middle stream MAPK kinase MKK4, not the other two 2K members MKK3 and MKK6; and followed by activation of the MAPK member p38, not the other two members ERK and JNK. These data suggest that triptonide is a selective MEKK4-MKK4-p38 axis agonist. Consequently, selective activation of the MEKK4-MKK4-p38 signaling axis by triptonide activated tumor suppressor p21 and inhibited CDK3 expression, resulting in cancer cell cycle arrest at G2/M phase and marked inhibition of pancreatic cancer cell tumorigenic capability in vitro and tumor growth in xenograft mice. Our findings support the notion that selective activation of tumor-suppressive MEKK4-MKK4-p38-p21signaling pathway by triptonide is a new approach for pancreatic cancer therapy, providing a new drug candidate for development of novel anti-cancer therapeutics.