Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase Inhibitor Research Articles

Lipopolysaccharide induced Interleukin-6 production is mediated through activation of ERK 1/2, p38 MAPK, MEK, and NFκB in chicken thrombocytes

Thrombocytes express Toll-like receptor 4 and apparently use both mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFKB) pathways for nuclear signaling. However, it is not well known if the same enzyme systems found in mammalian cells are fully functional in chickens. Therefore, kinase inhibitors were used with thrombocytes to block kinases in lipopolysaccharide (LPS) stimulated cells to determine if interleukin (IL)-6 expression and production would be diminished. Results demonstrated that extracellular-signal-regulated kinase (ERK)1/2 and p38 MAPK pathways influence gene expression of IL-6 through treatment with either ERK or p38 MAPK inhibitor. In addition, thrombocyte lysates from cells treated with ERK, p38, mitogen-activated protein kinase kinase (MEK)1/2 and inhibitor of nuclear factor kappa-B kinase (IKK) inhibitor showed different levels of the phosphorylated form of ERK1/2, p38 and NFκB. Furthermore, IL-6 gene expression and production were significantly upregulated in LPS stimulated thrombocytes relative to all inhibitor-treated cells.

Involvement of p38 mitogen-activated protein kinase in acquired gemcitabine-resistant human urothelial carcinoma sublines

Resistance to chemotherapeutic drugs is one of the major challenges in the treatment of cancer. A better understanding of how resistance arises and what molecular alterations correlate with resistance is the key to developing novel effective therapeutic strategies. To investigate the underlying mechanisms of gemcitabine (Gem) resistance and provide possible therapeutic options, three Gem-resistant urothelial carcinoma sublines were established (NG0.6, NG0.8, and NG1.0). These cells were cross-resistant to arabinofuranosyl cytidine and cisplatin, but sensitive to 5-fluorouracil. The resistant cells expressed lower values of [hENT1 × dCK/RRM1 × RRM2] mRNA ratio. Two adenosine triphosphate-binding cassette proteins ABCD1 as well as multidrug resistance protein 1 were elevated. Moreover, cyclin D1, cyclin-dependent kinases 2 and 4 were upregulated, whereas extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK) activity were repressed significantly. Administration of p38 MAPK inhibitor significantly reduced the Gem sensitivity in NTUB1 cells, whereas that of an extracellular signal-regulated kinase MAPK inhibitor did not. Furthermore, the Gem-resistant sublines also exhibited higher migration ability. Forced expression of p38 MAPK impaired the cell migration activity and augmented Gem sensitivity in NG1.0 cells. Taken together, these results demonstrate that complex mechanisms were merged in acquiring Gem resistance and provide information that can be important for developing therapeutic targets for treating Gem-resistant tumors.

Fluid Flow Stress Affects Peritoneal Cell Kinetics: Possible Pathogenesis of Peritoneal Fibrosis

Peritoneal fibrosis is an essential precursor condition to the development of encapsulating peritoneal sclerosis (EPS). This serious complication leads to a high mortality rate in peritoneal dialysis (PD) patients. Although several factors, including highly concentrated glucose in the dialysis solution, are believed to be potent agents for peritoneal fibrosis, the underlying mechanism remains unclear. During PD, the dialysis solution continuously generates fluid flow stress to the peritoneum under peristalsis and body motion. Fluid flow stress has been implicated as playing a critical role in the physiologic responses of many cell types. We therefore hypothesized that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis leading to EPS. To generate fluid flow stress, culture containers were placed on a rotatory shaker in a thermostatic chamber. In this system, the shaker rotated at a speed of 25 rpm with a radius of 1.5 cm. Mesothelial cells were cultured in low-glucose (1000 mg/L) or high-glucose (4500 mg/L) complete medium with and without flow stress. Fluid flow stress promoted hyperplasia and epithelial-mesenchymal transition (EMT) of mesothelial cells independent of glucose concentration. Fluid flow stress inhibited expression of ERK (extracellular signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) in mesothelial cells. Administration of ERK and p38 MAPK inhibitors replicated the stress-induced morphology of mesothelial cells. The present data indicate that fluid flow stress promotes hyperplasia and EMT of mesothelial cells via the MAPK axis, suggesting that fluid flow stress may be involved in the pathogenesis of peritoneal fibrosis.

The effect of extracellular signal-regulated kinases signaling pathway inhibition on histone phosphorylation and gene expression in colorectal canoer cells

Objective To study the effects of extracellular-signal regulated kinase mitogenactivated protein kinase (ERK-MAPK) signaling pathway inhibition on histone phosphorylation and the related gene expression in human colorectal cancer cells.Methods Two human colorectal cancer cell lines (SW1116 and HCT116) were cultured and treated with gradient(0,20,40/μmol/L) doses of ERK-MAPK signaling pathway inhibitor U0126.Cell viability was determined by cell counting kit 8 (CCK-8) assay.Cell cycle distribution was assessed by flow cytometry.The expression levels of histone H3 kinases including ribosomal S6 serine-threonine kinase (RSK-2) and mitogen-and stressactivated protein kinase 1 and 2 (MSK1 and MSK2),and the levels of histone H3 (Ser10) phosphorylation and c-Fos protein were detected using Western blotting.Results Treatment of these two human colorectal cancer cell lines with ERK-MAPK inhibitor resulted in a time and dose-dependent inhibition of cell proliferation significantly. Proliferation rate of HCT116 was reduced to 47% at 72 hours after 40/μmol/L U0126 treatment. Cell cycle analysis showed that the percentage of phase G0/G1 cells significantly increased (P<0. 01) and the percentage of phase S cells decreased (P<0.01) after treatment with ERK-MAPK inhibitor. The expression of MSK1 and RSK2 reduced obviously in both of human colorectal cancer cell lines treated with U0126, which resulted in a 28% and 40% reduction of levels of MSK1 and RSK2 as compared with control HCT116 cells respectively,while no detectable change in the expression of MSK2 was found. Consistent with this, the expression level of histone H3 (ser10) phosphorylation was markedly down-regulated by ERK-MAPK inhibitor, and the related protein c-Fos expression decreased accordantly. Conclusions Decreased ERK-MAPK signaling pathway may reduce histone H3 (Ser10) phosphorylation via suppression of the activity of histone H3 kinase including MSK1 and RSK2, but not MSK2, consequently decrease the expression of c-Fos protein, which results in the inhibition of colorectal cancer cells proliferation. Key words: Cell line,Tumor; Extracellular signal-regulated kinases; Cell cycle; Histone phosphorylation

Receptor for advanced glycation end-products (RAGE) modulates neutrophil adhesion and migration on glycoxidated extracellular matrix

AGEs (advanced glycation end-products) accumulate in collagen molecules during uraemia and diabetes, two diseases associated with high susceptibility to bacterial infection. Because neutrophils bind to collagen during their locomotion in extravascular tissue towards the infected area we investigated whether glycoxidation of collagen (AGE-collagen) alters neutrophil migration. Type I collagen extracted from rat tail tendons was used for in vitro glycoxidation (AGE-collagen). Neutrophils were obtained from peripheral blood of healthy adult volunteers and were used for the in vitro study of adhesion and migration on AGE- or control collagen. Glycoxidation of collagen increased adhesion of neutrophils to collagen surfaces. Neutrophil adhesion to AGE-collagen was inhibited by a rabbit anti-RAGE (receptor for AGEs) antibody and by PI3K (phosphoinositide 3-kinase) inhibitors. No effect was observed with ERK (extracellular-signal-regulated kinase) or p38 MAPK (mitogen-activated protein kinase) inhibitors. AGE-collagen was able to: (i) induce PI3K activation in neutrophils, and (ii) inhibit chemotaxis and chemokinesis of chemoattractant-stimulated neutrophils. Finally, we found that blocking RAGE with anti-RAGE antibodies or inhibiting PI3K with PI3K inhibitors restored fMLP (N-formylmethionyl-leucyl-phenylalanine)-induced neutrophil migration on AGE-collagen. These results show that RAGE and PI3K modulate adhesion and migration rate of neutrophils on AGE-collagen. Modulation of adhesiveness may account for the change in neutrophil migration rate on AGE-collagen. As neutrophils rely on their ability to move to perform their function as the first line of defence against bacterial invasion, glycoxidation of collagen may participate in the suppression of normal host defence in patients with diabetes and uraemia.

Inhibition of the Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway Decreases DNA Methylation in Colon Cancer Cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.

P38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes

Iron is essential for neoplastic cell growth, and iron chelators have been tested for potential anti-proliferative and anti-cancer effects, but the effects of iron chelators on oral cancer have not been clearly elucidated. To determine the mechanism of cell death induced by iron chelators, we explored the pathways of the three structurally related mitogen-activated protein (MAP) kinase subfamilies during iron chelator-induced apoptosis and differentiation of immortalized human oral keratinocytes (IHOK) and oral cancer cells (HN4). The iron chelator deferoxamine (DFO) exerted potent time- and dose-dependent inhibitory effects on the growth and apoptosis of IHOK and HN4 cells. DFO strongly activates p38 MAP kinase and extracellular signal-regulated kinase (ERK), but does not activate c-Jun N-terminal kinase/stress-activated protein kinase. Of the three MAP kinase blockers used, the selective p38 MAP kinase inhibitor SB203580 and ERK inhibitor PD98059 protected IHOK and HN4 cells against iron chelator-induced cell death, which indicates that the p38 and ERK MAP kinase is a major mediator of apoptosis induced by this iron chelator. Interestingly, treatment of IHOK and HN4 cells with SB203580 and PD98059 abolished cytochrome c release, as well as the activation of caspase-3 and caspase-8. DFO suppressed the expression of epithelial differentiation markers such as involucrin, CK6, and CK19, and this suppression was blocked by p38 and ERK MAP kinase inhibitors. Collectively, these data suggested that p38 and ERK MAP kinase plays an important role in iron chelator-mediated cell death and in the suppression of differentiation of oral immortalized and malignant keratinocytes, by activating a downstream apoptotic cascade that executes the cell death pathway.

Effects of neurosteroids on glucocorticoid receptor-mediated gene transcription in LMCAT cells—A possible interaction with psychotropic drugs

Aberrant activity of hypothalamic–pituitary–adrenal (HPA) axis is often observed in psychiatric disorders and both antidepressant and antipsychotic drugs are known to ameliorate some deleterious effects of glucocorticoids on brain function. Some neurosteroids possess antidepressant and neuroleptic-like properties and attenuate the stress-activated HPA axis activity. However, intracellular mechanism of neurosteroid interaction with glucocorticoids has not been elucidated. We evaluated effects of some neurosteroids on functional activity of glucocorticoid receptor (GR) in vitro. A combined treatment with antipsychotic drugs and involvement of some protein kinases in allopregnanolone effect on GR function were also studied. The effects of allopregnanolone, its two isomers (5β-pregnan-3α-ol-20-one and 5α-pregnan-3β-ol-20-one) and dehydroepiandrosterone sulfate (DHEAS) on the corticosterone-induced chloramphenicol acetyl transferase (CAT) activity were evaluated in mouse fibroblast cells stably transfected with mouse mammary tumor virus (MMTV)-CAT plasmid. We found that allopregnanolone (1–100 μM) and, to a lesser extent, both its isomers inhibited the GR-mediated gene transcription in a concentration-dependent manner. In contrast, DHEAS at the concentration up to 100 μM was inactive. Further experiments revealed that allopregnanolone and antipsychotic drugs (chlorpromazine and clozapine) showed a moderate, additive inhibitory effect on the GR function. With respect to intracellular mechanism of allopregnanolone action, we showed that this neurosteroid inhibited protein kinase C (PKC) activity, decreased the level of PKCα isoenzyme in the membrane fraction and decreased the amount of active phosphorylated form of extracellular signal-regulated kinase–mitogen-activated protein kinase (ERK-MAPK) in LMCAT cells. Since PKC and ERK-MAPK inhibitors attenuate the corticosterone-mediated gene transcription, the above findings suggest that allopregnanolone effect on GR function involves interaction with these kinase pathways. On the other hand, allopregnanolone had no effect on protein kinase A (PKA) activity. These data indicate that pregnanolone derivatives, like antidepressants and antipsychotic drugs, may attenuate some glucocorticoid effects via inhibition of GR-mediated gene transcription. Furthermore, the inhibitory effect of allopregnanolone on the corticosterone-induced gene transcription in LMCAT cells depended on the inhibition of PKC and ERK-MAPK pathways.

Differential activation of ERK, p38, and JNK MAPK by nociceptin/orphanin FQ in the potentiation of prostaglandin cerebrovasoconstriction after brain injury

Fluid percussion brain injury elevates the cerebrospinal fluid (CSF) concentration of the opioid nociceptin/orphanin FQ (N/OFQ), which potentiates vasoconstriction to the prostaglandins U 46619, a thromboxane A 2 mimic, and prostaglandin (PG)F 2a. This study investigated the role of the extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) isoforms of mitogen activated protein kinase (MAPK) in potentiated prostaglandin vasoconstriction after brain injury and the relationship of brain injury induced release of N/OFQ to MAPK. Pial artery diameter was measured with a video microscaler by observation through a glass coverslip cranial window placed in the parietal cortex of newborn pigs. Brain injury potentiated U 46619 induced pial artery vasoconstriction but U 0126 and SB 203580 (10 − 6 and 10 − 5 M, respectively) (ERK and p38 MAPK inhibitors) blocked the potentiation. In contrast, administration of SP 600125 (10 − 6 and 10 − 5 M) (JNK MAPK inhibitor) only attenuated brain injury induced U 46619 potentiation and such responses were significantly different than that in the presence of either U 0126 or SB 203580 after FPI. Co-administration of N/OFQ (10 − 10 M), the CSF concentration observed after brain injury, with U 46619 or PGF 2a under non brain injury conditions potentiated prostaglandin vasoconstriction but U 0126 and SB 203580 blocked such potentiation. Administration of SP 600125 modestly attenuated prostaglandin potentiation by N/OFQ. These data show that activation of ERK and p38 primarily contribute to potentiation of prostaglandin constriction after brain injury. These data suggest that N/OFQ differentially activates ERK, p38, and JNK MAPK to contribute to potentiated prostaglandin vasoconstriction after fluid percussion brain injury.

Burkholderia cepacia-induced IL-8 gene expression in an alveolar epithelial cell line: signaling through CD14 and mitogen-activated protein kinase.

Burkholderia cepacia is a prevalent pulmonary pathogen in patients with cystic fibrosis (CF). The lung pathology observed in patients with CF is postulated to be due to an overexpression of chemokines. This study investigated the induction of the neutrophil chemoattractant chemokine IL-8 and the signaling pathways activated by B. cepacia-infected human lung epithelial A549 (HLE) cells. Cells were infected with B. cepacia (genomovar III of the B. cepacia complex), and reverse transcriptase-PCR and ELISA for the cytokines were performed. B. cepacia (multiplicity of infection > or =4:1) induced HLE cells to significantly secrete IL-8 in a more potent manner than the predominant CF pathogen Pseudomonas aeruginosa (multiplicity of infection > or =64:1). IL-8 secretion by B. cepacia-infected HLE cells was abrogated by the gene transcription inhibitor actinomycin D and the protein translation inhibitor cycloheximide, confirming that B. cepacia-induced IL-8 secretion was mediated through de novo protein synthesis. Treatment of B. cepacia with proteinase K failed to down-regulate IL-8 secretion; furthermore, IL-8 secretion by B. cepacia-infected HLE cells was abrogated by > or =80% in the presence of anti-CD14 [specific lipopolysaccharide (LPS) receptor] antibody, thus suggesting that the IL-8-inducing component of B. cepacia was LPS and therefore dependent on CD14. The p38 mitogen-activated protein kinase (MAPK) inhibitor and the extracellular signal-regulated kinase MAPK inhibitor significantly abrogated IL-8 secretion by B. cepacia-infected HLE cells (SB203580, > or =80% inhibition; PD98059, > or =30% inhibition). In conclusion, B. cepacia-induced IL-8 secretion in A549 airway epithelial cells is more potent than P. aeruginosa; is mediated through LPS, which is CD14 dependent; and involves activation of the p38 and ERK MAPK pathways.