p38 mitogen-activated protein kinase mediates signal integration of TCR/CD28 costimulation in primary murine T cells.
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Optimal T cell activation requires two signals, one generated by TCR and another by the CD28 costimulatory receptor. In this study, we investigated the regulation of costimulation-induced mitogen-activated protein kinase (MAPK) activation in primary mouse T cells. In contrast to that reported for human Jurkat T cells, we found that p38 MAPK, but not Jun NH2-terminal kinase (JNK), is weakly activated upon stimulation with either anti-CD3 or anti-CD28 in murine thymocytes and splenic T cells. However, p38 MAPK is activated strongly and synergistically by either CD3/CD28 coligation or PMA/Ca2+ ionophore stimulation, which mimics TCR-CD3/CD28-mediated signaling. Activation of p38 MAPK correlates closely with the stimulation of T cell proliferation. In contrast, PMA-induced JNK activation is inhibited by Ca2+ ionophore. T cell proliferation and production of IL-2, IL-4, and IFN-gamma induced by both CD3 and CD3/CD28 ligation and the nuclear expression of the c-Jun and ATF-2 proteins are each blocked by the p38 MAPK inhibitor SB203580. Our findings demonstrate that p38 MAPK 1) plays an important role in signal integration during costimulation of primary mouse T cells, 2) may be involved in the induction of c-Jun activation and augmentation of AP-1 transcriptional activity, and 3) regulates whether T cells enter a state of functional unresponsiveness. (+info)
The functional synergy between IL-12 and IL-2 involves p38 mitogen-activated protein kinase and is associated with the augmentation of STAT serine phosphorylation.
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IL-12 and IL-2 can stimulate mitogen- or CD3-activated T cells to proliferate, produce IFN-gamma, and kill tumor cells. The magnitude of these functional responses is greatly augmented when T cells are activated by the combination of IL-12 and IL-2. Although peripheral blood T cells are largely unresponsive to these cytokines without prior activation, a small subset of CD8+ T cells (CD8+CD18bright) is strongly activated by the combination of IL-12 and IL-2. In this report we show that the functional synergy between IL-12 and IL-2 in CD8+CD18bright T cells correlates with the activation of the stress kinases, p38 mitogen-activated protein (MAP) kinase and stress-activated protein kinase (SAPK)/Jun N-terminal kinase, but not with the activation of the extracellular signal-regulated kinases. The functional synergy between IL-2 and IL-12 is also associated with a prominent increase in STAT1 and STAT3 serine phosphorylation over that observed with IL-12 or IL-2 alone. By contrast, STAT tyrosine phosphorylation is not augmented over that seen with either cytokine alone. A specific inhibitor of p38 MAP kinase completely inhibits the serine phosphorylation of STAT1 and STAT3 induced by IL-12 and IL-2 and abrogates the functional synergy between IL-12 and IL-2 without affecting STAT tyrosine phosphorylation. This suggests that p38 MAP kinase may play an important role in regulating STAT serine phosphorylation in response to the combination of IL-12 and IL-2. Furthermore, these findings indicate that the optimal activation of T cells by IL-12 and IL-2 may depend on an interaction between the p38 MAP kinase and Janus kinase/STAT signaling pathways. (+info)
Activation of p38 mitogen-activated protein kinase by oxidized LDL in vascular smooth muscle cells: mediation via pertussis toxin-sensitive G proteins and association with oxidized LDL-induced cytotoxicity.
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Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 microg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-s ulfophenyl)- 2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin-sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs. (+info)
p38 kinase mediates UV-induced phosphorylation of p53 protein at serine 389.
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The p53 tumor suppressor protein is a transcription factor that plays a key role in the process of apoptosis and the cell's defense against tumor development. Activation of p53 occurs, at least in part, by phosphorylation of its protein. Very recently it has been reported that UV induced a functional activation of p53 via phosphorylation at serine 389. Here, we report that the UV-induced phosphorylation of p53 at serine 389 is mediated by p38 kinase. UVC-induced phosphorylation of p53 at serine 389 was markedly impaired by either pretreatment of cells with p38 kinase inhibitor, SB202190, or stable expression of a dominant negative mutant of p38 kinase. In contrast, there was no inhibition observed in cells treated with specific MEK1 inhibitor, PD98059, or with stable expression of a dominant negative mutant of ERK2 or JNK1. Most importantly, p38 kinase could be co-immunoprecipitated with p53 by using antibodies against p53. Incubation of active p38 kinase with p53 protein caused the phosphorylation of p53 protein at serine 389 in vitro, while no phosphorylation of p53 at serine 389 was observed when p53 was incubated with activated JNK2 or ERK2. Furthermore, pretreatment of cells with SB202190 blocked the p53 DNA binding activity and p53-dependent transcription. These results strongly suggest that the p38 kinase is at least one of the most important mediators of p53 phosphorylation at serine 389 induced by UVC radiation. (+info)
A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory.
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Behavioral, biophysical, and pharmacological studies have implicated the hippocampus in the formation and storage of spatial memory. However, the molecular mechanisms underlying long-term spatial memory are poorly understood. In this study, we show that mitogen-activated protein kinase (MAPK, also called ERK) is activated in the dorsal, but not the ventral, hippocampus of rats after training in a spatial memory task, the Morris water maze. The activation was expressed as enhanced phosphorylation of MAPK in the pyramidal neurons of the CA1/CA2 subfield. In contrast, no increase in the percentage of phospho-MAPK-positive cells was detected in either the CA3 subfield or the dentate gyrus. The enhanced phosphorylation was observed only after multiple training trials but not after a single trial or after multiple trials in which the location of the target platform was randomly changed between each trial. Inhibition of the MAPK/ERK cascade in dorsal hippocampi did not impair acquisition, but blocked the formation of long-term spatial memory. In contrast, intrahippocampal infusion of SB203580, a specific inhibitor of the stress-activated MAPK (p38 MAPK), did not interfere with memory storage. These results demonstrate a MAPK-mediated cellular event in the CA1/CA2 subfields of the dorsal hippocampus that is critical for long-term spatial memory. (+info)
Receptor binding, behavioral, and electrophysiological profiles of nonpeptide corticotropin-releasing factor subtype 1 receptor antagonists CRA1000 and CRA1001.
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Receptor binding, behavioral, and electrophysiological profiles of 2-[N-(2-methylthio-4-isopropylphenyl)-N-ethylamino]-4-[4-(3-flu orophe nyl)-1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1000) and 2-[N-(2-bromo-4-isopropylphenyl)-N-ethylamino]-4-[4-(3-fluoropheny l)- 1,2,3,6-tetrahydropyridin-1-yl)-6-methylpyrimidine (CRA1001), putative novel and selective antagonists for corticotropin-releasing factor1 (CRF1) receptor were examined. Both CRA1000 and CRA1001 inhibited 125I-ovine CRF binding to membranes of rat frontal cortex with IC50 values of 20.6 and 22.3 nM, respectively. Likewise, CRA1000 and CRA1001 inhibited 125I-ovine CRF binding to membranes of rat pituitary. In contrast, both CRA1000 and CRA1001 were without affinity for the CRF2beta receptor when examined using rat heart. In mice orally administered CRA1000 and CRA1001 reversed the swim stress-induced reduction of the time spent in the light area in the light/dark exploration task. In nonstress conditions, CRA1000 and CRA1001 were without effect on the time spent in the light area in the same task in mice. Orally administered CRA1000 and CRA1001 dose dependently reversed the effects of i.c.v. infusion of CRF on time spent in the open arms in the elevated plus-maze in rats. Lesioning of olfactory bulbs induced hyperemotionality, and this effect was inhibited by either acute or chronic oral administration of CRA1000 and CRA1001 in rats. The firing rate of locus coeruleus neurons was increased by i.c.v.-infused CRF. This excitation of locus coeruleus neurons was significantly blocked by pretreatment with i.v. administration of CRA1000 and CRA1001. CRA1000 and CRA1001 had no effects on the hexobarbital-induced anesthesia in mice, the rotarod test in mice, the spontaneous locomotor activity in mice, and a passive avoidance task in rats. These observations indicate that both CRA1000 and CRA1001 are selective and competitive CRF1 receptor antagonists with potent anxiolytic- and antidepressant-like properties in various experimental animal models, perhaps through inhibition of CRF1 receptors. CRA1000 and CRA1001 may prove effective for treating subjects with depression- and/or anxiety-related disorders without the side effects seen in the related currently prescribed medications. (+info)
Insulin differentially affects xenobiotic-enhanced, cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A expression in primary cultured rat hepatocytes.
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Uncontrolled diabetes results in enhanced expression of cytochrome P-450 (CYP)2E1, CYP2B, CYP3A, and CYP4A. Because of the simultaneous and confounding metabolic and hormonal changes that occur in vivo as a consequence of diabetes, primary cultured rat hepatocytes provide an excellent model system for examination of the effects of insulin on P-450 expression and on xenobiotic-mediated P-450 expression. In the present study, we examined the effects of insulin on pyridine-, phenobarbital-, and ciprofibrate-mediated expression of CYP2E1, CYP2B, CYP3A, and CYP4A in primary cultured rat hepatocytes. Pyridine addition to primary rat hepatocytes cultured in the presence of 1 nM insulin or in the absence of insulin resulted in a 3.5-fold and 3-fold enhancement in CYP2E1 protein expression, respectively, in the absence of any pyridine-mediated increase in mRNA expression. In contrast, hepatocytes cultured in the standard concentration of 1 microM insulin resulted in only a 2-fold increase in protein expression. Thus, the fold-induction of CYP2E1 protein in response to pyridine was 1.5- to 1.8-fold greater in either the absence of insulin or in the presence of 1 nM insulin, respectively, than that monitored in the presence of 1 microM insulin. To examine whether insulin effects on xenobiotic-mediated CYP2E1 expression were selective, insulin effects on xenobiotic-mediated expression of transcriptionally regulated CYP2B, CYP3A, and CYP4A were examined. Pyridine- or phenobarbital-mediated induction of CYP2B mRNA and protein expression in hepatocytes was suppressed by as much as 80% at lower insulin levels (0 and 1 nM), relative to the level monitored in the presence of 1 microM insulin. Omitting insulin from the medium resulted in a 50% decrease in CYP3A mRNA levels in response to phenobarbital treatment and a 30% decrease in CYP4A mRNA levels in response to ciprofibrate treatment, relative to the level obtained in response to these treatments in the presence of 1 microM insulin. The results of this study demonstrate that decreasing the insulin level in the primary hepatocyte culture medium enhanced xenobiotic-mediated CYP2E1 expression, whereas lower insulin levels suppressed xenobiotic-mediated CYP2B, CYP3A, and CYP4A expression in this cell culture system. (+info)
Metabolic interactions between mibefradil and HMG-CoA reductase inhibitors: an in vitro investigation with human liver preparations.
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AIMS: To determine the effects of mibefradil on the nletabolism in human liver microsomal preparations of the HMG-CoA reductase inhibitors simvastatin, lovastatin, atorvastatin, cerivastatin and fluvastatin. METHODS: Metabolism of the above five statins (0.5, 5 or 10 microM), as well as of specific CYP3A4/5 and CYP2C8/9 marker substrates, was examined in human liver microsomal preparations in the presence and absence of mibefradil (0.1-50 microM). RESULTS: Mibefradil inhibited, in a concentration-dependent fashion, the metabolism of the four statins (simvastatin, lovastatin, atorvastatin and cerivastatin) known to be substrates for CYP3A. The potency of inhibition was such that the IC50 values (<1 microM) for inhibition of all of the CYP3A substrates fell within the therapeutic plasma concentrations of mibefradil, and was comparable with that of ketoconazole. However, the inhibition by mibefradil, unlike that of ketoconazole, was at least in part mechanism-based. Based on the kinetics of its inhibition of hepatic testosterone 6beta-hydroxylase activity, mibefradil was judged to be a powerful mechanism-based inhibitor of CYP3A4/5, with values for Kinactivation, Ki and partition ratio (moles of mibefradil metabolized per moles of enzyme inactivated) of 0.4 min(-1), 2.3 microM and 1.7, respectively. In contrast to the results with substrates of CYP3A, metabolism of fluvastatin, a substrate of CYP2C8/9, and the hydroxylation of tolbutamide, a functional probe for CYP2C8/9, were not inhibited by mibefradil. CONCLUSION: Mibefradil, at therapeutically relevant concentrations, strongly suppressed the metabolism in human liver microsomes of simvastatin, lovastatin, atorvastatin and cerivastatin through its inhibitory effects on CYP3A4/5, while the effects of mibefradil on fluvastatin, a substrate for CYP2C8/9, were minimal in this system. Since mibefradil is a potent mechanism-based inhibitor of CYP3A4/5, it is anticipated that clinically significant drug-drug interactions will likely ensue when mibefradil is coadministered with agents which are cleared primarily by CYP3A-mediated pathways. (+info)