Mitogen-activated Protein Kinase Extracellular Signal-related Kinase Research Articles

MircoRNA-322-5p promotes lipopolysaccharide-induced acute kidney injury mouse models and mouse primary proximal renal tubular epithelial cell injury by regulating T-box transcription factor 21/mitogen-activated protein kinase/extracellular signal-related kinase axis

Introduction and objectivesAcute kidney injury (AKI) is a common devastating complication characterized by an abrupt loss of renal function. It is of great significance to explore promising biomarkers for AKI treatment. Materials and methodsHere, we established LPS (lipopolysaccharide)-induced AKI mice models and LPS-induced AKI mouse renal tubular epithelial cell model. The severity of AKI was determined by the levels of BUN (blood urea nitrogen) and SCr (serum creatinine), the observation of pathological section as well as the renal tubular injury score. The apoptosis was determined by the measurement of Caspase-3 and Caspase-9 activities, and cell apoptosis assays. qRT-PCR (quantitative real-time PCR) and western blot revealed that miR-322-5p (microRNA-322-5p) was up-regulated in LPS -induced AKI models while Tbx21 (T-box transcription factor 21) was down-regulated in LPS-induced AKI models. Dual-luciferase reporter and RNA pulldown assays detected the interaction of Tbx21 with miR-322-5p. ResultsWe found that miR-322-5p was overtly over-expressed in the in vitro LPS-induced AKI model and promoted the apoptosis of AKI mouse renal tubular epithelial cells via inhibiting Tbx21, which suppressed the mitochondrial fission and cell apoptosis through MAPK/ERK (mitogen-activated protein kinase/extracellular signal-related kinase) pathway. ConclusionsWe demonstrated that miR-322-5p promotes LPS-induced mouse AKI by regulating Tbx21/MAPK/ERK axis, which might provide new sights for AKI research.

MicroRNA-322-5p promotes lipopolysaccharide-induced acute kidney injury mouse models and mouse primary proximal renal tubular epithelial cell injury by regulating T-box transcription factor 21/mitogen-activated protein kinase/extracellular signal-related kinase axis

Introduction and objectivesAcute kidney injury (AKI) is a common devastating complication characterized by an abrupt loss of renal function. It is of great significance to explore promising biomarkers for AKI treatment. Materials and methodsHere, we established LPS (lipopolysaccharide)-induced AKI mice models and LPS-induced AKI mouse renal tubular epithelial cell model. The severity of AKI was determined by the levels of BUN (blood urea nitrogen) and SCr (serum creatinine), the observation of pathological section as well as the renal tubular injury score. The apoptosis was determined by the measurement of Caspase-3 and Caspase-9 activities, and cell apoptosis assays. qRT-PCR (quantitative real-time PCR) and western blot revealed that miR-322-5p (microRNA-322-5p) was up-regulated in LPS -induced AKI models while Tbx21 (T-box transcription factor 21) was down-regulated in LPS-induced AKI models. Dual-luciferase reporter and RNA pulldown assays detected the interaction of Tbx21 with miR-322-5p. ResultsWe found that miR-322-5p was overtly over-expressed in the in vitro LPS-induced AKI model and promoted the apoptosis of AKI mouse renal tubular epithelial cells via inhibiting Tbx21, which suppressed the mitochondrial fission and cell apoptosis through MAPK/ERK (mitogen-activated protein kinase/extracellular signal-related kinase) pathway. ConclusionsWe demonstrated that miR-322-5p promotes LPS-induced mouse AKI by regulating Tbx21/MAPK/ERK axis, which might provide new sights for AKI research.

Berberine Hampers Influenza A Replication through Inhibition of MAPK/ERK Pathway.

Background: Berberine (BBR) is an isoquinoline alkaloid which exhibits a variety of biological and therapeutic properties, and has been reported by some to block replication of the influenza virus. However, contradictory results have also been presented, and the mechanistic explanation is lacking. Methods: A panel of cell lines (Madin–Darby canine kidney (MDCK), adenocarcinoma human alveolar basal epithelial cells (A549), lung epithelial type I (LET1)) and primary human airway epithelial cells (HAE) susceptible to influenza virus infection were infected with a seasonal influenza A virus in the presence or absence of BBR. Cytotoxicity towards cell lines was measured using XTT assay. The yield of the virus was analyzed using RT-qPCR. To study the molecular mechanism of BBR, confocal microscopy and Western blot analyses of cellular fractions were applied. Results and conclusions: Our results show cell-type-dependent anti-influenza properties of BBR in vitro which suggests that the compound acts on the cell and not the virus. Importantly, BBR hampers influenza replication in primary human airway epithelium 3D cultures that mimic the natural replication site of the virus. Studies show that the influenza A virus upregulates the mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathway and hijacks this pathway for nucleolar export of the viral ribonucleoprotein. Our results suggest that BBR interferes with this process and hampers influenza A replication.

The Role of M3 Muscarinic Receptor Ligand-Induced Kinase Signaling in Colon Cancer Progression.

Despite a reduction in incidence over the past decade, colon cancer remains the second most common cause of cancer death in the United States; recent demographics suggest this disease is now afflicting younger persons. M3 muscarinic receptor (M3R) mRNA and protein are over-expressed in colon cancer, and M3R can be activated by both traditional (e.g., acetylcholine) and non-traditional (e.g., bile acids) muscarinic ligands. In this review, we weigh the data supporting a prominent role for key protein kinases downstream of M3R activation in promoting colon cancer progression and dissemination. Specifically, we explore the roles that downstream activation of the mitogen activated protein kinase/extracellular signal-related kinase (MAPK/ERK), protein kinase C, p38 MAPK, and phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathways play in mediating colon cancer cell proliferation, survival, migration and invasion. We assess the impact of M3R-stimulated induction of selected matrix metalloproteinases germane to these hallmarks of colon cancer progression. In this context, we also critically review the reproducibility of findings derived from a variety of in vivo and in vitro colon cancer models, and their fidelity to human disease. Finally, we summarize the therapeutic potential of targeting various steps from ligand-M3R interaction to the activation of key downstream molecules.

Dysfunction in Brain-Derived Neurotrophic Factor Signaling Pathway and Susceptibility to Schizophrenia, Parkinson's and Alzheimer's Diseases.

Brain-Derived Neurotrophic Factor (BDNF) is a dominant neurotrophic factor in the brain which plays a crucial role in differentiation, regeneration and plasticity mechanisms. Binding of the BDNF to its high-affinity Tropomyosin-related kinase B (TrkB) receptor leads to phosphorylation of TrkB, thus activating the three important downstream intracellular signaling cascades within the neural cells including phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), Phospholipase C-γ (PLCγ), and mitogen-activated protein kinase/extracellular signal-related kinase (MAPK/ERK) pathways. Transcription of these pathways is regulated by cAMP Response Element-Binding protein (CREB) transcription factor, which can upregulate gene expression. In this review, we attempted to explore the role of BDNF and its associated pathways in susceptibility to Schizophrenia (Scz), Alzheimer's (AD), and Parkinson's (PD) diseases. Furthermore, we discuss dysfunction in BDNF signaling pathway and the therapeutic potential of BDNF in the treatment of these disorders. The review covers various therapeutic strategies including BDNF gene therapy, transplantation of BDNFexpressing cell grafts, epigenetic manipulation, and intraparenchymal BDNF protein infusion as well. This review seeks to achieve these goals by reviewing recent studies on BDNF and examining the details of BDNF pathway in any of the above-mentioned diseases.

BRAF and MEK Inhibitors: Use and Resistance in BRAF-Mutated Cancers.

The mitogen activated protein kinase/extracellular signal-related kinase (MAPK/ERK) signaling pathway serves an integral role in growth, proliferation, differentiation, migration, and survival of all mammalian cells. Aberrant signaling of this pathway is often observed in several types of hematologic and solid malignancies. The most frequent insult to this signaling cascade, leading to its constitutive activation, is to the serine/threonine kinase rapidly accelerating fibrosarcoma (RAF). Considering this, the development and approval of various small-molecule inhibitors targeting the MAPK/ERK pathway has become a mainstay of treatment as either mono- or combination therapy in these cancers. Although effective initially, a major clinical barrier with these inhibitors is the relapse of patients due to drug resistance. Knowledge of the mechanisms of resistance to these drugs is still premature, highlighting the need for a more in-depth understanding of how patients become insensitive to these pharmacologic interventions. Herein, we will succinctly summarize the milestones in the approval of select MAPK/ERK pathway inhibitors, their use in patients, and major modes of resistance.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

The Us2 Gene Product of Herpes Simplex Virus 2 Is a Membrane-Associated Ubiquitin-Interacting Protein

The Us2 gene encodes a tegument protein that is conserved in most members of the Alphaherpesvirinae. Previous studies on the pseudorabies virus (PRV) Us2 ortholog indicated that it is prenylated, associates with membranes, and spatially regulates the enzymatic activity of the MAP (mitogen-activated protein) kinase ERK (extracellular signal-related kinase) through direct binding and sequestration of ERK at the cytoplasmic face of the plasma membrane. Here we present an analysis of the herpes simplex virus 2 (HSV-2) Us2 ortholog and demonstrate that, like PRV Us2, HSV-2 Us2 is a virion component and that, unlike PRV Us2, it does not interact with ERK in yeast two-hybrid assays. HSV-2 Us2 lacks prenylation signals and other canonical membrane-targeting motifs yet is tightly associated with detergent-insoluble membranes and localizes predominantly to recycling endosomes. Experiments to identify cellular proteins that facilitate HSV-2 Us2 membrane association were inconclusive; however, these studies led to the identification of HSV-2 Us2 as a ubiquitin-interacting protein, providing new insight into the functions of HSV-2 Us2.

Distinct MAPK pathways are involved in IL-23 production in dendritic cells cocultured with NK cells in the absence or presence of angiotensin II

Accumulated evidence suggests that the crosstalk between dendritic cells (DCs) and natural killer (NK) cells enhances each other's capacity, and results in the production of a variety of soluble factors. However, little is known about the effect of DC–NK crosstalk in interleukin-23 (IL-23) production. In the present study we show that DC–NK coculture caused a high expression of IL-23, angiotensin II (Ang II) alone moderately increased IL-23 production in DCs, but decreased IL-23 secretion in the DC–NK coculture system. We found that Ang II does not influence DC maturation in DC–NK crosstalk. We next investigated the mitogen-activated protein kinase (MAPK) pathway in DCs. We found that Ang II increased IL-23 production through the extracellular signal-related kinase (ERK) pathway. All three MAPK members c-Jun N-terminal kinase (JNK), ERK and p38 were involved in IL-23 production in the DC–NK coculture system. In the presence of Ang II, only the JNK pathway seems to play a role in IL-23 production in DCs cocultured with NK. These data suggest that distinct MAPK pathways are involved in IL-23 production in DCs in response to different stimuli. This work demonstrates for the first time that IL-23 is produced in the DC–NK coculture system, and that Ang II is involved in DC–NK crosstalk. This data will act as a resource that allows further exploitation of role of immune response in atherosclerosis.

Nobiletin Attenuates VLDL Overproduction, Dyslipidemia, and Atherosclerosis in Mice With Diet-Induced Insulin Resistance

OBJECTIVEIncreased plasma concentrations of apolipoprotein B100 often present in patients with insulin resistance and confer increased risk for the development of atherosclerosis. Naturally occurring polyphenolic compounds including flavonoids have antiatherogenic properties. The aim of the current study was to evaluate the effect of the polymethoxylated flavonoid nobiletin on lipoprotein secretion in cultured human hepatoma cells (HepG2) and in a mouse model of insulin resistance and atherosclerosis.RESEARCH DESIGN AND METHODSLipoprotein secretion was determined in HepG2 cells incubated with nobiletin or insulin. mRNA abundance was evaluated by quantitative real-time PCR, and Western blotting was used to demonstrate activation of cell signaling pathways. In LDL receptor–deficient mice (Ldlr−/−) fed a Western diet supplemented with nobiletin, metabolic parameters, gene expression, fatty acid oxidation, glucose homeostasis, and energy expenditure were documented. Atherosclerosis was quantitated by histological analysis.RESULTSIn HepG2 cells, activation of mitogen-activated protein kinase-extracellular signal–related kinase signaling by nobiletin or insulin increased LDLR and decreased MTP and DGAT1/2 mRNA, resulting in marked inhibition of apoB100 secretion. Nobiletin, unlike insulin, did not induce phosphorylation of the insulin receptor or insulin receptor substrate-1 and did not stimulate lipogenesis. In fat-fed Ldlr−/− mice, nobiletin attenuated dyslipidemia through a reduction in VLDL-triglyceride (TG) secretion. Nobiletin prevented hepatic TG accumulation, increased expression of Pgc1α and Cpt1α, and enhanced fatty acid β-oxidation. Nobiletin did not activate any peroxisome proliferator–activated receptor (PPAR), indicating that the metabolic effects were PPAR independent. Nobiletin increased hepatic and peripheral insulin sensitivity and glucose tolerance and dramatically attenuated atherosclerosis in the aortic sinus.CONCLUSIONSNobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

Activation of p38 MAP kinase is involved in central neuropathic pain following spinal cord injury

Recent work regarding chronic central neuropathic pain (CNP) following spinal cord injury (SCI) suggests that activation of key signaling molecules such as members of the mitogen activated protein kinase (MAPK) family play a role in the expression of at-level mechanical allodynia. Previously, we have shown that the development of at-level CNP following moderate spinal cord injury is correlated with increased expression of the activated (and thus phosphorylated) forms of the MAPKs extracellular signal related kinase and p38 MAPK. The current study extends this work by directly examining the role of p38 MAPK in the maintenance of at-level CNP following spinal cord injury. Using a combination of behavioral, immunocytochemical, and electrophysiological measures we demonstrate that increased activation of p38 MAPK occurs in the spinal cord just rostral to the site of injury in rats that develop at-level mechanical allodynia after moderate SCI. Immunocytochemical analyses indicate that the increases in p38 MAPK activation occurred in astrocytes, microglia, and dorsal horn neurons in the spinal cord rostral to the site of injury. Inhibiting the enzymatic activity of p38 MAPK dose dependently reverses the behavioral expression of at-level mechanical allodynia and also decreases the hyperexcitability seen in thoracic dorsal horn neurons after moderate SCI. Taken together, these novel data are the first to demonstrate causality that increased activation of p38 MAPK in multiple cell types play an important role in the maintenance of at-level CNP following spinal cord injury.

Tumor Evasion of the Immune System: Inhibiting p38 MAP Kinase Signaling Restores the Function of Dendritic Cells.

Dendritic cells (DCs) from cancer patients are functionally defective, however, molecular mechanisms underlying are still poorly understood. In this study, we used the murine 5TGM1 myeloma model to examine the effect and mechanism of tumor-derived factors on the differentiation and function of DCs. Myeloma cells (5TGM1) or tumor culture conditioning medium (TCCM) were shown to inhibit differentiation and function of BM-derived DCs (BMDCs), evidenced by the downregulated expression of DC-related surface molecules, decreased IL-12 but increased IL-10 secretion, and compromised capacity of the cells to activate allospecific T cells in vitro. Similar results were obtained with other murine myeloma cells MOPC-315 and MPC-11. Moreover, TCCM-treated BMDCs were inferior to normal BMDCs at priming tumor-specific humoral and cellular immune responses in vivo (in the 5TGM1 mouse model). Neutralizing antibodies against IL-6, IL-10, and TGF-β partially abrogated the effects. TCCM treatment activated p38 mitogen-activated protein kinase (MAPK) and JNK but inhibited extracellular signal-related kinase (ERK). Inhibiting p38 MAPK by three different specific inhibitors was found to restore the phenotype, cytokine secretion, and function of TCCM-treated BMDCs. Vaccinating mice with BMDCs obtained from cultures in which both TCCM and p38 inhibitor were added was as efficacious as normal BMDCs at inducing tumor-specific antibody, type-1 (IFN-γ) T-cell, and CTL responses. Thus, our results suggest that tumor-induced p38 MAPK activation and ERK inhibition in DCs may be a new mechanism for tumor evasion, and regulating these signaling pathways in vivo or during DC differentiation may provide new strategies for generating potent DC vaccines for immunotherapy of multiple myeloma and other tumors.

Different signalling pathways regulate VEGF and IL-8 expression in breast cancer: implications for therapy

Elevated expression of pro-angiogenic cytokines is associated with aggressive tumour growth and decreased survival of patients with breast cancer. In general, the breast cancer cell lines with high vascular endothelial growth factor (VEGF) expression also express high levels of interleukin-8 (IL-8). The consequence of inhibiting mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K), both implicated in regulation of these cytokines, was examined in four cell lines. Treatment with the mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor U0126 reduced expression of VEGF and IL-8 in MDA-MB-231 cells, partially inhibited expression in MDA-MB-468 and Hs578T cells, with minimal effects in GI101A cells. Treatment with LY294002 reduced cytokine expression in GI101A and MDA-MB-468 cells, with partial reduction in Hs578T and less effect in MDA-MB-231 cells. Thus, IL-8 and VEGF were regulated by different signalling pathways in different cell lines; this suggests that inhibition of the dominantly active pathway can downregulate both angiogenic cytokines. Recognising which signalling pathway is active may identify targets for anti-angiogenic therapy of breast cancer.

Oncostatin M stimulates tissue inhibitor of metalloproteinase-1 via a MEK-sensitive mechanism in human myofibroblasts

Background/Aims : We previously showed that in cultured human myofibroblasts (hMFBs), Oncostatin M (OSM)-stimulated collagen accumulation is associated with increased tissue inhibitor of metalloproteinase (TIMP)1 message. However, the mechanism is unknown. Methods : hMFBs were isolated by outgrowth from cirrhotic liver explants and cultured. Using OSM (10 ng/ml) stimulation, with and without PD98059 (PD, a specific mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor), we measured: TIMP-1 protein in culture medium by Western blot, TIMP-1 mRNA levels and stability by Northern analysis, TIMP-1 promoter activity (including transcription site mutation analysis), DNA binding activity to nuclear proteins by electrophoretic mobility shift assay (EMSA), and total and phosphorylated MAP kinase in hMFB extracts by Western blot. Results : OSM stimulation of hMFBs increased TIMP-1 protein production 1.69-fold, TIMP-1 mRNA levels 2.36-fold, promoter activity 2.22-fold, TIMP-1 message stability, and phosphorylation of mitogen-activated protein kinase (MAPK). PD inhibited OSM-mediated stimulation of TIMP-1 protein, mRNA, promoter activity, phosphorylation of MAPK, and TIMP-1 message stability. An SP-1 transcription site of the TIMP-1 promoter is essential for OSM induction of TIMP-1 promoter activity. EMSA demonstrates that this site binds to transcriptional factors SP-1 and SP-3. Conclusions : OSM stimulates the TIMP-1 axis in hMFBs in vitro via a MEK-MAP kinase cascade.