Extracellular Signal-regulated Protein Kinase Activation Research Articles

LED Light-Induced ROS Differentially Regulates Focal Adhesion Kinase Activity in HaCaT Cell Viability.

In this study, changes in cell signaling mechanisms in skin cells induced by various wavelengths and intensities of light-emitting diodes (LED) were investigated, focusing on the activity of focal adhesion kinase (FAK) in particular. We examined the effect of LED irradiation on cell survival, the generation of intracellular reactive oxygen species (ROS), and the activity of various cell-signaling proteins. Red LED light increased cell viability at all intensities, whereas strong green and blue LED light reduced cell viability, and this effect was reversed by NAC or DPI treatment. Red LED light caused an increase in ROS formation according to the increase in the intensity of the LED light, and green and blue LED lights led to sharp increases in ROS formation. In the initial reaction to LEDs, red LED light only increased the phosphorylation of FAK and extracellular-signal regulated protein kinase (ERK), whereas green and blue LED lights increased the phosphorylation of inhibitory-κB Kinase α (IKKα), c-jun N-terminal kinase (JNK), and p38. The phosphorylation of these intracellular proteins was reduced via FAK inhibitor, NAC, and DPI treatments. Even after 24 h of LED irradiation, the activity of FAK and ERK appeared in cells treated with red LED light but did not appear in cells treated with green and blue LED lights. Furthermore, the activity of caspase-3 was confirmed along with cell detachment. Therefore, our results suggest that red LED light induced mitogenic effects via low levels of ROS–FAK–ERK, while green and blue LED lights induced cytotoxic effects via cellular stress and apoptosis signaling resulting from high levels of ROS.

Effects of Acute 2,3,7,8-Tetrachlorodibenzo-p-Dioxin Exposure on the Circulating and Cecal Metabolome Profile.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a polyhalogenated planar hydrocarbon belonging to a group of highly toxic and persistent environmental contaminants known as “dioxins”. TCDD is an animal teratogen and carcinogen that is well characterized for causing immunosuppression through activation of aryl hydrocarbon receptor (AHR). In this study, we investigated the effect of exposure of mice to an acute dose of TCDD on the metabolic profile within the serum and cecal contents to better define the effects of TCDD on host physiology. Our findings demonstrated that within the circulating metabolome following acute TCDD exposure, there was significant dysregulation in the metabolism of bioactive lipids, amino acids, and carbohydrates when compared with the vehicle (VEH)-treated mice. These widespread changes in metabolite abundance were identified to regulate host immunity via modulating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated protein kinase (ERK1/2) activity and work as biomarkers for a variety of organ injuries and dysfunctions that follow TCDD exposure. Within the cecal content of mice exposed to TCDD, we were able to detect changes in inflammatory markers that regulate NF-κB, markers of injury-related inflammation, and changes in lysine degradation, nicotinamide metabolism, and butanoate metabolism, which collectively suggested an immediate suppression of broad-scale metabolic processes in the gastrointestinal tract. Collectively, these results demonstrate that acute TCDD exposure results in immediate irregularities in the circulating and intestinal metabolome, which likely contribute to TCDD toxicity and can be used as biomarkers for the early detection of individual exposure.

Transcranial Laser Photobiomodulation Improves Intracellular Signaling Linked to Cell Survival, Memory and Glucose Metabolism in the Aged Brain: A Preliminary Study.

Aging is often accompanied by exacerbated activation of cell death-related signaling pathways and decreased energy metabolism. We hypothesized that transcranial near-infrared laser may increase intracellular signaling pathways beneficial to aging brains, such as those that regulate brain cell proliferation, apoptosis, and energy metabolism. To test this hypothesis, we investigated the expression and activation of intracellular signaling proteins in the cerebral cortex and hippocampus of aged rats (20 months old) treated with the transcranial near-infrared laser for 58 consecutive days. As compared to sham controls, transcranial laser treatment increased intracellular signaling proteins related to cell proliferation and cell survival, such as signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p70 ribosomal protein S6 kinase (p70S6K) and protein kinase B (PKB), also known as Akt that is linked to glucose metabolism. In addition, ERK is linked to memory, while ERK and JNK signaling pathways regulate glucose metabolism. Specifically, the laser treatment caused the activation of STAT3, ERK, and JNK signaling proteins in the cerebral cortex. In the hippocampus, the laser treatment increased the expression of p70S6K and STAT3 and the activation of Akt. Taken together, the data support the hypothesis that transcranial laser photobiomodulation improves intracellular signaling pathways linked to cell survival, memory, and glucose metabolism in the brain of aged rats.

Metabolic flexibility maintains proliferation and migration of FGFR signaling–deficient lymphatic endothelial cells

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid β-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal–regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator–activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.

Analgesic Effect of Combined Therapy with the Japanese Herbal Medicine "Yokukansan" and Electroacupuncture in Rats with Acute Inflammatory Pain.

Background: Japanese herbal medicine, called Kampo medicine, and acupuncture are mainly used in Japanese traditional medicine. In this experiment, the analgesic effect of Yokukansan (YKS) alone and a combination of YKS and electroacupuncture (EA) on inflammatory pain induced by formalin injection were examined. Methods: Animals were divided into four groups: a control group, formalin injection group (formalin), YKS-treated formalin group (YKS), and YKS- and EA-treated formalin group (YKS + EA). The duration of pain-related behaviors and extracellular signal-regulated protein kinase (ERK) activation in the spinal cord after formalin injection in the right hind paw were determined. Results: The duration of pain-related behaviors was dramatically prolonged in the late phase (10–60 min) in the formalin group. The YKS treatment tended to reduce (p = 0.08), whereas YKS + EA significantly suppressed the pain-related behaviors (p < 0.01). Immunohistochemical and Western blot analyses revealed that the number of phosphorylated ERK1/2 (pERK1/2)-positive cells and the pERK expression level, which were increased by formalin injection, were significantly inhibited by YKS (p < 0.05) and YKS + EA (p < 0.01). Conclusions: The YKS + EA combination therapy elicited an analgesic effect on formalin-induced acute inflammatory pain.

Nitric oxide induces HepG2 cell death via extracellular signal-regulated protein kinase activation by regulating acid sphingomyelinase.

Nitric oxide (NO) plays a vital role in the occurrence and development of tumours. Acid sphingomyelinase (ASM) participates in cell apoptosis, cell proliferation, metabolism and other biological processes. However, whether ASM has an effect on NO-treated HepG2 cells remains unknown, and the role of the extracellular signal-regulated protein kinase (ERK) pathway is also unclear. In the present study, the effects of NO on cell viability and apoptosis were assayed, followed by investigating the mRNA and protein levels of ASM and ERK phosphorylation in NO-treated HepG2 cells. The results showed that diethylenetriamine/NO (DETA-NO), an NO donor, promoted HepG2 cell death and apoptosis in a concentration-dependent manner and that the mRNA and protein expression levels of ASM were significantly decreased in DETA-NO-treated HepG2 cells. Moreover, ERK phosphorylation was significantly increased in DETA-NO-treated HepG2 cells. The inhibition of ERK phosphorylation increased DETA-NO-induced cell apoptosis. In summary, DETA-NO can promote HepG2 cell death in a concentration-dependent manner by activating ERK and NO might activate ERK by regulating ASM and then inducing HepG2 cell death.

Krill Oil Perturbs Proliferation and Migration of Mouse Colon Cancer Cells in vitro by Impeding Extracellular Signal-Regulated Protein Kinase Signaling Pathway.

The prevalence of colorectal cancer (CRC) continues to increase. Treatment of CRC remains a significant clinical challenge, and effective therapies for advanced CRC are desperately needed. Increasing attention and ongoing research efforts have focused on krill oil that may provide health benefits to the human body. Here we report that krill oil exerts in vitro anticancer activity through a direct inhibition on proliferation, colony formation, migration, and invasion of mouse colon cancer cells. Krill oil inhibited the proliferation and colony formation of CT-26 colon cancer cells by causing G0/G1 cell cycle arrest and apoptosis. Cell cycle arrest was attributable to reduction of cyclin D1 levels in krill oil-treated cells. Further studies revealed that krill oil induced mitochondrial-dependent apoptosis of CT-26 cells, including loss of mitochondrial membrane potential, increased cytosolic calcium levels, activation of caspase-3, and downregulation of anti-apoptotic proteins MCL-1 and BCL-XL. Krill oil suppressed migration of CT-26 cells by disrupting the microfilaments and microtubules. Extracellular signal-regulated protein kinase (ERK) plays crucial roles in regulating proliferation and migration of cancer cells. We found that krill oil attenuated the activation of ERK signaling pathway to exert the effects on cell cycle, apoptosis, and migration of colon cancer cells. We speculate that polyunsaturated fatty acids of krill oil may dampen ERK activation by decreasing the phospholipid saturation of cell membrane. Although findings from in vitro studies may not necessarily translate in vivo, our study provides insights into the possibility that krill oil or its components could have therapeutic potential in colon cancer.

Organophosphorus Compounds and MAPK Signaling Pathways.

The molecular signaling pathways that lead to cell survival/death after exposure to organophosphate compounds (OPCs) are not yet fully understood. Mitogen-activated protein kinases (MAPKs) including the extracellular signal-regulated protein kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and the p38-MAPK play the leading roles in the transmission of extracellular signals into the cell nucleus, leading to cell differentiation, cell growth, and apoptosis. Moreover, exposure to OPCs induces ERK, JNK, and p38-MAPK activation, which leads to oxidative stress and apoptosis in various tissues. However, the activation of MAPK signaling pathways may differ depending on the type of OPCs and the type of cell exposed. Finally, different cell responses can be induced by different types of MAPK signaling pathways after exposure to OPCs.

Preventive Effects of Gardenia jasminoides on Cerulein-Induced Chronic Pancreatitis.

Our previous report revealed that Gardenia jasminoides (GJ) has protective effects against acute pancreatitis. So, we examined whether aqueous extract of GJ has anti-inflammation and antifibrotic effects even against cerulein-induced chronic pancreatitis (CP). CP was induced in mice by an intraperitoneal injection of a stable cholecystokinin (CCK) analogue, cerulein, six times a day, four days per week for three weeks. GJ extract (0.1 or 1[Formula: see text]g/kg) or saline (control group) were intraperitoneally injected 1[Formula: see text]h before first cerulein injection. After three weeks of stimulation, the pancreas was harvested for the examination of several fibrotic parameters. In addition, pancreatic stellate cells (PSCs) were isolated using gradient methods to examine the antifibrogenic effects of GJ. In the cerulein-induced CP mice, the histological features of the pancreas showed severe tissue damage such as enlarged interstitial spaces, inflammatory cell infiltrate and glandular atrophy, and tissue fibrosis. However, treatment of GJ reduced the severity of CP such as pancreatic edema and inflammatory cell infiltration. Furthermore, treatment of GJ increased pancreatic acinar cell survival, and reduced pancreatic fibrosis and activation of PSC in vivo and in vitro. In addition, GJ treatment inhibited the activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) in the PSCs. These results suggest that GJ attenuated the severity of CP and the pancreatic fibrosis by inhibiting JNK and ERK activation during CP.

Lysophosphatidylcholine Activates Extracellular-Signal-Regulated Protein Kinase and Potentiates Vascular Contractile Responses in Rat Aorta

We previously reported that in the endothelium-denuded rat aorta, lysophosphatidylcholine (LPC) potentiates the contractile responses induced by high-K+, UK14,304 (a selective a2-adrenoceptor agonist), and phorbol ester with an associated tyrosine-phosphorylation of proteins. To further investigate this phenomenon, we examined the effects of extracellular-signal-regulated protein kinase (ERK)-kinase (MEK) inhibitors on the LPC-induced potentiation of the contractile responses to high-K+ and UK14,304 in this tissue. Although PD98059 (3 × 10-5 M) did not affect the high-K+-induced contractile response itself, it selectively inhibited the potentiating effect of LPC on the contraction and strongly inhibited the LPC-induced augmentation of the associated increase in [Ca2+]i. PD98059 also attenuated the LPC-induced augmentations of the increases in [Ca2+]i and contractile tension induced by UK14,304. U0126 (5 × 10-5 M), another MEK inhibitor, also attenuated the potentiating effect of LPC on high-K+-induced contractions. Western blot analysis revealed that LPC produced an increase in ERK-phosphorylation, and that this was inhibited by PD98059. Nicardipine inhibited the contractile response to 15 mM K+ in the LPC-treated aorta, but not the increase in ERK-phosphorylation induced by LPC. These results suggest that the LPC-induced augmentation of contractile responses in the rat aorta is due to activation of ERK, which in turn regulates Ca2+ influx.

Investigating the regulatory role of ORMDL3 in airway barrier dysfunction using in vivo and in vitro models.

The airway epithelium (AE) is the main protective barrier between the host and external environmental factors causing asthma. Allergens or pathogens induce AE dysfunction, including epithelial permeability alteration, trans-epithelial electrical resistance (TEER) reduction, upregulation of inflammatory mediators and downregulation of junctional complex molecules. Orosomucoid-like protein isoform 3 (ORMDL3), a gene closely associated with childhood onset asthma, is involved in airway inflammation and remodeling. It was hypothesized that ORMDL3 plays an important role in regulating AE barrier function. In vivo [chronic asthma induced by ovalbumin-respiratory syncytial virus (OVA-RSV)] in mice) and in vitro (human bronchial epithelial cells and 16HBE cells) models were used to assess ORMDL3′s role in AE function regulation, evaluating paracellular permeability, TEER and the expression levels of junctional complex molecules. The effects of ORMDL3 on the extracellular signal-regulated protein kinase (ERK) pathway were determined. In mice with OVA-RSV induced chronic asthma, ORMDL3 and sphingosine kinase 1 (SPHK1) were upregulated whereas the junction related proteins Claudin-18 and E-cadherin were downregulated. Overexpression of ORMDL3 resulted in decreased TEER, downregulation of junctional complex molecules and induced epithelial permeability. In contrast, ORMDL3 inhibition showed the opposite effects. In 16HBE cells, ORMDL3 overexpression induced SPHK1 distribution and activity, while SPHK1 inhibition resulted in increased TEER upon administration of an ORMDL3 agonist or ORMDL3 overexpression. In addition, ERK activation occurred downstream of SPHK1 activation in 16HBE cells. High levels of ORMDL3 result in damaged AE barrier function by inducing the SPHK1/ERK pathway.

Baicalein inhibits fibronectin-induced epithelial\u2013mesenchymal transition by decreasing activation and upregulation of calpain-2

The extracellular matrix protein fibronectin (FN) facilitates tumorigenesis and the development of breast cancer. Inhibition of the FN-induced cellular response is a potential strategy for breast cancer treatment. In the present study, we investigated the effects of the flavonoid baicalein on FN-induced epithelial–mesenchymal transition (EMT) in MCF-10A breast epithelial cells and in a transgenic mouse MMTV-polyoma middle T antigen breast cancer model (MMTV-PyMT). Baicalein inhibited FN-induced migration, invasion, and F-actin remodeling. Baicalein also suppressed FN-induced downregulation of the epithelial markers E-cadherin and ZO-1 and upregulation of the mesenchymal markers N-cadherin, vimentin, and Snail. Further investigation revealed that calpain-2 was involved in baicalein suppression of FN-induced EMT. Baicalein significantly decreased FN-enhanced calpain-2 expression and activation by suppressing its plasma membrane localization, substrate cleavage, and degradation of its endogenous inhibitor calpastatin. Overexpression of calpain-2 in MCF-10A cells by gene transfection partially blocked the inhibitory effect of baicalein on FN-induced EMT changes. In addition, baicalein inhibited calpain-2 by decreasing FN-increased intracellular calcium ion levels and extracellular signal-regulated protein kinases activation. Baicalein significantly decreased tumor onset, growth, and pulmonary metastasis in a spontaneous breast cancer MMTV-PyMT mouse model. Baicalein also reduced the expression of FN, calpain-2, and vimentin, but increased E-cadherin expression in MMTV-PyMT mouse tumors. Overall, these results revealed that baicalein markedly inhibited FN-induced EMT by inhibiting calpain-2, thus providing novel insights into the pharmacological action and mechanism of baicalein. Baicalein may therefore possess therapeutic potential for the treatment of breast cancer though interfering with extracellular matrix–cancer cell interactions.

Tanshinone IIA Ameliorates Spatial Learning and Memory Deficits by Inhibiting the Activity of ERK and GSK-3β.

Alzheimer disease (AD) is the most common type of dementia which is becoming a primary problem in the present society, but it lacks effective treatment methods and means of AD. Tanshinone IIA (Tan IIA) has been reported to have neuroprotective effects to restrain the Aβ25-35-mediated apoptosis. However, few studies try to understand how Aβ1-42 affects hyperphosphorylation of tau and how Tan IIA regulates this process at the molecular level. Fifty male Sprague-Dawley rats were randomly divided into 5 groups and infused through the lateral ventricle with Aβ1-42 except the control group. Then the rats were treated with Tan IIA through intragastric administration for 4 weeks. After the ability of learning and memory being measured, histomorphological examination and Western blot were used to detect the possible mechanism in the AD-associated model rats. We observed that Aβ1-42 infusion could induce spatial learning and memory deficits in rats. Simultaneously, Aβ1-42 also could reduce the neuron in cornu ammonis 1 and dentate gyrus of hippocampus, as well as increase the levels of cleaved caspase 3, hyperphosphorylated tau at the sites Ser396, Ser404, and Thr205 with enhancing staining of black granules in brain. We also found that Aβ1-42 could increase the activity of extracellular signal-regulated protein kinase (ERK) and glycogen synthase kinase-3β (GSK-3β). Meanwhile, these phenomena could be ameliorated when Tan IIA was used. We concluded that Tan IIA might have neuroprotective effect and improving learning and memory ability to be a viable candidate in AD therapy with mechanisms involving the ERK and GSK-3β signal pathway.

Nickel-induced VEGF expression via regulation of Akt, ERK1/2, NFκB, and AMPK pathways in H460 cells.

Prospective cohort studies have indicated that a highly nickel-polluted environment may severely affect human health, resulting in such conditions as respiratory tract cancers. Such exposure can trigger vascular endothelial growth factor (VEGF) expression. However, the signal transduction pathways leading to VEGF induction by nickel compounds are not well understood. This study revealed the occurrence of VEGF induction in human non-small-cell lung cancer H460 cells exposed to NiCl2 . Moreover, exposing H460 cells to NiCl2 activated extracellular signal-regulated protein kinase (ERK), nuclear factor kappa B (NFκB), and protein kinase B (Akt) as well as downregulated AMP activated protein kinase (AMPK) expression. The mitogen-activated protein kinase (MAPK) and ERK inhibitor significantly blocked NiCl2 -induced ERK activation and VEGF production. Pretreating H460 cells with a PI3K/Akt inhibitor substantially inhibited NiCl2 -induced VEGF expression and reduced Akt, ERK, and NFκB phosphorylation. Furthermore, 5-aminoimidazole-4-carboxamide ribonucleoside-induced AMPK activation improved VEGF expression in NiCl2 -treated H460 cells significantly. These results indicate that NiCl2 induces VEGF production through Akt, ERK, NFκB activation and AMPK suppression and mediates various types of pathophysiological angiogenesis.

Possible involvement of the μ opioid receptor in the antinociception induced by sinomenine on formalin-induced nociceptive behavior in mice

Sinomenine, an alkaloid originally isolated from the roots and the rhizome of Sinomenium acutum is used as a traditional Chinese herbal medicines for rheumatoid arthritis and neuralgia. The aims of this study were to investigate the effects of oral administration of shinomenine on formalin-induced nociceptive behavior in mice and the opioid receptor subtypes involved in the antinociceptive effects of sinomenine. Our findings showed that a single dose of oral-administrated sinomenine inhibited the formalin induced licking and biting responses in a dose-dependent manner. Intraperitoneal pretreatment with naloxone hydrochloride, an opioid receptor antagonist, and β-funaltrexamine hydrochloride (β-FNA), a selective μ-opioid receptor antagonist, significantly attenuated sinomenine induced antinociception, but not by naltrindole, a nonselective δ-opioid receptor antagonist and nor-binaltorphimine, a selective κ-opioid receptor antagonist. Furthermore, in western blot analysis, oral administration of sinomenine resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK1/2) activation induced by formalin. Naloxone hydrochloride and β-FNA significantly reversed the blockage of spinal ERK1/2 activation induced by sinomenine. These results suggest that sinomenine-induced anti nociceptive effect and blockage of spinal ERK1/2 activation may be triggered by activation of μ-opioid receptors.

Glycodelin-A stimulates the conversion of human peripheral blood CD16-CD56bright NK cell to a decidual NK cell-like phenotype.

Does glycodelin-A (GdA) induce conversion of human peripheral blood CD16-CD56bright natural killer (NK) cells to decidual NK (dNK) cells to facilitate placentation? GdA binds to blood CD16-CD56bright NK cells via its sialylated glycans and converts them to a dNK-like cells, which in turn regulate endothelial cell angiogenesis and trophoblast invasion via vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein 1 (IGFBP-1) secretion, respectively. dNK cells are the most abundant leucocyte population in the decidua. These cells express CD16-CD56bright phenotype. Peripheral blood CD16-CD56bright NK cells and hematopoietic precursors have been suggested to be capable of differentiating towards dNK cells upon exposure to the decidual microenvironment. These cells regulate trophoblast invasion during spiral arteries remodelling and mediate homoeostasis and functions of the endothelial cells. GdA is an abundant glycoprotein in the human decidua with peak expression between the 6th and 12th week of gestation, suggesting a role in early pregnancy. Indeed, GdA interacts with and modulates functions and differentiation of trophoblast and immune cells in the human feto-maternal interface. Aberrant GdA expression during pregnancy is associated with unexplained infertility, pregnancy loss and pre-eclampsia. CD16+CD56dim, CD16-CD56bright and dNK cells were isolated from human peripheral blood and decidua tissue, respectively, by immuno-magnetic beads or fluorescence-activated cell sorting. Human extravillous trophoblasts were isolated from first trimester placental tissue after termination of pregnancy. Biological activities of the cells were studied after treatment with GdA at a physiological dose of 5 μg/mL. GdA was purified from human amniotic fluid by immuno-affinity chromatography. Expression of VEGF, CD9, CD49a, CD151 and CD158a in the cells were determined by flow cytometry. Angiogenic proteins in the spent media of NK cells were determined by cytokine array and ELISA. Blocking antibodies were used to study the functions of the identified angiogenic proteins. Endothelial cell angiogenesis was determined by tube formation and trans-well migration assays. Cell invasion and migration were determined by trans-well invasion/migration assay. Binding of normal and de-sialylated GdA, and expression of L-selectin and siglec-7 on the NK cells were analysed by flow cytometry. The association between GdA and L-selectin on NK cells was confirmed by immunoprecipitation. Extracellular signal-regulated protein kinases (ERK) activation was determined by Western blotting and functional assays. GdA treatment enhanced the expression of dNK cell markers CD9 and CD49a and the production of the functional dNK secretory product VEGF in the peripheral blood CD16-CD56bright NK cells. The spent media of GdA-treated CD16-CD56bright NK cells promoted tube formation of human umbilical vein endothelial cells and invasiveness of trophoblasts. These stimulatory effects were mediated by the stimulatory activities of GdA on an ERK-activation dependent production of VEGF and IGFBP-1 by the NK cells. GdA had a stronger binding affinity to the CD16-CD56bright NK cells as compared to the CD16+CD56dim NK cells. This GdA-NK cell interaction was reduced by de-sialylation. GdA interacted with L-selectin, expressed only in the CD16-CD56bright NK cells, but not in the CD16+CD56dim NK cells. Anti-L-selectin functional blocking antibody suppressed the binding and biological activities of GdA on the NK cells. N/A. Some of the above findings are based on a small sample size of peripheral blood CD16-CD56bright NK cells. These results need to be confirmed with human primary dNK cells. This is the first study on the biological role of GdA on conversion of CD16-CD56bright NK cells to dNK-like cells. Further investigation on the glycosylation and functions of GdA will enhance our understanding on human placentation and placenta-associated complications with altered NK cell biology. This work was supported by the Hong Kong Research Grant Council Grant 17122415, Sanming Project of Medicine in Shenzhen, the Finnish Cancer Foundation, Sigrid Jusélius Foundation and the Finnish Society of Clinical Chemistry. The authors have no competing interests to declare.

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca2+ /calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1-7 cells). Recently, we found that GnRH treatment of GT1-7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIβ'e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1-7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.

Activation of c-Jun N-terminal kinase and p38 after cerebral ischemia upregulates cerebral sodium-glucose transporter type 1

Cerebral ischemic stress increases cerebral sodium-glucose transporter type 1 (SGLT-1). However, the mechanism by which cerebral ischemia leads to the up-regulation of SGLT-1 remains unclear. In peripheral tissue, the activation of mitogen-activated protein kinases (MAPKs) increases SGLT-1. MAPK pathways [c-Jun N-terminal kinase (JNK), p38 MAPK, and extracellular signal-regulated protein kinase (ERK)] are activated by cerebral ischemic stress. Therefore, we confirmed the involvement of MAPKs in the up-regulation of cerebral SGLT-1 after cerebral ischemia. Male ddY mice were subjected to middle cerebral artery occlusion (MCAO). Protein expression was assessed by western blotting. Mice received an intracerebroventricular (i.c.v.) injection of SP600125 (JNK inhibitor), SB203580 (p38 inhibitor), and PD98059 (MEK inhibitor) immediately after reperfusion. The infarction and behavioral abnormalities were assessed on days 1 and 3 after MCAO. The MAPK inhibitors suppressed the activation of JNK, p38, and ERK 3 h after MCAO. SP600125 and SB203580 administration ameliorated cerebral ischemic neuronal damage, whereas PD98059 administration exacerbated cerebral ischemic neuronal damage. SP600125 and SB203580 significantly suppressed the increase in SGLT-1 12 h after MCAO. PD98059 had no effect on SGLT-1 expression after MCAO. Our results indicate that the activation of JNK and p38 participate in the up-regulation of cerebral SGLT-1 after MCAO.

Possible involvement of the peripheral Mu-opioid system in antinociception induced by bergamot essential oil to allodynia after peripheral nerve injury

The essential oil of bergamot (BEO) is one of the most common essential oils and is most familiar to the general public. The aims of this study were to investigate the effect of intraplantar (i.pl.) BEO on neuropathic allodynia induced by partial sciatic nerve ligation (PSNL) in mice and the opioid receptor subtypes involved in the antiallodynic effects of BEO. Our findings showed that a single dose of i.pl. administration of BEO significantly inhibited the PSNL-induced neuropathic pain using the von Frey test. The i.pl pretreatment with naloxone methiodide, a peripherally acting μ-opioid receptor preferring antagonist, β-funaltrexamine hydrochloride (β-FNA), a selective μ-opioid receptor antagonist, and β-endorphin antiserum significantly reversed the antiallodynic effect of BEO in the von Frey test, but not by naltrindole, the nonselective δ-opioid receptor antagonist and nor-binaltorphimine, the selective κ-opioid receptor antagonist. Furthermore, in the western blotting analysis, i.pl. administration of BEO resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK) activation induced by PSNL. Naloxone methiodide and β-FNA significantly reversed the blockage of spinal ERK activation induced by BEO. These results suggest that i.pl. injection of BEO-induced antiallodynic effect and blockage of spinal ERK activation may be triggered by activation of peripheral μ-opioid receptors.

ERK inhibition promotes neuroectodermal precursor commitment by blocking self-renewal and primitive streak formation of the epiblast

BackgroundPluripotent stem cells hold great promise for regenerative medicine. However, before clinical application, reproducible protocols for pluripotent stem cell differentiation should be established. Extracellular signal-regulated protein kinase (ERK) signaling plays a central role for the self-renewal of epiblast stem cells (EpiSCs), but its role for subsequent germ layer differentiation is still ambiguous. We proposed that ERK could modulate differentiation of the epiblast.MethodsPD0325901 was used to inhibit ERK activation during the differentiation of embryonic stem cells and EpiSCs. Immunofluorescence, western blot analysis, real-time PCR and flow cytometry were used to detect germ layer markers and pathway activation.ResultsWe demonstrate that the ERK phosphorylation level is lower in neuroectoderm of mouse E7.5 embryos than that in the primitive streak. ERK inhibition results in neural lineage commitment of epiblast. Mechanistically, PD0325901 abrogates the expression of primitive streak markers by β-catenin retention in the cytoplasm, and inhibits the expression of OCT4 and NANOG during EpiSC differentiation. Thus, EpiSCs differentiate into neuroectodermal lineage efficiently under PD0325901 treatment. These results suggest that neuroectoderm differentiation does not require extrinsic signals, supporting the default differentiation of neural lineage.ConclusionsWe report that a single ERK inhibitor, PD0325901, can specify epiblasts and EpiSCs into neural-like cells, providing an efficient strategy for neural differentiation.