Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.     

G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH(2)-terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin II-induced activation of JNK and p38 MAPK was also inhibited by the expression of the Galpha(12/13)-specific regulator of G protein signaling (RGS) domain, a specific inhibitor of Galpha(12/13), but not by an RGS domain specific for Galpha(q). Constitutively active Galpha(12)- or Galpha(13)-induced activation of JNK and p38 MAPK, but not extracellular signal-regulated kinase (ERK), was inhibited by diphenyleneiodonium. Angiotensin II receptor stimulation rapidly activated Galpha(13), which was completely inhibited by the Galpha(12/13)-specific RGS domain. Furthermore, the Galpha(12/13)-specific but not the Galpha(q)-specific RGS domain inhibited angiotensin II-induced ROS production. Dominant negative Rac inhibited angiotensin II-stimulated ROS production, JNK activation, and p38 MAPK activation but did not affect ERK activation. Rac activation was mediated by Rho and Rho kinase, because Rac activation was inhibited by C3 toxin and a Rho kinase inhibitor, Y27632. Furthermore, angiotensin II-induced Rho activation was inhibited by Galpha(12/13)-specific RGS domain but not dominant negative Rac. An inhibitor of epidermal growth factor receptor kinase AG1478 did not affect angiotensin II-induced JNK activation cascade. These results suggest that Galpha(12/13)-mediated ROS production through Rho and Rac is essential for JNK and p38 MAPK activation.     

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.     

p38 MAPK信号传导通路

丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎ－ａｃｔｉｖａｔｅｄｐｏｒｏｔｅｉｎｋｉｎａｓｅ,ＭＡＰＫ）介导了生长、发育,分裂,死亡,以及细胞间的功能同步等多种细胞生理功能,在哺乳动物细胞中已发现和克隆了ＥＲＫ、ＪＮＫ／ＳＡＰＫ,ＥＲＫ５／ＢＭＫ１和ｐ３８／ＲＫ四个ＭＡＰＫ亚族,这些新的ＭＡＰＫ介导了物理,化学反激,细菌产物,炎性细胞因子等多种刺激引起的细胞反应,ｐ３８亚族至少包括ｐ３８（α）,ｐ３８β,ｐ     

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK.

These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis.     

ASK1 inhibitor treatment suppresses p38/JNK signalling with reduced kidney inflammation and fibrosis in rat crescentic glomerulonephritis

Activation of p38 mitogen‐activated protein kinase (MAPK) and c‐Jun amino terminal kinase (JNK) is prominent in human crescentic glomerulonephritis. p38 and JNK inhibitors suppress crescentic disease in animal models; however, the upstream mechanisms inducing activation of these kinases in crescentic glomerulonephritis are unknown. We investigated the hypothesis that apoptosis signal‐regulating kinase 1 (ASK1/MAP3K5) promote p38/JNK activation and renal injury in models of nephrotoxic serum nephritis (NTN); acute glomerular injury in SD rats, and crescentic disease in WKY rats. Treatment with the selective ASK1 inhibitor, GS‐444217 or vehicle began 1 hour before nephrotoxic serum injection and continued until animals were killed on day 1 (SD rats) or 14 (WKY rats). NTN resulted in phosphorylation (activation) of p38 and c‐Jun in both models which was substantially reduced by ASK1 inhibitor treatment. In SD rats, GS‐444217 prevented proteinuria and glomerular thrombosis with suppression of macrophage activation on day 1 NTN. In WKY rats, GS‐444217 reduced crescent formation, prevented renal impairment and reduced proteinuria on day 14 NTN. Macrophage activation, T‐cell infiltration and renal fibrosis were also reduced by GS‐444217. In conclusion, GS‐444217 treatment inhibited p38/JNK activation and development of renal injury in rat NTN. ASK1 inhibitors may have therapeutic potential in rapidly progressive glomerulonephritis.     

Involvement of p38 MAP kinase during iron chelator-mediated apoptotic cell death

Iron is an essential element for the neoplastic cell growth, and iron chelators have been tested for their potential anti-proliferative and cytotoxic effects. 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 apoptosis induced by iron chelators. We report that the chelator deferoxamine (DFO) strongly activates both p38 MAP kinase and extracellular signal-regulated kinase (ERK) at an early stage of incubation, but slightly activates c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) at a late stage of incubation. Among three MAP kinase blockers used, however, the selective p38 MAP kinase inhibitor SB203580 could only protect HL-60 cells from chelator-induced cell death, indicating that p38 MAP kinase serves as a major mediator of apoptosis induced by iron chelator. DFO also caused release of cytochrome c from mitochondria and induced activation of caspase 3 and caspase 8. Interestingly, treatment of HL-60 cells with SB203580 greatly abolished cytochrome c release, and activation of caspase 3 and caspase 8. Collectively, the current study reveals that p38 MAP kinase plays an important role in iron chelator-mediated cell death of HL-60 cells by activating downstream apoptotic cascade that executes cell death pathway.     

ASK1-p38 MAPK/JNK signaling cascade mediates anandamide-induced PC12 cell death

Anandamide is a neuroimmunoregulatory molecule that triggers apoptosis in a number of cell types including PC12 cells. Here, we investigated the molecular mechanisms underlying anandamide-induced cell death in PC12 cells. Anandamide treatment resulted in the activation of p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p44/42 MAPK in apoptosing cells. A selective p38 MAPK inhibitor, SB203580, or dn-JNK, JNK1(A-F) or SAPKbeta(K-R), blocked anandamide-induced cell death, whereas a specific inhibitor of MEK-1/2, U0126, had no effect, indicating that activation of p38 MAPK and JNK is critical in anandamide-induced cell death. An important role for apoptosis signal-regulating kinase 1 (ASK1) in this event was also demonstrated by the inhibition of p38 MAPK/JNK activation and death in cells overexpressing dn-ASK1, ASK1 (K709M). Conversely, the constitutively active ASK1, ASK1DeltaN, caused prolonged p38 MAPK/JNK activation and increased cell death. These indicate that ASK1 mediates anandamide-induced cell death via p38 MAPK and JNK activation. Here, we also found that activation of p38 MAPK/JNK is accompanied by cytochrome c release from the mitochondria and caspase activation (which can be inhibited by SB203580), suggesting that anandamide triggers a mitochondrial dependent apoptotic pathway. The caspase inhibitor, zVAD, and the mitochondrial pore opening inhibitor, cyclosporine A, blocked anandamide-induced cell death but not p38 MAPK/JNK activation, suggesting that activation of these kinases may occur upstream of mitochondrial associated events.     

Enhancement of fibroblast collagenase-1 (MMP-1) gene expression by tumor promoter okadaic acid is mediated by stress-activated protein kinases jun N-terminal kinase and p38

Collagenase-1 (matrix metalloproteinase-1, MMP-1) is expressed by several types of cells, including fibroblasts, and apparently plays an important role in the remodeling of collagenous extracellular matrix in various physiologic and pathologic situations. Here, we have examined the molecular mechanisms of the activation of fibroblast MMP-1 gene expression by a naturally occurring non-phorbol ester type tumor promoter okadaic acid (OA), a potent inhibitor of serine/threonine protein phosphatase 2A. We show that in fibroblasts OA activates three distinct subgroups of mitogen activated protein kinases (MAPKs): extracellular signal-regulated kinase 1,2 (ERK 1,2), c-Jun N-terminal-kinase/stress-activated protein kinase (JNK/SAPK) and p38. Activation of MMP-1 promoter by OA is entirely blocked by overexpression of dual-specificity MAPK phosphatase CL100. In addition, expression of kinase-deficient forms of ERK 1,2, SAPKβ, p38, or JNK/SAPK kinase SEK1 strongly inhibited OA-elicited activation of MMP-1 promoter. OA-elicited enhancement of MMP-1 mRNA abundance was also strongly prevented by two chemical MAPK inhibitors: PD 98059, a specific inhibitor of the activation of ERK1,2 kinases MEK1,2; and SB 203580, a selective inhibitor of p38 activity. Results of this study show that MMP-1 gene expression in fibroblasts is coordinately regulated by ERK1,2, JNK/SAPK, and p38 MAPKs and suggest an important role for the stress-activated MAPKs JNK/SAPK and p38 in the activation of MMP-1 gene expression. Based on these observations, it is conceivable that specific inhibition of stress-activated MAPK pathways may serve as a novel therapeutic target for inhibiting degradation of collagenous extracellular matrix.     

p38 MAP kinase regulates BMP-4-stimulated VEGF synthesis via p70 S6 kinase in osteoblasts

We previously reported that p70 S6 kinase takes part in bone morphogenetic protein-4 (BMP-4)-stimulated vascular endothelial growth factor (VEGF) synthesis in osteoblast-like MC3T3-E1 cells. Recently, we showed that BMP-4-induced osteocalcin synthesis is regulated by p44/p42 MAP kinase and p38 MAP kinase in these cells. In the present study, we investigated whether the MAP kinases are involved in the BMP-4-stimulated synthesis of VEGF in MC3T3-E1 cells. PD-98059 and U-0126, inhibitors of the upstream kinase of p44/p42 MAP kinase, failed to affect BMP-4-stimulated VEGF synthesis. SB-203580 and PD-169316, inhibitors of p38 MAP kinase, significantly reduced VEGF synthesis, whereas SB-202474, a negative control for p38 MAP kinase inhibitor, had little effect on VEGF synthesis. The BMP-4-stimulated phosphorylation of p38 MAP kinase was not affected by rapamycin, an inhibitor of p70 S6 kinase. On the contrary, SB-203580 and PD-169316 reduced the BMP-4-stimulated phosphorylation of p70 S6 kinase. In addition, anisomycin, an activator of p38 MAP kinase, phosphorylates p70 S6 kinase, and the phosphorylation was suppressed by SB-203580. LY-294002, an inhibitor of phosphatidylinositol 3-kinase, failed to suppress the phosphorylation of p38 MAP kinase induced by BMP-4. Not BMP-4 but anisomycin weakly induced the phosphorylation of phosphoinositide-dependent kinase-1. However, anisomycin had little effect on phosphorylation of either Akt or the mammalian target of rapamycin. Taken together, our results suggest that p38 MAP kinase functions in BMP-4-stimulated VEGF synthesis as a positive regulator at a point upstream from p70 S6 kinase in osteoblasts.     

Inhibition of p38 MAP kinase activity up-regulates multiple MAP kinase pathways and potentiates 1,25-dihydroxyvitamin D(3)-induced differentiation of human leukemia HL60 cells

Differentiation therapy for neoplastic diseases has potential for supplementing existing treatment modalities but its implementation has been slow. One of the reasons is the lack of full understanding of the complexities of cellular pathways through which signals for differentiation lead to cell maturation. This was addressed in this study using HL60 cells, a well-established model of differentiation of neoplastic cells. SB 203580 and SB 202190, specific inhibitors of a signaling protein p38 MAP kinase, were found to markedly accelerate monocytic differentiation of HL60 cells induced by low concentrations of 1,25-dihydroxyvitamin D(3) (1,25D(3)). Surprisingly, inhibition of p38 activity resulted in sustained enhancement of p38 phosphorylation and of its in vitro activity in the absence of the inhibitor, indicating up-regulation of the upstream components of the p38 pathway. In addition, SB 203580 or SB 202190 treatment of HL60 cells resulted in a prolonged activation of the JNK and, to a lesser extent, the ERK pathways. The data are consistent with the hypothesis that in HL60 cells an interruption of a negative feedback loop from a p38 target activates a common regulator of multiple MAPK pathways. The possibility also exists that JNK and/or ERK pathways amplify a differentiation signal provided by 1,25D(3).     

A dominant-negative p38 MAPK mutant and novel selective inhibitors of p38 MAPK reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes without affecting GLUT4 translocation

Participation of p38 mitogen-activated protein kinase (p38) in insulin-induced glucose uptake was suggested using pyridinylimidazole p38 inhibitors (e.g. SB203580). However, the role of p38 in insulin action remains controversial. We further test p38 participation in glucose uptake using a dominant-negative p38 mutant and two novel pharmacological p38 inhibitors related to but different from SB203580. We present the structures and activities of the azaazulene pharmacophores A291077 and A304000. p38 kinase activity was inhibited in vitro by A291077 and A304000 (IC(50) = 0.6 and 4.7 microm). At higher concentrations A291077 but not A304000 inhibited JNK2alpha (IC(50) = 3.5 microm). Pretreatment of 3T3-L1 adipocytes and L6 myotubes expressing GLUT4myc (L6-GLUT4myc myotubes) with A291077, A304000, SB202190, or SB203580 reduced insulin-stimulated glucose uptake by 50-60%, whereas chemical analogues inert toward p38 were ineffective. Expression of an inducible, dominant-negative p38 mutant in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. GLUT4 translocation to the cell surface, immunodetected on plasma membrane lawns of 3T3-L1 adipocytes or on intact L6-GLUT4myc myotubes, was not altered by chemical or molecular inhibition of p38. We propose that p38 contributes to enhancing GLUT4 activity, thereby increasing glucose uptake. In addition, the azaazulene class of inhibitors described will be useful to decipher cellular actions of p38 and JNK.     

Identification of arrestin-3-specific residues necessary for JNK3 kinase activation

Arrestins bind active phosphorylated G protein-coupled receptors, blocking G protein activation and channeling the signaling to G protein-independent pathways. Free arrestin-3 and receptor-bound arrestin-3 scaffold the ASK1-MKK4-JNK3 module, promoting JNK3 phosphorylation, whereas highly homologous arrestin-2 does not. Here, we used arrestin-2/3 chimeras and mutants to identify key residues of arrestin-3 responsible for its ability to facilitate JNK3 activation. Our data demonstrate that both arrestin domains are involved in JNK3 activation, with the C-terminal domain being more important than the N-terminal domain. We found that Val-343 is the key contributor to this function, whereas Leu-278, Ser-280, His-350, Asp-351, His-352, and Ile-353 play supporting roles. We also show that the arrestin-3-specific difference in the arrangement of the β-strands in the C-terminal domain that underlies its lower selectivity for active phosphoreceptors does not play an appreciable role in its ability to enhance JNK3 activation. Importantly, the strength of the binding of ASK1 or JNK3, as revealed by the efficiency of co-immunoprecipitation, does not correlate with the ability of arrestin proteins to promote ASK1-dependent JNK3 phosphorylation. Thus, multiple residues on the non-receptor-binding side of arrestin-3 are crucial for JNK3 activation, and this function and the receptor-binding characteristics of arrestin can be manipulated independently by targeted mutagenesis.     

alpha 2 integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK

The alpha(2) integrin subunit cytoplasmic domain uniquely supported epidermal growth factor (EGF)-stimulated migration on type I collagen. p38 MAP kinase- and phosphatidylinositol 3-kinase-specific inhibitors, but not a MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated alpha(2)-cytoplasmic domain-dependent migration. Following adhesion to collagenous matrices, cells expressing the full-length alpha(2) integrin subunit, but not cells expressing a chimeric alpha(2) integrin subunit in which the alpha(2)-cytoplasmic domain was replaced by the cytoplasmic domain of the alpha(1)-subunit, exhibited sustained and robust phosphorylation of p38 MAP kinase. Expression of dominant negative p38 MAP kinase inhibited alpha(2)-cytoplasmic domain-dependent, EGF-stimulated migration as well as unstimulated migration on collagen. Expression of constitutively active Rac1(Val-12) augmented p38 MAP kinase activation and alpha(2)-cytoplasmic domain-dependent migration. It also rescued the ability of cells expressing the alpha(1)-cytoplasmic domain to activate p38 MAPK and to migrate. These results suggest that the alpha(2) integrin cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that is required for unstimulated and EGF-stimulated migration on type I collagen.     

Flavonoids inhibit high glucose-induced up-regulation of ICAM-1 via the p38 MAPK pathway in human vein endothelial cells

Recently, several flavonoids have been shown to have cardioprotective, cancer preventive, or anti-inflammatory properties. However, the specific mechanisms underlying their protective effects remain unclear. We aimed to investigate the different effects of three representative flavonoids—hesperidin, naringin, and resveratrol—on intracellular adhesion molecule-1 (ICAM-1) induction in human umbilical vein endothelial cells (HUVECs) by using high-glucose (HG) concentrations and the possible underlying molecular mechanisms. In HG-induced HUVEC cultures, the effects of three different flavonoids on ICAM-1 production and p38 phosphorylation were examined in the presence or absence of inhibitors targeting the mitogen-activated protein kinase (MAPK) signal transduction pathway. HG stimulation of HUVECs increased the levels of the adhesion molecules ICAM-1 and endothelial selectin (E-selectin). Pretreatment with all the three flavonoids drastically inhibited ICAM-1 expression in a time-dependent manner, but did not alter VCAM-1 and E-selectin expressions. Moreover, we investigated the effects of flavonoids on the MAPK signal transduction pathway, because MAPK families are associated with vascular inflammation under stress. These flavonoids did not block HG-induced phosphorylation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but completely inhibited the HG-induced phosphorylation of p38 MAPK. SB202190, an inhibitor of p38 MAPK, also inhibited the HG-induced enrichment of ICAM-1. This study demonstrated that hesperidin, naringin, and resveratrol reduced the HG-induced ICAM-1 expression via the p38 MAPK signaling pathway, contributing to the inhibition of monocyte adhesion to endothelial cells.     

Specific inhibitor of MEK-mediated cross-talk between ERK and p38 MAPK during differentiation of human osteosarcoma cells

Osteosarcoma is the most common primary malignant bone tumor, accounting for approximately 20% of all primary sarcomas in bone. Although treatment modalities have been improved over the past decades, it is still a tumor with a high mortality rate in children and young adults. Based on histological considerations, osteosarcoma arises from impaired differentiation of these immature cells into more mature types and that correction of this impairment may reduce malignancy and increase the efficiency of chemotherapy. The purpose of this study was to determine the effect of specific inhibitors of MAPK extracellular signaling-regulated kinase (ERK) kinase (MEK) and p38 on the differentiation of human osteosarcoma cell line SaOS-2 cells. We found that PD98059, a specific inhibitor of MEK, inhibited the serum-stimulated proliferation of SaOS-2 cells; whereas SB203580, a specific inhibitor of p38 MAPK, had little effect on it. SB203580 suppressed ALPase activity, gene expression of type I collagen, and expression of ALP and BMP-2 mRNAs; whereas PD98059 upregulated them dose dependently. In addition, immunoblot and immunostaining analysis revealed that phosphorylation of ERK was increased by treatment with SB203580; whereas PD98059 increased the phosphorylation of p38, which implies a seesaw-like balance between ERK and p38 phosphorylation. We suggest that osteosarcoma cell differentiation is regulated by the balance between the activities of the ERK and p38 pathways and that the MEK/ERK pathway negatively regulates osteosarcoma cell differentiation, whereas the p38 pathway does so positively. MEK inhibitor may thus be a good candidate for altering the expression of the osteosarcoma malignant phenotype.     

Involvement of Rho-kinase in tumor necrosis factor-α-induced interleukin-6 release from C6 glioma cells

Tumor necrosis factor (TNF)-α stimulated interleukin (IL)-6 release and induced the phosphorylation of myosin phosphatase targeting subunit (MYPT)-1, a Rho-kinase substrate. The IL-6 release was significantly suppressed by Y-27632 and fasudil, Rho-kinase inhibitors. Although IκB inhibitor suppressed the TNF-α-induced IL-6 release, the Rho-kinase inhibitors did not affect the TNF-α-induced IκB phosphorylation. TNF-α induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), and p44/p42 MAP kinase. The TNF-α-induced IL-6 release was suppressed by SB203580, a p38 MAPK inhibitor, or SP600125, a SAPK/JNK inhibitor, but not by PD98059, a MAP kinase/extracellular signal-regulated kinase kinase inhibitor. The Rho-kinase inhibitors attenuated the TNF-α-induced phosphorylation of both p38 MAP kinase and SAPK/JNK.Rho-kinase, which has been used for the clinical treatment of cerebral vasospasms, may be involved in other central nervous system (CNS) disorders such as traumatic injury, stroke, neurodegenerative disease and neuropathic pain. TNF-α, a proinflammatory cytokine that affects the CNS through cytokines, such as IL-6, release from neurons, astrocytes and microglia. Therefore, we investigated the involvement of Rho-kinase in the TNF-α-induced IL-6 release from rat C6 glioma cells.These results strongly suggest that Rho-kinase regulates the TNF-α-induced IL-6 release at a point upstream from p38 MAPK and SAPK/JNK in C6 glioma cells. Therefore, Rho-kinase inhibitor may be considered to be a new clinical candidate for the treatment of CNS disorders in addition to cerebral vasospasms.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.