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.     

12-O-tetradecanoylphorbol-13-acetate may both potentiate and decrease the generation of apoptosis by the antileukemic agent arsenic trioxide in human promonocytic cells. Regulation by extracellular signal-regulated protein kinases and glutathione

Arsenic trioxide (As(2)O(3)) caused apoptosis in U-937 human promonocytic cells. This effect was potentiated by the simultaneous addition of the glutathione (GSH) synthesis inhibitor DL-buthionine-(R,S)-sulfoximine or the protein kinase C activators 12-O-tetradecanoylphorbol-13-acetate (TPA) and bryostatin 1. In addition TPA decreased the intracellular GSH content, caused ERK activation, and potentiated the As(2)O(3)-provoked activation of p38 and JNK. The addition of N-acetyl-L-cysteine, the PKC inhibitor GF109203X, and the MEK/ERK inhibitors PD98059 and U0126 attenuated both apoptosis induction and GSH decrease, whereas the p38 inhibitor SB203580 and the JNK inhibitor SP600125 were ineffective. TPA also potentiated ERK activation and GSH depletion when added simultaneously to cadmium chloride (CdCl(2)) and doxorubicin. However, TPA only enhanced apoptosis in the case of CdCl(2), which is a GSH-sensitive agent, whereas it reduced the toxicity of doxorubicin and other DNA-specific drugs. Finally, preincubation for 14-24 h with TPA did not potentiate but, instead, attenuated the As(2)O(3)- and CdCl(2)-provoked apoptosis. The same result was obtained by preincubation with bryostatin 1 and other differentiation inducers. It is concluded that TPA increases the apoptotic action of As(2)O(3), an effect mediated by ERK activation and GSH depletion. However, the increase in apoptosis is only effective in non-differentiated cells.     

The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.     

Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells

The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N,N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.     

p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease

Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.     

Distinct signaling pathways for MCP-1-dependent integrin activation and chemotaxis

Transmigration of monocytes to the subendothelial space is the initial step of atherosclerotic plaque formation and inflammation. Integrin activation and chemotaxis are two important functions involved in monocyte transmigration. To delineate the signaling cascades leading to integrin activation and chemotaxis by monocyte chemoattractant protein-1 (MCP-1), we have investigated the roles of MAPK and Rho GTPases in THP-1 cells, a monocytic cell line. MCP-1 stimulated beta1 integrin-dependent, but not beta2 integrin-dependent cell adhesion in a time-dependent manner. MCP-1-mediated cell adhesion was inhibited by a MEK inhibitor but not by a p38-MAPK inhibitor. In contrast, MCP-1-mediated chemotaxis was inhibited by the p38-MAPK inhibitor but not by the MEK inhibitor. The inhibitor of Rho GTPase, C3 exoenzyme, and a Rho kinase inhibitor abrogated MCP-1-dependent chemotaxis but not integrin-dependent cell adhesion. Further, C3 exoenzyme and the Rho kinase inhibitor blocked MCP-1-dependent p38-MAPK activation. These data indicate that ERK is responsible for integrin activation, that p38-MAPK and Rho are responsible for chemotaxis mediated by MCP-1, and that Rho and the Rho kinase are upstream of p38-MAPK in MCP-1-mediated signaling. This study demonstrates that two distinct MAPKs regulate two dependent signaling cascades leading to integrin activation and chemotaxis induced by MCP-1 in THP-1 cells.     

NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway

4-Hydroxyhexenal (HHE) is known to affect redox balance during aging, included are vascular dysfunctions. To better understand vascular abnormality through the molecular alterations resulting from HHE accumulation in aging processes, we set out to determine whether up-regulation of mitogen-activated protein kinase (MAPK) by HHE is mediated through nuclear factor kappa B (NF-kappaB) activation in endothelial cells. HHE induced NF-kappaB activation by inhibitor of kappaB (IkappaB) phosphorylation via the IkappaB kinase (IKK)/NF-kappaB inducing kinase (NIK) pathway. HHE increased the activity of p38 MAPK and extracellular signal regulated kinase (ERK), but not c-jun NH(2)-terminal kinase, indicating that p38 MAPK and ERK are closely involved in HHE-induced NF-kappaB transactivation. Pretreatment with ERK inhibitor PD98059, and p38 MAPK inhibitor SB203580, attenuated the induction of p65 translocation, IkappaB phosphorylation, and NF-kappaB luciferase activity. These findings strongly suggest that HHE induces NF-kappaB activation through IKK/NIK pathway and/or p38 MAPK and ERK activation associated with oxidative stress in endothelial cells.     

c-Jun N-terminal kinase negatively regulates lipopolysaccharide-induced IL-12 production in human macrophages: role of mitogen-activated protein kinase in glutathione redox regulation of IL-12 production

Although c-Jun N-terminal kinase (JNK) plays an important role in cytokine expression, its function in IL-12 production is obscure. The present study uses human macrophages to examine whether the JNK pathway is required for LPS-induced IL-12 production and defines how JNK is involved in the regulation of IL-12 production by glutathione redox, which is the balance between intracellular reduced (GSH) and oxidized glutathione (GSSG). We found that LPS induced IL-12 p40 protein and mRNA in a time- and concentration-dependent manner in PMA-treated THP-1 macrophages, and that LPS activated JNK and p38 mitogen-activated protein (MAP) kinase, but not extracellular signal-regulated kinase, in PMA-treated THP-1 cells. Inhibition of p38 MAP kinase activation using SB203580 dose dependently repressed LPS-induced IL-12 p40 production, as described. Conversely, inhibition of JNK activation using SP600125 dose dependently enhanced both LPS-induced IL-12 p40 production from THP-1 cells and p70 production from human monocytes. Furthermore, JNK antisense oligonucleotides attenuated cellular levels of JNK protein and LPS-induced JNK activation, but augmented IL-12 p40 protein production and mRNA expression. Finally, the increase in the ratio of GSH/GSSG induced by glutathione reduced form ethyl ester (GSH-OEt) dose dependently enhanced LPS-induced IL-12 p40 production in PMA-treated THP-1 cells. GSH-OEt augmented p38 MAP kinase activation, but suppressed the JNK activation induced by LPS. Our findings indicate that JNK negatively affects LPS-induced IL-12 production from human macrophages, and that glutathione redox regulates LPS-induced IL-12 production through the opposite control of JNK and p38 MAP kinase activation.     

Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori.

The aim of this study was to determine whether Helicobacter pylori activates mitogen-activated protein (MAP) kinases in gastric epithelial cells. Infection of AGS cells with an H. pylori cag+ strain rapidly (5 min) induced a dose-dependent activation of extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK) MAP kinases, as determined by Western blot analysis and in vitro kinase assay. Compared with cag+ strains, cag- clinical isolates were less potent in inducing MAP kinase, particularly JNK and p38, activation. Isogenic inactivation of the picB region of the cag pathogenicity island resulted in a similar loss of JNK and p38 MAP kinase activation. The specific MAP kinase inhibitors, PD98059 (25 microM; MAP kinase kinase (MEK-1) inhibitor) and SB203580 (10 microM; p38 inhibitor), reduced H. pylori-induced IL-8 production in AGS cells by 78 and 82%, respectively (p < 0.01 for each). Both inhibitors together completely blocked IL-8 production (p < 0.001). However, the MAP kinase inhibitors did not prevent H. pylori-induced IkappaBalpha degradation or NF-kappaB activation. Thus, H. pylori rapidly activates ERK, p38, and JNK MAP kinases in gastric epithelial cells; cag+ isolates are more potent than cag- strains in inducing MAP kinase phosphorylation and gene products of the cag pathogenicity island are required for maximal MAP kinase activation. p38 and MEK-1 activity are required for H. pylori-induced IL-8 production, but do not appear to be essential for H. pylori-induced NF-kappaB activation. Since MAP kinases regulate cell proliferation, differentiation, programmed death, stress, and inflammatory responses, activation of gastric epithelial cell MAP kinases by H. pylori cag+ strains may be instrumental in inducing gastroduodenal inflammation, ulceration, and neoplasia.     

Exaggerated human monocyte IL-10 concomitant to minimal TNF-alpha induction by heat-shock protein 27 (Hsp27) suggests Hsp27 is primarily an antiinflammatory stimulus

Unlike more well-studied large heat shock proteins (hsp) that induce both T cell antiinflammatory (IL-10, IL-4) and macrophage proinflammatory (TNF-alpha, IL-15, IL-12) cytokines, hsp27, a small hsp, has been primarily identified as a substrate of mitogen-activated protein kinase-activated protein kinase-2 involved in the p38 signaling pathway and activated during monocyte IL-10 production. Hsp27 can also act as an endogenous protein circulating in the serum of breast cancer patients and a protein whose induction correlates to protection from LPS shock. However, the cytokine-stimulating properties of hsp27 have been unexplored. In this study, exogenous hsp27 is demonstrated for the first time as a potent activator of human monocyte IL-10 production, but only a modest inducer of TNF-alpha. Although exogenous hsp27 stimulation activated all three monocyte mitogen-activated protein kinase pathways (extracellular signal-related kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38), only p38 activation was sustained and required for hsp27 induction of monocyte IL-10, while both ERK 1/2 and p38 activation were required for induction of TNF-alpha when using the p38 inhibitor SB203580 or the ERK inhibitor PD98059. Hsp27's transient activation of the c-Jun N-terminal kinase pathway, which can down-regulate IL-10, may contribute to its potent IL-10 induction. Hsp27's ERK 1/2 activation was also less sustained than activation by stimuli like LPS, possibly contributing to its modest TNF-alpha induction. The failure of either PD98059 or anti-TNF-alpha Ab to substantially inhibit IL-10 induction implied that hsp27 induces IL-10 via activation of p38 signaling independently of TNF-alpha activation and may be predominantly an antiinflammatory monokine stimulus.     

p21(Cip-1/SDI-1/WAF-1) expression via the mitogen-activated protein kinase signaling pathway in insulin-induced chondrogenic differentiation of ATDC5 cells

The embryonal carcinoma-derived cell line, ATDC5, differentiates into chondrocytes in response to insulin or insulin-like growth factor-I stimulation. In this study, we investigated the roles of mitogen-activated protein (MAP) kinases in insulin-induced chondrogenic differentiation of ATDC5 cells. Insulin-induced accumulation of glycosaminoglycan and expression of chondrogenic differentiation markers, type II collagen, type X collagen, and aggrecan mRNA were inhibited by the MEK1/2 inhibitor (U0126) and the p38 MAP kinase inhibitor (SB203580). Conversely, the JNK inhibitor (SP600125) enhanced the synthesis of glycosaminoglycan and expression of chondrogenic differentiation markers. Insulin-induced phosphorylation of ERK1/2 and JNK but not that of p38 MAP kinase. We have previously clarified that the induction of the cyclin-dependent kinase inhibitor, p21(Cip-1/SDI-1/WAF-1), is essential for chondrogenic differentiation of ATDC5 cells. To assess the relationship between the induction of p21 and MAP kinase activity, we investigated the effect of these inhibitors on insulin-induced p21 expression in ATDC5 cells. Insulin-induced accumulation of p21 mRNA and protein was inhibited by the addition of U0126 and SB203580. In contrast, SP600125 enhanced it. Inhibitory effects of U0126 or stimulatory effects of SP600125 on insulin-induced chondrogenic differentiation were observed when these inhibitors exist in the early phase of differentiation, suggesting that MEK/ERK and JNK act on early phase differentiation. SB202580, however, is necessary not only for early phase but also for late phase differentiation, indicating that p38 MAP kinase stimulates differentiation by acting during the entire period of cultivation. These results for the first time demonstrate that up-regulation of p21 expression by ERK1/2 and p38 MAP kinase is required for chondrogenesis, and that JNK acts as a suppressor of chondrogenesis by down-regulating p21 expression.     

ATX and LPA receptor 3 are coordinately up-regulated in lipopolysaccharide-stimulated THP-1 cells through PKR and SPK1-mediated pathways

Lysophosphatidic acid (LPA) is an important phospholipid mediator in inflammation and immunity. Previously, we showed that autotaxin (ATX), the enzyme producing LPA from lysophosphatidylcholine (LPC), is induced by LPS in THP-1 cells via the activation of PKR, JNK and p38 MAPK. In this study, we find that ATX and LPA receptor 3 (LPA(3)) are coordinately up-regulated in LPS-stimulated THP-1 cells. PKR-mediated activation of JNK1 and p38 MAPK is required for both ATX and LPA(3) up-regulation. SPK1-mediated activation of the PI3K-AKT-β-catenin pathway is essential for ATX induction, while SPK1-mediated ERK activation is required for LPA(3) up-regulation. Either ATX or LPA(3) knockdown inhibited CCL8 induction by LPS, suggesting that ATX and LPA(3) are involved in CCL8 induction during the inflammatory process against bacterial infection.