scholarly journals Requirement for RAR-mediated gene repression in skeletal progenitor differentiation

2002 ◽  
Vol 158 (1) ◽  
pp. 39-51 ◽  
Author(s):  
Andrea D. Weston ◽  
Roshantha A.S. Chandraratna ◽  
Joseph Torchia ◽  
T. Michael Underhill
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Retinoic Acid Receptor ◽  
Trichostatin A ◽  
Close Association ◽  
Primary Cultures ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Gene Repression ◽  
Retinoid Receptor

Chondrogenesis is a multistep process culminating in the establishment of a precisely patterned template for bone formation. Previously, we identified a loss in retinoid receptor–mediated signaling as being necessary and sufficient for expression of the chondroblast phenotype (Weston et al., 2000. J. Cell Biol. 148:679–690). Here we demonstrate a close association between retinoic acid receptor (RAR) activity and the transcriptional activity of Sox9, a transcription factor required for cartilage formation. Specifically, inhibition of RAR-mediated signaling in primary cultures of mouse limb mesenchyme results in increased Sox9 expression and activity. This induction is attenuated by the histone deacetylase inhibitor, trichostatin A, and by coexpression of a dominant negative nuclear receptor corepressor-1, indicating an unexpected requirement for RAR-mediated repression in skeletal progenitor differentiation. Inhibition of RAR activity results in activation of the p38 mitogen-activated protein kinase (MAPK) and protein kinase A (PKA) pathways, indicating their potential role in the regulation of chondrogenesis by RAR repression. Accordingly, activation of RAR signaling, which attenuates differentiation, can be rescued by activation of p38 MAPK or PKA. In summary, these findings demonstrate a novel role for active RAR-mediated gene repression in chondrogenesis and establish a hierarchical network whereby RAR-mediated signaling functions upstream of the p38 MAPK and PKA signaling pathways to regulate emergence of the chondroblast phenotype.

scholarly journals Role of MKK3 and p38 MAPK in cytokine-induced death of insulin-producing cells

2005 ◽  
Vol 393 (1) ◽  
pp. 129-139 ◽  
Author(s):  
Natalia Makeeva ◽  
Jason W. Myers ◽  
Nils Welsh
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Protein Kinase ◽  
P38 Mapk ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Nitric Oxide Donor ◽  
Cell Leukaemia ◽  
Dominant Negative Mutant

The aim of the present investigation was to elucidate further the importance of p38 MAPK (mitogen-activated protein kinase) in nitric oxide- and cytokine-induced β-cell death. For this purpose, isolated human islets were treated with d-siRNA (diced small interfering RNA) and then exposed to the nitric oxide donor DETA/NONOate [2,2′-(hydroxynitrosohydrazono)bis-ethanamine]. We observed that cells treated with p38α-specific d-siRNA, but not with d-siRNA targeting GL3 (a firefly luciferase siRNA plasmid) or PKCδ (protein kinase Cδ), were protected against nitric oxide-induced death. This was paralleled by an increased level of Bcl-XL (B-cell leukaemia/lymphoma-X long). For an in-depth study of the mechanisms of p38 activation, MKK3 (MAPK kinase 3), MKK6 and their dominant-negative mutants were overexpressed in insulin-producing RIN-5AH cells. In transient transfections, MKK3 overexpression resulted in increased p38 phosphorylation, whereas in stable MKK3-overexpressing RIN-5AH clones, the protein levels of p38 and JNK (c-Jun N-terminal kinase) were decreased, resulting in unaffected phospho-p38 levels. In addition, a long-term MKK3 overexpression did not affect cell death rates in response to the cytokines interleukin-1β and interferon-γ, whereas a short-term MKK3 expression resulted in increased cytokine-induced RIN-5AH cell death. The MKK3-potentiating effect on cytokine-induced cell death was abolished by a nitric oxide synthase inhibitor, and MKK3-stimulated p38 phosphorylation was enhanced by inhibitors of phosphatases. Finally, as the dominant-negative mutant of MKK3 did not affect cytokine-induced p38 phosphorylation, and as wild-type MKK3 did not influence p38 autophosphorylation, it may be that p38 is activated by MKK3/6-independent pathways in response to cytokines and nitric oxide. In addition, it is likely that a long-term increase in p38 activity is counteracted by both a decreased expression of the p38, JNK and p42 genes as well as an increased dephosphorylation of p38.

Differentiation regulates interleukin-1β-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase

2002 ◽  
Vol 362 (2) ◽  
pp. 367-373 ◽  
Author(s):  
Béatrice THOMAS ◽  
Sylvie THIRION ◽  
Lydie HUMBERT ◽  
Lujian TAN ◽  
Mary B. GOLDRING ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Articular Chondrocytes ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Human Articular Chondrocytes ◽  
Osteoarthritic Cartilage ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Cox 2

Chondrocyte dedifferentiation has been noted in osteoarthritic cartilage, but the contribution of this phenomenon is poorly understood. Interleukin (IL)-1β, the major pro-inflammatory cytokine found in osteoarthritic synovial fluid, induces the dedifferentiation of cultured articular chondrocytes, whereas E-series prostaglandins (PGE) are capable of inducing cell differentiation. Since PGE2 synthesis is up-regulated by IL-1β, we addressed the question of whether the state of chondrocyte differentiation may influence the production of IL-1-induced PGE2 by modulating cyclooxygenase (COX)-2 expression. Immortalized human articular chondrocytes, (tsT/AC62) cultured in monolayer after passage through alginate matrix (alg+) produced 5-fold greater amounts of PGE2 than continuous monolayer cultures (alg-) after stimulation with IL-1β. Moreover, IL-1β induced COX-2 expression at 0.01ng/ml in (alg+) cells, whereas a 100-fold higher dose of cytokine was necessary for stimulation in (alg-) cells. SB203580, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor, completely abolished the IL-1β-induced COX-2 mRNA. Overexpression of p38 MAPK induces a COX-2 reporter, whereas overexpression of dominant negative p38 MAPK represses IL-1β-induced promoter expression. Interestingly, IL-1β-induced p38 MAPK activity was greatly enhanced in (alg+) compared with (alg-) cells. Our results suggest that differentiated articular chondrocytes are highly responsive to IL-1β and that p38 MAPK mediates this response by inducing COX-2 gene expression.

scholarly journals Trophic Factor Withdrawal: p38 Mitogen-Activated Protein Kinase Activates NHE1, Which Induces Intracellular Alkalinization

2001 ◽  
Vol 21 (22) ◽  
pp. 7545-7557 ◽  
Author(s):  
Annette R. Khaled ◽  
Andrea N. Moor ◽  
Aiqun Li ◽  
Kyungjae Kim ◽  
Douglas K. Ferris ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Mapk Pathway ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Trophic Factor ◽  
Cytokine Signaling ◽  
Amino Acid Region ◽  
Mitogen Activated Protein ◽  
Intracellular Alkalinization

ABSTRACT Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

scholarly journals Novel Mechanism for Gonadotropin-Releasing Hormone Neuronal Migration Involving Gas6/Ark Signaling to p38 Mitogen-Activated Protein Kinase

2002 ◽  
Vol 22 (2) ◽  
pp. 599-613 ◽  
Author(s):  
Melissa P. Allen ◽  
Daniel A. Linseman ◽  
Hiroshi Udo ◽  
Mei Xu ◽  
Jerome B. Schaack ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Neuronal Migration ◽  
Neuronal Cells ◽  
Gonadotropin Releasing Hormone ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Gnrh Neuron ◽  
Releasing Hormone ◽  
Mitogen Activated Protein

ABSTRACT Gonadotropin-releasing hormone (GnRH) is the central regulator of the reproductive axis. Normal sexual maturation depends on the migration of GnRH neurons from the olfactory placode to the hypothalamus during development. Previously, we showed restricted expression of the membrane receptor adhesion-related kinase (Ark) in immortalized cell lines derived from migratory but not postmigratory GnRH neurons. In addition, Ark and GnRH transcripts were detected along the GnRH neuron migratory route in the E13 mouse cribriform plate. In the present study, we examined the role of Ark and its ligand, Gas6 (encoded by growth arrest-specific gene 6), in GnRH neuron migration. Gas6 stimulated lamellipodial extension, membrane ruffling, and chemotaxis of immortalized NLT GnRH neuronal cells via the Ark receptor. Gas6/Ark signaling promoted activation of the Rho family GTPase Rac, and adenoviral-mediated expression of dominant negative N17Rac abolished Gas6/Ark-induced actin cytoskeletal reorganization and migration of GnRH neuronal cells. In addition, p38 MAPK was activated downstream of Ark and Rac, and inhibition of p38 MAPK with either SB203580 or adenoviral dominant negative p38α also blocked Gas6/Ark-mediated migration. Finally, downstream of Rac and p38 mitogen-activated protein kinase (MAPK), Gas6/Ark signaling promoted activation of MAPK-activated protein kinase 2 and induced phosphorylation of HSP25, a known regulator of cortical actin remodeling. The data are the first to demonstrate a migratory signaling pathway downstream of Ark/Axl family receptors and suggest a previously unidentified role for p38 MAPK in neuronal migration. Furthermore, these studies support a potential role for Ark in the regulation of GnRH neuronal migration.

scholarly journals Transcriptional Regulators of theSchizosaccharomyces pombe fbp1Gene Include Two Redundant Tup1p-like Corepressors and the CCAAT Binding Factor Activation Complex

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1205-1215 ◽  
Author(s):  
Rozmin T K Janoo ◽  
Lori A Neely ◽  
Burkhard R Braun ◽  
Simon K Whitehall ◽  
Charles S Hoffman
Keyword(s):  
Protein Kinase ◽  
Fold Increase ◽  
Transcriptional Regulators ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Lacz Expression ◽  
Protein Subunit ◽  
Plasmid Library ◽  
Binding Factor ◽  
Mitogen Activated Protein

AbstractThe Schizosaccharomyces pombe fbp1 gene, which encodes fructose-1,6-bis-phosphatase, is transcriptionally repressed by glucose through the activation of the cAMP-dependent protein kinase A (PKA) and transcriptionally activated by glucose starvation through the activation of a mitogen-activated protein kinase (MAPK). To identify transcriptional regulators acting downstream from or in parallel to PKA, we screened an adh-driven cDNA plasmid library for genes that increase fbp1 transcription in a strain with elevated PKA activity. Two such clones express amino-terminally truncated forms of the S. pombe tup12 protein that resembles the Saccharomyces cerevisiae Tup1p global corepressor. These clones appear to act as dominant negative alleles. Deletion of both tup12 and the closely related tup11 gene causes a 100-fold increase in fbp1-lacZ expression, indicating that tup11 and tup12 are redundant negative regulators of fbp1 transcription. In strains lacking tup11 and tup12, the atf1-pcr1 transcriptional activator continues to play a central role in fbp1-lacZ expression; however, spc1 MAPK phosphorylation of atf1 is no longer essential for its activation. We discuss possible models for the role of tup11- and tup12-mediated repression with respect to signaling from the MAPK and PKA pathways. A third clone identified in our screen expresses the php5 protein subunit of the CCAAT-binding factor (CBF). Deletion of php5 reduces fbp1 expression under both repressed and derepressed conditions. The CBF appears to act in parallel to atf1-pcr1, although it is unclear whether or not CBF activity is regulated by PKA.

scholarly journals Interleukin-6 Inhibits Fas-Induced Apoptosis and Stress-Activated Protein Kinase Activation in Multiple Myeloma Cells

Blood ◽  
1997 ◽  
Vol 89 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Dharminder Chauhan ◽  
Surender Kharbanda ◽  
Atsushi Ogata ◽  
Mitsuyoshi Urashima ◽  
Gerrard Teoh ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Kinase ◽  
Dna Fragmentation ◽  
P38 Mapk ◽  
Interleukin 6 ◽  
Early Response ◽  
Mitogen Activated Protein Kinase ◽  
Serum Starvation ◽  
Mapk Activity ◽  
Induced Apoptosis

Abstract Fas belongs to the family of type-1 membrane proteins that transduce apoptotic signals. In the present studies, we characterized signaling during Fas-induced apoptosis in RPMI-8226 and IM-9 multiple myeloma (MM) derived cell lines as well as patient plasma cell leukemia cells. Treatment with anti-Fas (7C11) monoclonal antibody (MoAb) induced apoptosis, evidenced by internucleosomal DNA fragmentation and propidium iodide staining, and was associated with increased expression of c-jun early response gene. We also show that anti-Fas MoAb treatment is associated with activation of stress-activated protein kinase (SAPK) and p38 mitogen-activated protein kinase (MAPK); however, no detectable increase in extracellular signal-regulated kinases (ERK1 and ERK2) activity was observed. Because interleukin-6 (IL-6) is a growth factor for MM cells and inhibits apoptosis induced by dexamethasone and serum starvation, we examined whether IL-6 affects anti-Fas MoAb-induced apoptosis and activation of SAPK or p38 MAPK in MM cells. Culture of MM cells with IL-6 before treatment with anti-Fas MoAb significantly reduced both DNA fragmentation and activation of SAPK, without altering induction of p38 MAPK activity. These results therefore suggest that anti-Fas MoAb-induced apoptosis in MM cells is associated with activation of SAPK, and that IL-6 may both inhibit apoptosis and modulate SAPK activity.

scholarly journals Attenuation of CHOP-mediated Myocardial Apoptosis in Pressure-overloaded Dominant Negative p38α Mitogen-activated Protein Kinase Mice

2011 ◽  
Vol 27 (5) ◽  
pp. 487-496 ◽  
Author(s):  
Flori R. Sari ◽  
Bambang Widyantoro ◽  
Rajarajan A. Thandavarayan ◽  
Meilei Harima ◽  
Arun Prasath Lakshmanan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Myocardial Apoptosis ◽  
Mitogen Activated Protein

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

2012 ◽  
Vol 123 (3) ◽  
pp. 147-159 ◽  
Author(s):  
Ting-Hsing Chao ◽  
Shih-Ya Tseng ◽  
Yi-Heng Li ◽  
Ping-Yen Liu ◽  
Chung-Lung Cho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
No Production ◽  
Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Modification of ischemia/reperfusion induced infarct size by ischemic preconditioning in hypertrophied hearts

2021 ◽  
Vol 99 (2) ◽  
pp. 218-223
Author(s):  
Mohamad Nusier ◽  
Mohammad Alqudah ◽  
Vijayan Elimban ◽  
Naranjan S. Dhalla
Keyword(s):  
Protein Kinase ◽  
Cardiac Hypertrophy ◽  
Infarct Size ◽  
P38 Mapk ◽  
Ischemic Preconditioning ◽  
Creatine Phosphokinase ◽  
Ischemia Reperfusion ◽  
Mitogen Activated Protein Kinase ◽  
Normal Heart ◽  
Mitogen Activated Protein

This study examined the effects of ischemic preconditioning (IP) on the ischemia/reperfusion (I/R) induced injury in normal and hypertrophied hearts. Cardiac hypertrophy in rabbits was induced by L-thyroxine (0.5 mg/kg/day for 16 days). Hearts with or without IP (3 cycles of 5 min ischemia and 10 min reperfusion) were subjected to I/R (60 min ischemia followed by 60 min reperfusion). IP reduced the I/R-induced infarct size from 68% to 24% and 57% to 33% in the normal and hypertrophied hearts, respectively. Leakage of creatine phosphokinase in the perfusate from the hypertrophied hearts due to I/R was markedly less than that form the normal hearts; IP prevented these changes. Although IP augmented the increase in phosphorylated p38-mitogen-activated protein kinase (p38-MAPK) content due to I/R, this effect was less in the hypertrophied than in the normal heart. These results suggest that reduced cardioprotection by IP of the I/R-induced injury in hypertrophied hearts may be due to reduced activation of p38-MAPK in comparison with normal hearts.

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