Regulation and distribution of MAdCAM-1 in endothelial cells in vitro

2001 ◽  
Vol 281 (4) ◽  
pp. C1096-C1105 ◽  
Author(s):  
Tadayuki Oshima ◽  
Kevin P. Pavlick ◽  
F. Stephen Laroux ◽  
S. Kris Verma ◽  
Paul Jordan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Extracellular Signal ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Cell Adhesion Molecule 1

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a 60-kDa endothelial cell adhesion glycoprotein that regulates lymphocyte trafficking to Peyer's patches and lymph nodes. Although it is widely agreed that MAdCAM-1 induction is involved in chronic gut inflammation, few studies have investigated regulation of MAdCAM-1 expression. We used two endothelial lines [bEND.3 (brain) and SVEC (high endothelium)] to study the signal paths that regulate MAdCAM-1 expression in response to tumor necrosis factor (TNF)-α using RT-PCR, blotting, adhesion, and immunofluorescence. TNF-α induced both MAdCAM-1 mRNA and protein in a dose- and time-dependent manner. This induction was tyrosine kinase (TK), p42/44, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB/poly-ADP ribose polymerase (PARP) dependent. Because MAdCAM-1 is regulated via MAPKs, we examined mitogen/extracellular signal-regulated kinase (MEK)-1/2 activation in SVEC. We found that MEK-1/2 is activated by TNF-α within minutes and is dependent on TK and p42/44 MAPKs. Similarly, TNF-α activated NF-κB through TK, p42/44, p38 MAPKs, and PARP pathways in SVEC cells. MAdCAM-1 was also shown to be frequently distributed to endothelial junctions both in vitro and in vivo. Cytokines like TNF-α stimulate MAdCAM-1 in high endothelium via TK, p38, p42/22 MAPKs, and NF-κB/PARP. MAdCAM-1 expression requires NF-κB translocation through both direct p42/44 and indirect p38 MAPK pathways in high endothelial cells.

scholarly journals Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

2005 ◽  
Vol 25 (2) ◽  
pp. 854-864 ◽  
Author(s):  
Sandrine Marchetti ◽  
Clotilde Gimond ◽  
Jean-Claude Chambard ◽  
Thomas Touboul ◽  
Danièle Roux ◽  
...  
Keyword(s):  
Map Kinases ◽  
Fluorescent Protein ◽  
Proteasomal Degradation ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Double Mutation ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.

scholarly journals Effects of TNF-α on Leukocyte Adhesion Molecule Expressions in Cultured Human Lymphatic Endothelium

2007 ◽  
Vol 55 (7) ◽  
pp. 721-733 ◽  
Author(s):  
Yoshihiko Sawa ◽  
Yukitaka Sugimoto ◽  
Takeshi Ueki ◽  
Hiroyuki Ishikawa ◽  
Atuko Sato ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Leukocyte Adhesion ◽  
Dependent Manner ◽  
Lymphatic Endothelium ◽  
Leukocyte Adhesion Molecule ◽  
Tnf Α ◽  
Cell Adhesion Molecule 1

TNF-α alters leukocyte adhesion molecule expression of cultured endothelial cells like human umbilical vein endothelial cells (HUVEC). This study was designed to investigate the changes in vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) expression with TNF-α stimulation in cultured human neonatal dermal lymphatic endothelial cells (HNDLEC). The real-time quantitative PCR analysis on HNDLEC showed that TNF-α treatment leads to increases of VCAM-1 and ICAM-1 mRNAs to the 10.8- and 48.2-fold levels of untreated cells and leads to a reduction of PECAM-1 mRNA to the 0.42-fold level of untreated cells. Western blot and immunohistochemical analysis showed that TNF-α leads to VCAM-1 and ICAM-1 expressions that were inhibited by antiserum to human TNF receptor or by AP-1 inhibitor nobiletin. In flow cytometry analysis, the number of VCAM-1- and ICAM-1-positive cells increased, and PECAM-1-positive cells decreased with TNF-α treatment. Regarding protein amounts produced in cells and amounts expressed on the cell surface, VCAM-1 and ICAM-1 increased in HNDLEC and HUVEC, and PECAM-1 decreased in HNDLEC in a TNF-α concentration-dependent manner. VCAM-1, ICAM-1, and PECAM-1 protein amounts in TNF-α-stimulated cells were lower in HNDLEC than in HUVEC. This suggests that the lymphatic endothelium has the TNF-α-induced signaling pathway, resulting in increased VCAM-1 and ICAM-1 expression to a weaker extent than blood endothelium and PECAM-1 reduction to a stronger extent than blood endothelium.

scholarly journals Pharmacological Inhibition of p38 Mitogen-Activated Protein Kinases Affects KC/CXCL1-Induced Intraluminal Crawling, Transendothelial Migration, and Chemotaxis of NeutrophilsIn Vivo

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Najia Xu ◽  
Mokarram Hossain ◽  
Lixin Liu
Keyword(s):  
P38 Mapk ◽  
Neutrophil Recruitment ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Mitogen Activated Protein ◽  
Multistep Process ◽  
Inflammatory Processes ◽  
P38 Mapk Inhibitor

p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitmentin vivo.

scholarly journals ERK5 is targeted to myocyte enhancer factor 2A (MEF2A) through a MAPK docking motif

2004 ◽  
Vol 381 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Dalia BARSYTE-LOVEJOY ◽  
Alex GALANIS ◽  
Anne CLANCY ◽  
Andrew D. SHARROCKS
Keyword(s):  
Catalytic Domain ◽  
Mutational Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Substrate ◽  
Myocyte Enhancer Factor ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Enhancer Factor

One critical component in determining the specificity, and efficiency of MAPK (mitogen-activated protein kinase) substrate phophorylation is the presence of distinct docking domains in the substrate proteins. Docking domains have been shown to be important for the activities of members of the ERK (extracellular-signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 subfamilies of MAPKs towards their substrates. Here, we demonstrate that docking domains also play an important role in ERK5-mediated substrate phosphorylation. The presence of a docking domain promotes both phosphorylation of myocyte enhancer factor, MEF2A, in vitro and its activation in vivo by ERK5. Mutational analysis of the MEF2A docking domain demonstrates that the specificity determinants for ERK5 are similar to those observed with members of the p38 subfamily. A docking domain recognized by ERK5 can direct ERK5 to activate heterologous substrates. Deletion analysis demonstrates that as with other MAPKs, it is the catalytic domain of ERK5 that recognizes the docking domain. Our data therefore extend previous observations on other MAPKs and demonstrate that the requirement for specific docking domains in promoting MAPK action towards substrates is a general property of MAPKs.

scholarly journals Chinese herbal formulas for postmenopausal osteoporosis: A review of preclinical evidence on animal studies and molecular mechanism

2018 ◽  
Vol 01 (02) ◽  
pp. 75-83
Author(s):  
Jing Lin ◽  
Tian Lv ◽  
Fubo Tian ◽  
Yan Wang ◽  
Mingyan Wang ◽  
...  
Keyword(s):  
Postmenopausal Osteoporosis ◽  
Animal Studies ◽  
Mitogen Activated Protein Kinase ◽  
Chinese Herbal ◽  
Morphogenetic Protein ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Underlying Mechanisms

Whether Chinese herbal formulas are effective in treatment of postmenopausal osteoporosis remains unclear. The aim of this study is to explore the experimental evidence of both in vitro and in vivo preclinical studies using Chinese herbal formulas in postmenopausal osteoporosis. Searches were applied to various databases with relevant keywords. Original in vivo and in vitro studies using Chinese herbal formulas to treat postmenopausal osteoporosis, and with full text available, were included. Er-Xian Decoction, Bu-Shen-Ning-Xin Decoction, Qing E Formula, Liuwei Dihuang Wan, and Xian-Ling-Gu-Bao Decoction, the most commonly studied formulas, were selected from the pool of Chinese medicine. The preclinical data indicated the potential use of Chinese herbal formulas in postmenopausal osteoporosis. The underlying mechanisms included bone morphogenetic protein (BMP), Wnt/[Formula: see text]-catenin, extracellular-signal-regulated kinase/c-Jun [Formula: see text] terminal kinase/mitogen-activated protein kinase (ERK/JNK/MAPK), estrogen receptor (ER), and osteoprotegerin/receptor activator of nuclear factor [Formula: see text]B ligand (OPG/RANKL) signaling pathways. This study demonstrated the anti-osteoporotic effect of Chinese herbal formulas targeting different pathways in bone metabolism. Further study with adequate sample size and follow-up time, appropriate controls, and optimal blinding is required.

scholarly journals Optimizing photoswitchable MEK

2019 ◽  
Vol 116 (51) ◽  
pp. 25756-25763 ◽  
Author(s):  
Aleena L. Patel ◽  
Eyan Yeung ◽  
Sarah E. McGuire ◽  
Andrew Y. Wu ◽  
Jared E. Toettcher ◽  
...  
Keyword(s):  
Mitogen Activated Protein Kinase ◽  
Kinase Activation ◽  
Erk Activation ◽  
Signaling Systems ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Morphogenetic Event ◽  
Zebrafish Embryogenesis

Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in bothDrosophilaand zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

scholarly journals Control of Mycobacterial Replication in Human Macrophages: Roles of Extracellular Signal-Regulated Kinases 1 and 2 and p38 Mitogen-Activated Protein Kinase Pathways

2002 ◽  
Vol 70 (9) ◽  
pp. 4961-4967 ◽  
Author(s):  
Antje Blumenthal ◽  
Stefan Ehlers ◽  
Martin Ernst ◽  
Hans-Dieter Flad ◽  
Norbert Reiling
Keyword(s):  
Protein Kinase ◽  
Growth Control ◽  
Human Monocyte ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Activation ◽  
Extracellular Signal ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Bacterial Replication

ABSTRACT Intracellular persistence of mycobacteria may result from an intricate balance between bacterial replication and signaling events leading to antimicrobial macrophage activities. Using human monocyte-derived macrophages, we investigated the relevance of mitogen-activated protein kinase activation for the growth control of Mycobacterium avium isolates differing in their abilities to multiply intracellularly. The highly replicative smooth transparent morphotype of M. avium strain 2151 induced significantly less p38 and extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation than the smooth opaque morphotype of the same strain, which was gradually eliminated from macrophage cultures. Inhibition of the p38 pathway by highly specific inhibitors did not significantly affect mycobacterial replication within macrophages, regardless of the in vitro virulence of the M. avium strain. However, repression of the ERK1/2 pathway further enhanced intracellular growth of highly replicative M. avium strains, although it did not increase survival of the poorly replicating M. avium isolate. Inhibition of the ERK1/2 pathway resulted in decreased tumor necrosis alpha (TNF-α) secretion irrespective of the virulence of the M. avium isolate used for infection, revealing that TNF-α could have been only partially responsible for the control of intracellular M. avium growth. In conclusion, ERK1/2- and TNF-α-independent pathways are sufficient to limit intramacrophage growth of less-virulent M. avium strains, but early ERK1/2 activation in infected macrophages is critically involved in controlling the growth of highly replicative M. avium strains.

scholarly journals Phosphodiesterase 3A binds to 14-3-3 proteins in response to PMA-induced phosphorylation of Ser428

2005 ◽  
Vol 392 (1) ◽  
pp. 163-172 ◽  
Author(s):  
Mercedes Pozuelo Rubio ◽  
David G. Campbell ◽  
Nicholas A. Morrice ◽  
Carol Mackintosh
Keyword(s):  
Protein Kinase ◽  
Metal Ion ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Mitogen Activated Protein ◽  
Dependent Protein ◽  
Pkc Protein Kinase C

PDE3A (phosphodiesterase 3A) was identified as a phosphoprotein that co-immunoprecipitates with endogenous 14-3-3 proteins from HeLa cell extracts, and binds directly to 14-3-3 proteins in a phosphorylation-dependent manner. Among cellular stimuli tested, PMA promoted maximal binding of PDE3A to 14-3-3 proteins. While p42/p44 MAPK (mitogen-activated protein kinase), SAPK2 (stress-activated protein kinase 2)/p38 and PKC (protein kinase C) were all activated by PMA in HeLa cells, the PMA-induced binding of PDE3A to 14-3-3 proteins was inhibited by the non-specific PKC inhibitors Ro 318220 and H-7, but not by PD 184352, which inhibits MAPK activation, nor by SB 203580 and BIRB0796, which inhibit SAPK2 activation. Binding of PDE3A to 14-3-3 proteins was also blocked by the DNA replication inhibitors aphidicolin and mimosine, but the PDE3A–14-3-3 interaction was not cell-cycle-regulated. PDE3A isolated from cells was able to bind to 14-3-3 proteins after in vitro phosphorylation with PKC isoforms. Using MS/MS of IMAC (immobilized metal ion affinity chromatography)-enriched tryptic phosphopeptides and phosphospecific antibodies, at least five sites on PDE3A were found to be phosphorylated in vivo, of which Ser428 was selectively phosphorylated in response to PMA and dephosphorylated in cells treated with aphidicolin and mimosine. Phosphorylation of Ser428 therefore correlated with 14-3-3 binding to PDE3A. Ser312 of PDE3A was phosphorylated in an H-89-sensitive response to forskolin, indicative of phosphorylation by PKA (cAMP-dependent protein kinase), but phosphorylation at this site did not stimulate 14-3-3 binding. Thus 14-3-3 proteins can discriminate between sites in a region of multisite phosphorylation on PDE3A. An additional observation was that the cytoskeletal cross-linker protein plectin-1 coimmunoprecipitated with PDE3A independently of 14-3-3 binding.

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