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

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

The expression of hypoxia-inducible factor (HIF) is influenced by reactive oxygen species (ROS). Effect of bilirubin on HIF-1 expression in proximal tubular cells was investigated under physiological oxygen concentration, which is relative hypoxic condition mimicking oxygen content in the medulla of renal tissue. The human kidney (HK2) cells were cultured in 5% oxygen with or without bilirubin. HIF-1alpha protein expression was increased by bilirubin treatment at 0.01-0.2 mg/dL concentration. The messenger RNA expression of HIF-1alpha was increased by 1.69+/-0.05 folds in the cells cultured with 0.1 mg/dL bilirubin, compared to the control cells. The inhibitors of PI3K/mTOR, PI3K/AKT, and ERK 1/2 pathways did not attenuate increased HIF-1alpha expression by bilirubin. HIF-1alpha expression decreased by 10 microM exogenous hydrogen peroxide (H2O2); scavenger of ROS with or without bilirubin in the HK2 cells increased HIF-1alpha concentration more than that in the cells without bilirubin. Exogenous H2O2 decreased the phosphorylation of P70S6 kinase, which was completely reversed by bilirubin treatment. Knockdown of NOX4 gene by small interfering RNA (siRNA) increased HIF-1alpha mRNA expression. In coonclusion, bilirubin enhances HIF-1alpha transcription as well as the up-regulation of HIF-1alpha protein translation through the attenuation of ROS and subunits of NADPH oxidase.
Bilirubin/*pharmacology ; Cell Line ; Epithelial Cells/cytology/metabolism ; Humans ; Hydrogen Peroxide/toxicity ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Kidney Tubules, Proximal/cytology ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Oxygen/*pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphorylation/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism ; Signal Transduction/drug effects ; TOR Serine-Threonine Kinases/metabolism ; Transcriptional Activation/*drug effects ; Up-Regulation/drug effects

BACKGROUND/AIMS: 5'-Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cellular energy sensor that monitors intracellular AMP/adenosine triphosphate (ATP) ratios and is a key regulator of the proliferation and survival of diverse malignant cell types. In the present study, we investigated the effect of activating AMPK by 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) in thyroid cancer cells. METHODS: We used FRO thyroid cancer cells harboring the BRAF(V600E) mutation to examine the effect of AICAR on cell proliferation and cell survival. We also evaluated the involvement of mitogen-activated protein kinase (MAPK) pathways in this effect. RESULTS: We found that AICAR treatment promoted AMPK activation and suppressed cell proliferation and survival by inducing p21 accumulation and activating caspase-3. AICAR significantly induced activation of p38 MAPK, and pretreatment with SB203580, a specific inhibitor of the p38 MAPK pathway, partially but significantly rescued cell survival. Furthermore, small interfering RNA targeting AMPK-alpha1 abolished AICAR-induced activation of p38 MAPK, p21 accumulation, and activation of caspase-3. CONCLUSIONS: Our findings demonstrate that AMPK activation using AICAR inhibited cell proliferation and survival by activating p38 MAPK and proapoptotic molecules in FRO thyroid cancer cells. These results suggest that the AMPK and p38 MAPK signaling pathways may be useful therapeutic targets to treat thyroid cancer.
AMP-Activated Protein Kinases/genetics/metabolism ; Aminoimidazole Carboxamide/*analogs & derivatives/pharmacology ; Antineoplastic Agents/*pharmacology ; Caspase 3/metabolism ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Cell Survival/drug effects ; Cyclin-Dependent Kinase Inhibitor p21/metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; Enzyme Activators/pharmacology ; Humans ; Mutation ; Protein Kinase Inhibitors/pharmacology ; Proto-Oncogene Proteins B-raf/genetics ; RNA Interference ; Ribonucleotides/*pharmacology ; Signal Transduction/*drug effects ; Thyroid Neoplasms/*enzymology/genetics/pathology ; Time Factors ; Transfection ; p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors/*metabolism

OBJECTIVETo investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms.

METHODSNCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot.

RESULTSCell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group.

CONCLUSIONThe combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

Apoptosis ; drug effects ; Benzimidazoles ; administration & dosage ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; pathology ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin B1 ; metabolism ; Cyclin D1 ; metabolism ; Dose-Response Relationship, Drug ; Drug Synergism ; Humans ; Indazoles ; administration & dosage ; pharmacology ; Lung Neoplasms ; genetics ; pathology ; Mitogen-Activated Protein Kinase Kinases ; antagonists & inhibitors ; metabolism ; Mutation ; PTEN Phosphohydrolase ; genetics ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Poly(ADP-ribose) Polymerases ; metabolism ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins p21(ras) ; metabolism ; Signal Transduction ; Sulfonamides ; administration & dosage ; pharmacology ; bcl-2-Associated X Protein ; metabolism ; ras Proteins ; genetics

To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/ mTOR signal pathway inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells, mouse bone marrow-derived mesenchymal stem cell lines D1 cells were used as research object. And they were divided into control groups and salidroside (SD) groups. Different concentrations (5, 25, 50, 100 and 200 microg x mL(-1) of SD were used and SD (100 microg x mL(-1)) was used to induce at different time (0.5, 1, 3, 6, 9, 12, 24, 48 and 72 h). The immunofluorescence staining chemical technology, real-time PCR and Western blotting were used to detect the positive rates of NSE, MAP2, beta-Tubulin III, NES, GFAP and the expression levels of beta-Tubulin III, NSE, ERK1/2, AKT. The expression of ERK1/2 and NSE was detected when the ERK1/2 and PI3K/AKT/ mTOR signal pathway was blocked by PD98059 and LY294002. It indicated that the positive rates of NSE, MAP2, beta-Tubulin III, NES and GFAP were gradually enhanced with time increased. The expression level of NSE and beta-Tubulin III protein were significantly higher than those in control groups (P < 0.01). The expression of ERK1/2, AKT mRNA and protein were higher with concentration and time increased. When the ERK1/2 and PI3K/AKT/mTOR signal pathway were blocked, the expression levels of NSE, NES and beta-Tubulin III mRNA and NSE protein were inhibited significantly. It points out that SD can stimulate the ERK1/2 and PI3K/AKT/mTOR signal pathway to promote BMSCs differentiation into neural cells.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chromones ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Flavonoids ; pharmacology ; Glial Fibrillary Acidic Protein ; metabolism ; Glucosides ; antagonists & inhibitors ; isolation & purification ; pharmacology ; MAP Kinase Signaling System ; drug effects ; Mesenchymal Stromal Cells ; cytology ; Mice ; Microtubule-Associated Proteins ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Morpholines ; pharmacology ; Nestin ; metabolism ; Neurons ; cytology ; metabolism ; Phenols ; antagonists & inhibitors ; isolation & purification ; pharmacology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphopyruvate Hydratase ; genetics ; metabolism ; Plants, Medicinal ; chemistry ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rhodiola ; chemistry ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; metabolism ; Tubulin ; metabolism

OBJECTIVETo investigate the relationship between EGFR activation and down-regulation of miRNA-145 in lung cancer.

METHODSNormal human lung epithelia cell line (BEAS-2B), human lung adenocarcinoma cell lines with wild-type EGFR (A549 and H292) and human lung adenocarcinoma cell lines with EGFR mutation (H1975 and H1650) were chosen in this study. The levels of miRNA-145 and p-EGFR were determined by quantitative real-time PCR (qRT-PCR) and Western blotting, respectively, and the relationship between p-EGFR and miRNA-145 levels was analyzed. The miRNA-145 levels were determined by qRT-PCR after activating EGFR with EGF or blocking EGFR signal pathway with AG1478. In addition, ERK1/2 inhibitor U0126 was used to inhibit ERK1/2 activation and then the expression of miRNA-145 was detected.

RESULTSThe miRNA-145 levels were closely negatively related with p-EGFR in lung cancer cells (r = -0.926, P = 0.024). EGF down-regulated miRNA-145 expression, particularly in BEAS-2B cells (53.0%; t = 30.993, P = 0.001) and A549 cells (42.6%; t = 14.326, P = 0.005).The miRNA-145 was up-regulated after inhibiting p-EGFR with AG1478, and significantly enhanced by 67.5% in H1975 cells when treated with AG1478 (t = 8.269, P = 0.014). The ERK1/2 signal pathway was activated by p-EGFR. U0126 restored the miRNA-145 down-regulation induced by EGFR-activation in lung cancer cells.

CONCLUSIONThe activation of EGFR down-regulates miRNA-145 expression through ERK1/2 in lung cancer cells.

Butadienes ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line ; Cell Line, Tumor ; Down-Regulation ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Epidermal Growth Factor ; pharmacology ; Epithelial Cells ; metabolism ; Humans ; Lung ; cytology ; Lung Neoplasms ; metabolism ; pathology ; MAP Kinase Signaling System ; MicroRNAs ; metabolism ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Nitriles ; pharmacology ; Phosphorylation ; Quinazolines ; pharmacology ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism ; Tyrphostins ; pharmacology

The differentiation of periodontal ligament (PDL) progenitor cells is important for maintaining the homeostasis of PDL tissue and alveolar bone. Vitamin C (VC), a water-soluble nutrient that cannot be biosynthesized by humans, is vital for mesenchymal stem cells differentiation and plays an important role in bone remodeling. Therefore, the objective of this study was to determine the function and mechanism of VC in PDL progenitor cells osteogenic differentiation at the molecular level. We demonstrated that VC could induce the osteogenic differentiation and maturation of PDL progenitor cell without other osteogenic agents. During the process, VC preferentially activated ERK1/2 but did not affect JNK or p38. Co-treatment with ERK inhibitor effectively decreased the Vitamin C-induced expression of Runx2. ERK inhibitor also abrogated Vitamin C-induced the minimized nodules formation. PELP1, a nuclear receptor co-regulator, was up-regulated under VC treatment. PELP1 knockdown inhibited ERK phosphorylation. The overexpression of PELP1 had a positive relationship with Runx2 expression. Taken together, we could make a conclude that VC induces the osteogenic differentiation of PDL progenitor cells via PELP1-ERK axis. Our finding implies that VC may have a potential in the regeneration medicine and application to periodontitis treatment.
Ascorbic Acid ; pharmacology ; Butadienes ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Co-Repressor Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Core Binding Factor Alpha 1 Subunit ; genetics ; metabolism ; Humans ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; Phosphorylation ; drug effects ; RNA Interference ; RNA, Small Interfering ; metabolism ; Stem Cells ; cytology ; Transcription Factors ; antagonists & inhibitors ; genetics ; metabolism ; Up-Regulation ; drug effects

Currently, about 300 million people worldwide are affected by asthma. Most of these sufferers inhale immunosuppressants (ie corticosteroids) and beta-adrenergic receptor agonists for their asthma treatment. However, about 5%-10% of patients of asthma have poor response to such treatment. Investigation of kinase signaling pathway and nuclear transcription factor as a target molecule in the treatment of allergic asthma has been the concern of scholars home and abroad. This paper reviewed inhibitors of kinase signaling pathway and nuclear transcription factors for the treatment of asthma.
Animals ; Asthma ; drug therapy ; enzymology ; Humans ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; Phosphatidylinositol 3-Kinase ; antagonists & inhibitors ; Protein Kinase Inhibitors ; therapeutic use ; Protein-Tyrosine Kinases ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Transcription Factors ; antagonists & inhibitors

NMDA-induced excitotoxicity cause severe neuronal damage including apoptosis and necrosis. The present study was aimed to evaluate the proportion of NMDA-induced apoptosis of rat cortical neurons and discover signal transduction mechanism. Caspase inhibitor and lactate dehydrogenase (LDH) assay were used to study the NMDA-induced apoptosis. To explore the involved signal pathways, the primary culture of rat cortical neurons were pretreated by the inhibitors of three MAPK pathways, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. With 2 h of NMDA treatment, cellular apoptosis was measured by caspase-3 activity, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and Annexin V staining. The results showed that: (1) Caspase-dependent apoptosis accounted for 22.49% in NMDA-induced neuronal death; (2) Pretreatment with p38 MAPK inhibitor SB203580 (10 μmol/L) significantly decreased NMDA-mediated caspase-3 activity by 30.43% (P < 0.05). However, ERK inhibitor PD98059 (20 μmol/L) or JNK inhibitor SP600125 (20 μmol/L) did not influence caspase-3 activity; (3) Pretreatment with SB203580 significantly reduced the number of NMDA-induced TUNEL-positive cells by 33.10% (P < 0.05). PD98059 (20 μmol/L) or SP600125 (20 μmol/L) did not show obvious effect; (4) Pretreatment with SB203580 (10 μmol/L) significantly reduced the number of NMDA-induced early apoptotic neurons by 55.56% (P < 0.05). Also, SP600125 (20 μmol/L) significantly decreased the amount of late apoptotic/dead cells by 67.59% (P < 0.05). There was no effect of PD98059 (20 μmol/L). These results indicate that: (1) NMDA induces neuronal apoptosis besides necrosis; (2) p38 MAPK, but not JNK and ERK, is involved in NMDA-induced neuronal apoptosis, and inhibition of the apoptotic signaling pathway contributes to neuroprotection; (3) JNK activation might contribute to NMDA-induced neuronal necrosis rather than apoptosis.
Animals ; Anthracenes ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Imidazoles ; pharmacology ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; MAP Kinase Signaling System ; N-Methylaspartate ; pharmacology ; Neurons ; cytology ; Primary Cell Culture ; Pyridines ; pharmacology ; Rats ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors