BACKGROUNDSurfactant protein-A (SP-A) contributes to the regulation of sepsis-induced acute kidney injury. In a previous study, we demonstrated that the expression of SP-A in the human renal tubular epithelial (HK-2) cells can be stimulated by lipopolysaccharide (LPS). The present study evaluated the possible signal-transducing mechanisms of LPS-induced SP-A biosynthesis in the HK-2 cells.

METHODSTetrazolium salt colorimetry (MTT) assay was used to detect cell viability of HK-2 cells after LPS stimulation on different time points. HK-2 cells were stimulated with 100 ng/ml of LPS for different durations to determine the effects of LPS on SP-A and toll-like receptor 4 (TLR4) messenger RNA (mRNA) expression, as well as phosphorylation of mitogen-activated/extracellular signal-regulated kinase (MEK) 1, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (p38MAPK), and nuclear factor-kappa B (NF-κB) inhibitor-alpha (IkB-α). Then, HK-2 cells were pretreated with CLI-095, a TLR4 inhibitor, to analyze mRNA and protein levels of SP-A and TLR4 and expression of NF-κB in the cytoplasm and nucleus of HK-2 before LPS exposure.

RESULTSHK-2 cells exposed to 100 ng/ml of LPS for 1, 6, and 24 h did not affect cell viability which showed no toxic effect of 100 ng/ml LPS on cells (P = 0.16); however, the biosynthesis of SP-A mRNA and protein in HK-2 cells was significantly increased (P = 0.02). As to the mechanism, LPS enhanced transmembrane receptor TLR4 protein expression. Sequentially, LPS time dependently augmented phosphorylation of MEK1, ERK1/2, and p38MAPK. In addition, levels of phosphorylated IκB-α and nuclear NF-κB were augmented with LPS exposure for 2 h. LPS-induced SP-A and TLR4 mRNA as well as NF-κB expression were significantly inhibited by pretreatment with CLI-095.

CONCLUSIONSThe present study exhibited that LPS can increase SP-A synthesis in human renal epithelial cells through sequentially activating the TLR4-related MEK1-ERK1/2-NF-κB-dependent pathway.

Cell Line ; Cell Survival ; drug effects ; physiology ; Colorimetry ; Humans ; Kidney ; cytology ; metabolism ; Lipopolysaccharides ; toxicity ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; NF-kappa B ; metabolism ; Pulmonary Surfactant-Associated Protein A ; metabolism ; Sulfonamides ; pharmacology ; Tetrazolium Salts ; chemistry ; Toll-Like Receptor 4 ; antagonists & inhibitors ; metabolism

OBJECTIVETumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance greatly limits the clinical therapeutic efficacy of TRAIL. Elucidating the molecular mechanism underlying TRAIL resistance will be fundamental to resolving this problem.

METHODSNuclear and cytoplasmic protein extraction and immuno？uorescence (IF) assay were used to detect changes in heterogeneous nuclear ribonucleoprotein K (hnRNPK) localization in H1299 cells. The evaluation of cell apoptosis in cells transfected with GFP-hnRNPK, GFP-hnRNPK S284/353A, or GFP-hnRNPK S284/353D mutant was performed using cleaved caspase-3 antibody. The gene expression of XIAP was tested by quantitative RT-PCR.

RESULTSPreviously, we reported that hnRNPK antagonized TRAIL-induced apoptosis through inhibition of PKC-mediated GSK3β phosphorylation. In this study, we further demonstrate that TRAIL treatment induces cytoplasmic accumulation of hnRNPK in H1299 cells. The hnRNPK localized in the cytoplasm has a higher capacity to antagonize TRAIL-induced apoptosis. Both ERK1/2 signaling inhibitor U0126 and ERK-phosphoacceptor-site mutant (GFP-hnRNPK S284/353A) diminish cytoplasmic accumulation of hnRNPK induced by TRAIL. Moreover, we show that XIAP is involved in hnRNPK-mediated TRAIL resistance in H1299 cells.

CONCLUSIONTaken together, these results give new insights into the understanding of the molecular mechanism associated with TRAIL resistance in lung adenocarcinoma.

Apoptosis ; physiology ; Cell Line, Tumor ; Gene Expression Regulation ; physiology ; Heterogeneous-Nuclear Ribonucleoprotein K ; genetics ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; genetics ; metabolism ; Up-Regulation ; physiology ; X-Linked Inhibitor of Apoptosis Protein ; genetics ; metabolism

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

OBJECTIVEThis paper aims to investigate the apoptotic effect of inactivated Sendai virus (hemagglutinating virus of Japan-enveloped, HVJ-E) on murine melanoma cells (B16F10) and the possible mechanisms involved in the putative apoptotic reactions.

METHODSB16F10 cells were treated with HVJ-E at various multiplicities of infection (MOI), and the reactive oxygen species (ROS), cell viability, and apoptosis were measured. Next, the roles of ROS in the regulation of Bcl-2/Bax and the activation of mitogen-activated protein kinase (MAPK) pathways in HVJ-E-treated B16F10 cells were analyzed. To further evaluate the cytotoxic effect of HVJ-E-generated ROS on B16F10 cells, HVJ-E was intratumorally injected, both with and without N-acetyl-L-cysteine (NAC), into melanoma tumors on BALB/c mice. Tumor volume was then monitored for 3 weeks, and the tumor proteins were separated for immunoblot assay.

RESULTSTreatment of B16F10 cells with HVJ-E resulted in a dose-dependent inhibition of cell-viability and an induction of apoptosis. The latter effect was associated with the generation of ROS. Inhibition of ROS generation by NAC resulted in a significant reduction of HVJ-E-induced Erk1/2, JNK, and p38 MAPK activation. Additionally, ROS inhibition caused a decrease in the Bcl-2/Bax ratio as well as promoting activation of apoptosis both in vitro and in vivo.

CONCLUSIONThese results suggest that HVJ-E possesses potential anticancer activity in B16F10 cells through ROS-mediated mitochondrial dysfunction involving the MAPK pathway.

Animals ; Apoptosis ; Cell Line, Tumor ; Mice ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Respirovirus Infections ; virology ; Sendai virus ; physiology ; Virus Inactivation

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase (MAPK) signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β (IL-1β)-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-1β restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.
3T3 Cells ; Animals ; Cartilage, Articular ; cytology ; Cell Survival ; physiology ; Cells, Cultured ; Chondrocytes ; drug effects ; enzymology ; Coculture Techniques ; Culture Media, Conditioned ; Gelatinases ; drug effects ; Interleukin-1beta ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; physiology ; Matrix Metalloproteinase 2 ; drug effects ; Matrix Metalloproteinase 9 ; drug effects ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; drug effects ; Monocytes ; cytology ; NF-kappa B ; antagonists & inhibitors ; Osteoclasts ; physiology ; Protease Inhibitors ; analysis ; Tissue Inhibitor of Metalloproteinase-1 ; drug effects ; Tissue Inhibitor of Metalloproteinase-2 ; drug effects ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors

BACKGROUNDAbnormal neuronal differentiation plays an important role in central nervous system (CNS) development abnormalities such as Down syndrome (DS), a disorder that results directly from overexpression of genes in trisomic cells. Receptor-interacting protein 140 (RIP140) is significantly upregulated in DS brains, suggesting its involvement in DS CNS development abnormalities. However, the role of RIP140 in neuronal differentiation is still not clear. The current study aimed to investigate the effect of RIP140 overexpression on the differentiation of neuro-2a (N2a) neuroblastoma cells, in vitro.

METHODSStably RIP140-overexpressing N2a (N2a-RIP140) cells were used as a neurodevelopmental model, and were constructed by lipofection and overexpression validated by real-time polymerase chain reaction and Western blot. Retinoic acid (RA) was used to stimulate N2a differentiation. Combining the expression of Tuj1 at the mRNA and protein levels, the percentage of cells baring neurites, and the number of neurites per cell body was semi-quantified to determine the effect of RIP140 on differentiation of N2a cells. Furthermore, western blot and the ERK1/2 inhibitor U0126 were used to identify the specific signaling pathway by which RIP140 induces differentiation of N2a cells. Statistical significance of the differences between groups was determined by one-way analysis of variance followed by the Dunnett test.

RESULTSCompared to untransfected N2a cells RIPl40 expression in N2a-RIP140 cells was remarkably upregulated at both the mRNA and protein levels. N2a-RIP140 cells had a significantly increased percentage of cells baring neurites, and numbers of neurites per cell, as compared to N2a cells, in the absence and presence of RA (P < 0.05). In addition, Tuj1, a neuronal biomarker, was strongly upregulated in N2a-RIP140 cells (P < 0.05) and phosphorylated ERK1/2 (p-ERK1/2) levels in N2a-RIP140 cells were dramatically increased, while differentiation was inhibited by the ERK1/2-specific inhibitor U0126.

CONCLUSIONSRIP140 overexpression promotes N2a cell neuronal differentiation by activating the ERK1/2 pathway.

Blotting, Western ; Cell Differentiation ; physiology ; Cell Line ; Humans ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neurons ; cytology ; metabolism ; Nuclear Receptor Co-Repressor 1 ; metabolism ; Signal Transduction ; physiology

OBJECTIVETo explore the effect of drug-containing serum of Chinese herbal compounds [Xiongshao Capsule (XS, for activating blood) and Huanglian Capsule (HL, for dispelling toxin)] on tumor necrosis factor-alpha (TNF-alpha)-induced adherence between human umbilical vein endothelial cells (HUVECs) and polymorphonuclear neutrophils (PMN), inflammatory reaction and expression of related proteins in mitogen-activated protein kinase (MAPK) pathway.

METHODSThirty-two rats were randomly divided into four groups (8 in each group) using random digit table: the blank control group treated with distilled water, the test group I treated with Chinese herbal compound of XS (0.135 g/kg), the test group II treated with Chinese herbal compound of HL (0.135 g/kg), and the test group Ill treated with Chinese herbal compound of XS (0.135 g/kg) and HL (0.135 g/kg). All medication was given by gastrogavage once a day for a week. Rats' blood serum was harvested 1 h after the last administration to prepare drug-containing serum. HUVECs were exposed to TNF-alpha (100 ng/mL) to induce cell injury model and incubated with corresponding drug-containing serum (10%) for 24 h. Normal rats' serum was given to cells in the blank control group and the model group, while XC + HL containing serum was given to cells in the rest 3 groups. The adherence of HUVECs and PMN cells was detected by using rose bengal strain. Levels of E-selectin, intercellular adhesion molecule-1 (ICAM-1), and interleukin-1beta (IL-1P) in the supernatant of cultured HU-VECs were determined by ELISA. Protein expressions of mitogen-activated protein kinases p38 (p38MAPK) and extracellular signal-regulated kinase 1/2 (ERK 12) were determined by Western blot.

RESULTSCompared with the blank control group, HUVECs were seriously injured; PMN adherence amount significantly increased; levels of E-selectin, ICAM-1, and IL-1beta increased; expression levels of p-p38MAPK and p-ERK 1/2 in the supernatant of HUVECs significantly increased in the model group (all P < 0.01). Compared with the model group, HUVECs-PMN adherence amount decreased (P < 0.05); levels of E-selectin, ICAM-1, and IL-1 beta in the supernatant of HUVECs decreased (P < 0.01, P < 0.05); expression levels of p-p38MAPK and p-ERK 1/2 of endothelial cells decreased in the test group I, II, and III (P < 0.01).

CONCLUSIONSDrug-containing serums of activating blood, activating blood and dispelling toxin could attenuate TNF-alpha induced injury of HUVECs, inhibit HUVECs-PMN adherence and the release of adhesion factors. Its mechanism might be involved with protein phosphorylation of p38MAPK and ERK 1/2 in the MAPK pathway.

Animals ; Drugs, Chinese Herbal ; therapeutic use ; E-Selectin ; Endothelial Cells ; physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Inflammation ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; Mitogen-Activated Protein Kinase 3 ; Neutrophils ; Rats ; Serum ; Tumor Necrosis Factor-alpha ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

BACKGROUNDOur previous studies have indicated that the beneficial effects of grafting neural stem cells (NSCs) overexpressing glial cell line-derived neurotrophic factor (GDNF) in rats after stroke. However, the underlying mechanisms are highly debatable. In this study, we investigated whether neurogenesis, Akt, and extracellular signal-regulated kinase 1/2 (Erk1/2) signaling were involved in this process.

METHODSTransient ischemic stroke were induced by occluding middle cerebral artery for 2 hours and reperfusion. At 3 days after reperfusion, GDNF/NSCs, NSCs, and vehicle were administered. Immunohistochemical staining was used to evaluate neurogenesis by nestin antibody; phosphorylation of Akt and Erk1/2 was investigated by Western blotting analysis.

RESULTSTransplantation of GDNF/NSCs and NSCs significantly increased nestin-positive cells compared to control group (vehicle) from 1 to 7 weeks after reperfusion, and GDNF/NSCs showed stronger effect than NSCs at 2 and 3 weeks after reperfusion. Meanwhile, enhanced phosphorylation level of Erk1/2 was observed in the GDNF/NSCs and NSCs groups compared with control group, and phosphorylation level of Erk1/2 in GDNF/NSCs group was remarkably higher than that of NSCs group at any given time. In contrast, expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), known as inhibitor of Erk1/2 signaling, was significantly decreased in the GDNF/NSCs and NSCs groups compared with the control group. Moreover, much enhanced and prolonged phosphorylation level of Akt of GDNF/NSCs group was detected compared with control and NSCs group.

CONCLUSIONGrafting GDNF/NSCs enhances neurogenesis and activates Akt and Erk1/2 signaling, that may provide the potential for GDNF/NSCs in stroke treatment.

Animals ; Glial Cell Line-Derived Neurotrophic Factor ; metabolism ; Male ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Neurogenesis ; physiology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stem Cell Transplantation ; Stroke ; metabolism ; therapy

The p38 mitogen-activated protein kinase (MAPK) plays an evolutionarily conserved role in the cellular response to microbial infection and environmental stress. Activation of p38 is mediated through phosphorylation by upstream MAPKK, which in turn is activated by MAPKKK. In the Caenorhabditis elegans, the p38 MAPK (also called PMK-1) signaling pathway has been shown to be required in its resistance to bacterial infection. However, how different upstream MAP2Ks and MAP3Ks specifically contribute to the activation of PMK-1 in response to bacterial infection still is not clearly understood. By using double-stranded RNA-mediated interference (RNAi) and genetic mutants of C. elegans, we demonstrate that C. elegans MOM-4, a mammalian TAK1 homolog, is required for the resistance of C. elegans to a P. aeruginosa infection. We have also found that the MKK-4 of C. elegans is required for P. aeruginosa resistance, but not through the regulation of DLK-1. In summary, our results indicate that different upstream MAPKKKs or MAPKKs regulate the activation of PMK-1 in response to P. Aeruginosa.
Animals ; Caenorhabditis elegans ; enzymology ; genetics ; immunology ; microbiology ; Caenorhabditis elegans Proteins ; genetics ; metabolism ; Disease Resistance ; Enzyme Activation ; MAP Kinase Kinase 1 ; metabolism ; MAP Kinase Signaling System ; Membrane Proteins ; deficiency ; genetics ; metabolism ; Mutation ; Pseudomonas Infections ; enzymology ; Pseudomonas aeruginosa ; physiology ; RNA Interference ; p38 Mitogen-Activated Protein Kinases ; metabolism