Phosphorylation Of Extracellular Signal-regulated Protein Kinase Research Articles

ATP promotes resident CD34+ cell migration mainly through P2Y2-Stim1-ERK/p38 pathway.

Extracellular adenosine triphosphate (ATP) is one of the most abundant biochemical constitutes within the stem cell microenvironment and is postulated to play critical roles in cell migration. However, it is unclear whether ATP regulates the cell migration of CD34+ vascular wall-resident stem/progenitor cells (VW-SCs) and participates in angiogenesis. Therefore, the biological mechanisms of cell migration mediated by ATP was determined by in vivo subcutaneous matrigel plug assay, ex vivo aortic ring assay, in vitro transwell migration assay, and other molecular methods. In the present study, ATP dose-dependently promoted CD34+ VW-SCs migration, which was more obviously attenuated by inhibiting or knocking down P2Y2 than P2Y6. Furthermore, it was confirmed that ATP potently promoted the migration of resident CD34+ cells from cultured aortic artery rings and differentiation into endothelial cells in matrigel plugs by using inducible lineage tracing Cd34-CreERT2; R26-tdTomato mice, whereas P2Y2 and P2Y6 blocker greatly inhibited the effect of ATP. In addition, ATP enhanced the protein expression of stromal interaction molecule 1 (STIM1) on cell membrane, blocking the calcium release-activated calcium (CRAC) channel with shSTIM1 or BTP2 apparently inhibited ATP-evoked intracellular Ca2+ elevation and channel opening, thereby suppressing ATP-driven cell migration. Moreover, extracellular signal-regulated protein kinase (ERK) inhibitor PD98059 and p38 inhibitor SB203580 remarkably inhibited ERK and p38 phosphorylation, cytoskeleton rearrangement, and subsequent cell migration. Unexpectedly, it was found that knocking down STIM1 greatly inhibited ATP-triggered ERK/p38 activation. Taken together, it was suggested that P2Y2 signaled through the CRAC channel mediated Ca2+ influx and ERK/p38 pathway to reorganize the cytoskeleton and promoted the migration of CD34+ VW-SCs.NEW & NOTEWORTHY In this study, we observed that the purinergic receptor P2Y2 is critical in the regulation of vascular wall-resident CD34+ cells' migration. ATP could activate STIM1-mediated extracellular Ca2+ entry by triggering STIM1 translocation to the plasma membrane, and knockdown of STIM1 prevented ERK/p38 activation-mediated cytoskeleton rearrangement and cell migration.

COX-2/PGE2/VEGF signaling promotes ERK-mediated BMSCs osteogenic differentiation under hypoxia by the paracrine action of ECs.

Understanding the crosstalk between endothelial cells (ECs) and bone-marrow mesenchymal stem cells (BMSCs) in response to hypoxic environments and deciphering of the underlying mechanisms are of great relevance for better application of BMSCs in tissue engineering. Here, we demonstrated that hypoxia promoted BMSCs proliferation, colony formation, osteogenic markers expression, mineralization, and extracellular signal-regulated protein kinase (ERK) phosphorylation, and that PD98059 (ERK inhibitor) blocked hypoxia-induced osteogenic differentiation. Hypoxia enhanced ECs migration, cyclooxygenase 2 (COX-2) and integrin αvβ3 expression, and prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF) secretion. NS398 (selective COX-2 inhibitor) and LM609 (integrin αvβ3 specific inhibitor) impaired the ECs response to hypoxia, and exogenous PGE2 partially reversed the effects of NS398. BMSCs: ECs co-culture under hypoxia upregulated BMSCs osteogenesis and ERK phosphorylation, as well as ECs migration, integrin αvβ3 expression, and PGE2 and VEGF secretion. NS398 (pretreated ECs) lessened PGE2, VEGF concentrations of the co-culture system. NS398-treated ECs and AH6809 (combined EP1/2 antagonist)/L-798106 (selective EP3 antagonist)/L-161982 (selective EP4 antagonist)/SU5416 [VEGF receptor (VEGFR) inhibitor]-treated BMSCs impaired the co-cultured ECs-induced enhancement of BMSCs osteogenic differentiation. In conclusion, hypoxia enhances BMSCs proliferation and ERK-mediated osteogenic differentiation, and augments the COX-2-dependent PGE2 and VEGF release, integrin αvβ3 expression, and migration of ECs. COX-2/PGE2/VEGF signaling is involved in intercellular BMSCs: ECs communication under hypoxia.

Study on the mechanism of Mycoplasma gallisepticum infection on chicken tracheal mucosa injury

ABSTRACT Mycoplasma gallisepticum (MG) is a pathogenic microorganism that causes serious harm to the poultry industry. It is mainly adsorbed on the cilia and mucosa of respiratory epithelial cells, causing tracheal mucosal damage or cilia loss, causing chronic respiratory disease (CRD). In order to study the effect of MG infection on chicken tracheal mucosa injury and explore its possible mechanism, specific-pathogen-free (SPF) chickens were challenged with Mycoplasma gallisepticum wild-type strain MG-HY. Then, transcriptome sequencing analysis was performed to study the mechanism of MG tracheal mucosal damage. During infection, MG localizes and proliferates in the chicken trachea, and induces mucosal damage. A total of 3112 significantly (P < 0.01) differentially expressed genes (DEGs) were selected by RNA-seq, including 1646 up-regulated genes and 1466 down-regulated genes. Functional analysis showed increased expression levels of genes involved in immune defense response and mechanical barrier of tracheal mucosa in infected chicks. The expression level of pro-inflammatory cytokines (TNF-α) increased, activating the upstream protein Ras of the ERK-MLCK signaling pathway, Ras causing ERK phosphorylation levels to rise and MLCK activation, thus causing phosphationalization of MLC, and further regulating the expression and mucous distribution of tight junction protein (TJ), leading to tracheal mucosal injury in chicks. The results of qRT-PCR assay and immunohistochemical analysis were consistent with the results of transcriptome analysis. Overall, Our findings provide a basis for further research on the underlying mechanism of chick tracheal mucosal damage caused by MG infection, and help to understand how MG induces respiratory immune damage in birds.

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.

Effect of Danshen on TLR2-triggered inflammation in macrophages

BackgroundDanshen (Salvia Miltiorrhiza Radix et Rhizoma) is a valued herbal plant widely used to treat cardiovascular diseases in Asian countries. In modern medicine, innate immunity-induced inflammation is considered a risk factor for cardiovascular diseases. However, little is known about the anti-inflammatory effects and molecular mechanism of Danshen. PurposeTo evaluate the molecular mechanisms of Danshen on Toll-like receptor (TLR) 2-triggered inflammation in macrophages and identify its bioactive components. MethodsPam3CSK4-stimulated bone marrow-derived macrophages (BMMs) were treated with Danshen water extract (DSE), and the levels of proinflammatory cytokines (interleukin (IL)-6, IL-12 and tumor necrosis factor (TNF)-α) were measured by both real-time quantitative PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). RNA sequencing (RNA-seq)-based bioinformatics analyses were applied to reveal the novel molecular mechanisms of DSE, followed by western blotting for verification. Additionally, HPLC-UV analysis along with bioassays was performed to identify the bioactive ingredients of DSE. ResultsThe results of RT-qPCR and ELISA showed that DSE significantly inhibited proinflammatory cytokine expression in a dose-dependent manner. Transcriptome analyses revealed that a wider panel of inflammatory cytokines responded to the regulatory effect of DSE, and that the TNF signaling pathway might have played a vital role. Western blotting data confirmed the involvement of extracellular signal-regulated protein kinases (ERK) and Jun N-terminal Kinase (JNK) related singling pathway. Among the seven components identified in DSE, Danshensu (DSS) and protocatechuic aldehyde (PA) were confirmed as bioactive ones with anti-inflammatory effects. ConclusionDSE showed a promising effect against TLR2-triggered inflammation associated with the inhibition of the TNF cascade down-streamed mitogen-activated protein kinase (MAPK) signaling pathway, in which IL-6 acts as the key effective molecule, and ERK and JNK phosphorylation was inhibited. Notably, DSS and PA were considered bioactive components with anti-inflammatory bioactivity.

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.

Ganglioside Hp-s1 Analogue Inhibits Amyloidogenic Toxicity in Alzheimer's Disease Model Cells.

Alzheimer's disease (AD) is characterized by extracellular deposition of amyloid plaques, which are predominantly composed of amyloid-β (Aβ) peptide derived from amyloid precursor protein (APP) cleavage. APP interacts with tropomyosin receptor kinase A, a neurotrophic receptor associated with gangliosides and mediating neuronal survival and differentiation through the extracellular signal-regulated protein kinase (ERK) pathway. The ganglioside Hp-s1's analogue Hp-s1A exerts neuritogenic activity; however, its effect on AD pathology remains unknown. To test the hypothesis that Hp-s1A is a potential candidate to treat AD, we established the AD-modeled cell line by expressing human Swedish and Indiana APP gene (APP-Swe/Ind) in N2a mouse neuroblastoma cells. The cells were treated with Hp-s1A or monosialoganglioside GM1 for comparison. The AD model cells expressing APP-Swe/Ind exhibited a significant reduction in viability, as well as neurite outgrowth rate, in comparison to the control cells expressing APP-695. APP C-terminal fragment-β (CTFβ) and Aβ42 were increased in the AD cell lysates and the culture media, respectively. With the treatment of either Hp-s1A or GM1 at 1 μM, the AD model cells showed a significant increase in viability; however, only Hp-s1A reduced CTFβ levels in these cells. Further analysis of the culture media revealed that Hp-s1A also reduced Aβ42 production from AD model cells. The phosphorylation of ERK was elevated and the neurite outgrowth rate was restored with Hp-s1A treatment. In conclusion, the ganglioside analogue Hp-s1A inhibited amyloidogenic processing of APP and promoted neurotrophic activity and survival of AD model cells. Hp-s1A has great potential in AD therapeutic development.

Phytol suppresses melanogenesis through proteasomal degradation of MITF via the ROS-ERK signaling pathway

Phytol (3,7,11,15-tetramethyl-2-hexadecen-1-ol) is an acyclic monounsaturated diterpene alcohol generated from chlorophyll metabolism that exerts anti-inflammatory, antithrombotic, antimicrobial, and antitumor effects. However, the effect of phytol on melanogenesis and the underlying molecular mechanisms of its inhibition remain unknown. Here, we found that phytol suppressed α-melanocyte-stimulating hormone-induced melanogenesis in B16F10 murine melanoma cells without any toxic effects. Phytol significantly attenuated melanin production by reducing the expression of tyrosinase and tyrosinase related protein 1. Treatment with phytol inhibited the expression of microphthalmia-associated transcription factor (MITF) by phosphorylating extracellular signal-regulated protein kinase (ERK). The ERK inhibitor PD98059 restored MITF expression and prevented the anti-melanogenic effect of phytol. We found that the ERK inhibitor coincidently abrogated MITF ubiquitination and degradation, suggesting that the ERK pathway is involved in phytol-induced ubiquitination of MITF. Furthermore, our data show that reactive oxygen species (ROS) production was increased in cells treated with phytol. Consistently, a ROS scavenger inhibited ERK phosphorylation and restored MITF degradation. Accordingly, the intermediary role of ROS was confirmed in phytol-induced MITF degradation. Taken together, these results demonstrate that phytol stimulates ROS production and modulates ERK-mediated proteasomal degradation of MITF in B16F10 murine melanoma cells. These findings suggest that phytol may have potential to be utilized as a whitening agent in cosmetics and as a therapy for skin hyperpigmentation.

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.

The Rho-associated kinase inhibitors Y27632 and fasudil promote microglial migration in the spinal cord via the ERK signaling pathway.

Rho-associated kinase (ROCK) is a key regulatory protein involved in inflammatory secretion in microglia in the central nervous system. Our previous studies showed that ROCK inhibition enhances phagocytic activity in microglia through the extracellular signal-regulated kinase (ERK) signaling pathway, but its effect on microglial migration was unknown. Therefore, in this study, we investigated the effects of the ROCK inhibitors Y27632 and fasudil on the migratory activity of primary cultured microglia isolated from the spinal cord, and we examined the underlying mechanisms. The microglia were treated with Y27632, fasudil and/or the ERK inhibitor U0126. Cellular morphology was observed by immunofluorescence. Transwell chambers were used to assess cell migration. ERK levels were measured by in-cell western blot assay. Y27632 and fasudil increased microglial migration, and the microglia were irregularly shaped and had many small processes. These inhibitors also upregulated the levels of phosphorylated ERK protein. The ERK inhibitor U0126 suppressed these effects of Y27632 and fasudil. These findings suggest that the ROCK inhibitors Y27632 and fasudil promote microglial migration in the spinal cord through the ERK signaling pathway.

Ketamine, a Clinically Used Anesthetic, Inhibits Vascular Smooth Muscle Cell Proliferation via PP2A-Activated PI3K/Akt/ERK Inhibition.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) gives rise to major pathological processes involved in the development of cardiovascular diseases. The use of anti-proliferative agents for VSMCs offers potential for the treatment of vascular disorders. Intravenous anesthetics are firmly established to have direct effects on VSMCs, resulting in modulation of blood pressure. Ketamine has been used for many years in the intensive care unit (ICU) for sedation, and has recently been considered for adjunctive therapy. In the present study, we investigated the effects of ketamine on platelet-derived growth factor BB (PDGF-BB)-induced VSMC proliferation and the associated mechanism. Ketamine concentration-dependently inhibited PDGF-BB-induced VSMC proliferation without cytotoxicity, and phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated protein kinase (ERK) inhibitors, LY294002 and PD98059, respectively, have similar inhibitory effects. Ketamine was shown to attenuate PI3K, Akt, and ERK1/2 phosphorylation induced by PDGF-BB. Okadaic acid, a selective protein phosphatase 2A (PP2A) inhibitor, significantly reversed ketamine-mediated PDGF-BB-induced PI3K, Akt, and ERK1/2 phosphorylation; a transfected protein phosphatse 2a (pp2a) siRNA reversed Akt and ERK1/2 phosphorylation; and 3-O-Methyl-sphingomyeline (3-OME), an inhibitor of sphingomyelinase, also significantly reversed ERK1/2 phosphorylation. Moreover, ketamine alone significantly inhibited tyrosine phosphorylation and demethylation of PP2A in a concentration-dependent manner. In addition, the pp2a siRNA potently reversed the ketamine-activated catalytic subunit (PP2A-C) of PP2A. These results provide evidence of an anti-proliferating effect of ketamine in VSMCs, showing activation of PP2A blocks PI3K, Akt, and ERK phosphorylation that subsequently inhibits the proliferation of VSMCs. Thus, ketamine may be considered a potential effective therapeutic agent for reducing atherosclerotic process by blocking the proliferation of VSMCs.

Tyrosine kinase Fyn regulates iNOS expression in LPS-stimulated astrocytes via modulation of ERK phosphorylation

The high concentrations of nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in activated glial cells in response to neuroinflammatory stimuli have neurotoxic effects on the brain. At basal levels, iNOS expression is low, and proinflammatory stimuli induce iNOS expression in astrocytes, microglia, and oligodendrocytes. Fyn, a non-receptor tyrosine kinase, regulates iNOS expression in several types of immune cells. However, its role in stimulated astrocytes is less clear. In this study, we investigated the role of Fyn in the regulation of lipopolysaccharide (LPS)-induced iNOS expression in astrocytes from mice and rats. Intracerebroventricular LPS injections in cortical regions enhanced iNOS mRNA and protein levels, which were increased in Fyn-deficient mice. Accordingly, LPS-induced nitrite production was enhanced in primary astrocytes cultured from Fyn-deficient mice or rats. Similar results were observed in cultured astrocytes after the siRNA-induced knockdown of Fyn expression. Finally, we observed increased LPS-induced extracellular signal-regulated protein kinase (ERK) activation in Fyn-deficient astrocytes. These results suggested that Fyn has a regulatory role in iNOS expression in astrocytes during neuroinflammatory responses.

Signal-Regulated Protein Kinases/Protein Kinase B-p53-BH3-Interacting Domain Death Agonist Pathway Regulates Gingipain-Induced Apoptosis in Osteoblasts.

Gingipains are cysteine proteases produced by Porphyromonas gingivalis, the predominant pathogen in chronic periodontitis. The present study aims to examine the role of gingipains in promoting apoptosis in osteoblasts. Human calvarial osteoblasts and osteoblast MC3T3-E1 cells were treated with 8.348 U/L gingipains. Flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining were used to detect cell apoptosis. Protein expression was examined by Western blotting, and gene expression was detected by real-time polymerase chain reaction. Small interfering (si)RNA transfection was used to knock down BH3-interacting domain death agonist (Bid) expression. Treatment with 8.348 U/L gingipains from 4 to 72 hours increased apoptosis, accompanied by elevated cleaved caspase-3 levels. Notably, gingipain-induced apoptosis was associated with increase of Bid and its truncated form, tBid, as well as p53. Transfection with Bid siRNA resulted in suppression of gingipain-induced apoptosis. The p53 inhibitor, Pifithrin-α, blocked the gingipain-induced Bid. The ability of gingipains to stimulate p53 and Bid expression was mimicked by PD-0325901 and MK-2206, the specific extracellular signal-regulated protein kinases (ERK) and protein kinase B (PKB) inhibitors, respectively. Furthermore, treatment with gingipains reduced phospho-ERK and phospho-PKB levels, an effect correlated to gingipain-induced increase in p53 and tBid expression. The present findings suggest that Bid plays an essential role in gingipain-induced osteoblast apoptosis, which is dependent on inhibition of ERK and PKB phosphorylation, followed by the activation of p53.

Collagen triple helix repeat containing 1 promotes tumor angiogenesis in gastrointestinal stromal tumors.

Collagen triple helix repeat containing 1 (Cthrc1) is a secreted protein that has been observed to lead to poorer prognosis by inducing the invasion and metastasis in different tumors; however, it has not been demonstrated that Cthrc1 is involved in tumor angiogenesis. Immunohistochemical staining of Cthrc1 and CD31 in gastrointestinal stromal tumor tissue demonstrated that Cthrc1 is associated with microvascular density. Overexpression of Cthrc1 protein may alter the properties of human umbilical vein endothelial cells (HUVECs), including migration, invasion, tubule formation and aortic ring sprouting. Small interfering RNA-mediated knockdown of Cthrc1 was performed to verify the opposite effects. Migration and tubule formation induced by Cthrc1 overexpression in HUVECs was attenuated by inhibition of phosphorylation in extracellular-signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. The pro-angiogenic effect of Cthcr1 is associated with increased phosphorylation of ERK and JNK in HUVECs. Silencing the expression of Cthrc1 protein may be a promising strategy to inhibit tumor angiogenesis.

Effects of small interfering RNA silencing metastasis-associated in colon cancer-1 gene on the proliferation, migration and invasion of liver cancer cells

Objective To investigate the effects of small interfering RNA (siRNA) silencing metastasis-associated in colon cancer-1 (MACC1) gene on the proliferation, migration and invasion of liver cancer cells, and the possible mechanism. Methods HepG2 cells were cultured. According to the different transfectants, the cells were divided into siRNA-MACC1 group, siRNA-NC group and blank control group. The cell proliferations in the different transfected groups were measured by methyl thiazol tetrazolium (MTT) assay. The cell apoptosis in the different transfected groups was examined by flow cytometry. The cell migration and invasion abilities in the different transfected groups were tested by Transwell methods. The expression levels of MACC1, hepatocyte growth factor (HGF) and c-Met genes in the different transfected groups were detected by real-time fluorescent quantitative polymerase chain reaction(FQ-PCR). The expression levels of MACC1, HGF, c-Met, MEK, phosphorylated MEK (p-MEK), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) proteins in the different transfected groups were detected by Western blotting. Results (1) MTT results showed that absorptivity (A) values in the siRNA-MACC1 group at 24, 48, 72 and 96 h (0.27±0.05, 0.36±0.06, 0.46±0.05, 0.58±0.07) were lower than the siRNA-NC group (0.39±0.06, 0.47±0.05, 0.64±0.07, 0.75±0.08) and blank control group (0.41±0.05, 0.49±0.07, 0.62±0.06, 0.77±0.09), the differences were statistically significant (F=25.945, 32.286, 40.264, 38.165, P=0.000, 0.000, 0.000, 0.000). (2) Flow cytometry results showed that the apoptosis rate in the siRNA-MACC1 group was (13.52±2.61)%, which was significantly higher than the siRNA-NC group and blank control group [(4.94±1.84)%, (5.10±2.13)%, F=73.918, P=0.000]. (3) The number of migration cells and the number of invasive cells in the siRNA-MACC1 group (73.62±12.71, 59.23±11.53) were significantly lower than those in the siRNA-NC group and blank control group (98.53±14.22, 85.44±13.12 and 102.51±15.13, 87.62±14.24, F=11.149, 9.500, P=0.000, 0.000). (4) Compared with siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF and c-Met mRNA in the siRNA-MACC1 group were significantly lower than the siRNA-NC group and blank control group (F=51.235, 137.507, 139.708, P=0.000, 0.000, 0.000). (5) Compared with the siRNA-NC group and blank control group, the relative expression levels of MACC1, HGF, c-Met, p-MEK and p-ERK proteins in the siRNA-MACC1 group were decreased (F=49.803, 33.564, 24.391, 228.400, 137.410, P=0.000, 0.000, 0.000, 0.000, 0.000), while the the relative expression levels of MEK and ERK proteins were increased (F=12.751, 23.829, P=0.000, 0.000). Conclusion Specific inhibition of MACC1 gene expression in HepG2 cells could effectively inhibit cell proliferation, decrease cell migration and invasion, and induce cell apoptosis. The mechanism might be related to HGF/Met and MEK/ERK signaling pathways. Key words: Liver cancer; Metastasis-associated in colon cancer-1; Small interfering; Biological characteristics

Chelidonic acid evokes antidepressant-like effect through the up-regulation of BDNF in forced swimming test.

Depression is usually accompanied by neuro-inflammatory reactions. Chelidonic acid, in particular, has shown anti-inflammatory effects. The objective of this study was to evaluate the anti-depressant effects of chelidonic acid and to discuss the potential mechanisms of a forced swimming test. Chelidonic acid was administered orally once a day for 14 days. On the 14th day, chelidonic acid resulted in a significant decrease in immobility time during the forced swimming test without alteration of locomotor activity, in an open field test. Chelidonic acid also increased the number of nissl bodies in the hippocampus. Brain-derived neurotrophic factor expression and extracellular signal-regulated protein kinase phosphorylation in the hippocampus were up-regulated by the administration of chelidonic acid. Chelidonic acid administration significantly increased the mRNA expression of hippocampal estrogen receptor-β. The levels of hippocampal interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were effectively attenuated by the administration of chelidonic acid. In addition, chelidonic acid significantly increased the levels of 5-hydroxytryptamine (serotonin), dopamine, and norepinephrine compared with those levels for the mice that were administered distilled water in the hippocampus. These results suggest that chelidonic acid might serve as a new therapeutic strategy for the regulation of depression associated with inflammation.

Calcitonin gene-related peptide protects rats from cerebral ischemia/reperfusion injury via a mechanism of action in the MAPK pathway.

The aim of the present study was to investigate the protective function and underlying mechanism of calcitonin gene-related peptide (CGRP) on cerebral ischemia/reperfusion damage in rats. Adult male Wistar rats were selected for the establishment of an ischemia/reperfusion injury model through the application of a middle cerebral artery occlusion. Animals were randomly divided into 6 groups of 24 animals. Drugs were administered according to the design of each group; animals were administered CGRP, CGRP8–37, PD98059 and SB20358. The neurobehavioral scores of the rat cerebral ischemia model in each group were calculated. The infarction range of the rat brain tissues was observed by 2,3,5-triphenyltetrazolium chloride staining. The expression levels of three proteins, phosphorylated c-Jun N-terminal kinase (JNK)/JNK, phosphorylated extracellular signal-regulated protein kinase (ERK)/ERK and p-p38/p38, in the mitogen-activated protein kinase (MAPK) pathway in the brain tissues was detected by western blotting. The results showed that CGRP could improve the neurobehavioral function of the ischemic rats and reduce the infarction range. Western blotting results confirmed that the function of the CGRP was mediated mainly through the reduction of the JNK and p38 phosphorylation and the promotion of ERK phosphorylation. Therefore, the present study confirmed that an increase in the exogenous CRGP could effectively improve ischemia/reperfusion injury of the brain tissue. The mechanisms of action were achieved through a reduction in JNK and p38 phosphorylation and an increase in ERL phosphorylation in the MAPK pathway. These mechanisms were interdependent.

β-Arrestins Negatively Regulate the Toll Pathway in Shrimp by Preventing Dorsal Translocation and Inhibiting Dorsal Transcriptional Activity

The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that β-arrestins negatively regulate Toll signaling in two different ways. β-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of β-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. β-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that β-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.

Pterygium epithelium abnormal differentiation related to activation of extracellular signal-regulated kinase signaling pathway in vitro.

To investigate whether the abnormal differentiation of the pterygium epithelium is related to the extracellular signal-regulated kinase (ERK) signaling pathway in vitro. The expression levels of phosphorylated ERK (P-ERK), keratin family members including K19 and K10 and the ocular master control gene Pax-6 were measured in 16 surgically excised pterygium tissues and 12 eye bank conjunctiva. In colony-forming cell assays, the differences in clone morphology and in K10, K19, P-ERK and Pax-6 expression between the head and body were investigated. When cocultured with the ERK signaling pathway inhibitor PD98059, the changes in clone morphology, colony-forming efficiency, differentiated marker K10, K19 and Pax-6 expression and P-ERK protein expression level were examined by immunoreactivity and Western blot analysis. The expression of K19 and Pax-6 decreased in the pterygium, especially in the head. No staining of K10 was found in the normal conjunctiva epithelium, but it was found to be expressed in the superficial cells in the head of the pterygium. Characteristic upregulation of P-ERK was observed by immunohistochemistry. The clone from the head with more differentiated cells in the center expressed more K10, and the clone from the body expressed more K19. The P-ERK protein level increased in the pterygium epithelium compared with conjunctiva and decreased when cocultured with PD98059. The same medium with the ERK inhibitor PD98059 was more effective in promoting clonal growth than conventional medium with 3T3 murine feeder layers. It was observed that the epithelium clone co-cultured with the inhibitor had decreased K10 expression and increased K19 and Pax-6 expression. We suggest ERK signaling pathway activation might play a role in the pterygium epithelium abnormal differentiation.

Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways

Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling.