P38 Mitogen-activated Protein Kinase Inhibitor SB203580 Research Articles

Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells

Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5–2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.

Doxorubicin induces cardiomyocyte dysfunction via a p38 MAP kinase-dependent oxidative stress mechanism

Doxorubicin, an anthracycline used for cancer therapy, is known to elicit an irreversible cardiotoxicity. Several mechanisms were postulated for its cardiac toxicity including generation of reactive oxygen species (ROS). This study was designed to determine the acute effect of doxorubicin on cardiac mechanical and intracellular Ca 2+ properties in isolated ventricular myocytes. Contractile properties of male adult rat ventricular myocytes were analyzed including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90) and maximal velocity of shortening/relengthening (±d L/d t). Intracellular Ca 2+ transients and generation of ROS were measured with fura-2 and fluoroprobe 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, respectively. Acute (5 min) incubation of myocytes with doxorubicin (10 −9–10 −4 M) significantly prolonged TPS, TR 90 and intracellular Ca 2+ transient decay rate without affecting PS, ±d L/d t, resting intracellular Ca 2+ levels and electrically triggered intracellular Ca 2+ rise. Interestingly, the doxorubicin-induced prolongation of TPS and TR 90 was ablated by treatment of the antioxidant Vitamin C (100 μM) or the p38 MAP kinase inhibitor SB203580 (10 μM). Both Vitamin C and SB203580 unmasked a doxorubicin-induced positive response in PS. Vitamin C itself enhanced basal ±d L/d t, whereas, SB203580 unmasked a doxorubicin-induced positive response of ±d L/d t. The doxorubicin-induced response of intracellular Ca 2+ transients was essentially unaffected by Vitamin C. The role of ROS in doxorubicin-induced cardiac contractile response was confirmed with the ability of doxorubicin to enhance ROS generation, which was prevented by Vitamin C and SB203580. These data provide evidence that doxorubicin impairs cardiac contractile property in single myocytes through an oxidative stress-mediated pathway.

Ligation of CD44 by A3D8 Monoclonal Antibody Induces Terminal Differentiation of THP-1 Leukemia Cells by Promoting Cross-Talk between Extracellular Signal-Regulated Kinase and Phosphoinositide-3 Kinase/Akt Signaling Pathway.

Ligation of CD44 with anti-CD44 monoclonal antibody A3D8 induces terminal differentiation of human leukemia cells. However, the underlying molecular mechanisms remain largely unknown. In this study, we examined the importance of MEK/ERK, p38 MAPK, and phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in A3D8-induced monocytic differentiation of THP-1 leukemia cells. THP-1 cells displayed cytologic changes typical of mature monocytes and an increased expression of monocyte-specific antigen CD14 (49% ± 4%) and of myeloid-specific antigen CD11b (68% ± 6%) after 3 days of A3D8 treatment. The level of CD15 was also increased. The increase in the expression of these differentiation antigens was dose- and time-dependent. We found that CD44 ligation with A3D8 led to rapid and sustained activation of the essential kinases in the extracellular signal-regulated kinase pathway such as phospho-Raf-1, phospho-MEK1/2, and phospho-ERK1/2. However, the total levels of these kinases were not affected during the course of differentiation. Ser473 Akt phosphorylation was also observed shortly after the cells were exposed to A3D8 and sustained thereafter. In contrast, the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and C-Jun N-terminal kinase were not increased by CD44 ligation. Pretreatment of cells with 20mM of MEK1 inhibitor PD98059 abrogated the A3D8-induced activation of MEK1/2 and ERK1/2 and potently inhibited the THP-1 differentiation. However, a p38 MAPK inhibitor SB203580 did not abolished the CD44 ligation-induced terminal differentiation of THP-1 cells. Pretreatment of cells with PI3-K inhibitor LY294002 resulted in a nearly complete inhibition of A3D8-induced terminal differentiation. Overexpression of dominant-negative Akt also reduced the A3D8-induced THP-1 differentiation, suggesting that A3D8-induced THP-1 differentiation is coupled to PI3-K/Akt activation. Surprisingly, the pharmacological inhibition of PI3-K blocked not only A3D8-induced Akt activation but also A3D8-induced activation of Raf, MEK and ERK pathway. By contrast, pretreatment of cells with PD98059 did not inhibit the A3D8-induced Akt activation, suggesting that PI3-K/Akt is the upstream of Raf/MEK/ERK pathway in A3D8-induced terminal differentiation of THP-1 leukemia cells. Taken together, our findings demonstrated that the cross-talk between the activation of A3D8-inducible PI3-K/Akt pathway and the Raf/MEK/ERK pathway in THP-1 cell exists, and plays a critical role during CD44 ligation-induced THP-1 differentiation.

C-Myc-mediated expression of nucleophosmin/B23 decreases during retinoic acid-induced differentiation of human leukemia HL-60 cells

The retinoic acid-induced differentiation of human leukemia HL-60 cells towards mature granulocytic cells was accompanied by the decline in the protein levels of c-myc, nucleophosmin/B23 and its promoter activity. These RA-induced effects were further enhanced by the concurrent treatment of HL-60 cells with p38 map kinase inhibitor SB203580 (SB). It seems that there is a strong correlation of nucleophosmin/B23 and c-Myc expressions in cells under RA treatment. Furthermore, nucleophosmin/B23 promoter activity decreased upon c-Myc antisense-mediated reduction of intracellular amount of c-Myc. CHIP assays showed that binding of c-Myc to the nucleophosmin/B23 promoter decreased in RA-treated cells. Thus, nucleophosmin/B23 expression is targeted by c-Myc during RA-induced differentiation. These results provide evidence for a novel mechanism of transcriptional downregulation of nucleophosmin/B23 and the functional role of c-Myc in RA-induced differentiation.

Regulation of corticotropin-releasing factor receptor type 2β mRNA by mitogen-activated protein kinases in aortic smooth muscle cells

The actions of the corticotropin-releasing factor (CRF) family of peptides are mediated by the seven transmembrane-domain G-protein-coupled receptors, the CRF receptors. CRF receptor type 2β (CRFR2β) messenger RNA (mRNA) is expressed primarily in the cardiovascular system, where its levels are decreased by urocortin 1 (Ucn1), a novel peptide in the CRF family. In a previous study, we reported that CRFR2β mRNA levels were partially down-regulated via the cAMP-protein kinase A pathway. This study focused on the involvement of the intracellular mitogen-activated protein (MAP) kinase pathway in the modulation of CRFR2β mRNA levels. Ribonuclease protection assays showed that decreases in CRFR2β mRNA levels induced by Ucn1 and cAMP were attenuated by the p38 MAP kinase inhibitor SB202190 or SB203580. This finding suggested that the p38 MAP kinase pathway was involved in this regulation. Anisomycin, a classic p38 kinase activator, increased CRFR2β mRNA levels in A7r5 cells. This effect of anisomycin was completely reversed by H7, a serine/threonine kinase inhibitor, while both p38 kinase and MAP kinase kinase inhibitors failed to block the increase in CRFR2β mRNA levels caused by anisomycin. As anisomycin can activate Jun amino terminal kinases, as well as p38 MAP kinase, it is possible that other MAP kinases, such as Jun amino terminal kinases, also contribute to the increase in gene levels. Alternatively, anisomycin may increase CRFR2β mRNA levels indirectly as a consequence of blocking protein synthesis.

Angiotensin II-Induced Akt Activation through the Epidermal Growth Factor Receptor in Vascular Smooth Muscle Cells Is Mediated by Phospholipid Metabolites Derived by Activation of Phospholipase D

Angiotensin II (Ang II) activates cytosolic Ca(2+)-dependent phospholipase A(2) (cPLA(2)), phospholipase D (PLD), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR) and Akt in vascular smooth muscle cells (VSMC). This study was conducted to investigate the relationship between Akt activation by Ang II and other signaling molecules in rat VSMC. Ang II-induced Akt phosphorylation was significantly reduced by the PLD inhibitor 1-butanol, but not by its inactive analog 2-butanol, and by brefeldin A, an inhibitor of the PLD cofactor ADP-ribosylation factor, and in cells infected with retrovirus containing PLD(2) siRNA or transfected with PLD(2) antisense but not control LacZ or sense oligonucleotide. Diacylglycerol kinase inhibitor II diminished Ang II-induced and diC8-phosphatidic acid (PA)-increased Akt phosphorylation, suggesting that PLD-dependent Akt activation is mediated by PA. Ang II-induced EGFR phosphorylation was inhibited by 1-butanol and PLD(2) siRNA and also by cPLA(2) siRNA. In addition, the inhibitor of arachidonic acid (AA) metabolism 5,8,11,14-eicosatetraynoic acid (ETYA) reduced both Ang II- and AA-induced EGFR transactivation. Furthermore, ETYA, cPLA(2) antisense, and cPLA(2) siRNA attenuated Ang II-elicited PLD activation. p38 MAPK inhibitor SB202190 [4-(4-flurophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole] reduced PLD activity and EGFR and Akt phosphorylation elicited by Ang II. Pyrrolidine-1, a cPLA(2) inhibitor, and cPLA(2) siRNA decreased p38 MAPK activity. These data indicate that Ang II-stimulated Akt activity is mediated by cPLA(2)-dependent, p38 MAPK regulated PLD(2) activation and EGFR transactivation. We propose the following scheme of the sequence of events leading to activation of Akt in VSMC by Ang II: Ang II-->cPLA(2)-->AA-->p38 MAPK-->PLD(2)-->PA-->EGFR-->Akt.

The p38 MAPK Inhibitor SB203580 Blocks Adenosine A1 Receptor-Induced Attenuation of In Vivo Myocardial Stunning

There is considerable evidence implicating a key role for p38 mitogen-activated protein kinase (MAPK) in ischemic and pharmacological preconditioning against myocardial infarction. However, there have been few, if any, studies examining the role of p38 MAPK in the protection of stunned myocardium. The purpose of this study was to determine whether p38 MAPK plays a role in the adenosine A(1) receptor anti-stunning effect in in vivo porcine myocardium. Regional myocardial stunning in anesthetized, open-chest pigs was induced by 15 min of left anterior descending coronary artery (LAD) occlusion and 3 h of reperfusion (RP). Animals were treated with either vehicle (n = 5), AMP579 (70 microg/kg i.v.; 25 microg/kg bolus + 1.5 microg/kg/min for 30 min prior to ischemia, n = 5), the p38 MAPK inhibitor SB203580 (0.25 mg/kg i.v. bolus, n = 4) or a combination of SB203580 plus AMP579 (n = 5). Regional ventricular function was monitored by measurements of segment shortening and load insensitive parameters including preload recruitable stroke work (PRSW) and PRSW area (PRSWA). The ischemic area at risk was similar in all groups and there was no necrosis in any heart. Treatment with AMP579 significantly improved reperfusion regional PRSW and PRSWA compared to vehicle controls. The p38 inhibitor SB203580 alone did not alter the extent of myocardial stunning, but it abolished the beneficial effect of AMP579 pretreatment. These results provide the first evidence that p38 MAPK activation may play an important role in the mechanism by which adenosine agonists attenuate myocardial stunning.

Altering dietary nutrient intake that reduces glycogen content leads to phosphorylation of nuclear p38 MAP kinase in human skeletal muscle: association with IL-6 gene transcription during contraction.

To determine the effect of glycogen availability and contraction on intracellular signaling and IL-6 gene transcription, eight males performed 60 min of exercise on two occasions: either with prior ingestion of a normal (Con) or low carbohydrate (LCHO) diet that reduced pre-exercise muscle glycogen content. Muscle biopsies were obtained and analyzed for IL-6 mRNA. In addition, nuclear proteins were isolated from the samples and analyzed for the mitogen- activated protein kinases (MAPK) c-jun amino-terminal kinase (JNK) 1 and 2 and p38 MAPK. Nuclear fractions were also analyzed for the phosphorylated forms of JNK (p-JNK) and p38 MAPK (p-p38 MAPK) and the abundance of the nuclear transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor kappa-beta (NF-kappabeta). No differences were observed in the protein abundance of total JNK 1/2, p38 MAPK, NFAT, or NF-kappabeta before exercise, but the nuclear abundance of p-p38 MAPK was higher (P<0.05) in LCHO. Contraction resulted in an increase (P<0.05) in nuclear p-JNK 1/2, but there were no differences when comparing CON with LCHO. The fold increase in IL-6 mRNA with contraction was potentiated (P<0.05) in LCHO. A correlation between pre-exercise nuclear phosphorylated p38 MAPK and contraction-induced fold increase in IL-6 mRNA was performed, revealing a highly significant correlation (r=0.96; P<0.01). We next incubated L6 myotubes in ionomycin (a compound known to induce IL-6 mRNA) with or without the pyridinylimidazole p38 MAPK inhibitor SB203580. Treatments did not affect total nuclear p38 MAPK, but ionomycin increased (P<0.05) both nuclear p-p38 MAPK and IL-6 mRNA. The addition of SB203580 to ionomycin decreased (P<0.05) nuclear p-p38 MAPK and totally abolished (P<0.05) the ionomycin- induced increase in IL-6 mRNA. These data suggest that reduced carbohydrate intake that results in low intramuscular glycogen leads to phosphorylation of p38 MAPK at the nucleus. Furthermore, phosphorylation of p38 MAPK in the nucleus appears to be an upstream target for IL-6, providing new insights into the regulation of IL-6 gene transcription.

Mitogen-activated protein kinase regulates neurofilament axonal transport

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.

Ligation of the lactosylceramide receptor (CDw17) promotes neutrophil migration

Abstract Introduction: B-Glucan, a β-(1–3) linked glucose polymer, enhances neutrophil anti-infective activity by binding to specific receptors including the lectin site of the CR3 integrin (CD11b/CD18) and the glycosphingolipid lactosylceramide. B-glucan promotes neutrophil chemotaxis towards fMLP and C5a. Enhancement of neutrophil migration to fMLP occurs via CR3 through a p38 dependent mechanism. We investigated the mechanism by which β-glucan promotes neutrophil chemotaxis towards C5a. Methods: Migration was assessed by serum agarose overlay technique using chambered slides precoated or not with β-glucan (100μg/ml). Migration of human neutrophils at 37 C for 2 hours was established towards C5a (10 pmol) using PBS as control. Antibody blocking experiments included neutrophil pretreatment with mAbs specific for CR3 or for CDw17. In some experimental groups neutrophils were treated with the p38 MAP kinase inhibitor SB203580 (10mM) prior to assay. The number of chemotaxing cells was quantified by microscopy. Results: The anti-CDw17 mAb but not anti-CR3 mAb obviated the β-glucan promotion. This finding indicates that lactosylceramide is the β-glucan receptor that mediates enhanced chemotaxis towards C5a. Non-specific isotype antibodies did not affect neutrophil migration. Further, p38-inhibited neutrophils did not demonstrate enhanced migration to C5a in the presence of β-glucan. Conclusions: Ligation of lactosylceramide by β-glucan promoted neutrophil chemotaxis towards C5a signaling through a p38 dependent pathway. This is the first demonstration of the involvement of lactosylceramide in neutrophil chemotaxis. This receptor may prove to be a novel therapeutic target in enhancing neutrophil anti-infective activity.

Gender-specific effects of P38 mitogen-activated protein kinase activation in myocardial ischemia-reperfusion injury

Abstract Introduction: Gender differences have been demonstrated in myocardial functional recovery following ischemia-reperfusion (I/R) injury. A local inflammatory response accompanies myocardial ischemia, and activation of p38 mitogen-activated protein kinase (MAPK) plays an important role in the production of inflammatory mediators. However, it is unknown whether gender differences exist in the inflammatory signal transduction pathway following I/R. Thus, we hypothesized that females have increased myocardial function following I/R compared to males, and that p38 MAPK inhibition provides gender-specific protection. Methods: Isolated perfused rat hearts (Langendorff with continuous recording of left ventricular developed pressure, LVDP) from normal age-matched males and females were subjected to 25 minutes of ischemia followed by 40 minutes of reperfusion in the presence or absence of the p38 MAPK inhibitor SB203580 (2 × 10 −5 M). Results: Females had significantly increased recovery of LVDP following I/R compared to males (66.87 +/− 7.07% vs. 34.28 +/− 5.02%, p Conclusions: We conclude that females have a relative cardioprotection against I/R compared to males, and that p38 MAPK activation has a more pronounced effect during I/R injury in females than in males. This suggests that the inflammatory signaling mechanisms are gender-specific, and that females and males may respond differently to therapies targeted at limiting myocardial inflammation and injury.

Decreased copper-zinc superoxide dismutase activity and increased resistance to oxidative stress in glia maturation factor-null astrocytes.

Glia maturation factor (GMF) is a highly conserved protein found mainly in the nervous system. The current work was undertaken to investigate the effect of GMF expression in astrocytes on CuZn superoxide dismutase (CuZnSOD or SOD I) and on the vulnerability of the cells to H2O2 toxicity. Primary astrocyte cultures were derived from mice in which the GMF gene was completely deleted by homologous recombination (knockout). Astocytes derived from knockout animals displayed a lower level of CuZnSOD activity and protein. The reduction in CuZnSOD was restored by transfection with a GMF/adenovirus construct, and the resulting increase was blocked by the p38 MAP kinase inhibitor SB203580. There was no change in the other isoform of SOD (MnSOD or SOD II). Endogenous H2O2 was lower in the knockout cells, and the cells became more resistant to H2O2 toxicity compared to the wild type. In the GMF-null cells, concurrent with a decrease in CuZnSOD, the function of which is to convert superoxide to H2O2, there was an increase in the activity of the two enzymes that degrade H2O2: catalase and glutathione peroxidase. By regulating the redox state of the cell, GMF may be involved in a wide spectrum of cellular events ranging from survival, proliferation, differentiation, to death.

LDL oxidized by hypochlorous acid causes irreversible platelet aggregation when combined with low levels of ADP, thrombin, epinephrine, or macrophage-derived chemokine (CCL22)

AbstractThe in vitro oxidation of low-density lipoprotein (LDL) by hypochlorous acid produces a modified form (HOCl-LDL) capable of stimulating platelet function. We now report that HOCl-LDL is highly effective at inducing platelet function, causing stable aggregation and α-granule secretion. Such stimulation depended on the presence of low levels of primary agonists such as adenosine diphosphate (ADP) and thrombin, or others like epinephrine (EPI) and macrophage-derived chemokine (MDC, CCL22). Agonist levels, which by themselves induced little or reversible aggregation, caused strong stable aggregation when combined with low levels of HOCl-LDL. Platelet activation by HOCl-LDL and ADP (1 μM) caused P-selectin (CD62P) exposure, without serotonin or adenosine triphosphate (ATP) secretion. Intracellular calcium levels rose slowly (from 100 to 200 nM) in response to HOCl-LDL alone and rapidly when combined with ADP to about 300 nM. p38 mitogen-activated protein kinase (MAPK) became phosphorylated in response to HOCl-LDL alone. This phosphorylation was not blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide, which reduced the extent of aggregation and calcium increase. However, the p38 MAPK inhibitor SB203580 blocked platelet aggregation and phosphorylation of p38 MAPK. These findings suggest that HOCl-LDL exposed during atherosclerotic plaque rupture, coupled with low levels of primary agonists, can rapidly induce extensive and stable thrombus formation. (Blood. 2004;104:380-389)

Oncogenic Raf-1 regulates epithelial to mesenchymal transition via distinct signal transduction pathways in an immortalized mouse hepatic cell line.

The epithelial to mesenchymal transition (EMT) is considered to be an important event during malignant tumor progression and metastasis. Although Raf/MEK/ERK signaling causes EMT, the mechanisms, including the signaling pathways, are as yet unclear. In the present study we have examined the effects of signal transduction pathways on oncogenic Raf-1-induced EMT, using an immortalized mouse hepatic cell line. Oncogenic Raf-1-induced EMT is characterized by down-regulation of adherens and tight junctions and the reorganization of actin. An active Raf-1 gene was introduced into a mouse hepatic cell line which was then treated with the MAP kinase inhibitor PD98059, the p38 MAP kinase inhibitor SB203580, the PI3 kinase inhibitor LY294002 or the c-Src tyrosine kinase inhibitor PP2. The expression and localization of the adherens and tight junction proteins E-cadherin, occludin, ZO-1, claudin-1 and claudin-2 were determined by western blotting, RT-PCR and immunocytochemistry. The barrier function of tight junctions was assessed by measurements of transepithelial electric resistance (TER) and permeability in terms of fluxes of [(14)C]mannitol and [(14)C]inulin. In Raf-1-transfected cells expression of occludin and claudin-2 was markedly down-regulated at the protein and mRNA levels and the TER value was decreased, while the permeability was increased. The distribution of ZO-1, pancadherin and F-actin was changed from linear to zipper-like structures at cell borders. In Raf-1-transfected cells treated with PD98059 and SB203580, but not LY294002, expression and localization of claudin-2, but not occludin, recovered, together with barrier function, measured as the TER value. The distributions of ZO-1, pancadherin and F-actin also recovered on treatment with PD98059 and SB203580, but not LY294002. Expression and localization of occludin recovered slightly on treatment with PP2. Thus, oncogenic Raf-1 regulates EMT via distinct MAP kinase, p38 MAP kinase and c-Src tyrosine kinase signal pathways in the mouse hepatic cell line.

Interleukin 1beta-induced production of H2O2 contributes to reduced sigmoid colonic circular smooth muscle contractility in ulcerative colitis.

We have shown that neurokinin A-induced contraction of human sigmoid circular muscle (HSCM) is reduced in patients with ulcerative colitis and that interleukin (IL)-1beta may play a role in this change. We now examine changes in the signal transduction pathway mediating neurokinin A-induced contraction of HSCM and explore the role of IL-1beta and of H(2)O(2) in these changes. In Fura 2-AM-loaded ulcerative colitis HSCM cells, neurokinin A- and caffeine-induced peak Ca(2+) increase and cell shortening were significantly reduced. In normal cells, neurokinin A-induced contraction was decreased by protein kinase C inhibitor chelerythrine and by calmodulin inhibitor CGS9343B [1,3-dihydro-1-[1-[(4-methyl-4H,6H-pyrrolo[1,2-a][4,1]-benzoxazepin-4-yl)methyl]-4-piperidinyl]-2H-benzimidazol-2-one (1:1) maleate]. In ulcerative colitis muscle cells, contraction was inhibited only by chelerythrine but not by CGS9343B. IL-1beta treatment of normal HSCM strips and cells reproduced the changes observed in ulcerative colitis. IL-1beta-induced reduction in caffeine-induced peak Ca(2+) increase and contraction was reversed by catalase, suggesting a role of H(2)O(2). IL-1beta-induced H(2)O(2) production was inhibited by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059 (2'-amino-3'-methoxyflavone) and by cytosolic phospholipase A2 (cPLA(2)) inhibitor AACOCF3 (arachidonyltrifluoromethyl ketone), but neither by p38 MAPK inhibitor SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole] nor by nuclear factor-kappaB (NF-kappaB) inhibitory peptide NF-kappaB SN50 (H-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-Val-Gln-Arg-Lys-Arg-Gln-Lys-Leu-Met-Pro-OH). IL-1beta significantly increased the phosphorylation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 MAPKs and cPLA(2) and IL-1beta-induced cPLA(2) phosphorylation was blocked by PD98059. We conclude that Ca(2+) stores of HSCM cells may be reduced in ulcerative colitis and that the signal transduction pathway of neurokinin A-induced contraction switches from calmodulin- and protein kinase C-dependent in normal cells to protein kinase C-dependent in ulcerative colitis cells. IL-1beta reproduces these changes, possibly by production of H(2)O(2) via sequential activation of MAPKs (ERK1/ERK2) and cPLA(2).

P38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x L

The role of p38 mitogen-activated protein kinase (MAPK) in apoptosis is a matter of debate. Here, we investigated the involvement of p38 MAPK in endothelial apoptosis induced by tumor necrosis factor α (TNF). We found that activation of p38 MAPK preceded activation of caspase-3, and the early phase of p38 MAPK stimulation did not depend on caspase activity, as shown by pretreatment with the caspase inhibitors z-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk) and Boc-Asp(OMe)-fluoromethylketone (BAF). The p38 MAPK inhibitor SB203580 significantly attenuated TNF-induced apoptosis in endothelial cells, suggesting that p38 MAPK is essential for apoptotic signaling. Furthermore, we observed a time-dependent increase in active p38 MAPK in the mitochondrial subfraction of cells exposed to TNF. Notably, the level of Bcl-x L protein was reduced in cells undergoing TNF-induced apoptosis, and this reduction was prevented by treatment with SB203580. Immunoprecipitation experiments revealed p38 MAPK-dependent serine–threonine phosphorylation of Bcl-x L in TNF-treated cells. Exposure to lactacystin prevented both the downregulation of Bcl-x L and activation of caspase-3. Taken together, our results suggest that TNF-induced p38 MAPK-mediated phosphorylation of Bcl-x L in endothelial cells leads to degradation of Bcl-x L in proteasomes and subsequent induction of apoptosis.

German Cockroach Extract Induces Activation of Human Eosinophils to Release Cytotoxic Inflammatory Mediators

Background: Eosinophils play an important role in the pathogenesis of allergic diseases. Sensitization and exposure to cockroach allergen have been demonstrated to be one of the major risk factors for the development of bronchial asthma. However, little is known regarding the functional capacity of cockroach extract antigen to activate human eosinophils. Objective: We investigated whether German cockroach extract can activate human eosinophils to release cytotoxic inflammatory mediators. Methods: Purified eosinophils from the peripheral blood were incubated with various concentrations (0–200 µg/ml) of German cockroach extract antigen. Effector functions of eosinophils were checked by degranulation and superoxide anion production. In addition, we examined surface expression of CD11b and CD69, and intracellular activation of p38 mitogen-activated protein kinase (p38 MAP kinase) in cockroach-stimulated eosinophils. Results: German cockroach extract induced degranulation and superoxide production from human eosinophils. In addition, incubation of eosinophils for 3 h with the cockroach extract resulted in an increased level of the surface expression of CD11b and CD69. Furthermore, cockroach-induced superoxide production from eosinophils was significantly inhibited by the pretreatment of cells with a p38 MAP kinase inhibitor SB202190. Indeed, a large amount of phosphorylated forms of p38 MAP kinase was detected in cockroach-stimulated eosinophils. Conclusions: Our results suggest that German cockroach extract induces activation of human eosinophils to release cytotoxic inflammatory mediators such as superoxide and granular proteins.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90 ), and maximal velocities of shortening/relengthening (± d L /d t ). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9 –10 −5 M) depressed PS, prolonged TR 90 , and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L /d t , resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′ ,7 ′ -Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR 90), and maximal velocities of shortening/relengthening (± d L/d t). Intracellular Ca 2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10 −9–10 −5 M) depressed PS, prolonged TR 90, and decreased electrically stimulated intracellular Ca 2+ rise in a concentration-dependent manner. TPS, ± d L/d t, resting intracellular Ca 2+ level, and intracellular Ca 2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 μM) and the p38 MAP kinase inhibitor SB203580 (1 μM). 2 ′,7 ′-Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.

The role of p38 MAP kinase in the synergistic cytotoxic action of calcitriol and TNF-α in human breast cancer cells

Calcitriol, the hormonal form of Vitamin D, potentiates the activity of some agents of the anti-cancer immune system including tumor necrosis factor-α (TNF-α). Different signaling pathways activated by TNF-α may be targets for calcitriol action. Activation of p38 MAP kinase was shown to have both pro- and anti-apoptotic actions in TNF-α-induced programmed cell death depending on cell context. Treatment of MCF-7 breast cancer cells with TNF-α resulted in activation of p38 MAP kinase that persisted for at least 24 h. Whereas calcitriol had no effect on the earlier phase of p38 MAP kinase activation (up to 1 h), it inhibited the activation of this pathway between one and 24 h after exposure to TNF-α. Both calcitriol and the p38 MAP kinase inhibitor SB203580 enhanced TNF-α-induced cytotoxicity and drop in mitochondrial membrane potential, but their combined effect was sub-additive. Taken together, these findings suggest that p38 MAP kinase plays an anti-apoptotic role in TNF-α-induced cytotoxicity in MCF-7 cells and that the synergistic interaction between TNF-α and calcitriol, leading to mitochondrial damage and subsequent cell death, is partially due to modulation of this signaling pathway.