Induced Phosphorylation (induced + phosphorylation)

Distribution by Scientific Domains


Selected Abstracts


Negative Regulation by p70 S6 Kinase of FGF-2,Stimulated VEGF Release Through Stress-Activated Protein Kinase/c- Jun N-Terminal Kinase in Osteoblasts,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2007
Shinji Takai
Abstract To clarify the mechanism of VEGF release in osteoblasts, we studied whether p70 S6 kinase is involved in basic FGF-2,stimulated VEGF release in osteoblast-like MC3T3-E1 cells. In this study, we show that p70 S6 kinase activated by FGF-2 negatively regulates VEGF release through SAPK/JNK in osteoblasts. Introduction: Vascular endothelial growth factor (VEGF) plays an important role in bone metabolism. We have previously reported that fibroblast growth factor-2 (FGF-2) stimulates the release of VEGF through p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c- Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells and that FGF-2,activated p38 MAP kinase negatively regulates VEGF release. However, the mechanism behind VEGF release in osteoblasts is not precisely known. Materials and Methods: The levels of VEGF released from MC3T3-E1 cells were measured by enzyme immunoassay. The phosphorylation of each protein kinase was analyzed by Western blotting. To knock down p70 S6 kinase in MC3T3-E1 cells, the cells were transfected with siRNA to target p70 S6 kinase. Results: FGF-2 time-dependently induced the phosphorylation of p70 S6 kinase. Rapamycin significantly enhanced the FGF-2,stimulated VEGF release and VEGF mRNA expression. The FGF-2,induced phosphorylation of p70 S6 kinase was suppressed by rapamycin. Rapamycin markedly enhanced the FGF-2,induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. SP600125, a specific inhibitor of SAPK/JNK, suppressed the amplification by rapamycin of the FGF-2,stimulated VEGF release similar to the levels of FGF-2 with SP600125. Finally, downregulation of p70 S6 kinase by siRNA significantly enhanced the FGF-2,stimulated VEGF release and phosphorylation of SAPK/JNK. Conclusions: These results strongly suggest that p70 S6 kinase limits FGF-2,stimulated VEGF release through self-regulation of SAPK/JNK, composing a negative feedback loop, in osteoblasts. [source]


Vascular endothelial growth factor protects hepatoma cells against oxidative stress-induced cell death

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 6 2006
Shinji Osada
Abstract Background:, The aim of the present study was to examine coordination of the vascular endothelial growth factor (VEGF) and VEGF receptor (Flk-1) system and to study control of VEGF expression by oxidative stress, which is considered a model for chronic liver disease. Methods:, Cell viability was determined by test method with 3-[4, 5-dimethylthiazol-2-yl]-2, 5-dephenyl tetrazolium bromide (MTT). Expressions of cellular proteins were evaluated by western blot analysis. Results:, The c-Met tyrosine phosphorylation in PLC/PRF/5 hepatoma cells was increased by treatment with 20 ng/mL hepatocyte growth factor (HGF), and extracellular signal-regulated kinase (ERK) was also activated. Although Flk-1 was phosphorylated in response to VEGF (>50 ng/mL), phosphorylated ERK was not detected at these concentrations. A total of 5.0 and 10 µmol/L hydrogen peroxide (H2O2) caused cell death in a dose-dependent manner after 24 h. On western blot analysis at 1 h with H2O2, rapid phosphorylation of both ERK1/2 and c-Jun NH2 -terminal kinase (JNK) was observed. In the first 6 h, H2O2 induced cell death for 58.4 ± 6.8%, whereas the presence of 100 ng/mL VEGF improved the survival rate to 77.2 ± 4.2%. The VEGF significantly decreased H2O2 -induced cell death after 12 h, whereas HGF (20 ng/mL) did not have a similar effect. When cells were incubated with 5 µmol/L H2O2, expression of VEGF protein was detected. Furthermore, H2O2 -induced phosphorylation of ERK and JNK was also reduced by VEGF (100 ng/mL). In contrast, HGF did not induce phosphorylation of ERK and JNK. Conclusion:, Hepatoma cells might be able to survive under continuous oxidative stress through expression of VEGF. [source]


Mitogen-activated protein kinase pathway mediates N -(4-hydroxyphenyl)retinamide-induced neuronal differentiation in the ARPE-19 human retinal pigment epithelial cell line

JOURNAL OF NEUROCHEMISTRY, Issue 2 2008
William Samuel
Abstract We have shown previously that N -(4-hydroxyphenyl)retinamide (4HPR, fenretinide), a retinoic acid derivative, induces neuronal differentiation in cultured human retinal pigment epithelial (RPE) cells [Chen et al., J. Neurochem., 84 (2003), 972]. We asked the question whether the mitogen-activated protein kinase (MAPK) pathway is involved in the regulation of the 4HPR-induced neuronal differentiation of RPE (ARPE-19) cells. When we treated ARPE-19 cells with 4HPR, c-Raf and MEK1/2 kinase were activated resulting in activation of the downstream effector ERK1/2 and of SAPK/JNK. By blocking the upstream kinase MEK1/2 with specific inhibitor U0126 we abrogated the 4HPR-induced phosphorylation of ERK1/2 and SAPK/JNK, indicating that the neuronal differentiation occurs through a positive cross-talk between the ERK and the SAPK/JNK pathways. Both U0126 and the suppression of ERK1/2 expression with small interfering RNA effectively blocked the 4HPR-induced neuronal differentiation of RPE cells and the expression of calretinin. The activated ERK1/2 then induced a sequential activation of p90RSK, and increase in phosphorylation of transcription factors c- fos and c- jun leading to transcriptional activation of AP-1. Taken together, our results clearly demonstrate that c-Raf/MEK1/2 signaling cascade involving ERK1/2 plays a central role in mediating the 4HPR-induced neuronal differentiation and calretinin expression in the human ARPE-19 retinal pigment epithelial cell line. [source]


Manganese potentiates nuclear factor-,B-dependent expression of nitric oxide synthase 2 in astrocytes by activating soluble guanylate cyclase and extracellular responsive kinase signaling pathways

JOURNAL OF NEUROSCIENCE RESEARCH, Issue 9 2008
Julie A. Moreno
Abstract Inflammatory activation of glial cells is associated with neuronal injury in several degenerative movement disorders of the basal ganglia, including manganese neurotoxicity. Manganese (Mn) potentiates the effects of inflammatory cytokines on nuclear factor-,B (NF-,B)-dependent expression of nitric oxide synthase 2 (NOS2) in astrocytes, but the signaling mechanisms underlying this effect have remained elusive. It was postulated in the present studies that direct stimulation of cGMP synthesis and activation of mitogen-activated protein (MAP) kinase signaling pathways underlies the capacity of Mn to augment NF-,B-dependent gene expression in astrocytes. Exposure of primary cortical astrocytes to a low concentration of Mn (10 ,M) potentiated expression of NOS2 mRNA and protein along with production of NO in response to interferon-, (IFN,) and tumor necrosis factor-, (TNF,), which was prevented by overexpression of dominant negative I,B,. Mn also potentiated IFN,- and TNF,-induced phosphorylation of extracellular response kinase (ERK), p38, and JNK, as well as cytokine-induced activation of a fluorescent NF-,B reporter construct in transgenic astrocytes. Activation of ERK preceded that of NF-,B and was required for maximal activation of NO synthesis. Independently of IFN,/TNF,, Mn-stimulated synthesis of cGMP in astrocytes and inhibition of soluble guanylate cyclase (sGC) abolished the potentiating effect of Mn on MAP kinase phosphorylation, NF-,B activation, and production of NO. These data indicate that near-physiological concentrations of Mn potentiate cytokine-induced expression of NOS2 and production of NO in astrocytes via activation of sGC, which promotes ERK-dependent enhancement of NF-,B signaling. © 2008 Wiley-Liss, Inc. [source]


S -Allyl- L -Cysteine Sulfoxide Inhibits Tumor Necrosis Factor-Alpha Induced Monocyte Adhesion and Intercellular Cell Adhesion Molecule-1 Expression in Human Umbilical Vein Endothelial Cells

THE ANATOMICAL RECORD : ADVANCES IN INTEGRATIVE ANATOMY AND EVOLUTIONARY BIOLOGY, Issue 3 2010
Chai Hui
Abstract Garlic and its water-soluble allyl sulfur-containing compound, S -Allyl- L -cysteine Sulfoxide (ACSO), have shown antioxidant and anti-inflammatory activities, inhibiting the development of atherosclerosis. However, little is known about the mechanism(s) underlying the therapeutic effect of ACSO in inhibiting the formation of atherosclerostic lesion. This study aimed to investigate whether ACSO could modulate tumor necrosis factor-alpha (TNF-,)-induced expression of intercellular cell adhesion molecule-1, monocyte adhesion and TNF-,-mediated signaling in human umbilical vein endothelial cells. While TNF-, promoted the intercellular cell adhesion molecule-1 mRNA transcription in a dose- and time-dependent manner, ACSO treatment significantly reduced the levels of TNF-,-induced intercellular cell adhesion molecule-1 mRNA transcripts (P < 0.01). Furthermore, ACSO dramatically inhibited TNF-, triggered adhesion of THP-1 monocytes to endothelial cells and porcine coronary artery rings. Moreover, ACSO mitigated TNF-, induced depolarization of mitochondrial membrane potential and overproduction of superoxide anion, associated with the inhibition of NOX4, a subunit of nicotinamide adenine dinucleotide phosphate-oxidase, mRNA transcription. In addition, ACSO also inhibited TNF-,-induced phosphorylation of JNK, ERK1/2 and I,B, but not p38. Apparently, ACSO inhibited proinflammatory cytokine-induced adhesion of monocytes to endothelial cells by inhibiting the mitogen-activated protein kinase signaling and related intercellular cell adhesion molecule-1 expression, maintaining mitochondrial membrane potential, and suppressing the overproduction of superoxide anion in endothelial cells. Therefore, our findings may provide new insights into ACSO on controlling TNF-,-mediated inflammation and vascular disease. Anat Rec, 2010. © 2010 Wiley-Liss, Inc. [source]


Regulation of bone morphogenetic protein signalling in human pulmonary vascular development,

THE JOURNAL OF PATHOLOGY, Issue 1 2008
M Southwood
Abstract The bone morphogenetic protein (BMP) type II receptor (BMPR-II) is predominantly expressed on the vascular endothelium in the adult lung. Although mutations in BMPR-II are known to underlie many cases of familial pulmonary arterial hypertension (FPAH), little is known regarding the expression of BMPs and their signalling pathways during normal lung development or the impact of BMPR-II mutations on endothelial cell function. We determined the cellular localization and expression levels of BMP4, BMP receptors, and activation of downstream signalling via phospho-Smad1 in a developmental series of human embryonic and fetal lungs by immunohistochemistry. The expression of BMP4 and BMP receptors was temporally and spatially regulated during lung development. BMPR-II expression correlated with phosphorylation of tissue Smad1 and was highest during the late pseudoglandular and early canalicular stage of lung development, when vasculogenesis is intense. Phospho-Smad1 expression was associated with markers of proliferation in endothelial cells. In vitro studies confirmed that BMPs 2 and 4 induced phosphorylation of Smad1/5 and pulmonary artery endothelial cell (PAEC) migration and proliferation. Adenoviral transfection of PAECs with mutant kinase-deficient BMPR-II, or siRNA knockdown of BMPR-II, inhibited Smad signalling and the proliferative response to BMP4. Our findings support a critical role for BMPs in lung vasculogenesis. Dysfunctional BMP signalling in PAECs during development may lead to abnormal pulmonary vascular development and contribute to the pathogenesis of FPAH. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source]


Aurothiomalate inhibits cyclooxygenase 2, matrix metalloproteinase 3, and interleukin-6 expression in chondrocytes by increasing MAPK phosphatase 1 expression and decreasing p38 phosphorylation: MAPK phosphatase 1 as a novel target for antirheumatic drugs

ARTHRITIS & RHEUMATISM, Issue 6 2010
Riina Nieminen
Objective Aurothiomalate is a disease-modifying antirheumatic drug that suppresses inflammation and retards cartilage degradation and bone erosion in arthritis. The molecular mechanisms of action of aurothiomalate are not known in detail. MAPK pathways are major signaling pathways in inflammation that regulate the production of many inflammatory and destructive factors in arthritis. The purpose of the present study was to investigate the effects of aurothiomalate on the activity of p38 MAPK and on the expression of MAPK phosphatase 1 (MKP-1), cyclooxygenase 2 (COX-2), matrix metalloproteinase 3 (MMP-3), and interleukin-6 (IL-6) in immortalized murine H4 chondrocytes and in intact human and murine cartilage. Methods Protein expression was examined by Western blotting or by enzyme-linked immunosorbent assay, and messenger RNA (mRNA) expression was examined by real-time reverse transcription,polymerase chain reaction analysis. The mediator role of MKP-1 was investigated by using small interfering RNA (siRNA) methods to down-regulated MKP-1 expression in chondrocytes in culture and by comparing the responses in intact cartilage from MKP-1,deficient and wild-type mice. The effects of aurothiomalate were also confirmed in human rheumatoid cartilage by using tissue samples obtained at the time of total knee replacement surgery. Results Aurothiomalate inhibited IL-1,,induced COX-2 expression and prostaglandin E2 production by destabilizing COX-2 mRNA, as did the p38 MAPK inhibitor SB203580. Interestingly, aurothiomalate also increased the expression of MKP-1 and reduced the IL-1,,induced phosphorylation of p38 MAPK. Knockdown of MKP-1 by siRNA significantly impaired the ability of aurothiomalate to inhibit the phosphorylation of p38 MAPK and the expression of COX-2, MMP-3, and IL-6. Likewise, aurothiomalate reduced COX-2, MMP-3, and IL-6 expression in articular cartilage from patients with rheumatoid arthritis, as well as in articular cartilage from wild-type mice but not from MKP-1,/, mice. Conclusion Our findings indicate a novel mechanism for the antiinflammatory and antierosive actions of aurothiomalate, through increased expression of MKP-1, which leads to reduced activation of p38 MAPK and suppressed expression of COX-2, MMP-3, and IL-6. The results suggest that manipulation of MKP-1 levels is a promising new mechanism to be directed in the search and development of novel antiinflammatory and antierosive compounds that have the good efficacy of gold compounds but not their toxicity. [source]


A novel inhibitor of Smad-dependent transcriptional activation suppresses tissue fibrosis in mouse models of systemic sclerosis

ARTHRITIS & RHEUMATISM, Issue 11 2009
Minoru Hasegawa
Objective Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc), and an increasing number of promising molecular targets for antifibrotic therapies have been described recently. Transforming growth factor , (TGF,) is well known to be the principal factor that leads to tissue fibrosis. The present study was undertaken to investigate the ability of HSc025, a novel small compound that antagonizes TGF,/Smad signaling through the activation of nuclear translocation of Y-box binding protein 1, to prevent tissue fibrosis in vitro or in mouse models of SSc. Methods Human dermal fibroblasts were exposed to HSc025 at various concentrations in the presence of TGF,, and levels of collagen or fibronectin expression were determined. HSc025 (15 mg/kg/day for 14 days) was administered orally to tight skin mice and to mice with bleomycin-induced pulmonary fibrosis. Improvement of tissue fibrosis was evaluated by histologic or biochemical examination in each model. Results Pretreatment with HSc025 prevented Smad-dependent promoter activation, in a dose-dependent manner; however, HSc025 had no effect on TGF,-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGF,-induced collagen or fibronectin expression were also confirmed in vitro. Orally administered HSc025 significantly reduced hypodermal thickness and hydroxyproline content in tight skin mice, and markedly decreased the histologic score and hydroxyproline content in the lungs of bleomycin-treated mice. Conclusion These results demonstrate that HSc025 is a novel inhibitor of TGF,/Smad signaling, resulting in the improvement of skin and pulmonary fibrosis. Orally available HSc025 might therefore be useful in the treatment of SSc. [source]


Chondroitin Sulfate Inhibits the Nuclear Translocation of Nuclear Factor-,B in Interleukin-1,-Stimulated Chondrocytes

BASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2008
Claudia Jomphe
In addition, chondroitin sulfate prevents joint space narrowing of the knee. We hypothesized that the anti-inflammatory effect of chondroitin sulfate is associated to a decrease in the activation of mitogen-activated protein kinases (MAPK) and of the transcription factors nuclear factor-,B (NF-,B) and activator protein-1 (AP-1). Cultured rabbit chondrocytes were stimulated with interleukin-1, (IL-1,) in presence of chondroitin sulfate. Nuclear translocation of NF-,B and AP-1, and nitrite concentrations (as an index for nitric oxide) was assessed 48 hr later. The effect of chondroitin sulfate on IL-1, activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and p38MAPK was documented by immunoblot. The effect of chondroitin sulfate on sodium nitroprusside-induced apoptosis was evaluated with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling assay. Chondroitin sulfate reduced IL-1,-induced NF-,B nuclear translocation, but not AP-1 translocation, it decreased IL-1,-induced phosphorylation of Erk1/2 and abrogated p38MAPK phosphorylation, but did not prevent IL-1,-induced increase in nitrite. Finally, chondroitin sulfate decreased nitroprusside-induced apoptosis of the chondrocytes. These results suggest that some of the biological activities of chondroitin sulfate may be associated to the reduction in Erk1/2 and p38MAPK phosphorylation and nuclear transactivation of NF-,B. [source]


Activation of protein kinase B by the A1 -adenosine receptor in DDT1MF-2 cells

BRITISH JOURNAL OF PHARMACOLOGY, Issue 4 2000
Renée Germack
In this study the effect of insulin and A1 -adenosine receptor stimulation on protein kinase B (PKB) activation has been investigated in the hamster vas deferens smooth muscle cell line DDT1MF-2. Increases in PKB phosphorylation were determined by Western blotting using an antibody that detects PKB phosphorylation at Ser473. Insulin, a recognized activator of PKB, stimulated a concentration-dependent increase in PKB phosphorylation in DDT1MF-2 cells (EC50 5±1 pM). The selective A1 -adenosine receptor agonist N6 -cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in PKB phosphorylation in DDT1MF-2 cells (EC50 1.3±0.5 nM). CPA-mediated increases in PKB phosphorylation were antagonized by the A1 -adenosine receptor selective antagonist 1,3-dipropylcyclopentylxanthine (DPCPX) yielding an apparent KD value of 2.3 nM. Pre-treatment of DDT1MF-2 cells with pertussis toxin (PTX, 100 ng ml,1 for 16 h), to block Gi/Go -dependent pathways, abolished CPA (1 ,M) induced phosphorylation of PKB. In contrast, responses to insulin (100 nM) were resistant to PTX pre-treatment. The phosphatidylinositol 3-kinase (PI-3K) inhibitors wortmannin (IC50 10.3±0.6 nM) and LY 294002 (IC50 10.3±1.2 ,M) attenuated the phosphorylation of PKB elicited by CPA (1 ,M) in a concentration-dependent manner. Wortmannin (30 nM) and LY 294002 (30 ,M) also blocked responses to insulin (100 nM). Removal of extracellular Ca2+ and chelation of intracellular Ca2+ with BAPTA had no significant effect on CPA-induced PKB phosphorylation. Similarly, pretreatment (30 min) with inhibitors of protein kinase C (Ro 31-8220; 10 ,M), tyrosine kinase (genistein; 100 ,M), mitogen-activated protein (MAP) kinase kinase (PD 98059; 50 ,M) and p38 MAPK (SB 203580; 20 ,M) had no significant effect on CPA-induced PKB phosphorylation. In conclusion, these data demonstrate that A1 -adenosine receptor stimulation in DDT1MF-2 cells increases PKB phosphorylation through a PTX and PI-3K-sensitive pathway. British Journal of Pharmacology (2000) 130, 867,874; doi:10.1038/sj.bjp.0703396 [source]