Dual-frequency ultrasound enhances functional neuron differentiation from neural stem cells by modulating Ca2+ dynamics and the ERK1/2 signaling pathway.

Our previous study demonstrated that ultrasound is able to promote differentiation on neural stem cells (NSCs), and dual-frequency ultrasound promotes this effect due to enhanced acoustic cavitation compared with single-frequency ultrasound. However, the underlying biological reasons have not been well disclosed. The purpose of this study was to investigate the underlying bioeffects, mechanisms and signaling pathways of dual-frequency ultrasound on NSC differentiation. The morphology, neurite outgrowth, and differentiation percentages were investigated under various dual-frequency simulation parameters with exposure periods varying from 5 to 15 min. Morphological observations identified that dual-frequency ultrasound stimulation promoted ultrasound dose-dependent neurite outgrowth. In particular, cells exposed for 10 min/2 days showed optimal neurite outgrowth and neuron differentiation percentages. In addition, live cell calcium images showed that dual-frequency ultrasound enhanced the internal calcium content of the cells, and calcium ions entering cells from the extracellular environment could be observed. Dual frequency ultrasound exposure enhanced extracellular calcium influx and upregulated extracellular signal-regulated kinases 1/2 (ERK1/2) expression. Observations from immunostaining and protein expression examinations also identified that dual-frequency ultrasound promoted brain-derived neurotrophic factor (BDNF) secretion from astrocytes derived from NSCs. In summary, evidence supports that dual-frequency ultrasound effectively enhances functional neuron differentiation via calcium channel regulation via the downstream ERK1/2 pathway and promotes BDNF secretion to serve as feedback to cascade neuron differentiation. The results may provide an alternative for cell-based therapy in brain injury.

Involvement of Hypoxia-Inducible Factor 1-α in Experimental Testicular Ischemia and Reperfusion: Effects of Polydeoxyribonucleotide and Selenium.

Polydeoxyribonucleotide (PDRN) is an agonist of the A2A adenosine receptor derived from salmon trout sperm. Selenium (Se) is a trace element normally present in the diet. We aimed to investigate the long-term role of PDRN and Se, alone or in association, after ischemia-reperfusion (I/R) in rats. The animals underwent 1 h testicular ischemia followed by 30 days of reperfusion or a sham I/R and were treated with PDRN or Se alone or in association for 30 days. I/R significantly increased hypoxia-inducible factor 1-α (HIF-1α) in Leydig cells, malondialdehyde (MDA), phosphorylated extracellular signal-regulated kinases 1/2 (pErk 1/2), and apoptosis decreased testis weight, glutathione (GSH), testosterone, nuclear factor erythroid 2-related factor 2 (Nrf2), induced testicular structural changes, and eliminated HIF-1α spermatozoa positivity. The treatment with either PDRN or Se significantly decreased MDA, apoptosis, and HIF-1α positivity of Leydig cells, increased testis weight, GSH, testosterone, and Nrf2, and improved the structural organization of the testes. PDRN and Se association showed a higher protective effect on all biochemical, structural, and immunohistochemical parameters. Our data suggest that HIF-1α could play important roles in late testis I/R and that this transcriptional factor could be modulated by PDRN and Se association, which, together with surgery, could be considered a tool to improve varicocele-induced damages.

Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca2+-Regulated ERK1/2-Activating Pathway.

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01-0.5 mM) exhibited greater cytotoxicity than ketamine (0.1-3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca2+]i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca2+ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca2+]i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca2+]i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca2+]i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.

Transcriptome analysis reveals transforming growth factor-ß1 prevents extracellular matrix degradation and cell adhesion during the follicular-luteal transition in cows.

Ovarian angiogenesis is an extremely rapid process that occurs during the transition from follicle to corpus luteum (CL) and is crucial for reproduction. It is regulated by numerous factors including transforming growth factor-ß1 (TGFB1). However, the regulatory mechanism of TGFB1 in ovarian angiogenesis is not fully understood. To address this, in this study we obtained high-throughput transcriptome analysis (RNA-seq) data from bovine luteinizing follicular cells cultured in a system mimicking angiogenesis and treated with TGFB1, and identified 455 differentially expressed genes (DEGs). Quantitative real-time PCR results confirmed the differential expression patterns of the 12 selected genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified that the MAPK and ErbB pathways, cell adhesion molecules (CAMs), and extracellular matrix (ECM)-receptor interactions may play pivotal roles in TGFB1-mediated inhibition of CL angiogenesis. TGFB1 phosphorylated ERK1/2 (MAPK1/3) and Akt, indicating that these pathways may play an important role in the regulation of angiogenesis. Several genes with specific functions in cell adhesion and ECM degradation were identified among the DEGs. In particular, TGFB1-induced upregulation of syndecan-1 (SDC1) and collagen type I alpha 1 chain (COL1A1) expression may contribute to the deposition of type I collagen in luteinizing follicular cells. These results indicate that TGFB1 inhibits cell adhesion and ECM degradation processes involving ERK1/2, ErbB, and PI3K/Akt signaling pathways, and leads to inhibition of angiogenesis during the follicular-luteal transition. Our results further reveal the molecular mechanisms underlying the actions of TGFB1 in early luteinization.

MC2R/MRAP2 activation could affect bovine ovarian steroidogenesis potential after ACTH treatment.

Stressors activate the hypothalamic-pituitary-adrenal (HPA) axis, reducing fertility by interfering with the mechanisms that regulate the timing of events within the follicular phase of the estrous cycle. In the HPA axis, melanocortin 2 receptor (MC2R) mediates responses to adrenocorticotropic hormone (ACTH) in concert with melanocortin receptor accessory protein 2 (MRAP2). The aims of the present study were: (1) to evaluate the effects of ACTH administered in cows in the preovulatory period on the expression of the MC2R/MRAP2 complex in the dominant follicle; and (2) to analyze the involvement of Extracellular signal Regulated Kinase 1 (ERK1) signaling in the activation of MC2R and the expression of key enzymes involved in the biosynthesis of glucocorticoids (GCs) in the dominant follicle. To this end, 100 IU ACTH was administered to Holstein cows from a local dairy farm during pro-estrus every 12 h for four days until ovariectomy, which was performed before ovulation. Protein immunostaining of MC2R was higher in the dominant follicles of ACTH-treated cows (p < 0.05). Also, Western blot analysis showed higher activation of the ERK1 signaling pathway in ACTH-treated cows (p < 0.05). Finally, immunohistochemistry performed in the dominant follicles of ACTH-treated cows detected higher expression of CYP17A1 and CYP21A2 (p < 0.05). These results suggest that the bovine ovary is able to respond locally to ACTH as a consequence of stress altering the expression of relevant steroidogenic enzymes. The results also confirm that the complete GC biosynthesis pathway is present in bovine dominant follicle and therefore GCs could be produced locally.

Lipopolysaccharide-induced inflammation in human peritoneal mesothelial cells is controlled by ERK1/2-CDK5-PPARγ axis.

BACKGROUND: Peritonitis is a common complication in which the peritoneum becomes inflamed. Peroxisome proliferator-activated receptor (PPAR)γ agonists and extracellular signal-regulated kinases 1/2 (ERK1/2) inactivation have been found to restore damage caused by lipopolysaccharide-induced (LPS) inflammation. This study aimed to investigate the association between PPARγ and ERK1/2 in LPS-induced inflammation in peritonitis. METHODS: Human peritoneal mesothelial cells were maintained in Dulbecco's Modified Eagle Medium and treated with LPS under a series of different concentrations and treatment times. Cellular interleukins-1ßeta (IL-1ß), cellular interleukins-6 (IL-6), cellular interleukins-12 (IL-12) were measured by enzyme-linked immunosorbent assay (ELISA) assay. Expression or activation of cyclin-dependent kinase (CDK)5, ERK1/2, and PPARγ was detected using quantitative real-time PCR and/or western blot. RESULTS: LPS induced dose- and time-dependent increments in the cellular IL-1ß, IL-6, and IL-12 contents, cyclin-dependent kinase 5 (CDK5) expression, and PPARγSer273 phosphorylation. Treatment with 1 µg/mL LPS for 12 hours was the optimal experimental design for inflammation stimulation. The concentration of LPS over 1 µg/mL or treatment more than 12 hours reduced the inflammatory status. LPS stimulation also activated ERK1/2 and increased its interaction with CDK5. Further, ERK1/2 inhibition by AZD0364 prevented IL-1ß, IL-6, IL-12, and CDK5 expression, as well as activation of ERK1/2 and phosphorylation of PPARγ, induced by LPS. Knockdown of CDK5 using its siRNA caused similar changes as AZD0364, minus ERK1/2 inactivation. CONCLUSIONS: Our results suggested that LPS-induced inflammation in human peritoneal mesothelial cells can be partly suppressed by inhibiting the ERK1/2/CDK5/PPARγ axis.

Surface Phenotype Changes and Increased Response to Oxidative Stress in CD4+CD25high T Cells.

Conversion of CD4+CD25+FOXP3+ T regulatory cells (Tregs) from the immature (CD45RA+) to mature (CD45RO+) phenotype has been shown during development and allergic reactions. The relative frequencies of these Treg phenotypes and their responses to oxidative stress during development and allergic inflammation were analysed in samples from paediatric and adult subjects. The FOXP3lowCD45RA+ population was dominant in early childhood, while the percentage of FOXP3highCD45RO+ cells began increasing in the first year of life. These phenotypic changes were observed in subjects with and without asthma. Further, there was a significant increase in phosphorylated ERK1/2 (pERK1/2) protein in hydrogen peroxide (H2O2)-treated CD4+CD25high cells in adults with asthma compared with those without asthma. Increased pERK1/2 levels corresponded with increased Ca2+ response to T cell receptor stimulation. mRNA expression of peroxiredoxins declined in Tregs from adults with asthma. Finally, CD4+CD25high cells from paediatric subjects were more sensitive to oxidative stress than those from adults in vitro. The differential Treg sensitivity to oxidative stress observed in children and adults was likely dependent on phenotypic CD45 isoform switching. Increased sensitivity of Treg cells from adults with asthma to H2O2 resulted from a reduction of peroxiredoxin-2, -3, -4 and increased pERK1/2 via impaired Ca2+ response in these cells.

ERK/Drp1-dependent mitochondrial fission contributes to HMGB1-induced autophagy in pulmonary arterial hypertension.

OBJECTIVES: High-mobility group box-1 (HMGB1) and aberrant mitochondrial fission mediated by excessive activation of GTPase dynamin-related protein 1 (Drp1) have been found to be elevated in patients with pulmonary arterial hypertension (PAH) and critically implicated in PAH pathogenesis. However, it remains unknown whether Drp1-mediated mitochondrial fission and which downstream targets of mitochondrial fission mediate HMGB1-induced pulmonary arterial smooth muscle cells (PASMCs) proliferation and migration leading to vascular remodelling in PAH. This study aims to address these issues. METHODS: Primary cultured PASMCs were obtained from male Sprague-Dawley (SD) rats. We detected RNA levels by qRT-PCR, protein levels by Western blotting, cell proliferation by Cell Counting Kit-8 (CCK-8) and EdU incorporation assays, migration by wound healing and transwell assays. SD rats were injected with monocrotaline (MCT) to establish PAH. Hemodynamic parameters were measured by closed-chest right heart catheterization. RESULTS: HMGB1 increased Drp1 phosphorylation and Drp1-dependent mitochondrial fragmentation through extracellular signal-regulated kinases 1/2 (ERK1/2) signalling activation, and subsequently triggered autophagy activation, which further led to bone morphogenetic protein receptor 2 (BMPR2) lysosomal degradation and inhibitor of DNA binding 1 (Id1) downregulation, and eventually promoted PASMCs proliferation/migration. Inhibition of ERK1/2 cascade, knockdown of Drp1 or suppression of autophagy restored HMGB1-induced reductions of BMPR2 and Id1, and diminished HMGB1-induced PASMCs proliferation/migration. In addition, pharmacological inhibition of HMGB1 by glycyrrhizin, suppression of mitochondrial fission by Mdivi-1 or blockage of autophagy by chloroquine prevented PAH development in MCT-induced rats PAH model. CONCLUSIONS: HMGB1 promotes PASMCs proliferation/migration and pulmonary vascular remodelling by activating ERK1/2/Drp1/Autophagy/BMPR2/Id1 axis, suggesting that this cascade might be a potential novel target for management of PAH.

ERK1/2 kinases and dopamine D2 receptors participate in the anticonvulsant effects of a new derivative of benzoylpyridine oxime and valproic acid.

Inhibition of the activity of extracellular signal-regulated kinases (ERK1/2) induced by the activation of the dopamine D2 receptor signalling cascade may be a promising pharmacological target. The aim of this work was to study the involvement of ERK1/2 and dopamine D2 receptor in the mechanism of the anticonvulsant action of valproic acid (VA) and a new benzoylpyridine oxime derivative (GIZH-298), which showed antiepileptic activity in different models of epilepsy. We showed that subchronic exposure to maximal electroshock seizures (MES) for 5 days reduced the density of dopamine D2 receptors in the striatum of mice. GIZH-298 counteracted the decrease in the number of dopamine D2 receptors associated with MES and increased the number of ligand binding sites of dopamine D2 receptors in mice without MES. The affinity of dopamine D2 receptors to the ligand was not changed by GIZH-298. MES caused an increase in ERK1/2 and synapsin I phosphorylation in the striatum while GIZH-298, similar to VA, reduced the levels of both phospho-ERK1/2 and phosphosynapsin I after MES, which correlated with the decrease in the intensity of seizure in mice. In addition, GIZH-298 suppressed ERK1/2 phosphorylation in SH-SY5Y human neuroblastoma cells at therapeutic concentrations, while VA inhibited ERK1/2 phosphorylation in vivo but not in vitro. The data obtained expand the understanding of the mechanisms of action of VA and GIZH-298, which involve regulating the activity of ERK1/2 kinases, probably by modulating dopamine D2 receptors in limbic structures, as well as (in the case of GIZH-298) directly inhibiting of the ERK1/2 cascade.

CLDN3 expression and function in pregnancy-induced hypertension.

This aim of the present study was to investigate the expression and function of claudin 3 (CLDN3) in pregnancy-induced hypertension. The mRNA expression levels of CLDN3 in the placental tissue and peripheral blood of patients with pregnancy-induced hypertension were measured using reverse transcription-quantitative PCR. Human trophoblast HTR8/SVneo cells overexpressing CLDN3 were generated using a lentiviral vector. Cell Counting kit-8 (CCK-8) assay, flow cytometry, Transwell chamber assays, confocal laser scanning microscopy and western blot analysis were performed to detect cell proliferation, invasion, migration and apoptosis, in addition to matrix metalloproteinase (MMP) expression and ERK1/2 phosphorylation. The mRNA expression levels of CLDN3 were significantly reduced in the placental tissues and peripheral blood samples of patients with pregnancy-induced hypertension compared with healthy pregnant controls. CLDN3 overexpression significantly increased HTR8/SVneo cell proliferation, invasion and migration whilst reducing apoptosis. HTR8/SVneo cells overexpressing CLDN3 also exhibited increased myofiber levels, increased MMP-2 and MMP-9 expression and increased ERK1/2 signaling activity. CLDN3 downregulation may be associated with the pathogenesis of pregnancy-induced hypertension. In conclusion, CLDN3 promotes the proliferative and invasive capabilities of human trophoblast cells, with the underlying mechanisms possibly involving upregulation of MMP expression via the ERK1/2 signaling pathway.

Diabetes promotes invasive pancreatic cancer by increasing systemic and tumour carbonyl stress in KrasG12D/+ mice.

BACKGROUND: Type 1 and 2 diabetes confer an increased risk of pancreatic cancer (PaC) of similar magnitude, suggesting a common mechanism. The recent finding that PaC incidence increases linearly with increasing fasting glucose levels supports a central role for hyperglycaemia, which is known to cause carbonyl stress and advanced glycation end-product (AGE) accumulation through increased glycolytic activity and non-enzymatic reactions. This study investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanisms involved. METHODS: Pdx1-Cre;LSL-KrasG12D/+ mice were interbred with mitosis luciferase reporter mice, rendered diabetic with streptozotocin and treated or not with carnosinol (FL-926-16), a selective scavenger of reactive carbonyl species (RCS) and, as such, an inhibitor of AGE formation. Mice were monitored for tumour development by in vivo bioluminescence imaging. At the end of the study, pancreatic tissue was collected for histology/immunohistochemistry and molecular analyses. Mechanistic studies were performed in pancreatic ductal adenocarcinoma cell lines challenged with high glucose, glycolysis- and glycoxidation-derived RCS, their protein adducts AGEs and sera from diabetic patients. RESULTS: Cumulative incidence of invasive PaC at 22 weeks of age was 75% in untreated diabetic vs 25% in FL-926-16-gtreated diabetic and 8.3% in non-diabetic mice. FL-926-16 treatment suppressed systemic and pancreatic carbonyl stress, extracellular signal-regulated kinases (ERK) 1/2 activation, and nuclear translocation of Yes-associated protein (YAP) in pancreas. In vitro, RCS scavenging and AGE elimination completely inhibited cell proliferation stimulated by high glucose, and YAP proved essential in mediating the effects of both glucose-derived RCS and their protein adducts AGEs. However, RCS and AGEs induced YAP activity through distinct pathways, causing reduction of Large Tumour Suppressor Kinase 1 and activation of the Epidermal Growth Factor Receptor/ERK signalling pathway, respectively. CONCLUSIONS: An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINs progression to invasive PaC, showing that hyperglycaemia promotes PaC mainly through increased carbonyl stress. In vitro experiments demonstrated that both circulating RCS/AGEs and tumour cell-derived carbonyl stress generated by excess glucose metabolism induce proliferation by YAP activation, hence providing a molecular mechanism underlying the link between diabetes and PaC (and cancer in general).

Pharmacological stimulation of sigma-1 receptor promotes activation of astrocyte via ERK1/2 and GSK3ß signaling pathway.

Astrocyte is considered to be a type of passive supportive cells that preserves neuronal activity and survival. The dysfunction of astrocytes is involved in the pathological processes of major depression. Recent studies implicate sigma-1 receptors as putative therapeutic targets for current available antidepressant drugs. However, it is absent of direct evidences whether sigma-1 receptor could promote activation of astrocyte. In the present study, we took advantage of primary astrocyte culture and a highly selective agonist of sigma-1 receptor, (+)SKF-10047 to determine the effect of sigma-1 receptor on Brdu (bromodeoxyuridine) labeling positive cells, migration as well as GFAP (glial fibrillary acidic protein) expression of astrocyte. The results showed that (+)SKF-10047 notably increased the number of Brdu labeling positive cells, migration, and the expression of GFAP in primary astrocytes, which were blocked by antagonist of sigma-1 receptor. Moreover, we also found that (+)SKF-10047 increased the phosphorylation of ERK1/2 (extracellular signal-regulated kinases 1/2) and GSK3ß (glycogen synthase kinase 3ß) (ser 9) in the primary astrocytes. In addition, pharmacological inhibition of ERK1/2 and GSK3ß (ser 9) abolished sigma-1 receptor-promoted activation of astrocyte. Therefore, sigma-1 receptor could be considerate as a new pattern for modulating astrocytic function might emerge as therapeutic strategies.

Treadmill exercise with bone marrow stromal cells transplantation facilitates neuroprotective effect through BDNF-ERK1/2 pathway in spinal cord injury rats.

Transplantation of bone marrow stromal cells (BMSCs) has been known as one of the effective therapeutic methods for functional recovery of spinal cord injury (SCI). Treadmill exercise also facilitates the functional recovery of SCI. Previously, we reported that combination of BMSCs transplantation with treadmill exercise potentiated the locomotor function in SCI rats. In the present study, we investigated whether recovery effect of BMSCs transplantation or treadmill exercise appears through the brain-derived neurotrophic factor (BDNF)-extracellular signal-regulated kinases 1/2 (ERK1/2) pathway. The spinal cord contusion injury was performed at the T9-T10 level using the impactor. Cultured BMSCs were transplanted directly into the lesion 1 week after SCI. Treadmill exercise was performed 6 days per a week for 6 weeks. Western blot for Bax, Bcl-2, BDNF, tyrosine kinase B (TrkB), and phosphorylated ERK1/2 (p-ERK1/2), phosphorylated JNK was performed. In the present results, combination of BMSCs transplantation with tread-mill exercise potently decreased Bax expression, potently increased Bcl-2 expression, and potently enhanced BDNF and TrkB expressions in the injured spinal cord. Combination of BMSCs transplantation with treadmill exercise further facilitated p-ERK1/2 and p-c-Jun expression levels. The present findings demonstrated the synergistic effect of treadmill exercise on neuroregenerative effect of BMSCs transplantation appeared through the activation of BDNF-ERK1/2 pathway in SCI.

Hyperoxia Disrupts Extracellular Signal-Regulated Kinases 1/2-Induced Angiogenesis in the Developing Lungs.

Hyperoxia contributes to the pathogenesis of bronchopulmonary dysplasia (BPD), a chronic lung disease of infants that is characterized by interrupted alveologenesis. Disrupted angiogenesis inhibits alveologenesis, but the mechanisms of disrupted angiogenesis in the developing lungs are poorly understood. In pre-clinical BPD models, hyperoxia increases the expression of extracellular signal-regulated kinases (ERK) 1/2; however, its effects on the lung endothelial ERK1/2 signaling are unclear. Further, whether ERK1/2 activation promotes lung angiogenesis in infants is unknown. Hence, we tested the following hypotheses: (1) hyperoxia exposure will increase lung endothelial ERK1/2 signaling in neonatal C57BL/6J (WT) mice and in fetal human pulmonary artery endothelial cells (HPAECs); (2) ERK1/2 inhibition will disrupt angiogenesis in vitro by repressing cell cycle progression. In mice, hyperoxia exposure transiently increased lung endothelial ERK1/2 activation at one week of life, before inhibiting it at two weeks of life. Interestingly, hyperoxia-mediated decrease in ERK1/2 activation in mice was associated with decreased angiogenesis and increased endothelial cell apoptosis. Hyperoxia also transiently activated ERK1/2 in HPAECs. ERK1/2 inhibition disrupted angiogenesis in vitro, and these effects were associated with altered levels of proteins that modulate cell cycle progression. Collectively, these findings support our hypotheses, emphasizing that the ERK1/2 pathway is a potential therapeutic target for BPD infants with decreased lung vascularization.

Upregulation of seladin-1 and nestin expression in bone marrow mesenchymal stem cell transplantation via the ERK1/2 and PI3K/Akt signaling pathways in an Alzheimer's disease model.

The aim of the present study was to determine the roles of bone marrow mesenchymal stem cell (BM-MSC) transplantation in a model of Alzheimer's disease (AD) and determine the underlying mechanism. The expression of selective Alzheimer's disease indicator-1 (Seladin-1) and nestin was detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. The phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK)1/2 inhibitors, LY294002 and PD98059, were employed to evaluate the molecular mechanism. The results indicated that the mRNA and protein expression of Seladin-1 and nestin was lower in the AD group when compared with the control group. BM-MSC transplantation reversed this decrease in expression, potentially by increasing the protein expression of phosphorylated (p)-protein kinase B (Akt) and p-ERK1/2. In addition, LY294002 (the PI3K inhibitor) and/or PD98059 (the ERK1/2 inhibitor) blocked the enhancement of BM-MSC transplantation on the expression of Seladin-1 and nestin in the hippocampus. These results indicated that BM-MSC transplantation enhanced Seladin-1 and nestin expression potentially via a mechanism associated with the activation of the PI3K/Akt and ERK1/2 signaling pathways. The present study offers preliminary evidence that treatment with BM-MSCs may represent a potential therapeutic approach against brain lesions in AD.

Involvement of CGRP-RCP in the caveolin-1/ERK1/2 signal pathway in the static pressure-induced proliferation of vascular smooth muscle cells.

Previous study suggested that the receptor component protein (RCP), one of the components of calcitonin gene-related peptide (CGRP) receptor, plays a multiple role in the cellular signal transduction. The study was designed to investigate whether or not the RCP involved in the regulation of caveolin-1/extracellular signal-regulated kinases-1 and -2 (ERK1/2) signal pathway in the vascular smooth muscle cells (VSMCs) proliferation induced by static pressure. Mouse-derived VSMCs line A10 (A10 VSMCs) was served as project in this experiment. Results showed that the A10 VSMCs viability and proliferating cell nuclear antigen (PCNA) expression which were increased by static pressure were inhibited by pretreatment of CGRP. In like manner, the expressions of the decreased-caveolin-1 and the increased-phosphorylated ERK1/2 (p-ERK1/2) induced by static pressure were significantly reversed by pretreatment of CGRP, respectively. Meanwhile, the expression of RCP was up-regulated by the static pressure. Silence of RCP gene with the small interrupt RNA (siRNA) not only significantly increased A10 VSMC proliferation but also increased the expression of p-ERK1/2 in response to static pressure. When treatment of A10 VSMCs with 120-mmHg static pressure for different time, however, the protein band of caveolin-1 and RCP was the least at time point of 10 min, but the p-ERK1/2 expression was the most maximum. In conclusion, RCP maybe involved in the static pressure-induced A10 VSMCs proliferation by regulation of caveolin-1/ERK1/2 signal pathway.

Dissecting the signaling features of the multi-protein complex GPCR/ß-arrestin/ERK1/2.

G protein-coupled receptors (GPCRs) have emerged as key biological entities that regulate a plethora of physiological processes and participate in the onset and development of many diseases. Moreover, these receptors are important targets of almost 25% of the current therapeutic drugs in the market. Upon agonist binding, GPCRs activate a great number of signaling pathways, resulting in important cellular events like gene transcription, survival, proliferation and differentiation. In order to activate such events, GPCRs interact with a variety of scaffold and molecular entities, particularly with G proteins, but also with ß-arrestins and the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, forming unique signaling modules. The aim of this review is to analyze the signaling features of the multi-protein complex GPCR-ß-arrestin-ERK1/2, a unique signaling module that has received considerable attention from different research groups due to its molecular and physiological roles in diverse cellular contexts.

TGFBR1*6A is a potential modifier of migration and invasion in colorectal cancer cells.

Type 1 transforming growth factor ß receptor (TGFBR1)*6A, a common hypomorphic variant of TGFBR1, may act as a susceptibility allele in colorectal cancer. However, the contribution of TGFBR1*6A to colorectal cancer development is largely unknown. To test the hypothesis that TGFBR1*6A promotes colorectal cancer invasion and metastasis via Smad-independent transforming growth factor-ß (TGF-ß) signaling, the effect of TGFBR1*6A on the invasion of colorectal cancer cells was assessed. pCMV5-TGFBR1*6A-HA plasmids were transfected into SW48 and DLD-1 cells by Lipofectamine-mediated DNA transfection. The effect of TGF-ß1 on the proliferation of SW48 and DLD-1 cells transfected with TGFBR1*6A was determined by MTT assay. The effects of the TGF-ß1 on the invasion of the transfected SW48 and DLD-1 cells were determined using Matrigel-coated plates. Transforming migrating chambers were used to determine the effects of TGF-ß1 on the migration of the transfected SW48 and DLD-1 cells. Western blot analysis was used to determine the expression of phosphorylated (p-) extracellular-signal-regulated kinase (ERK), p-P38 and p-SMAD family member 2 in SW48 cells. Using transfected TGFBR1*6A SW48 and DLD-1 cell lines our group demonstrated that, in comparison with TGFBR1*9A, TGFBR1*6A is capable of switching TGF-ß1 growth-inhibitory signals into growth-stimulatory signals which significantly increased the invasion of SW48 and DLD-1 cells. Functional assays indicated that TGFBR1*6A weakened Smad-signaling but increased ERK and p38 signaling, which are crucial mediators of cell migration and invasion. From this, it was possible to conclude that TGFBR1*6A enhanced SW48 cell migration and invasion through the mitogen-activated protein kinase pathway and that it may contribute to colorectal cancer progression in a TGF-ß1/Smad signaling-independent manner. This suggests that TGFBR1*6A may possess oncogenic properties and that it may affect the migration and invasion of colorectal cancer cells.

Effects of ß-blockers on house dust mite-driven murine models pre- and post-development of an asthma phenotype.

BACKGROUND: Our previous studies suggested certain ß-adrenoceptor blockers (ß-blockers) attenuate the asthma phenotype in ovalbumin driven murine models of asthma. However, the ovalbumin model has been criticized for lack of clinical relevance. METHODS: We tested the non-selective ß-blockers, carvedilol and nadolol, in house dust mite (HDM) driven murine asthma models where drugs were administered both pre- and post-development of the asthma phenotype. We measured inflammation, mucous metaplasia, and airway hyper-responsiveness (AHR). We also measured the effects of the ß-blockers on extracellular-signal regulated kinase (ERK 1/2) phosphorylation in lung homogenates. RESULTS: We show that nadolol, but not carvedilol, attenuated inflammation and mucous metaplasia, and had a moderate effect attenuating AHR. Following HDM exposure, ERK1/2 phosphorylation was elevated, but the level of phosphorylation was unaffected by ß-blockers, suggesting ERK1/2 phosphorylation becomes dissociated from the asthma phenotype. CONCLUSION: Our findings in HDM models administering drugs both pre- and post-development of the asthma phenotype are consistent with previous results using ovalbumin models and show differential effects for nadolol and carvedilol on the asthma phenotype. Lastly, our data suggest that ERK1/2 phosphorylation may be involved in development of the asthma phenotype, but may have a limited role in maintaining the phenotype.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity.

The Na⁺/H⁺ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H⁺ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-ß (PKC-ß) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 µM glutamate or 20 mM NH4Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-ß, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for 〉3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH4Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-ß, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-ß/ERK1/2/p90RSK pathway in neuronal cells.