Mitogen-activated protein kinase modulation of nuclear factor-kappaB-induced granulocyte macrophage-colony-stimulating factor release from human alveolar macrophages

Granulocyte macrophage-colony-stimulating factor (GM-CSF), released from alveolar macrophages (AM), is an important regulator of eosinophil, T cell, and macrophage function and survival. We determined the mechanisms of GM-CSF regulation in AM from normal volunteers activated by lipopolysaccharide (LPS) by examining the role of nuclear factor-kappaB (NF-kappaB), and of p38 mitogen-activated protein (MAP) kinase and MAP kinase kinase (MKK-1). PD 098059 (10 microM), an inhibitor of upstream activator of MKK-1, inhibited GM-CSF expression, but the expression of GM-CSF was not inhibited by SB 203580 (10 microM), an inhibitor of p38-MAP kinase. Phosphorylation of extracellular signal-regulated kinase-1 (ERK-1), ERK-2, and p38 MAP kinase by LPS were demonstrated on Western blot analysis. LPS increased NF-kappaB:DNA binding as examined by electrophoretic mobility shift assay, but this was not suppressed by PD 098059 or by SB 203580. LPS induced an increase in NF-kappaB activation as examined by p50 translocation assay without suppression by PD 098059 or by SB 203580. SN50 (100 microM), an inhibitor of NF-kappaB translocation and the specific IKK-2-Inhibitor (AS602868; 10 microM), also prevented GM-CSF expression and release induced by LPS, indicating that GM-CSF release is NF-kappaB-dependent. PD 098059, but not SB 203580, inhibited LPS-induced histone acetyltransferase (HAT) activity, indicating chromatin modification. Furthermore, AS602868 and SN 50 suppressed LPS-induced HAT activity. TSA (10 ng/ml), an inhibitor of histone deacetylase (HDAC), reversed the inhibitory effect of PD 098059, SB 203580, SN 50 and AS602868 on GM-CSF release. GM-CSF expression and release in AM is controlled by NF-kappaB activation, and this is modulated by phosphorylation of MKK-1 and p38 MAP kinase acting on histone acetylation.     

Inhibition of p38 mitogen-activated protein kinase augments lipopolysaccharide-induced cell proliferation in CD14-expressing Chinese hamster ovary cells

CD14-expressing Chinese hamster ovary (CD14-CHO) cells, established by transfection of human CD14 DNA, acquired high responsiveness to lipopolysaccharide (LPS) through membrane-bound CD14 expression. LPS induced DNA synthesis and activated a series of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2 (Erk1/2), p38, and c-Jun N-terminal kinase/stress-activated protein kinase, in CD14-CHO cells but not in mock-transfected CHO cells. Anti-CD14 antibody completely abrogated both LPS-induced DNA synthesis and LPS-induced phosphorylation of those MAP kinases, suggesting a critical role of membrane-bound CD14 in LPS signaling. A p38 MAP kinase inhibitor, SB203580, markedly augmented LPS-induced DNA synthesis in CD14-CHO cells, whereas an Erk1/2 inhibitor, PD98059, had no affect. On the other hand, SB203580 exhibited no effect on epidermal growth factor-induced DNA synthesis in CD14-CHO cells, although PD98059 inhibited it significantly. The activation and inactivation of p38 MAP kinase with dominant negative and dominant positive mutants also suggested the participation of p38 MAP kinase in LPS-induced DNA synthesis. It was therefore suggested that the activation of p38 MAP kinase can negatively regulate LPS-induced cell proliferation in CD14-CHO cells.     

Regulation by intracellular glutathione of TNF-alpha-induced p38 MAP kinase activation and RANTES production by human pulmonary vascular endothelial cells

BACKGROUND: We have previously shown that p38 mitogen-activated protein (MAP) kinase regulates tumor necrosis factor-alpha (TNF-alpha)-induced RANTES production by human pulmonary vascular endothelial cells, and that sensitivity to TNF-alpha is inversely correlated with cellular reduction and oxidation (redox) state. However, a regulatory role of intracellular glutathione (GSH) in TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production has not been determined. In the present study, therefore, we extended our previous studies and focused on redox regulation on p38 MAP kinase activation. METHODS: Human pulmonary vascular endothelial cells were exposed to N-acetylcysteine (NAC) or buthionine sulfoximine (BSO), and then TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production were determined. RESULTS: The results showed that 1) NAC attenuated TNF-alpha-induced p38MAP kinase activation and RANTES production 2) SB 203580 as the specific inhibitor of p38 MAP kinase activity attenuated TNF-alpha-induced RANTES production 3) BSO facilitated TNF-alpha-induced p38 MAP kinase activation and RANTES production 4) SB 203580 attenuated BSO-mediated facilitation of TNF-alpha-induced RANTES production. CONCLUSIONS: These results indicated that TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human pulmonary vascular endothelial cells are inversely regulated by intracellular GSH levels.     

Intracellular glutathione regulates tumour necrosis factor-α-induced p38 MAP kinase activation and RANTES production by human bronchial epithelial cells

BACKGROUND: RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. The cellular reduction and oxidation (redox) changes are involved in the activation of p38 mitogen-activated protein (MAP) kinase and the induction of cytokine expression. It has previously been shown that tumour necrosis factor (TNF)-MA activates p38 mitogen-activated protein (MAP) kinase to produce cytokine, including RANTES, that N-acetylcysteine (NAC) attenuates cytokine production by human bronchial epithelial cells (BECs), and that sensitivity to TNFalpha is inversely correlated with cellular redox state. However, a role of cellular redox regulated by intracellular glutathione (GSH) in TNFalpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs has not been determined. OBJECTIVE: Human BECs were exposed to NAC or buthionine sulfoximine (BSO). TNFalpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs were then examined in order to clarify these issues. RESULTS: The results showed that: NAC attenuated TNFalpha-induced p38 MAP kinase activation and RANTES production; SB 203580 as the specific inhibitor of p38 MAP kinase activity attenuated TNF-alpha-induced RANTES production; BSO facilitated TNF-alpha-induced p38 MAP kinase activation and RANTES production; SB 203580 attenuated BSO-mediated facilitation of TNF-alpha-induced RANTES production; and the intracellular GSH increased in NAC-treated cells, whereas the intracellular GSH was reduced in BSO-treated cells. CONCLUSIONS: These results indicate that cellular redox regulated by GSH is critical for TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated RANTES production by human BECs.     

Roles of p38 mitogen-activated protein kinase,NF-kappaB,and protein kinase C in proinflammatory cytokine mRNA expression by human peripheral blood leukocytes,monocytes, and neutrophils in response to Anaplasma phagocytophila

Anaplasma phagocytophila, an obligately intracellular bacterium of granulocytes, causes human granulocytic ehrlichiosis. Within 2 h after addition of A. phagocytophila, interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and IL-6 mRNAs are induced in human peripheral blood leukocytes (PBLs) or monocytes in vitro. However, neutrophils generate only IL-1beta mRNA. In the present study, signaling pathways for induction of these three cytokines were examined. TNF-alpha and IL-6 mRNA expression by PBLs was inhibited with SB 203580 (a p38 mitogen-activated protein kinase [MAPK] inhibitor), MG-132 (a proteasome inhibitor), and SN-50 (an NF-kappaB inhibitor). Activation of p38 MAPK and NF-kappaB mRNAs in monocytes was detectable within 15 to 30 min after addition of A. phagocytophila. Expression of these two cytokine mRNAs in PBLs and monocytes was also dependent on protein kinase C (PKC), protein kinase A (PKA), and protein tyrosine kinase (PTK). IL-1beta mRNA expression by neutrophils was not dependent on p38 MAPK, and p38 MAPK was not activated in neutrophils incubated with A. phagocytophila. IL-1beta mRNA induction by PBLs, monocytes, and neutrophils was dependent on PKC and PKA. Neutrophil expression of IL-1beta mRNA was dependent on transglutaminase, phospholipase C, and PTK, all of which are also required for internalization of A. phagocytophila. However, monocyte expression of IL-1beta mRNA was less dependent on these enzymes. These results suggest that A. phagocytophila transduces different signals between its host neutrophils and monocytes for proinflammatory cytokine generation.     

p38 MAP kinase regulates RANTES production by TNF-alpha-stimulated human pulmonary vascular endothelial cells

BACKGROUND: RANTES plays an important role in the production of allergic inflammation of the airway through its chemotactic activity for eosinophils. However, the intracellular signal regulating RANTES expression in human pulmonary vascular endothelial cells has not been determined. In the present study, therefore, we examined the role of p38 mitogen-activated protein (MAP) kinase in RANTES production by tumor necrosis factor (TNF)-alpha-stimulated pulmonary vascular endothelial cells in order to clarify the signal transduction pathway regulating RANTES production by pulmonary vascular endothelial cells. METHODS: We examined p38 MAP kinase activation, and the effect of SB 203580, as the specific inhibitor for p38 MAP kinase, on p38 MAP kinase activity and RANTES production by TNF-alpha-stimulated human pulmonary vascular endothelial cells. RESULTS: The results showed that TNF-alpha induced RANTES production and p38 MAP kinase activity in human pulmonary vascular endothelial cells. Abrogation of p38 MAP kinase activity by SB 203580 repressed TNF-alpha-induced p38 MAP kinase activity and RANTES production. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in the TNF-alpha-activated signaling pathway which regulates RANTES production by human pulmonary vascular endothelial cells.     

TGF-β1激活的p38MAPK在TGF-β1上调人卵巢癌细胞PAI-1表达中的作用

目的:纤溶酶原激活物抑制剂1(PAI-1)在凝血、创伤修复、炎症和肿瘤转移中起重要作用。已有报道转化生长因子β1(TGF-β1)能通过Smad通路诱导PAI-1表达,但TGF-β1能否通过激活非Smad通路诱导PAI-1表达尚不清楚,因此本研究探讨了在卵巢癌细胞中TGF-β1激活的非Smad通路p38丝裂原活化蛋白激酶(p38MAPK)和细胞外信号调节激酶(ERK)与TGF-β1上调PAI-1表达的关系。方法:用10μg/L TGF-β1处理卵巢癌SKOV3细胞和HO-8910细胞后,采用real-time PCR和Western blotting的方法检测PAI-1的表达,用磷酸化p38MAPK的抗体和磷酸化ERK的抗体检测p38 MAPK和ERK的激活情况,用p38 MAPK和ERK的特异性抑制剂SB203580和PD98059分别抑制其活性后,检测PAI-1的表达。结果:TGF-β1在卵巢癌细胞中可明显上调PAI-1mRNA和蛋白的表达,并可快速激活p38 MAPK和ERK。用p38 MAPK的抑制剂可以明显抑制TGF-β1上调PAI-1表达,但是抑制ERK活性对TGF-β1上调PAI-1表达没有明显影响。结论:TGF-β1激活的p38 MAPK通路参与了TGF-β1上调PAI-1的表达。     

p38 MAP kinase regulates TNFα-, IL-1α- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

BACKGROUND: RANTES and granulocyte macrophage-colony stimulating factor (GM-CSF) play an important role in the production of allergic inflammation of the airway through their chemotactic activity for eosinophils. Recent studies have indicated that p38 mitogen-activated protein (MAP) kinase regulates cytokine expression in various cells; however, the role of p38 MAP kinase in RANTES and GM-CSF production in human bronchial epithelial cells (BECs) has not yet been determined. OBJECTIVE: In the present study, we examined serine phosphorylation of MKK3 and MKK6 which is the upstream regulator of p38 MAP kinase and p38 MAP kinase activation in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha and platelet-activating factor (PAF)-stimulated BECs and the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on RANTES and GM-CSF expression in order to clarify the intracellular signal regulating RANTES and GM-CSF production by human BECs. RESULTS: The results showed that TNF alpha, IL-1 alpha and PAF induced serine phosphorylation of MKK3 and MKK6, and p38 MAP kinase activation in BECs. SB 203580 inhibited p38 MAP kinase activity and RANTES and GM-CSF production by TNF alpha-, IL-1 alpha- or PAF-stimulated human BECs. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in TNF alpha-, IL-1 alpha- or PAF-activated signalling pathway which regulates RANTES and GM-CSF production by BECs and that the specific inhibitor for p38 MAP kinase activity might be useful for the treatment of allergic inflammation of the airway.     

p38 mitogen-activated protein kinase regulates growth factor-induced mitogenesis of rat pulmonary myofibroblasts

Myofibroblast proliferation is a central feature of pulmonary fibrogenesis. Several growth factors, including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), stimulate myofibroblast growth by activating extracellular signal regulated kinases 1 and 2 (ERK1/2). In this report, we demonstrate that PDGF-BB and EGF also activate the p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 activity with the pyridinylimidazole compound SB203580 enhanced both PDGF-BB and EGF-stimulated DNA synthesis in rat lung myofibroblasts. ERK1/2 phosphorylation in response to either PDGF-BB or EGF treatment was significantly increased by pretreatment of cells with SB203580. We also demonstrated that ERK1/2-induced phosphorylation of PHAS-1 substrate was enhanced by inhibition of p38 MAP kinase with SB203580. However, SB203580 did not significantly increase growth factor-induced activation of MEK, the upstream kinase that phosphorylates ERK1/2. p38 MAP kinase was co-immunoprecipitated with ERK-1/2 following growth factor stimulation. Collectively, these data demonstrate that p38 MAP kinase activation negatively regulates PDGF- and EGF-mediated growth responses by directly interacting with ERK1/2 and suppressing its phosphorylation.     

The p38 mitogen-activated protein kinases can have opposing roles in the antigen-dependent or endotoxin-stimulated production of IL-12 and IFN-gamma.

The p38 mitogen-activated protein kinases (p38 MAPK) are activated in lymphocytes and acessory cells during innate and antigen-specific responses. We show that an inhibitor of two isoforms of p38 MAPK, SB 203580, inhibited the antigen-initiated production of IL-12, and IFN-gamma by cultures of splenic APC and naive CD4(+) T cells. Paradoxically, SB 203580 enhanced the LPS plus IFN-gamma-initiated production of IL-12 by peritoneal exudate macrophages, and the LPS-initiated of the production of both IL-12 and IFN-gamma by non-T non-B (scid) splenocytes. The enhancing effect of SB 203580 on the production of IL-12 by peritoneal exudate macrophages stimulated by LPS and IFN-gamma was dose dependent (EC(50) 0.3 microM), was only seen at lower concentrations of IFN-gamma and was due, at least in part, to a dose-dependent (IC(50) 0.3 microM) inhibition of the production of IL-10. These results indicate first, that p38 MAP kinase activity is required for the production of IL-10, as well as that of proinflammatory cytokines such as IL-12 and IFN-gamma, and, second, that the net effects of SB 203580 on the production of IL-12 and IFN-gamma can be positive or negative, depending on stimuli, cell populations, and levels of cytokines such as IFN-gamma and IL-10.     

Induction of cytokine mRNA expression in U937 cells by Salmonella typhimurium porins is regulated by different phosphorylation pathways

Lipopolysaccharide (LPS) and porins of Gram-negative outer membranes are the main pathogenic factors implicated in the clinical syndrome of septic shock. The biological activity of porins and LPS are similar, but they occur by different mechanisms. It seems that porins act through different intracellular pathways with respect to LPS. In this study we analyzed the role of several inhibitors of the MEK/ERK signal pathway on the induction of proinflammatory and immunological cytokines in U937 cell line stimulated by Salmonella typhimurium porins and compared it to the cytokine induction after LPS stimulation. We investigated the effects of p38 MAP kinase inhibitor SB-203580, MEK/ERK kinase inhibitor PD-098059 and Raf-1 inhibitor forskolin, and demonstrated that they modulate cytokine mRNA expression in a different manner as a consequence of the use of porins or LPS as stimuli. TNF- and IL-1 mRNA expression is decreased by PD-098059 after stimulation with LPS but not with porins in differentiated U937 cells. IL-10 mRNA expression is inhibited by SB-203580 and PD-098059 after stimulation with porins in U937 cells. IL-6 and IL-8 mRNA expression is not changed by PD-098059 or SB-203580, after stimulation either with porins or LPS. Furthermore, mRNA expression of the studied cytokines, except for GM-CSF, is not changed using forskolin.     

p38 MAP kinase mediates stress-induced leukotriene synthesis in a human B-lymphocyte cell line.

5-Lipoxygenase (5-LO), which catalyzes the first two steps in leukotriene biosynthesis, is a target for pharmacological treatment of inflammatory disorders. Previous studies have shown that B-lymphocytes express 5-LO. Here we demonstrate that several stimuli of cell stress such as osmotic shock (sorbitol, NaCl), oxidative stress (hydrogen peroxide, diamide), chemical stress sodium arsenite, and inflammatory cytokines enhanced cellular 5-LO activity in a B cell line (BL41-E95-A), when added simultaneously with ionophore plus arachidonate. It is interesting that sorbitol alone was sufficient for 5-LO product formation in the presence of exogenous arachidonic acid. These stimuli also activated p38 mitogen-activated protein (MAP) kinase and downstream MAP kinase-activated protein kinases in BL41-E95-A cells, which could phosphorylate 5-LO or heat shock protein 27 in vitro. The p38 MAP kinase inhibitor SB203580 abolished stress-induced leukotriene synthesis in B cells, without inhibition of 5-LO catalytic activity in cell-free systems. Our results indicate that p38 MAP kinase activation by cell stress is required for efficient leukotriene synthesis in B-lymphocytes.