Mediated Mitogen-activated Protein Kinase Research Articles

The deubiquitinase OTUD1 deubiquitinates TIPE2 and plays a protective role in sepsis-induced lung injury by targeting TAK1-mediated MAPK and NF-κB signaling

Ovarian tumor domain-containing protease 1 (OTUD1) is a critical negative regulator that promotes innate immune homeostasis and is extensively involved in the pathogenesis of sepsis. In this study, we performed a powerful integration of multiomics analysis and an experimental mechanistic investigation to elucidate the immunoregulatory role of OTUD1 in sepsis at the clinical, animal and cellular levels. Our study revealed the upregulation of OTUD1 expression and the related distinctive alterations observed via multiomics profiling in clinical and experimental sepsis. Importantly, in vivo and in vitro, OTUD1 was shown to negatively regulate inflammatory responses and play a protective role in sepsis-induced pathological lung injury by mechanistically inhibiting the activation of the transforming growth factor-beta-activated kinase 1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling pathways in the present study. Subsequently, we probed the molecular mechanisms underlying OTUD1′s regulation of NF-κB and MAPK pathways by pinpointing the target proteins that OTUD1 can deubiquitinate. Drawing upon prior research conducted in our laboratory, it has been demonstrated that tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) performs a protective function in septic lung injury and septic encephalopathy by suppressing the NF-κB and MAPK pathways. Hence, we hypothesized that TIPE2 might be a target protein of OTUD1. Additional experiments, including Co-IP, immunofluorescence co-localization, and Western blotting, revealed that OTUD1 indeed has the ability to deubiquitinate TIPE2. In summary, OTUD1 holds potential as an immunoregulatory and inflammatory checkpoint agent, and could serve as a promising therapeutic target for sepsis-induced lung injury.

Genomic DNA Extracted from Lactiplantibacillus plantarum Attenuates Porphyromonas gingivalis Lipopolysaccharide (LPS)-Induced Inflammatory Responses via Suppression of Toll-Like Receptor (TLR)-Mediated Mitogen-Activated Protein Kinase (MAPK) and Nuclear Factor-κB (NF-κB) Signaling Pathways.

In the present study, we aimed to examine the inhibition of genomic DNA from Lactiplantibacillus plantarum (LpDNA) on Porphyromonas gingivalis lipopolysaccharide (PgLPS)-induced inflammatory responses in RAW264.7 cells. Pretreatment with LpDNA for 15 h significantly inhibited PgLPS-induced mRNA expression and protein secretion of interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein-1. LpDNA pretreatment also reduced the mRNA expression of Toll-like receptor (TLR)2 and TLR4. Furthermore, LpDNA inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and the activation of nuclear factor-κB (NF-κB) induced by PgLPS. Taken together, these findings demonstrate that LpDNA attenuates PgLPS-induced inflammatory responses by regulating MAPKs and NF-κB signaling pathways through the suppression of TLR2 and TLR4 expression.

A novel mechanism of 6-methoxydihydroavicine in suppressing ovarian carcinoma by disrupting mitochondrial homeostasis and triggering ROS/ MAPK mediated apoptosis.

Introduction: Alkaloids derived from M. cordata (Papaveraceae family), have been found to display antineoplastic activity in several types of cancer. However, the antitumor effects and mechanisms of a new alkaloid extracted from the fruits of M. cordata, named 6-Methoxydihydroavicine (6-ME), remains unclear in the case of ovarian cancer (OC). Methods: CCK-8 assay was employed to analyze the cell viabilities of OC cells. RTCA, and colony-formation assays were performed to measure OC cell growth. Alterations in apoptosis and ROS levels were detected by flow cytometry in accordance with the instructions of corresponding assay kits. A Seahorse XFe96 was executed conducted to confirm the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect alterations in target proteins. The subcutaneous xenografted tumor model of OC was used to further validate the anti-tumor activity of 6-ME in vivo. Results: Here, we reported for the first time that 6-ME inhibits OC cells growth in vitro and in vivo. Meanwhile, we found that 6-ME showed great antineoplastic activities by disrupting mitochondria homeostasis and promoting apoptosis in OC cells. Further investigation of the upstream signaling of apoptosis revealed that 6-ME-triggered apoptosis was induced by reactive oxygen species (ROS)-mediated mitogen-activated protein kinase (MAPK) activation and mitochondria dysfunction in OC cells. Furthermore, we found oxaloacetic acid (OAA), a crucial metabolite has been proved to be related to NADPH production, can block the cytotoxicity and accumulation of ROS caused by 6-ME in OC cells. Discussion: In summary, our data show that 6-ME exhibits cytotoxicity to OC cells in a ROS-dependent manner by interrupting mitochondrial respiration homeostasis and inducing MAPK-mediated apoptosis. This evidence suggests that 6-ME is a promising remedy for OC intervention.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

Downregulation of TREM2 expression exacerbates neuroinflammatory responses through TLR4-mediated MAPK signaling pathway in a transgenic mouse model of Alzheimer’s disease

Activation of glial cells and neuroinflammation play an important role in the onset and development of Alzheimer’s disease (AD). Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the brain that is involved in regulating neuroinflammation. However, the precise effects of TREM2 on neuroinflammatory responses and its underlying molecular mechanisms in AD have not been studied in detail. Here, we employed a lentiviral-mediated strategy to downregulation of TREM2 expression on microglia in the brain of APPswe/PS1dE9 (APP/PS1) transgenic mice and BV2 cells. Our results showed that downregulation of TREM2 significantly aggravated AD-related neuropathology including Aβ accumulation, peri-plaque microgliosis and astrocytosis, as well as neuronal and synapse-associated proteins loss, which was accompanied by a decline in cognitive ability. The further mechanistic study revealed that downregulation of TREM2 expression initiated neuroinflammatory responses through toll-like receptor 4 (TLR4)-mediated mitogen-activated protein kinase (MAPK) signaling pathway and subsequent stimulating the production of pro-inflammatory cytokines in vivo and in vitro. Moreover, blockade of p38, JNK, and ERK1/2 inhibited the release of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) induced by Aβ1–42 in TREM2-knocked down BV2 cells. Taken together, these findings indicated that TREM2 might be a potential therapeutic target for AD and other neuroinflammation-related diseases.

Characterization of p38 Inflammatory Response in Human Brain Vascular Pericyte Cells

G protein-coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro-inflammatory and pro-angiogenic responses. Prior studies have revealed an atypical pathway for p38 activation that induces auto-phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein-1 (TAB1) in endothelial cells. During angiogenesis and inflammation, endothelial cells are known for their rapid proliferation, migration, and expression of cytokines. Pericyte cells serve to guide these endothelial cells, modulating vascular stability and orchestrating vascular inflammatory responses. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells. We hypothesize that atypical p38 signaling regulates G protein-coupled receptor-mediated vascular function through control of pericyte signaling. Our preliminary data shows for the first time that a family of GPCR ligands, including thrombin and histamine, can activate p38 via the atypical p38 signaling pathway in primary human brain vascular pericytes. Chemical inhibition and siRNA depletion of TAB1-TAB2 abrogates inflammatory signaling by brain pericyte cells. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co-cultured model of pericyte and endothelial cells. Using this model, we are the first to define atypical p38 signaling in pericyte cytokine expression, migration, and endothelial vascular formation. These studies represent an unexplored branch of neurovascular inflammatory signaling, which we predict is a critical driver of neurovascular disease.

Epigallocatechin-3-Gallate Alleviates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease via Inhibition of Apoptosis and Promotion of Autophagy through the ROS/MAPK Signaling Pathway.

Nonalcoholic fatty liver disease (NAFLD) represents one of the most common chronic liver diseases in the world. It has been reported that epigallocatechin-3-gallate (EGCG) plays important biological and pharmacological roles in mammalian cells. Nevertheless, the mechanism underlying the beneficial effect of EGCG on the progression of NAFLD has not been fully elucidated. In the present study, the mechanisms of action of EGCG on the growth, apoptosis, and autophagy were examined using oleic acid- (OA-) treated liver cells and the high-fat diet- (HFD-) induced NAFLD mouse model. Administration of EGCG promoted the growth of OA-treated liver cells. EGCG could reduce mitochondrial-dependent apoptosis and increase autophagy possibly via the reactive oxygen species- (ROS-) mediated mitogen-activated protein kinase (MAPK) pathway in OA-treated liver cells. In line with in vitro findings, our in vivo study verified that treatment with EGCG attenuated HFD-induced NAFLD through reduction of apoptosis and promotion of autophagy. EGCG can alleviate HFD-induced NAFLD possibly by decreasing apoptosis and increasing autophagy via the ROS/MAPK pathway. EGCG may be a promising agent for the treatment of NAFLD.

Protective effect of MAPK signaling pathway mediated by ITGB3 gene silencing on myocardial ischemia-reperfusion injury in mice and its mechanism.

To explore the effect of integrin β3 (ITGB3) gene silencing mediated mitogen-activated protein kinase (MAPK) signaling pathway on myocardial ischemia-reperfusion injury (MIRI) in mice. MIRI mice model was established, and myocardial tissues of MIRI mice and sham operation group mice were extracted. Hematoxylin-Eosin (HE) staining was used to observe the pathological changes of myocardial tissue; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was used to detect the apoptosis of myocardial cells; ELISA method was used to detect the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-α in the two groups. The infarct size was measured by TTC staining. Myocardial cells of MIRI model mice were isolated and cultured, and then grouped and transfected. The cells were transfected with the grouping of MIRI group, negative control (NC) group, MAPK signal pathway agonist Anisomycin group, MAPK signal pathway inhibitor SB203580 group, ITGB3-siRNA group, SB203580 + ITGB3-siRNA group. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot were used to detect the mRNA and protein expressions of ITGB3, p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43, pro-apoptotic factor Bax and anti-apoptotic factor Bcl-2. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay was used to detect cell proliferation and flow cytometry to detect cell apoptosis. The expression of ITGB3 in myocardial tissue of MIRI mice was significantly higher than that of sham operated mice (p<0.05). Compared with the sham operation group, the apoptosis rate of myocardial cells in MIRI group was significantly increased, the expression levels of IL-1, IL-6 and TNF-α were significantly increased, and the myocardial infarction area was significantly increased (all p<0.05). Compared with MIRI and NC groups, ITGB3 mRNA and protein expression levels in ITGB3-siRNA group and SB203580 + ITGB3-siRNA group were significantly decreased (all p<0.05), but no significant change was found in Anisomycin group and SB203580 group (p>0.05). Furthermore, ITGB3-siRNA group and Anisomycin group had markedly decreased mRNA and protein expressions of ITGB3 and Bax, increased mRNA and protein expressions of p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43 and Bcl-2, as well as increased cell proliferation and decreased cell apoptosis (all p<0.05); SB203580 group indicated an opposite result with Anisomycin group; while SB203580 + ITGB3-siRNA revealed none significant statistical difference. In addition, compared with ITGB3-siRNA group, SB203580 + ITGB3-siRNA group showed significantly upregulated mRNA and protein expressions of Bax, downregulated mRNA and protein expressions of p38MAPK/p-p38MAPK, GSK-3β/p-GSK-3β, Cx43/p-Cx43 and Bcl-2, as well as decreased cell proliferation and increased cell apoptosis (all p<0.05). Silencing ITGB3 gene expression can promote the activation of MAPK signaling pathway, elevate the phosphorylation of GSK-3β and Cx43 in the downstream, promote the proliferation of mouse myocardial cells, inhibit myocardial cell apoptosis and inflammatory reaction, and thus have protective effect on MIRI in mice.

Characterization of p38 Inflammatory Response in Human Pericytes

G protein‐coupled receptor (GPCR) mediated mitogen activated protein kinase (MAPK) p38 activation is a crucial regulator of pro‐inflammation and pro‐angiogenic responses during chronic lung disease. Our studies have revealed an atypical (non‐canonical) pathway for p38 activation that induces auto‐phosphorylation of p38 through the direct binding of transforming growth factor β activated kinase 1 binding protein‐1 (TAB1) in endothelial cells. However, during angiogenesis, endothelial cells are known for their rapid reproduction, migration, and tubule formation. Pericyte cells serve to guide these endothelial cells, providing vascular stability and cytokine signaling. During inflammation, pericytes migrate away from the vasculature leaving endothelial tubules exposed and fragile. Atypical p38 activation has not yet been examined in pericyte cells, but is hypothesized to play a key role in regulating G protein‐coupled receptor‐mediated control of vascular function. Very little is known about the cellular mechanisms that control atypical p38 activation, representing a novel therapeutic target. Our preliminary data shows that thrombin and histamine can activate p38 via the atypical p38 signaling pathway in primary human pericytes. As the role of GPCR induced p38 signaling in pericytes has not been examined, our current studies focus on defining this role, including establishing a co‐cultured model of pericyte and endothelial cells. Using this model, we are the first to define the role of atypical p38 signaling in pericytes, along with their vascular formation and function. These studies represent a novel approach to regulate vascular p38 activity, pro‐inflammatory signaling, and chronic lung disease, and this model will provide us with the tools necessary to identify the role of p38 induced inflammation in the migration of pericyte cells.

Chitooligosaccharide inhibits RANKL-induced osteoclastogenesis and ligation-induced periodontitis by suppressing MAPK/ c-fos/NFATC1 signaling.

Considering the high rate of osteoclast-related diseases worldwide, research targeting osteoclast formation/function is crucial. In vitro, we demonstrated that chitooligosaccharide (CS) dramatically inhibited osteoclastogenesis as well as osteoclast function dose-dependently. CS suppressed osteoclast-specific genes expression during osteoclastogenesis. Furthermore, we found that CS attenuated receptor activator of nuclear factor kappa B ligand (RANKL)-mediated mitogen-activated protein kinase (MAPK) pathway involving p38, erk1/2, and jnk, leading to the reduced expression of c-fos and nuclear factor of activated T cells c1 (NFATc1) during osteoclast differentiation. In vivo, we found CS protected rats from periodontitis-induced alveolar bone loss by micro-computerized tomography and histological analysis. Overall, CS inhibited RANKL-induced osteoclastogenesis and ligature-induced rat periodontitis model, probably by suppressing the MAPK/c-fos/NFATc1 signaling pathway. Therefore, CS may be a safe and promising treatment for osteoclast-related diseases.

MicroRNA-326 Inhibits Apoptosis and Promotes Proliferation of Dopaminergic Neurons in Parkinson's Disease Through Suppression of KLK7-Mediated MAPK Signaling Pathway.

Parkinson's disease (PD), one of the motor system disorders, is characterized by the loss of dopamine-producing brain cells. Accumulating evidence has highlighted the involvement of microRNAs (miRs) in the development and progression of PD. Hence, we aimed at exploring possible effects of miR-326 on the progression of PD in mice in an attempt to elucidate the underlying mechanism associated with the kallikrein-related peptidase 7 (KLK7)-mediated mitogen-activated protein kinase (MAPK) signaling pathway. In order to identify the regulatory relationship between miR-326 and KLK7 and its biological significance in PD, PD mouse models were established and subsequently treated with mimics or inhibitors of miR-326 or siRNA-KLK7. The content of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyrosine (3-MT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA); positive expression of tyrosine hydroxylase (TH) and inducible nitric oxide synthase (iNOS); and the levels of IL-1, IL-6, TNF-α, INF-γ, and MAPK signaling pathway-related genes were determined accordingly. The results obtained indicated that KLK7 was negatively targeted by miR-326, with lower miR-326 and higher KLK7 detected among PD mice. The overexpression of miR-326 or silencing of KLK7 was demonstrated to increase the content of DA, DOPAC, HVA, 3-MT, SOD, GSH-Px, and TH positive expression, while reducing iNOS positive expression, MDA content and cell apoptosis, as well as inhibited levels of IL-1, IL-6, TNF-α, INF-γ, and mRNA and protein levels of p38, ERK, JNK, and caspase-3. Taken together, these results provided evidence suggesting that miR-326 could inhibit iNOS activation and apoptosis of dopaminergic neurons through inhibiting the MAPK signaling pathway and negatively regulating KLK7 in mice with PD. These findings highlight the potential of miR-326 as a novel target for future PD treatment.

MicroRNA-141-3p inhibits retinal neovascularization and retinal ganglion cell apoptosis in glaucoma mice through the inactivation of Docking protein 5-dependent mitogen-activated protein kinase signaling pathway.

Retinal neovascularization occurs in various ocular disorders including proliferative diabetic retinopathy and secondary neovascular glaucoma, resulting in blindness. This paper aims to investigate the effect of microRNA-141-3p (miR-141-3p) on retinal neovascularization and retinal ganglion cells (RGCs) in glaucoma mice through the Docking protein 5 (DOK5)-mediated mitogen-activated protein kinase (MAPK) signaling pathway. Chip retrieval and difference analysis were used for the potential mechanism of miR-141-3p on glaucoma. All modeled mice were transfected with different expression of mimic or inhibitor. The expressions of miR-141-3p, DOK5, and related genes and proteins of the MAPK signaling pathway were detected by Reverse transcription quantitative polymerase chain reaction and western blot analysis. Cell proliferation, lumen formation, and apoptosis in the retinal vascular epithelial cells and RGCs were detected using Matrigel angiogenesis and terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling assays. Moreover, a total of 63 and 294 differentially expressed genes were obtained in GSE2378 and GSE9944 chips, and 4 genes were within the intersection of the chips. In addition, the results showed that miR-141-3p was found to inhibit the DOK5 gene and activate the MAPK pathway. The number of RGCs, the expression of p38, extracellular-signal-regulated kinases (ERK), Jun N-terminal kinase (JNK), IGF-1, VEGF, HIF1-α, Bax, caspase-3, and the extent of p38, ERK, and JNK phosphorylated were decreased with miR-141-3p upregulation. Lastly, the results obtained showed that miR-141-3p inhibited the proliferation of retinal vascular epithelial cells and inhibited angiogenesis, as well as promoted apoptosis of RGCs. The study suggests that miR-141-3p inhibits retinal neovascularization in glaucoma mice by impeding the activation of the DOK5-mediated MAPK signaling pathway.

Melatonin: A Multifunctional Molecule That Triggers Defense Responses against High Light and Nitrogen Starvation Stress in Haematococcus pluvialis.

Melatonin (MLT), a ubiquitously distributed small molecule, functions in plant responses to various biotic and abiotic stresses. However, the interactions between melatonin and other important molecules in Haematococcus pluvialis response stresses are largely unknown. In the present study, exogenous melatonin improved H. pluvialis resistance to nitrogen starvation and high light. We concluded that exogenous melatonin treatment prevented the reactive oxygen species (ROS) burst and limited cell damage induced by abiotic stress through activation of antioxidant enzymes and antioxidants. Astaxanthin, a major antioxidant in H. pluvialis cells, exhibited a 2.25-fold increase in content after treatment with melatonin. The maximal astaxanthin content was 32.4 mg g-1. The functional roles of the nitric oxide (NO)-mediated mitogen activated protein kinase (MAPK) signaling pathway and cyclic adenosine monophosphate (cAMP) signaling pathway induced by melatonin were also evaluated. The results clearly indicate that cAMP signaling pathways are positively associated with microalgal astaxanthin biosynthesis. Additionally, the NO-dependent MAPK signaling cascade is activated in response to astaxanthin accumulation induced by melatonin, confirming that MAPK is a target of NO action in physiological processes. This work is the first to use H. pluvialis as in vivo model and documents the influence of melatonin on the physiological response to abiotic stress in this microalgae.

Quinalizarin Induces Apoptosis through Reactive Oxygen Species (ROS)-Mediated Mitogen-Activated Protein Kinase (MAPK) and Signal Transducer and Activator of Transcription 3 (STAT3) Signaling Pathways in Colorectal Cancer Cells.

BackgroundQuinalizarin (1,2,5,8-tetrahydroxyanthraquinone) exhibits potentially useful anticancer effects by inducing apoptosis in several types of cancer, but its underlying mechanism of action remains unknown. The present study examined the effects of quinalizarin on the induction of cell cycle arrest, apoptosis, the generation of reactive oxygen species (ROS), other underlying mechanisms, and its role in modifying colorectal cancer cell lines.Material/MethodsThe MTT assay was used to evaluate the viability of SW480 and HCT-116 cells that had been treated with quinalizarin and 5-fluorouracil (5-FU). Cell cycle arrest and apoptosis were analyzed by flow cytometry. Western blotting was used to investigate the mitochondrial pathway; Akt, MAPK, and STAT3 signaling pathways were also investigated. The relationship between ROS generation and apoptosis was analyzed by flow cytometry and western blotting.ResultsThe results indicated that quinalizarin significantly inhibits the viability of SW480 and HCT-116 cells in a dose-dependent manner. Quinalizarin induced SW480 cell cycle arrest at G2/M by regulating cyclin B1 and CDK1/2. The apoptosis-related protein expression levels of p-p53, Bad, cleaved caspase-3, cleaved PARP and p-JNK were increased in quinalizarin-treated cells, while protein expression levels Bcl-2, p-Akt, p-ERK, and p-STAT3 were decreased. Quinalizarin induced apoptosis in colorectal cancer cells by regulating MAPK and STAT3 signaling pathways via ROS generation.ConclusionsQuinalizarin induces apoptosis via ROS-mediated MAPK/STAT3 signaling pathways.

Polyphenols from Lonicera caerulea L. Berry Inhibit LPS-Induced Inflammation through Dual Modulation of Inflammatory and Antioxidant Mediators.

Lonicera caerulea L. berry polyphenols (LCBP) are considered as major components for bioactivity. This study aimed to clarify the molecular mechanisms by monitoring inflammatory and antioxidant mediator actions in lipopolysaccharide (LPS)-induced mouse paw edema and macrophage cell model. LCBP significantly attenuated LPS-induced paw edema (3.0 ± 0.1 to 2.8 ± 0.1 mm, P < 0.05) and reduced (P < 0.05) serum levels of monocyte chemotactic protein-1 (MCP-1, 100.9 ± 2.3 to 58.3 ± 14.5 ng/mL), interleukin (IL)-10 (1596.1 ± 424.3 to 709.7 ± 65.7 pg/mL), macrophage inflammatory protein (MIP)-1α (1761.9 ± 208.3 to 1369.1 ± 56.4 pg/mL), IL-6 (1262.8 ± 71.7 to 499.0 ± 67.1 pg/mL), IL-4 (93.3 ± 25.7 to 50.7 ± 12.5 pg/mL), IL-12(p-70) (580.4 ± 132.0 to 315.2 ± 35.1 pg/mL), and tumor necrosis factor-α (TNF-α, 2045.5 ± 264.9 to 1270.7 ± 158.6 pg/mL). Cell signaling analysis revealed that LCBP inhibited transforming growth factor β activated kinase-1 (TAK1)-mediated mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, and enhanced the expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and manganese-dependent superoxide dismutase (MnSOD) in earlier response. Moreover, cyanidin 3-glucoside (C3G) and (-)-epicatechin (EC), two major components of LCBP, directly bound to TAK1. These data demonstrated that LCBP might inhibit LPS-induced inflammation by modulating both inflammatory and antioxidant mediators.

Trained immunity or tolerance: opposing functional programs induced in human monocytes after engagement of various pattern recognition receptors.

Upon priming with Candida albicans or with the fungal cell wall component β-glucan, monocytes respond with an increased cytokine production upon restimulation, a phenomenon termed “trained immunity.” In contrast, the prestimulation of monocytes with lipopolysaccharide has long been known to induce tolerance. Because the vast majority of commensal microorganisms belong to bacterial or viral phyla, we sought to systematically investigate the functional reprogramming of monocytes induced by the stimulation of pattern recognition receptors (PRRs) with various bacterial or viral ligands. Monocytes were functionally programmed for either enhanced (training) or decreased (tolerance) cytokine production, depending on the type and concentration of ligand they encountered. The functional reprogramming of monocytes was also associated with cell shape, granulocity, and cell surface marker modifications. The training effect required p38- and Jun N-terminal protein kinase (JNK)-mediated mitogen-activated protein kinase (MAPK) signaling, with specific signaling patterns directing the functional fate of the cell. The long-term effects on the function of monocytes were mediated by epigenetic events, with both histone methylation and acetylation inhibitors blocking the training effects. In conclusion, our experiments identify the ability of monocytes to acquire adaptive characteristics after prior activation with a wide variety of ligands. Trained immunity and tolerance are two distinct and opposing functional programs induced by the specific microbial ligands engaging the monocytes.

Correction: LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide–Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors

Correction: LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide–Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors

LY303511 Enhances TRAIL Sensitivity of SHEP-1 Neuroblastoma Cells via Hydrogen Peroxide–Mediated Mitogen-Activated Protein Kinase Activation and Up-regulation of Death Receptors

We recently reported that LY294002 (LY29) and LY303511 (LY30) sensitized tumor cells to drug-induced apoptosis independent of the phosphoinositide 3-kinase/Akt pathway. Here, we investigated the mechanism of LY30-induced sensitization of human neuroblastoma cells to TRAIL-mediated apoptosis. We provide evidence that LY30-induced increase in intracellular H(2)O(2) up-regulates the expression of TRAIL receptors (DR4 and DR5) in SHEP-1 cells by activating mitogen-activated protein kinases, resulting in a significant amplification of TRAIL-mediated caspase-8 processing and activity, cytosolic translocation of cytochrome c, and cell death. Involvement of the death receptors was further confirmed by the ability of blocking antibodies against DR4 and/or DR5 to inhibit LY30-induced TRAIL sensitization. Pharmacologic inhibition of c-Jun NH(2) terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation by SP600125 and PD98059, respectively, blocked LY30-induced increase in sensitization to TRAIL-mediated death. Finally, small interfering RNA-mediated gene silencing of JNK and ERK inhibited LY30-induced increase in surface expression of DR4 and DR5, respectively. These data show that JNK and ERK are two crucial players involved in H(2)O(2)-mediated increase in TRAIL sensitization of tumor cells upon exposure to LY30 and underscore a novel mode of action of this inactive analogue of LY29. Our findings could have implications for the use of LY30 and similar compounds for enhancing the apoptotic sensitivity of neuroblastoma cells that often become refractory to chemotherapy.

Stretch-induced MAP kinase activation in cardiac myocytes: Differential regulation through β 1-integrin and focal adhesion kinase

Mitogen-activated protein (MAP) kinases have been implicated in hemodynamic load induced heart failure. Both angiotensin II (Ang II) and mechanical stretch activate MAP kinases in cardiac myocytes. In this study, we used a neonatal rat ventricular myocyte (NRVM) model to determine the role of focal-adhesion kinase (FAK) in β 1 integrin mediated MAP kinase activation in response to mechanical stretch in presence and absence of Ang II receptor blockade (ATB). NRVM plated on deformable membranes coated with collagen IV were exposed to 20% equiaxial static-stretch. β 1 integrin signaling was blocked by adenovirus-mediated expression of a dominant-negative form of β 1D integrin (tac-β 1D). FAK signaling was disrupted by infecting NRVM with adenovirus expressing FAK-related non-kinase (FRNK). Western blot analysis was used to assess the phosphorylation of MAP kinases. In the presence and absence of ATB, mechanical stretch caused maximal phosphorylation of ERK, p38 and JNK at 5 min, which was significantly attenuated in NRVM expressing tac-β 1D. In the presence of ATB, FRNK overexpression significantly increased basal phosphorylation of ERK (40.2 ± 8.6% P < 0.05), p38 (39.5 ± 11.7%, P < 0.05), JNK (86 ± 29.4%, P < 0.05) and stretch-induced p38 (48.1 ± 8.7%, P < 0.05) and JNK (85.0 ± 19.4%, P < 0.05) phosphorylation. However, in the absence of ATB, FRNK overexpression significantly reduced basal and stretch-induced phosphorylation of only ERK. Examination of FAK activation revealed that β 1 integrin was required for stretch-induced phosphorylation of FAK at Y 397 and Y 925, but not Y 861. In summary, mechanical stretch-activated ERK1/2, p38 and JNK through FAK independent and dependent mechanisms. β 1 integrin was required for FAK independent activation of all three MAP kinases, whereas cross-talk between β 1 integrin and Ang II receptors mediated FAK dependent regulation of ERK1/2.

Redox regulation of reactive oxygen species-induced p38 MAP kinase activation and barrier dysfunction in lung microvascular endothelial cells.

Reactive oxygen species (ROS)-mediated compromise of endothelial barrier integrity has been implicated in a number of pulmonary disorders, including adult respiratory distress syndrome, pulmonary edema, and vasculitis. The mechanisms by which ROS increase endothelial permeability are unclear. We hypothesized that ROS-induced changes in cellular redox status (thiols) may contribute to endothelial barrier dysfunction. To test this hypothesis, we used N-acetylcysteine (NAC) and diamide to modulate intracellular levels of cellular glutathione (GSH) and investigated hydrogen peroxide (H(2)O(2))-mediated mitogen-activated protein kinase (MAPK) activation and transendothelial electrical resistance (TER). Exposure of bovine lung microvascular endothelial cells (BLMVECs) to H(2)O(2), in a dose- and time-dependent fashion, increased endothelial permeability. Pretreatment of BLMVECs with NAC (5 mM) for 1 h resulted in partial attenuation of H(2)O(2)-induced TER (a measure of increase in permeability) and GSH. Furthermore, treatment of BLMVECs with diamide, which is known to reduce the intracellular GSH, resulted in significant reduction in TER, which was prevented by NAC. To understand further the role of MAPKs in ROS-induced barrier dysfunction, we examined the role of extracellular signal-regulated kinase (ERK) and p38 MAPK on H(2)O(2)- and diamide-mediated permeability changes. Both H(2)O(2) and diamide, in a dose-dependent manner, activated ERK and p38 MAPK in BLMVECs. However, SB203580, an inhibitor of p38 MAPK, but not PD98059, blocked H(2)O(2)- and diamide-induced TER. Also, NAC prevented H(2)O(2)- and diamide-induced p38 MAPK, but not ERK activation. These results suggest a role for redox regulation of p38 MAPK in ROS-dependent endothelial barrier dysfunction.