Emodin, a naturally occurring anthraquinone derivative isolated from Polygoni cuspidati radix, has several beneficial pharmacologic effects, which include anti-cancer, anti-diabetic, and anti-inflammatory activities. In this study, the authors examined the effect of emodin on the production of proinflammatory cytokines, such as, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6, in mouse bone marrow-derived mast cells (BMMCs) stimulated with phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187. To investigate the mechanism responsible for the regulation of pro-inflammatory cytokine production by emodin, the authors assessed its effects on the activations of transcriptional factor nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs). Emodin attenuated the nuclear translocation of (NF)-kappaB p65 and its DNA-binding activity by reducing the phosphorylation and degradation of IkappaBalpha and the phosphorylation of IkappaB kinase B (IKK). Furthermore, emodin dose-dependently attenuated the phosphorylations of MAPKs, such as, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAP kinase, and the stress-activated protein kinases (SAPK)/c-Jun-N-terminal kinase (JNK). Taken together, the findings of this study suggest that the anti-inflammatory effects of emodin on PMA plus A23187-stimulated BMMCs are mediated via the inhibition of NF-kappaB activation and of the MAPK pathway.
Animals ; Calcimycin* ; Calcium ; Cytokines ; Emodin* ; I-kappa B Kinase ; Interleukin-6* ; Interleukins ; Mast Cells* ; Mice ; Mitogen-Activated Protein Kinases ; NF-kappa B* ; p38 Mitogen-Activated Protein Kinases ; Phosphorylation ; Phosphotransferases* ; Protein Kinases ; Tumor Necrosis Factor-alpha*

OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the human growth hormone (hGH) gene expression in a rat somatotropic pituitary cell line MtT/S.

METHODSThe plasmids containing various lengths of hGH gene 5'-promoter fragments were constructed. Stably transfected MtT/S cells were created by cotransfecting the above plasmids and pcDNA3. 1(+) with DMRIE-C transfection reagent After the administration of these cells with IL-6 and/or various inhibitors of signaling transduction pathways, the luciferase activities in MtT/S cells lysis were assayed to demonstrate the effects of IL-6 on hGH gene promoter activity and possibly involved mechanism.

RESULTSThe 10(3) U/mL IL-6 stimulated GH secretion and synthesis, and promoted the 5'-promoter activity of GH gene in stably transfected MtT/SGL cells with the action of 1.69 times above the control. Among inhibitors of signaling transduction pathways, mitogen-activated protein kinase kinase (MAPKK/MEK) inhibitor PD98059 (40 micromol/L) and p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 micromol/L) completely blocked the stimulatory effect of IL-6. Western blot analysis further confirmed the activation of phosphorylated MEK and p38 MAPK in MtT/SGL cells. Neither over-expression of Pit-1 nor inhibition of Pit-1 expression affected IL-6 induction of hGH promoter activity. A series of deletion constructs of hGH promoter were created to identify the DNA sequence that mediated the effect of IL-6. The results showed that the stimulatory effect of IL-6 was abolished following deletion of the -196 to - 132 bp fragment.

CONCLUSIONSIL-6 promotes GH secretion and synthesis by rat MtT/S somatotroph cells. The stimulatory effect of IL-6 on hGH gene promoter appears to require the activation of MEK and p38 MAPK, and a fragment of promoter sequence that spans the - 196 to - 132 bp of the gene, but may be unlinked with Pit-1 protein.

Animals ; Cell Line ; Gene Expression Regulation ; Human Growth Hormone ; genetics ; metabolism ; Humans ; Interleukin-6 ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; genetics ; metabolism ; MAP Kinase Signaling System ; physiology ; Mitogen-Activated Protein Kinase Kinases ; genetics ; metabolism ; Promoter Regions, Genetic ; Rats ; Somatotrophs ; cytology ; metabolism ; p38 Mitogen-Activated Protein Kinases ; genetics ; metabolism

OBJECTIVETo quantify the interleukin (IL)-1beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides ([PS) extracted from Porphyromonoas endodontalis (P. endodontalis) in osteoblasts, and to relate P. endodontalis LPS to the bone resorptive pathogenesis in the lesions of chronic apical periodontitis.

METHODSMG63 cells was pretreated with PD98059 or SB203580 for 1 h and then treated with P. endodontolis LPS for 6 h. The expression of IL-1beta mRNA and IL-6 mRNA were detected by reverse transcription polymerase chain reaction (RT-PCR) technique.

RESULTSThe production of IL-1beta mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with PD98059. Both of the production of IL-1beta mRNA and JL-6 mRNA induced by P. endodontalis LPS decreased in osteoblasts pretreated with SB203580.

CONCLUSIONThe synthesis of IL-1beta mRNA stimulated by Pendodontalis LPS in MG63 probably occur via extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen activated protein kinase (MAPK) signal transduction system. The synthesis of IL-6 mRNA stimulated by P.endodontalis LPS in MG63 probahly occur via p38MAPK signal transduction system.

Humans ; Imidazoles ; Interleukin-6 ; Lipopolysaccharides ; MAP Kinase Signaling System ; Osteoblasts ; Porphyromonas endodontalis ; Pyridines ; RNA, Messenger ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases

OBJECTIVETo study the effects of 50 Hz power-frequency magnetic fields on signal transduction pathway of P38 mitogen-activated protein kinase (P38 MAPK), and explore the cellular signal transduction mechanism of the biological effects induced by power-frequency magnetic fields.

METHODSChinese hamster lung (CHL) cell line was exposed to power-frequency magnetic fields with two intensities(0.1 and 0.4 mT) for different exposure durations. The cytoplasmic protein was extracted. The phosphorylated(activated) and non-phosphorylated P38 MAPK and MKK3/MKK6 were measured by Western blotting analysis with their specific corresponding antibodies.

RESULTSPower-frequency magnetic fields at 0.4 mT for 10 min could transitorily induce the activation of P38 MAPK and after 15 min the phosphorylation of P38 MAPK restored to control level, while 0.1 mT power-frequency magnetic fields could not induce the activation of P38 MAPK within 24 h. However, both 0.1 mT and 0.4 mT power-frequency magnetic fields could not phosphorylate(activate) the MKK3/MKK6, which is a general upstream kinase of P38 MAPK.

CONCLUSIONPower-frequency magnetic fields could transitorily activate the P38 MAPK, but not MKK3/MKK6. The activation mechanism of P38 MAPK needs to be further identified.

Animals ; Cell Line ; Cricetinae ; Cricetulus ; Enzyme Activation ; radiation effects ; Lung ; enzymology ; radiation effects ; MAP Kinase Kinase 3 ; metabolism ; MAP Kinase Kinase 6 ; metabolism ; Magnetics ; p38 Mitogen-Activated Protein Kinases ; metabolism ; radiation effects

The aim of the present study was to investigate the role of mitogen-activated protein kinase kinase 6 (MKK6)-P38 signaling pathway in cyclic mechanical stretch-induced high mobility group box 1 protein (HMGB1) expression in alveolar macrophages. In the study, Sprague-Dawley rats were anesthetized and then sacrificed by bloodletting. The lungs were lavaged six times with prechilled PBS. Alveolar macrophages were isolated from lavage samples. Recombinant plasmids were transfected into alveolar macrophages with liposome DOTAP. Alveolar macrophages transfected with P38(AF)/pGFP and MKK6b(E)/pGFP plasmids were taken as treated groups, while the groups that transfected with pcDNA3 plasmid and pGFP plasmid served as blank transfection group and control group, respectively. All the groups were then cultured in 6-well Bioflex cell culture plates and exposed to cyclic mechanical stretch at 20% elongation using Flexercell 4000T cell stretching unit. The results showed that the transfection of MKK6b(E) led to a marked increases in P38 kinase activity compared with control group. In contrast, the transfection of P38(AF) significantly inhibited P38 kinase activity. Compared with control group, HMGB1 protein and mRNA expression in MKK6b(E) transfected cells increased markedly, while HMGB1 expression in P38(AF) transfected cells decreased markedly. These results suggest that MKK6-P38 MAPK signaling pathway regulates the expression of HMGB1 induced by cyclic mechanical stretch in alveolar macrophages.
Animals ; Cells, Cultured ; HMGB1 Protein ; metabolism ; MAP Kinase Kinase 6 ; metabolism ; Macrophages, Alveolar ; enzymology ; Phosphorylation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Stress, Mechanical ; p38 Mitogen-Activated Protein Kinases ; metabolism

AIM: (-)-Epigallocatechin-3-gallate (EGCG), a major compound of tea polyphenols, exhibited antitumor activity in previous studies. In these studies, EGCG usually inhibits EGFR, and impairs the ERK1/2 phosphorylation in tumor cells. The aim was to clarify the mechanism of ERK1/2 activation induced by EGCG. METHOD: Jurkat and 293T cells were treated with EGCG in different culture conditions. Western Blotting (WB) was employed to analyze ERK1/2 and MEK phosphorylation. Cetuximab and FR180204 were used to inhibit cell signaling. The stability of EGCG was assessed by HPLC. The concentration of hydrogen peroxide generated by the auto-oxidation of EGCG was determined by photocolorimetric analysis. RESULTS: Activation of ERK1/2 was observed to be both time-and dose-dependent. Stimulation of cell signaling was dependent on MEK activity, but independent of EGFR activity. Unexpectedly, EGCG was depleted within one hour of incubation under traditional culture conditions. Auto-oxidation of EGCG generated a high level of hydrogen peroxide in the medium. Addition of catalase and SOD to the acidic medium inhibited the oxidation of EGCG. However, this particular condition also prevented the phosphorylation of ERK1/2. The generation of ROS by hydrogen peroxide may also induce ERK1/2 activation in Jurkat cells. CONCLUSION ERK1/2 phosphorylation was caused by auto-oxidation of EGCG. Traditional culture conditions were determined to be inappropriate for EGCG research.
Camellia sinensis ; chemistry ; Catalase ; metabolism ; Catechin ; analogs & derivatives ; pharmacology ; Humans ; Hydrogen Peroxide ; metabolism ; Jurkat Cells ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 6 ; metabolism ; Oxidation-Reduction ; Phosphorylation ; Plant Extracts ; pharmacology ; Polyphenols ; pharmacology ; Superoxide Dismutase ; metabolism

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.
Butadienes ; Chemokine CCL2 ; Chromones ; Humans ; Imidazoles ; Indoles ; Inflammation ; Leukemia ; Macrophages ; Maleimides ; Mitogen-Activated Protein Kinases ; Morpholines ; Nitriles ; Phosphatidylcholines ; Phosphorylation ; Protein Kinase C ; Pyridines ; Toll-Like Receptor 6 ; Toll-Like Receptors ; Up-Regulation

PURPOSE: Adriamycin(R) is one of the most commonly used drugs in the treatment of breast cancer. This study was performed to understand the molecular mechanisms of drug resistance in breast cancer cells. MATERIALS AND METHODS: We have analyzed the MCF-7 breast cell line and its adriamycin-resistant variants, MCF-7/ADR using human 10 K element cDNA microarrays. RESULTS: We defined 68 genes that were up-regulated (14 genes) or down-regulated (54 genes) in adriamycin resistant breast cancer cells. Several genes, such as G protein-coupled receptor kinase 5, phospholipase A2, guanylate cyclase 1, vimentin, matrix metalloproteinase 1 are up-regulated in drug resistant cells. Several genes, such as interferon, alpha-inducible protein 27, forkhead box M1, mitogen-activated protein kinase 6, regulator of mitotic spindle assembly 1 and tumor necrosis factor superfamily are down-regulated in adriamycin resistant cells. The altered expression of genes observed in microarray was verified by RT-PCR. CONCLUSION: These findings show that cDNA microarray analysis can be used to obtain gene expression profiles reflecting the effect of anticancer drugs on breast cancer cells. Such data may lead to the assigning of signature expression profiles of drug-resistant tumors which may help predict responses to drugs and assist in the design of tailored therapeutic regimens to overcome drug resistance.
Breast Neoplasms* ; Breast* ; Cell Line ; DNA, Complementary* ; Doxorubicin* ; Drug Resistance ; Gene Expression* ; Guanylate Cyclase ; Humans ; Interferons ; Matrix Metalloproteinase 1 ; Mitogen-Activated Protein Kinase 6 ; Oligonucleotide Array Sequence Analysis* ; Phospholipases A2 ; Phosphotransferases ; Spindle Apparatus ; Transcriptome* ; Tumor Necrosis Factor-alpha ; Vimentin

We have previously shown that the specific phosphatidylinositol 3-kinase inhibitor LY294002 (LY29), and its inactive analog LY303511 (LY30), inhibit a monocyte chemoattractant protein-1 (MCP-1) expression in human umbilical vein endothelial cells; these results suggest the potential of LY30 as an anti-inflammatory drug. In this study, we determined the effects of LY30 on the production of various inflammatory cytokines in human macrophagic THP-1 cells which were stimulated with lipopolysaccharide (LPS). LY30 selectively suppressed the mRNA expression of IL-12 p40, TNF-α, and MCP-1 without affecting the expression of IL-1α, IL-6, and IL-8. Inhibition of the production of IL-12 and TNF-α by LY30 was also demonstrated using ELISA assays. In order to elucidate the mechanisms of the action of LY30, we examined the role played by the mitogen-activated protein kinases and the key transcription factors, AP-1 and NF-κB in LPS-stimulated THP-1 cells. The results revealed that LY30 inhibited LPS-induced activation of ERK, but not p38 or JNK. Furthermore, the AP-1 DNA binding activity was suppressed by LY30 based upon the dosage, whereas NF-κB DNA binding was not affected. These results suggest that LY30 selectively inhibits cytokine production in the LPS-stimulated macrophagic THP-1 cells by downregulating the activation of ERK and AP-1.
Chemokine CCL2 ; Cytokines ; DNA ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; Humans* ; Interleukin-12 ; Interleukin-6 ; Interleukin-8 ; Mitogen-Activated Protein Kinases ; Phosphatidylinositol 3-Kinase ; RNA, Messenger ; Transcription Factor AP-1 ; Transcription Factors

The present study explored the anti-inflammatory and anti-thrombotic mechanism of Jingfang Granules on tail thrombosis induced by carrageenan in mice. Thirty-two male ICR mice were randomly divided into a control group, a model group, a Jingfang Granules group, and a positive drug(aspirin) group, with eight mice in each group. The thrombosis model was induced by intraperitoneal injection of carrageenan(45 mg·kg~(-1)) combined with low-temperature stimulation, and the mice were treated with drugs for 7 days before modeling. Twenty-four hours after modeling, blood was detected for four blood coagulation indices in each group. The enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of plasma interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and other inflammatory factors. The tails of mice in each group were cut off to observe tail lesions and measure the length of the thrombus. The protein expression and phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and p38 mitogen-activated protein kinase(p38 MAPK) in spleen tissues were detected by Western blot. The results showed that dark red thrombus appeared in the tails of mice in each group. The length of the black part accounted for about 40% of the total tail in the model group. Additionally, the model group showed prolonged prothrombin time(PT), increased fibrinogen(FIB) content, and shortened activated partial thromboplastin time(APTT). Compared with the model group, the groups with drug intervention displayed shortened black parts in the tail and improved four blood coagulation indices(P<0.05). As revealed by ELISA, the expression levels of TNF-α, IL-1β, and IL-6 in the mouse plasma were significantly up-regulated in the model group, and those in the groups with drug intervention were reduced as compared with the model group(P<0.05). As demonstrated by Western blot, the protein expression and phosphorylation levels of ERK1/2 and p38 MAPK in the spleen tissues were significantly elevated in the model group, while those in the Jingfang Granules group were down-regulated as compared with the model group with a significant difference. Jingfang Granules can inhibit tail thrombosis of mice caused by carrageenan presumedly by inhibiting the activation of ERK1/2 and p38 MAPK signaling pathways.
Animals ; Carrageenan/adverse effects* ; Interleukin-6/metabolism* ; MAP Kinase Signaling System ; Male ; Mice ; Mice, Inbred ICR ; Signal Transduction ; Thrombosis/drug therapy* ; Tumor Necrosis Factor-alpha/metabolism* ; p38 Mitogen-Activated Protein Kinases/metabolism*