OBJECTIVETo study the effects of benzo(a)pyrene (BaP) on the cell cycle distribution and activities of mitogen-activated protein kinase (MAPK) signal molecules (ERK1/2, JNK1/2 and p38) in human embryo lung cells (HELF), and to investigate the relationship between alterations of MAPK protein phosphorylation and the cell cycle distributions.

METHODSThe phosphorylation of MAPK were induced by exposing HELF cells to BaP at 0.1, 0.5, 2.5 and 12.5 micromol/L. The phosphorylation and protein expression levels of ERK1/2, JNK1/2 and p38 were determined through western-blotting assay. And the flow cytometry assay was used to measure the cell cycle effects in HELF cells after treatment with 2.5 micromol/L BaP for 24 h.

RESULTSThe phosphorylation levels of ERK1/2, JNK1/2 and p38 were significantly increased through BaP exposure. In addition, the phosphorylation of these three MAPKs has similar alteration pattern. We found that exposure of cells to 2.5 microM of BaP for 24 h resulted in a decrease of G(0) and G(1) population by 11.9% (F = 41.38, P < 0.01) and an increase of S population by 17.2% (F = 68.13, P < 0.01). Three chemical inhibitors of MAPK (AG126, SP600125 and SB203580) could significantly inhibit the cell cycle alteration because of BaP treatment.

CONCLUSIONERK1/2, JNK1/2 and p38 could positively regulate the BaP independently induced cell cycle alterations.

Benzo(a)pyrene ; toxicity ; Cell Cycle ; drug effects ; Cells, Cultured ; Fibroblasts ; drug effects ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lung ; cytology ; embryology ; MAP Kinase Kinase 4 ; metabolism ; MAP Kinase Signaling System ; drug effects ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinase 8 ; metabolism ; Mitogen-Activated Protein Kinase 9 ; metabolism ; Signal Transduction ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo investigate the effect of SP600125, a specific c-jun N-terminal protein kinase (JNK) inhibitor, on Staphylococcus aureus (S. aureus)-induced U937 cell death and the underlying mechanism.

METHODSThe human monocytic U937 cells were treated with S. aureus at different time with or without SP600125. Cell apoptosis was analyzed by flow cytometry. JNK, Bax, and caspase-3 activities were detected by Western blotting.

RESULTSS. aureus induced apoptosis in cultured U937 cells in a time-dependent manner. Expression of Bax and phospho-JNK significantly increased in S. aureus-treated U937 cells, and the level of activated caspase-3 also increased in a time-dependent manner. Inhibition of JNK with SP600125 significantly inhibited S. aureus-induced apoptosis in U937 cells.

CONCLUSIONSS. aureus can induce apoptosis in U937 cells by phosphorylation of JNK and activation of Bax and caspase-3. SP600125 protects U937 cells from apoptosis induced by S. aureus via inhibiting the activity of JNK.

Anthracenes ; pharmacology ; Apoptosis ; physiology ; Caspase 3 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Macrophages ; cytology ; metabolism ; microbiology ; Mitogen-Activated Protein Kinase 8 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 9 ; antagonists & inhibitors ; metabolism ; Phosphorylation ; drug effects ; Protein Kinase Inhibitors ; pharmacology ; Signal Transduction ; physiology ; Staphylococcus aureus ; physiology ; U937 Cells ; bcl-2-Associated X Protein ; metabolism

The aim of this study was to investigate the apoptosis-inducing effect of gambogic acid (GA) on Jurkat cells and its underlying signaling pathway. Apoptosis induced by GA and some inhibitors was assayed by Annexin V/PI doubling staining. The levels of caspase 3, caspase 8 and caspase 9 activated in living Jurkat cells were measured by flow cytometry. The expressions of caspase 3, caspase 9, p-JNK and P38 were detected by Western blot. The results showed that GA induced apoptosis of Jurkat cells in a dose-dependent manner. The positive cell number of activated caspase 3, caspase 8, caspase 9 and the levels of activated caspase 3, caspase 9, p-JNK, P38 increased after Jurkat cells were treated with GA. ROS, CaMKII, caspase 3, caspase 9, MAPKK, JNK1/2 and P38 inhibitors had some significant effect on GA-induced apoptosis. ROS, CaMKII, MAPKK, JNK1/2 and P38 inhibitors decreased the levels of activated caspase 3, caspase 9 by GA.ROS, CaMKII, MAPKK, JNK1/2 inhibitors decreased the levels of p-JNK by GA. ROS, CaMKII, MAPKK, P38 inhibitors decreased the levels of P38 by GA. It is concluded that GA induced apoptosis of Jurkat cells by activated caspases through activating of ROS-CaMKII-MAPKK-JNK/P38 pathway.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Jurkat Cells ; MAP Kinase Signaling System ; drug effects ; Xanthones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

Nerve growth factor (NGF) is known to regulate both cancer cell survival and death signaling, depending on the cellular circumstances, in various cell types. In this study, we showed that NGF strongly upregulated the protein level of tropomyosin-related kinase A (TrkA) in TrkA-inducible SK-N-MC cancer cells, resulting in increases in various TrkA-dependent cellular processes, including the phosphorylation of c-Jun N-terminal kinase (JNK) and caspase-8 cleavage. In addition, NGF enhanced TrkA-induced morphological changes and cell death, and this effect was significantly suppressed by the JNK inhibitor SP600125, but not by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin. To investigate novel targets associated with the enhancement of TrkA-induced SK-N-MC cell death caused by NGF, we performed Coomassie Brilliant Blue staining and two-dimensional (2D) proteomic analysis in TrkA-inducible SK-N-MC cells. We identified 31 protein spots that were either greatly upregulated or downregulated by TrkA during NGF treatment using matrix-associated laser desorption/ionization time of flight/time of flight mass spectrometry, and we analyzed the effects of SP600125 and wortmannin on the spots. Interestingly, 11 protein spots, including heterogeneous nuclear ribonucleoprotein K (hnRNP K), lamin B1 and TAR DNA-binding protein (TDP43), were significantly influenced by SP600125, but not by wortmannin. Moreover, the NGF/TrkA-dependent inhibition of cell viability was significantly enhanced by knockdown of hnRNP K using small interfering RNA, demonstrating that hnRNP K is a novel target associated with the regulation of TrkA-dependent SK-N-MC cancer cell death enhanced by NGF.
Caspase 8 ; Cell Death* ; Cell Survival ; Heterogeneous-Nuclear Ribonucleoprotein K ; JNK Mitogen-Activated Protein Kinases ; Mass Spectrometry ; Nerve Growth Factor* ; Phosphatidylinositol 3-Kinase ; Phosphorylation ; Phosphotransferases ; RNA, Small Interfering

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

Activation of c-Jun N-terminal kinase (JNK) is associated with a wide range of disparate cellular responses to extracellular stimuli. In mammals, three JNK isoforms are known, and their differential regulation occurs in a stimulus- or a cell type-dependent manner. However, the underlying mechanism of this differential regulation has not been clearly elucidated. Here we demonstrated that JNK1 and JNK3 were activated in SH-SY5Y cells after treatment with H2O2 or UV. In SH-SY5Y cells overexpressing mJIP1, a splicing variant of a JNK scaffold protein JIP1, the H2O2-induced activities of both JNK1 and JNK3 were significantly suppressed. In the same cell line, however, UV-induced JNK1 activity was significantly suppressed, but JNK3 activity was not. During the RA-induced differentiation of SH-SY5Y cells, JNK1 was activated, whereas JNK3 was not, and this JNK1 activation was completely abolished in the cells overexpressing mJIP1. These results suggest that JIP1 plays a role in the regulation of the isoform-specific activation of JNKs in stimulus-dependent manner.
Cell Line ; Humans* ; JNK Mitogen-Activated Protein Kinases ; Mammals ; Neuroblastoma* ; Protein Isoforms

Middle cerebral artery occlusion (MCAO) results in cell death by activation of complex signal pathways for cell death and survival. Hypothermia is a robust neuroprotectant, and its effect has often been attributed to various mechanisms, but it is not yet clear. Upstream from the cell death promoters and executioners are several enzymes that may activate several transcription factors involved in cell death and survival. In this study, we immunohistochemically examined the phosphorylation of mitogen- activated protein kinase, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 kinase during early period of the ischemic injury, following 2 hours (h) of transient MCAO. Increased phosphorylation of ERK and p38 was observed in the vessels at 3 h, neuron-like cells at 6 and 12 h and glia-like cells at 12 h. Activation of JNK was not remarkable, and a few cells showed active JNK following ischemia. Phosphorylation of Elk-1, a transcription factor, was reduced by ischemic insult. Hypothermia attenuated the activation of ERK, p38 and JNK, and inhibited reduction of Elk-1. These data suggest that signals via different MAPK family members converge on the cell damage process and hypothermia protects the brain by interfering with these pathways.
Brain ; Cell Death ; Humans ; Hypothermia* ; Infarction, Middle Cerebral Artery ; Ischemia ; JNK Mitogen-Activated Protein Kinases ; Mitogen-Activated Protein Kinases* ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Signal Transduction ; Stroke* ; Transcription Factors

The epithelial cytokine response, associated with reactive oxygen species (ROS), is important in Helicobacter pylori (H. pylori)-induced inflammation. H. pylori induces the production of ROS, which may be involved in the activation of mitogen-activated protein kinases (MAPK), janus kinase/signal transducers and activators of transcription (Jak/Stat), and oxidant-sensitive transcription factor, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), and thus, expression of interleukin-8 (IL-8) in gastric epithelial cells. alpha-lipoic acid, a naturally occurring thiol compound, is a potential antioxidant. It shows beneficial effects in treatment of oxidant-associated diseases including diabetes. The present study is purposed to investigate whether alpha-lipoic acid inhibits expression of inflammatory cytokine IL-8 by suppressing activation of MAPK, Jak/Stat, and NF-kappaB in H. pylori-infected gastric epithelial cells. Gastric epithelial AGS cells were pretreated with or without alpha-lipoic acid for 2 h and infected with H. pylori in a Korean isolate (HP99) at a ratio of 300:1. IL-8 mRNA expression was analyzed by RT-PCR analysis. IL-8 levels in the medium were determined by enzyme-linked immunosorbent assay. NF-kappaB-DNA binding activity was determined by electrophoretic mobility shift assay. Phospho-specific and total forms of MAPK and Jak/Stat were assessed by Western blot analysis. ROS levels were determined using dichlorofluorescein fluorescence. As a result, H. pylori induced increases in ROS levels, mRNA, and protein levels of IL-8, as well as the activation of MAPK [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase 1/2 (JNK1/2), p38], Jak/Stat (Jak1/2, Stat3), and NF-kappaB in AGS cells, which was inhibited by alpha-lipoic acid. In conclusion, alpha-lipoic acid may be beneficial for prevention and/or treatment of H. pylori infection-associated gastric inflammation.
Enzyme-Linked Immunosorbent Assay ; Epithelial Cells/metabolism ; Gastric Mucosa/*drug effects/metabolism/microbiology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/immunology/*metabolism ; Helicobacter pylori/drug effects/*pathogenicity ; Humans ; Interleukin-8/genetics/*metabolism ; JNK Mitogen-Activated Protein Kinases ; Janus Kinase 1 ; Mitogen-Activated Protein Kinases/*biosynthesis ; NF-kappa B/*metabolism ; RNA, Messenger/isolation & purification/metabolism ; Reactive Oxygen Species/metabolism ; STAT3 Transcription Factor ; Stomach/metabolism/*microbiology ; Thioctic Acid/*pharmacology

JNK signaling pathway plays an important role in regulation of cell growth, proliferation, differentiation, migration and apoptosis. Studies have shown that JNK signaling pathway is closely related to autophagy, but the underlying mechanisms are not clear. Clarification of the correlation between the JNK signaling pathway and autophagy may provide opportunities for identifying potential molecular targets for the treatment of diseases associated with autophagy.
Autophagy ; Humans ; JNK Mitogen-Activated Protein Kinases ; MAP Kinase Signaling System

BACKGROUNDMesangial hypercellularity is a critical early histopathological finding in human and experimental glomerular diseases. Hyperlipidemia and the glomerular deposition of lipoproteins are commonly associated with mesangial hypercellularity and play an important pathobiological role in the development of glomerular diseases. The activated cytoplasmic mitogen-activated protein kinase (MAPK), including mainly extracellular-signal regulated protein kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, has been thought to translocate into the nucleus and activate various transcription factors and protooncogenes associated with cell growth and proliferation. Lipoprotein (a) (Lp(a)) has been shown to stimulate proliferation of mesangial cells, but the events of Lp(a) signaling have not yet been characterized. The purpose of this study was to investigate the signal transduction pathways involved in Lp(a)-induced cell proliferation and provide an evidence for the participation of Lp(a) in intracellular signaling pathways for mesangial cell proliferation.

METHODSLp(a) was isolated from a patient who was being treated with low density lipoprotein (LDL)-apheresis by density gradient ultracentrifugation and then chromatography. Human mesangial cells (HMCs) were isolated by the sequential sieving technique and stimulated with Lp(a) in different concentration and time course. The DNA synthesis of the cells was measured by [3H] thymidine incorporation for detecting the proliferation. The expression of all the three members of MAPK family, including ERK1/ERK2, JNK, and p38, and their phosphorylation were detected by Western blotting.

RESULTSLp(a) could induce a significant dose-dependent proliferation of HMCs. The 3H-TdR incorporation was 1.64+/-0.31, 1.69+/-0.48, 3.59+/-0.68 (P<0.01), 4.14+/-0.78 (P<0.01), and 4.05+/-0.55 (P<0.01) (10(3) cpm) at the Lp(a) concentration of 0, 5, 10, 25, and 50 microg/ml, respectively. Lp(a) induced an increase in ERK1/ERK2 phosphorylation between 5 and 60 minutes, and in JNK phosphorylation between 15 and 30 minutes after incubating with HMCs, whereas the level of p38 and its phosphorylation was not changed.

CONCLUSIONSLp(a) could stimulate the proliferation of HMCs by activiating the phosphorylation of ERK1/ERK2 and JNK MAPK signaling pathway, whereas p38 pathway had no effect on the Lp(a)-induced HMC proliferation, which indicated that three MAPKs seem to be distinctly involved in the effect. In particular, it also provides the evidence that Lp(a) may act as one of the major endogenous modulators for mitogenic signaling response and cell proliferation within the glomerulus.

Blotting, Western ; Cells, Cultured ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Lipoprotein(a) ; pharmacology ; Mesangial Cells ; Mitogen-Activated Protein Kinase 1 ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphorylation ; drug effects ; p38 Mitogen-Activated Protein Kinases ; metabolism