Association Of Mitogen-activated Protein Kinase Research Articles

Anti-inflammatory effects of dexmedetomidine on human amnion-derived WISH cells.

Background: To maintain the normal pregnancy, suppression of inflammatory signaling pathway is a crucial physiologic response. Dexmedetomidine has been used for labor analgesia or supplement of inadequate regional analgesia during delivery. And it has been reported that dexmedetomidine has an anti-inflammatory effect. In this study, we examined the influence of dexmedetomidine on the expression of cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2) and inflammatory cytokines in lipopolysaccharide (LPS)-stimulated human amnion-derived WISH cells. In addition, we evaluated the association of mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathway in anti-inflammatory effect of dexmedetomidine.Methods: Human amnion-derived WISH cells were pretreated with various concentrations of dexmedetomidine (0.001-1 µg/ml) for 1 h and after then treated with LPS (1 µg/ml) for 24 h. MTT assay was conducted to evaluate the cytotoxicity. Nitric oxide (NO) production was analyzed using Griess-reaction microassay. RT-PCR was performed for analysis of mRNA expressions of COX-2, PGE2, tumor necrosis factor (TNF)-α and interlukin (IL)-1β. Protein expressions of COX-2, PGE2, p38 and NF-κB were analyzed by western blotting.Results: LPS and dexmedetomidine had no cytotoxic effect on WISH cells. There was no difference in NO production after dexmedetomidine pretreatment. The mRNA and protein expressions of COX-2 and PGE2 were decreased by dexmedetomidine pretreatment in LPS-treated WISH cells. Dexmedetomidine also attenuated the LPS-induced mRNA expression of TNF-α and IL-1β. The activation of p38 and NF-κB was suppressed by dexmedetomidine pretreatment in LPS-treated WISH cells.Conclusion: We demonstrated that dexmedetomidine pretreatment suppressed the expressions of inflammatory mediators increased by LPS. In addition, this study suggests that anti-inflammatory effect of dexmedetomidine on WISH cells was mediated by the inhibitions of p38 and NF-κB activation.

Nongenomic actions of l-thyroxine and 3,5,3′-triiodo-l-thyronine. Focus on “l-Thyroxine vs. 3,5,3′-triiodo-l-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase”

<i>Nongenomic actions of <scp>l</scp>-thyroxine and 3,5,3′-triiodo-<scp>l</scp>-thyronine.</i> Focus on “<scp>l</scp>-Thyroxine vs. 3,5,3′-triiodo-<scp>l</scp>-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase”

Association of mitogen-activated protein kinase with upregulation of plasminogen activator inhibitor-1 in rat astrocytes stimulated with plasminogen

Association of mitogen-activated protein kinase with upregulation of plasminogen activator inhibitor-1 in rat astrocytes stimulated with plasminogen

Resveratrol Induced Serine Phosphorylation Of p53 Causes Apoptosis In A Mutant p53 Prostate Cancer Cell Line

Resveratrol (Calbiochem, La Jolla, California) is a naturally occurring stilbene reported to cause apoptosis in various cultured cancer cells. In the current study the effect of resveratrol was determined in the androgen insensitive DU 145 prostate cancer cell line. Induction of apoptosis and activation of apoptosis related signal transduction pathways were measured. DU 145 cells were treated with resveratrol and apoptosis was measured by determining nucleosome content. Activation of mitogen activated protein kinase (MAPK) (extracellular signal-regulated kinase 1/2), p53 content and serine-15 phosphorylation of p53 were measured by immunoblot. Electrophoretic mobility shift assay of p53 binding to DNA, and measurement of p21 and glyceraldehyde-3-phosphate dehydrogenase messenger RNA were also done. Resveratrol induced apoptosis in DU 145 cells. The stilbene activated MAPK and caused increased abundance of p53 and serine-15 phosphorylated p53. Resveratrol induced serine-15 phosphorylation of p53 was blocked by PD 98059 (Calbiochem), a MAPK kinase inhibitor, implicating MAPK activation in the phosphorylation of p53. PD 98059 also inhibited resveratrol induced apoptosis. These results suggest that apoptosis induction by resveratrol in DU 145 cells requires serine-15 phosphorylation of p53 by MAPK. Inhibition of MAPK dependent serine-15 phosphorylation resulted in reduced p53 binding to a p53 specific oligonucleotide on electrophoretic mobility shift assay. Pifithrin-alpha (Calbiochem), a p53 inhibitor, blocked resveratrol induced serine-15 phosphorylation of p53 and p53 binding to DNA. Resveratrol caused a p53 stimulated increase in p21 messenger RNA. Transfection of additional wild-type p53 into DU 145 cells induced apoptosis, which was further enhanced by resveratrol treatment. Resveratrol causes apoptosis in DU 145 prostate cancer cells. This action depends on the activation of MAPK, increase in cellular p53 content, serine-15 phosphorylation of p53 and increased p53 binding to DNA.

Thyroid hormone promotes serine phosphorylation of p53 by mitogen-activated protein kinase.

L-Thyroxine (T(4)) nongenomically promotes association of mitogen-activated protein kinase (MAPK) and thyroid hormone receptor TRbeta1 (TR) in the cell nucleus, leading to serine phosphorylation of the receptor. The oncogene suppressor protein, p53, is serine phosphorylated by several kinases and is known to interact with TRbeta1. We studied whether association of p53 and TR is modulated by T(4) and involves serine phosphorylation of p53 by MAPK. TR-replete 293T human kidney cells were incubated with a physiological concentration of T(4) for 10-90 min. Nuclear fractions were immunoprecipitated and the resulting proteins separated and immunoblotted for co-immunoprecipitated proteins. Activated MAPK immunoprecipitates of nuclei from T(4)-treated cells accumulated p53 in a time-dependent manner; T(4) and T(4)-agarose were more effective than T(3). T(4)-induced nuclear complexing of p53 and MAPK was inhibited by PD 98059 (PD) and U0126, two MAPK kinase (MEK) inhibitors, and was absent in cells treated with MEK antisense oligonucleotide and in dominant negative Ras cells. T(4) also caused nuclear co-immunoprecipitation of TRbeta1 and p53, an effect also inhibited by PD. Nuclear complexing of p53 and MAPK also occurred in HeLa cells, which lack functional TR. Constitutively activated MAPK caused phosphorylation of a recombinant p53-GST fusion protein in vitro; thus, p53 is a substrate for MAPK. An indicator of p53 transcriptional activity, accumulation of the immediate-early gene product, c-Jun, was inhibited by T(4). This T(4) effect was reversed by PD, indicating that the transcriptional activity of p53 was altered by T(4)-directed MAPK-p53 interaction.

Thyroxine Promotes Association of Mitogen-activated Protein Kinase and Nuclear Thyroid Hormone Receptor (TR) and Causes Serine Phosphorylation of TR

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.

Association of mitogen-activated protein kinase with the microtubule cytoskeleton.

Using indirect immunofluorescence microscopy and biochemical techniques, we have determined that approximately one-third of the total mitogen-activated protein kinase (MAPK) is associated with the microtubule cytoskeleton in NIH 3T3 mouse fibroblasts. This population of enzyme can be separated from the soluble form that is found distributed throughout the cytosol and is also present in the nucleus after mitogen stimulation. The microtubule-associated enzyme pool constitutes half of all detectable MAPK activity after mitogenic stimulation. These findings extend the known in vivo associations of MAPK with microtubules to include the entire microtubule cytoskeleton of proliferating cells, and they suggest that a direct association of MAPK with microtubules may be in part responsible for the observed correlations between MAPK activities and cytoskeletal alteration.

MAP Kinase Becomes Stably Activated at Metaphase and Is Associated with Microtubule-Organizing Centers during Meiotic Maturation of Mouse Oocytes

Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate Mr 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation. These forms remained throughout maturation and in oocytes arrested at metaphase II. Following oocyte activation, which induces a transition to interphase, the slow-migrating forms were replaced by the fast-migrating forms observed in prophase oocytes. MAP kinase activity also increased after oocytes entered metaphase, and this increase required protein synthesis and phosphorylation. To investigate the intracellular distribution of the immunoreactive species, spindles were purified from metaphase II eggs. Both the 42- and the 44-kDa species were detected in immunoblots, and bright staining of the spindle poles was observed by immunofluorescence. When intact oocytes undergoing maturation were examined by immunofluorescence, foci of staining were initially detected on opposing sides of the condensing chromosomes and then became congregated at each pole of the first meiotic spindle. No localized staining was observed during the first meiotic division, but stained foci were present at the poles of the second meiotic spindle. In addition, several cytoplasmic foci of staining often could be seen. When oocytes were exposed to taxol, which permits nonspindle microtubule-organizing centers (MTOCs) present in the cytoplasm to nucleate microtubule assembly, the cytoplasmic foci labeled by the MAP kinase antibodies were found to contain tubulin. We conclude that mouse oocytes contain 42- and 44-kDa species of MAP kinase and that, after maturing oocytes enter metaphase, MAP kinase activity is stimulated by means of a process requiring protein synthesis and phosphorylation. MAP kinase is present in the spindle and is specifically associated with the MTOCs present at the spindle poles and in the cytoplasm. Evidence from cell-free systems suggests that the alterations in MTOC activity that normally occur at metaphase in oocytes may be regulated by MAP kinase. The association of MAP kinase with MTOCs provides a potential structural basis for this cell cycle-dependent change in MTOC activity.