MAP Kinase Activation Research Articles

Changes in Nup62 content affect contact-induced differentiation of cultured myoblasts

Differentiation of cultured skeletal myoblasts is induced by extrinsic signals that include reduction in ambient mitogen concentration and increased cell density. Using an established murine myoblast cell line (C2C12), we have found that experimental reduction of the nucleoporin p62 (Nup62) content of myoblasts enhances differentiation in high-mitogen medium, while forced expression of Nup62 inhibits density-induced differentiation. In contrast, differentiation of myoblasts induced by low-mitogen medium was unaffected by ectopic Nup62 expression. Further analyses suggested that Nup62 content affects density-induced myoblast differentiation through a mechanism involving activation of p38 MAP kinase. Nuclear pore complex (NPC) composition, in particular changes in NUP62 content, may be altered during viral infection, differentiation, and in neoplastic growth. The results support a functional role for changes in Nup62 composition in NPCs and density-induced myogenic differentiation, and suggest a link between loss of Nup62 content and induction of an intracellular stress signaling pathways.

KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with a dismal prognosis. Currently, there is no effective therapy for PDAC, and a detailed molecular and functional evaluation of PDACs is needed to identify and develop better therapeutic strategies. Here we show that the transcription factor Krüppel-like factor 7 (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks PDAC tumor growth and metastasis in cell culture and in mice. KLF7 expression in PDACs can be up-regulated due to activation of a MAP kinase pathway or inactivation of the tumor suppressor p53, two alterations that occur in a large majority of PDACs. ShRNA-mediated knockdown of KLF7 inhibits the expression of IFN-stimulated genes (ISGs), which are necessary for KLF7-mediated PDAC tumor growth and metastasis. KLF7 knockdown also results in the down-regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), resulting in Golgi complex fragmentation, and reduced protein glycosylation, leading to reduced secretion of cancer-promoting growth factors, such as chemokines. Genetic or pharmacologic activation of Golgi complex fragmentation blocks PDAC growth and metastasis similar to KLF7 inhibition. Our results demonstrate a therapeutically amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex integrity.

Dissociation of TRIF bias and adjuvanticity

Toll-like receptors (TLRs), a family of “pattern recognition receptors,” bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-β (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a “TRIF bias”) than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity.

MEKK2 inhibits activation of MAP kinases in Arabidopsis.

The Arabidopsis MEKK1-MKK1/MKK2-MPK4 kinase cascade is monitored by the nucleotide-binding leucine-rich-repeat immune receptor SUMM2. Disruption of this kinase cascade leads to activation of SUMM2-mediated immune responses. MEKK2, a close paralog of MEKK1, is required for defense responses mediated by SUMM2, the molecular mechanism of which is unclear. In this study, we showed that MEKK2 serves as a negative regulator of MPK4. It binds to MPK4 to directly inhibit its phosphorylation by upstream MKKs. Activation of SUMM2-mediated defense responses induces the expression of MEKK2, which in turn blocks MPK4 phosphorylation to further amplify immune responses mediated by SUMM2. Intriguingly, MEKK2 locates in a tandem repeat consisting of MEKK1, MEKK2 and MEKK3, which was generated from a recent gene duplication event, suggesting that MEKK2 evolved from a MAPKKK to become a negative regulator of MAP kinases.

The protective effects of moscatilin against methylglyoxal-induced neurotoxicity via the regulation of p38/JNK MAPK pathways in PC12 neuron-like cells

Methylglyoxal (MGO) is an endogenous toxic compound that plays a vital role in diabetic complications such as diabetic neuropathy. Moscatilin is a bibenzyl component from Dendrobium species, has been shown to possess a wide range of pharmacological activities. To clarify whether moscatilin prevents rat pheochromocytoma cells (PC12 cells) from damage induced by MGO, cells were pre-treated with moscatilin and then stimulated with MGO. Moscatilin inhibited MGO associated cytotoxicity in a concentration (0.1, 0.5, or 1.0 μmol/L)-dependent manner and downregulated the formation of advanced glycation end products and reactive oxygen species. Moscatilin attenuated MGO-induced mitochondrial dysfunction involving the loss of mitochondrial membrane potential and depletion of adenosine triphosphate. MGO induced cell apoptosis via the upregulation of p53, caspases 3 and poly(ADP-ribose)polymerase, enhancement of cytochrome c release, and interruption of the Bax/Bcl-2 balance; these detrimental effects were ameliorated by moscatilin. Furthermore, moscatilin inhibited MGO-induced activation of MAP kinase (MAPK) superfamily, including p38 and c-Jun N-terminal kinases (JNKs). In conclusion, we found that the neuroprotective effect of moscatilin is due to a reduction of MGO-induced damage to mitochondria function through modulating the p38 and JNK stress-activated MAPK cascades pathway. Thus, it might be a potent compound for preventing/counteracting diabetic neuropathy.

S1PR1 interacts with endoplasmic reticulum stress sensor, BiP, to regulate calcium homeostasis and endothelial barrier function

Endothelial–cell surface localized sphingosine 1 phosphate receptor 1 (S1PR1) is well known to enhance barrier function upon ligating S1P. We recently showed that S1P phosphorylation of S1PR1 at tyrosine (Y) 143 residue induces S1PR1 internalization and compromises barrier function. Here, we assessed the mechanism internalizing the Y143‐S1PR1 and how Y143‐S1PR1 disrupts barrier function. Herein, using TIRF and confocal imaging, we show that phosphorylated (Y143D‐S1PR1) S1PR1 mutant localizes at endoplasmic reticulum (ER). Inhibition of dynamin activity, a GTP‐dependent pinchase, rescued Y143D‐S1PR1 expression at the cell surface. However, dynamin inhibition had no effect on cell‐surface retention of S1PR1 or Y143F‐S1PR1. In other studies, we show that Y143D‐S1PR1 bound WT‐dynamin strongly while weakly with GTPase defective dynamin. Cell‐surface retained S1PR1 induces signaling including calcium mobilization and activation of MAP kinases by binding Gi heterotrimeric protein. Intriguingly, we show that internalized Y143D‐S1PR1 augmented Ca2+ entry which was blocked by pertussis toxin. Furthermore, phosphorylated receptor bound Gi indicating that internalized S1PR1 binds cytosolic Gi which then increases Ca2+ entry. We further showed Y143D‐S1PR1 mutant augmented Ca2+ entry by organizing stromal‐interaction molecule 1 (STIM1) dependent ER‐PM junction formation. To address the mediator involved in retaining Y143D‐S1PR1 at the ER, we performed mass spectrometric analysis and identified GRP78 (BiP), an endoplasmic resident protein, as the novel interacting partner of S1PR1. Depletion of BiP or STIM1 rescued barrier function even in presence of Y143D‐S1PR1. Thus, BiP recruits phosphorylated Y143D‐S1PR1 at the ER where S1PR1 mutant organizes ER‐PM junctions to mediate Ca2+ entry disrupting endothelial barrier function.Support or Funding InformationNIH

Sex differences in MAP kinase activation in the periaqueductal gray after morphine treatment

Morphine is a potent opioid analgesic, but its long term use can lead to negative side effects, including tolerance, which is a decrease in the effectiveness of the opioid. An area of active interest is looking into the molecular effects of chronic morphine treatment in the Periaqueductal gray (PAG), a brain region that controls descending pain modulation. One such molecular target within the PAG is extracellular‐signal regulated kinase 1/2 (ERK). Previous studies have shown that pharmacological inhibition of ERK enhanced morphine tolerance, indicating that ERK activity is associated with better responsiveness to morphine. The PAG is known to contain a heterogenous population of neurons including GABA and glutamate subtypes. However, which neurons ERK is activated in within the PAG following morphine tolerance is unknown. Further, there are known differences in PAG activity between male and female mice. These sex‐differences have not been well studied after morphine tolerance using acute pain tests. The purpose of this research is to investigate differences in ERK activation following morphine tolerance in male and female mice. We treated wild‐type male and female mice with morphine (10 mg/kg, i.p.) or saline for 5 days to induce morphine tolerance, following which both behavior and protein immunofluorescence were assessed. We observe sex‐specific differences in ERK activation levels and morphine antinociceptive tolerance in mice. We also assessed co‐localization of ERK with GABA and glutamate neurons after morphine tolerance. The study will help us understand the cell‐type specificity of kinase activation following morphine tolerance. Further this will give us more information about the nature of neurons that are contributing to sex‐differences in opioid functions within the PAG.

Chronic Intermittent Hypoxia Blunts Phrenic Motor Plasticity: Role of Inflammation

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). Preconditioning with a single day of chronic intermittent hypoxia (8 hours, 2 min hypoxic episodes with 2 min intervals) abolishes AIH‐induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activation (Huxtable et al., 2015). pLTF is restored by high‐dose, systemic administration of the non‐steroidal anti‐inflammatory drug, ketoprofen, or spinal p38 MAP kinase inhibition. Similarly, preconditioning with 7 days of CIH (8 hours per day, 2 min episodes, 2 min intervals) abolishes pLTF, but we observed that phospho‐p38 within phrenic motor neurons is unchanged from control rats (Gonzalez‐Rothi and colleagues, unpublished). Thus, mechanisms whereby 7 days of CIH undermines AIH‐induced pLTF are unclear. Since CIH induces time‐dependent spinal inflammatory gene expression profiles over time (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single day) blunts LTF through an inflammation‐dependent mechanism independent of p‐38MAP kinase. In ongoing experiments, we are evaluating whether anti‐inflammatory drugs restore AIH‐induced pLTF in rats preconditioned with prolonged CIH (7–28 days). Preliminary analyses in rats preconditioned with CIH for 7 days indicate that pLTF is expressed when ketoprofen is given on each day of the CIH exposure (67% baseline), but not when a single ketoprofen dose is given 3 hours prior to assessment of pLTF (−6% baseline). These experiments are the first to explore shifting mechanisms whereby prolonged CIH and associated inflammation undermine phrenic motor plasticity. Such experiments are essential since CIH is a hallmark of sleep apnea, a condition highly prevalent in individuals living with neurological disorders (spinal cord injury, ALS, etc.). It is therefore important to identify and mitigate factors which may undermine the potential for therapeutic interventions (i.e. repetitive AIH) to harness endogenous plasticity and ultimately improve breathing.Support or Funding InformationSupported by: Department of Defense: SCI160123 and NIH R01 HL147554

A MAP kinase‐driven mechanical stress response mediates the hypertrophic response in skeletal muscle

When cells are exposed to unfavorable conditions they cope by activating cellular stress responses. Such signaling depends on activation of the p38 and JNK MAP kinases and is crucial for both general tissue homeostasis and a range of physiological reactions. However, the mechanisms linking diverse stress stimuli to MAP kinase activation remain poorly understood.Here we show how a specific MAP3 kinase mediates activation of p38 and JNK upon hyperosmotic‐ and mechanical stress. Employing fluorescence microscopy techniques, we show that this MAP3 kinase translocates from the cytoplasm to the nuclear lamina and other detergent‐resistant structures in the nucleus upon osmotic stress induction. Here, it co‐localizes with structures rich in DNA damage response factors such as 53BP1, but void of DNA strand breaks, which were shown to constitute nuclear sensor domains for mechanical stress.Mechanical stress is experienced in skeletal muscle due to the repeated contraction of muscle fibers upon physical exercise. The signaling from these events underlie the hypertrophic response, where muscles respond to exercise by growing larger and stronger. We show that the above signaling pathway operates during exercise‐associated stimulus of C2C12 myotubes, where it is responsible for the production of a range of inflammatory transcripts and myokines (such as IL6). Consequently, in a mouse model deficient for the MAP3 kinase, we see clear signs of pathology associated with decreased muscle repair and adaptation to exercise.In conclusion, our work provides novel insight into the mechanisms by which cells sense and react to mechanical stress stimuli and identify skeletal muscle as a major target organ upon deficiency in these responses.

Mutant Huntingtin affects toll-like receptor 4 intracellular trafficking and cytokine production in mast cells

BackgroundHuntington’s disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases.MethodsBone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed.ResultsR6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice.ConclusionsOur data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells.

Surface protein CD20 serves as the critical target of immunotherapy in various B-cell malignancies for decades, however its biological function and regulation remain largely elusive. Better understanding of CD20 function may help to design improved rational therapies to prevent development of resistance. Using CRISPR/Cas9 technique, we have abrogated CD20 expression in five different malignant B-cell lines. We show that CD20 deletion has no effect upon B-cell receptor signaling or calcium flux. Also B-cell survival and proliferation is unaffected in the absence of CD20. On the contrary, we found a strong defect in actin cytoskeleton polymerization and, consequently, defective cell adhesion and migration in response to homeostatic chemokines SDF1α, CCL19 and CCL21. Mechanistically, we could identify a reduction in chemokine-triggered PYK2 activation, a calcium-activated signaling protein involved in activation of MAP kinases and cytoskeleton regulation. These cellular defects in consequence result in a severely disturbed homing of B cells in vivo.

High-Density Lipoprotein (HDL) Inhibits Serum Amyloid A (SAA)-Induced Vascular and Renal Dysfunctions in Apolipoprotein E-Deficient Mice.

Serum amyloid A (SAA) promotes endothelial inflammation and dysfunction that is associated with cardiovascular disease and renal pathologies. SAA is an apoprotein for high-density lipoprotein (HDL) and its sequestration to HDL diminishes SAA bioactivity. Herein we investigated the effect of co-supplementing HDL on SAA-mediated changes to vascular and renal function in apolipoprotein E-deficient (ApoE−/−) mice in the absence of a high-fat diet. Male ApoE−/− mice received recombinant human SAA or vehicle (control) by intraperitoneal (i.p.) injection every three days for two weeks with or without freshly isolated human HDL supplemented by intravenous (i.v.) injection in the two weeks preceding SAA stimulation. Aorta and kidney were harvested 4 or 18 weeks after commencement of treatment. At 4 weeks after commencement of treatment, SAA increased aortic vascular cell adhesion molecule (VCAM)-1 expression and F2-isoprostane level and decreased cyclic guanosine monophosphate (cGMP), consistent with SAA stimulating endothelial dysfunction and promoting atherosclerosis. SAA also stimulated renal injury and inflammation that manifested as increased urinary protein, kidney injury molecule (KIM)-1, and renal tissue cytokine/chemokine levels as well as increased protein tyrosine chlorination and P38 MAPkinase activation and decreased in Bowman’s space, confirming that SAA elicited a pro-inflammatory phenotype in the kidney. At 18 weeks, vascular lesions increased significantly in the cohort of ApoE−/− mice treated with SAA alone. By contrast, pretreatment of mice with HDL decreased SAA pro-inflammatory activity, inhibited SAA enhancement of aortic lesion size and renal function, and prevented changes to glomerular Bowman’s space. Taken together, these data indicate that supplemented HDL reduces SAA-mediated endothelial and renal dysfunction in an atherosclerosis-prone mouse model.

Clinical and Survival Impact of Sex-Determining Region Y-Box 2 in Colorectal Cancer: An Integrated Analysis of the Immunohistochemical Study and Bioinformatics Analysis.

Transcription factor sex-determining region Y-box 2 (SOX2) involves in the maintenance of cancer stem cells. However, the role of SOX2 in colorectal cancer (CRC) remains unclear. This study was conducted to investigate the effect of SOX2 on CRC. Studies were searched using electronic databases. The combined odds ratios (ORs) or hazard ratios (HRs: multivariate Cox survival analysis) with their 95% confidence intervals (CIs) were calculated. The Cancer Genome Atlas (TCGA) and GEO datasets were further applied to validate the survival effect. The functional analysis of SOX2 was investigated. In this work, 13 studies including 2337 patients were identified, and validation data were enrolled from TCGA and GEO datasets. SOX2 expression was not significantly related to age, gender, microsatellite instability (MSI) status, clinical stage, histological grade, tumor size, pT-stage, lymph node metastasis, distal metastasis, and cancer-specific survival (CSS) but was correlated with worse overall survival (OS: n = 536 patients) (P < 0.05). Furthermore, TCGA data demonstrated similar results, with no significant correlation between SOX2 and pathological characteristics. Further validation data (OS: n = 1408 and disease-free survival (DFS): n = 1367) showed that SOX2 expression was correlated with worse OS (HR = 1.35, 95% CI: 1.11–1.65, P=0.004) and DFS (HR = 1.30, 95% CI: 1.04–1.62, P=0.02). The functional analyses showed that SOX2 involved in cell-cell junction, focal adhesion, extracellular matrix- (ECM-) receptor interaction, and MAP kinase activity. Our findings suggest that SOX2 expression may be correlated with the worse prognosis of CRC.

Bergamot Polyphenols Improve Dyslipidemia and Pathophysiological Features in a Mouse Model of Non-Alcoholic Fatty Liver Disease

There is a need for continued drug development for nonalcoholic steatohepatitis (NASH). Bergamot is a plant whose fruit juice is enriched with flavonoids and phenolic compounds which improves dyslipidemia and markers of systemic inflammation in patients with Metabolic Syndrome. The aim of this study was to perform a preclinical “proof of concept” study of Bergamot polyphenolic formulation (BPF99) for the treatment of NASH. A disease reversal study was performed in the diet-induced animal model of NAFLD (DIAMOND). Groups of 8 weeks old mice were randomly assigned to receive chow diet, high fat diet with sugar in drinking water (Western diet- WD). Mice on WD were further randomized to continue on WD gavaged with vehicle or continue on WD with additional gavage of BPF99 (50 mg/kg) after 16 weeks of diet. Mice were euthanized after 11 additional weeks. The primary endpoint was resolution of NASH. Secondary endpoints included changes in individual histological features, body weight, liver enzymes, dyslipidemia, markers of oxidative stress and molecular markers of disease activity and fibrosis. The results showed that BPF99 reduced ALT (mean 71.6 vs 44.6 IU/l, p < 0.01), triglycerides (38.8 vs 28.1 mg/dl, p < 0.05), LDL-C (39.2 vs 23.7 mg/dl, p < 0.001). It significantly improved NASH resolution (p < 0.001) and the SAF scores (p < 0.05) while the NAS improvement approached significance. BPF99 reduced markers of oxidative stress, along with reduced JNK and p38 MAP kinase activity. BPF99 did not reduce the number of mice with fibrosis but improved collagen proportional area (p < 0.04) and procollagen I and III expression. Collectively our results showed that BPF99 resolves NASH and ameliorates key histological and pathophysiological features of NASH along with improvement in ALT and dyslipidemia in the DIAMOND mice.

Intratracheal GLP-1 receptor agonist treatment up-regulates mucin via p38 and exacerbates emphysematous phenotype in mucus hypersecretory obstructive lung diseases

Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that stimulates glucose-mediated insulin production by pancreatic beta cells. It is also associated with protective effects in multiple tissues. GLP-1 receptor is highly expressed in pulmonary tissue, hinting possible pulmonary delivery of GLP-1 drugs. However, little is known about the role of GLP-1 signaling in the lung, especially in mucus hypersecretory obstructive lung diseases. Here, we showed that treatment with exendin-4, a clinically available GLP-1 receptor agonist, up-regulates mucin expression in normal airway epithelial cells and in the lung of normal mice, indicating mucus stimulatory effect of GLP-1 under physiological condition. Exendin-4 also increased mucin expression in in vitro cellular and in vivo murine models of obstructive lung diseases via the activation of p38 MAP kinase. Notably, mucin induction in vivo exacerbated key pulmonary abnormalities including emphysematous phenotypes, implying that GLP-1 signaling in the lung is detrimental under pulmonary obstructive condition. Another GLP-1 receptor agonist liraglutide had similar induction of mucin. Together, our studies not only demonstrate novel physiological and pathological roles of GLP-1 in the lung but may also caution against the clinical use of inhaled GLP-1 receptor agonists in the patients with obstructive lung diseases.

Macrophage migration inhibitory factor regulates integrin-β1 and cyclin D1 expression via ERK pathway in podocytes

AimsMacrophage migration inhibitory factor (MIF) is found to increase in proliferative glomerulonephritis. MIF binds to the MIF receptor (CD74) that activates MAP kinase (ERK and p38). Integrins and cyclinD1 regulate cell proliferation, differentiation and adhesion. This study evaluates whether MIF can regulate integrin-β1/cyclin D1 expression and cell adhesion of podocytes. Main methodsExpression of integrin-β1 mRNA/protein and cyclin D1 mRNA under stimulation of MIF was evaluated by real-time PCR and Western blotting. MIF receptor (CD74) and MAP kinase under MIF treatment were examined to determine which pathway regulated integrin-β1 and cyclin D1 expression. Cell adhesion was evaluated under MIF treatment and/or anti-integrin-β1 antibody by cell adhesion assay. Key findingsProtein levels of integrin-β1 were up-regulated under MIF treatment in a dosage-dependent manner. CD74 protein levels were not changed after MIF treatment. Integrin-β1 and cyclin D1 mRNA levels were up-regulated after MIF 100 ng/ml treatment. ERK inhibitor U0126 reduced MIF-induced the increase in integrin-β1 mRNA and protein expression following MIF stimulation. However, p38 inhibitor SB 203580 did not inhibit MIF-induced increase in integrin-β1 mRNA and protein expression following MIF stimulation. MIF-induced increase in cyclin D1 mRNA level also was inhibited only by U0126 following MIF stimulation. Podocyte adhesion was increased after MIF treatment, but, anti-integrin-β1 antibody decreased MIF-enhanced podocyte adhesion. SignificanceMIF increases integrin-β1 and cyclin D1 expression through the ERK pathway in podocytes, and the up-regulated expression of integrin-β1 increases podocyte adhesion. These results provide further understanding for the role of MIF in developing proliferative glomerulonephritis.

Spinal AMP kinase activity differentially regulates phrenic motor plasticity.

Acute intermittent hypoxia (AIH) elicits phrenic motor plasticity via multiple distinct cellular mechanisms. With moderate AIH, phrenic motor facilitation (pMF) requires Gq protein-coupled serotonin type 2 receptor activation, ERK MAP kinase activity, and new synthesis of brain-derived neurotrophic factor. In contrast, severe AIH elicits pMF by an adenosine-dependent mechanism that requires exchange protein activated by cAMP, Akt, and mammalian target of rapamycin (mTOR) activity, followed by new tyrosine receptor kinase B protein synthesis; this same pathway is also initiated by Gs protein-coupled serotonin 7 receptors (5-HT7). Because the metabolic sensor AMP-activated protein kinase (AMPK) inhibits mTOR-dependent protein synthesis, and mTOR signaling is necessary for 5-HT7 but not 5-HT2 receptor-induced pMF, we hypothesized that spinal AMPK activity differentially regulates pMF elicited by these distinct receptor subtypes. Serotonin type 2A receptor [5-HT2A; (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride] or 5-HT7 (AS-19) receptor agonists were administered intrathecally at C4 (3 injections, 5-min intervals) while recording integrated phrenic nerve activity in anesthetized, vagotomized, paralyzed, and ventilated rats. Consistent with our hypothesis, spinal AMPK activation with 2-deoxyglucose or metformin blocked 5-HT7, but not 5-HT2A receptor-induced pMF; in both cases, pMF inhibition was reversed by spinal administration of the AMPK inhibitor compound C. Thus, AMPK differentially regulates cellular mechanisms of serotonin-induced phrenic motor plasticity.NEW & NOTEWORTHY Spinal AMP-activated protein kinase (AMPK) overactivity, induced by local 2-deoxyglucose or metformin administration, constrains serotonin 7 (5-HT7) receptor-induced (but not serotonin type 2A receptor-induced) respiratory motor facilitation, indicating that metabolic challenges might regulate specific forms of respiratory motor plasticity. Pharmacological blockade of spinal AMPK activity restores 5-HT7 receptor-induced respiratory motor facilitation in the presence of either 2-deoxyglucose or metformin, showing that AMPK is an important regulator of 5-HT7 receptor-induced respiratory motor plasticity.

The Cytoprotective and Anti-cancer Potential of Bisbenzylisoquinoline Alkaloids from Nelumbo nucifera.

Natural product therapy has been gaining therapeutic importance against various diseases, including cancer. The failure of chemotherapy due to its associated adverse effects promoted adjunct therapy with natural products. Phytochemicals exert anti-carcinogenic activities through the regulation of various cell signaling pathways such as cell survival, inflammation, apoptosis, autophagy and metastasis. The 'small molecule-chemosensitizing agents' from plants induce apoptosis in drug-resistant and host-immune resistant cancer cells in in vitro as well as in vivo models. For example, alkaloids from Nelumbo nucifera, liensinine, isoliensinine and neferine exert the anticancer activity through enhanced ROS generation, activation of MAP kinases, followed by induction of autophagy and apoptotic cell death. Likewise, these alkaloids also exert their cytoprotective action against cerebrovascular stroke/ischemic stroke, diabetes, and chemotherapy-induced cytotoxicity. Therefore, the present review elucidates the pharmacological activities of these bisbenzylisoquinoline alkaloids which include the cytoprotective, anticancer and chemosensitizing abilities against various diseases such as cardiovascular diseases, neurological diseases and cancer.

Network Pharmacology-Based Strategy for Predicting Therapy Targets of Traditional Chinese Medicine Xihuang Pill on Liver Cancer.

Objective To investigate the potential therapy targets and pharmacological mechanism of traditional Chinese medicine (TCM) Xihuang pill in liver cancer based on network pharmacology. Methods Drug ingredients-target network was constructed based on the target sets of Xihuang pill and liver cancer. The overlapping genes between Xihuang pill targets and liver cancer-related molecular targets were investigated using comparative analysis. Moreover, the PPI network and module was constructed based on overlapping genes and hub nodes, respectively, followed by the pathway enrichment analysis. Results A drug ingredients-target network was established with 1184 nodes and 11035 interactions. Moreover, a total of 106 overlapping genes were revealed between drug targets and liver cancer molecular targets. Furthermore, a PPI network and 4 modules were further investigated based on overlapping genes, respectively. These hub nodes such as VEGFA and EGFR were mainly enriched in GO functions including positive regulation of MAP kinase activity, activation of protein kinase activity, regulation of MAP kinase activity, and pathways like proteoglycans in cancer, bladder cancer, and estrogen signaling. Conclusion VEGFA and EGFR might be potential therapy targets of Xihuang pill in liver cancer. Furthermore, the effect of Xihuang pill on liver cancer might be realized by targeting VEGFA and EGFR in pathways like proteoglycans in cancer and estrogen signaling.

Antiproliferative effect of mifepristone (RU486) on human neuroblastoma cells (SK-N-SH): in vitro and in vivo studies.

RU486 (mifepristone), a glucocorticoid and progesterone receptor antagonist, has been reported to exert antiproliferative effects on tumor cells. Experiments were performed to analyze the effects of RU486 on the proliferation of the human neuroblastoma, both in vitro and in vivo, using the human neuroblastoma SK-N-SH cell line. The exposure in vitro of SK-N-SH cells to RU486 revealed a dose-dependent inhibition of 3H-thymidine incorporation due to a rapid but persistent inhibition of MAPKinase activity and ERK phosphorylation. A significant decrease of SK-N-SH cell number was evident after 3, 6, and 9 days of treatment (up to 40% inhibition), without evident cell death. The inhibitory effect exerted by RU486 was not reversed by the treatment of the cells with dexamethasone or progesterone. Moreover, RU486 induced a shift in SK-N-SH cell phenotypes, with an almost complete disappearance of the neuronal-like and a prevalence of the epithelial-like cell subtypes. Finally, the treatment with RU486 of nude mice carrying a SK-N-SH cell xenograft induced a strong inhibition (up to 80%) of tumor growth. These results indicated a clear effect of RU486 on the growth of SK-N-SH neuroblastoma cells that does not seem to be mediated through the classical steroid receptors. RU486 acted mainly on the more aggressive component of the SK-N-SH cell line and its effect in vivo was achieved at a concentration already used to inhibit oocyte implantation.