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2.
Int J Mol Sci ; 21(22)2020 Nov 20.
Article in English | MEDLINE | ID: mdl-33233528

ABSTRACT

Head and neck squamous cell carcinoma (HNSCC) is a widespread disease with a low survival rate and a high risk of recurrence. Nowadays, immune checkpoint inhibitor (ICI) treatment is approved for HNSCC as a first-line treatment in recurrent and metastatic disease. ICI treatment yields a clear survival benefit, but overall response rates are still unsatisfactory. As shown in different cancer models, hepatocyte growth factor/mesenchymal-epithelial transition (HGF/Met) signaling contributes to an immunosuppressive microenvironment. Therefore, we investigated the relationship between HGF and programmed cell death protein 1 (PD-L1) expression in HNSCC cell lines. The preclinical data show a robust PD-L1 induction upon HGF stimulation. Further analysis revealed that the HGF-mediated upregulation of PD-L1 is MAP kinase-dependent. We then hypothesized that serum levels of HGF and soluble programmed cell death protein 1 (sPD-L1) could be potential markers of ICI treatment failure. Thus, we determined serum levels of these proteins in 20 HNSCC patients before ICI treatment and correlated them with treatment outcomes. Importantly, the clinical data showed a positive correlation of both serum proteins (HGF and sPD-L1) in HNSCC patient's sera. Moreover, the serum concentration of sPD-L1 was significantly higher in ICI non-responsive patients. Our findings indicate a potential role for sPD-L1 as a prognostic marker for ICI treatment in HNSCC.


Subject(s)
Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/genetics , Gene Expression Regulation, Neoplastic , Head and Neck Neoplasms/genetics , Hepatocyte Growth Factor/genetics , Neoplasm Recurrence, Local/genetics , Squamous Cell Carcinoma of Head and Neck/genetics , Adult , Aged , Antibodies, Monoclonal/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , B7-H1 Antigen/blood , Cohort Studies , Epithelial-Mesenchymal Transition/drug effects , Female , Head and Neck Neoplasms/diagnosis , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/mortality , Hepatocyte Growth Factor/blood , Hepatocyte Growth Factor/pharmacology , Humans , Ipilimumab/therapeutic use , Lymphatic Metastasis , Male , Middle Aged , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/blood , Mitogen-Activated Protein Kinase 3/genetics , Neoplasm Recurrence, Local/diagnosis , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/mortality , Nivolumab/therapeutic use , Prognosis , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/blood , Proto-Oncogene Proteins c-met/genetics , RNA, Small Interfering/blood , RNA, Small Interfering/genetics , Signal Transduction , Squamous Cell Carcinoma of Head and Neck/diagnosis , Squamous Cell Carcinoma of Head and Neck/drug therapy , Squamous Cell Carcinoma of Head and Neck/mortality , Survival Analysis , Treatment Outcome , Tumor Microenvironment/drug effects , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
3.
Clin Immunol ; 219: 108570, 2020 10.
Article in English | MEDLINE | ID: mdl-32791312

ABSTRACT

Chronic hepatitis B (CHB) is a life-threatening disease caused by HBV infection. Our previous work proved that activation of ERK1/2 and STAT3 signaling was involved in HBV tolerance. We herein investigated clinical significances of serum ERK1/2 and STAT3 proteins in CHB. Results showed that ERK1/2 and STAT3 were fluctuated with natural history of CHB. In addition, STAT3 was found to be positively correlated to the elevation of ALT, AST and GGT, while ERK1 was negatively correlated to decreases of TP and ALB. Also, there was a positive correlation between the anti-HBc antibody and ERK1, ERK2 or STAT3 in HBeAg-negative patients. Strikingly, serum ERK1 and ERK2 could reflect level of HBsAg-specific CD8+ T cells. A model composed with baseline ERK1 and ERK2 levels had a high accuracy to predict the effect of IFNα treatment. In conclusion, serum ERK1, ERK2 and STAT3 could serve as novel biomarkers in chronic HBV infections.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Hepatitis B, Chronic , Interferon-alpha/therapeutic use , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3/blood , Hepatitis B Antibodies/blood , Hepatitis B, Chronic/blood , Hepatitis B, Chronic/drug therapy , Hepatitis B, Chronic/immunology , Humans , Mitogen-Activated Protein Kinase 1/immunology , Mitogen-Activated Protein Kinase 3/immunology , STAT3 Transcription Factor/blood , STAT3 Transcription Factor/immunology , Treatment Outcome
4.
Zhonghua Yu Fang Yi Xue Za Zhi ; 47(9): 788-93, 2013 Sep.
Article in Chinese | MEDLINE | ID: mdl-24351557

ABSTRACT

OBJECTIVE: To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal. METHODS: 70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR). RESULTS: A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P < 0.01). Compared with the control group ((1.58 ± 1.07)µg/g), arsenic level in the patients' hair ((4.45 ± 2.78) µg/g) increased significantly, the differences were significant (F = 48.22, P < 0.01). The relative expression amount of the median(quartile) for ERK2, JNK1 mRNA were 0.0667 (0.0378-0.1371) and 0.0013 (0.0009-0.0025), respectively. Compared with the control group 0.1744 (0.1009-0.1985) and 0.0022 (0.0017-0.0030) , only the decreases of ERK2, JNK1 mRNA expression was significant (χ(2) = 15.10, 14.25, P < 0.01), and no significance in the other index. ERK2 mRNA relative expression for mild, medium and severe groups were separately 0.0818 (0.0408-0.1509) ,0.0582 (0.0154-0.1699) and 0.0588 (0.0399-0.1034) . Compared with the control group (0.1744 (0.1099-0.1985) ), there was significant difference (Z = -2.89, -3.19, -2.67, P < 0.01). JNK1 mRNA relative expression were 0.0012 (0.0007-0.001 57), 0.0019 (0.0011-0.0035), 0.0013 (0.0010-0.0026), respectively. Compared with the control group (0.0022 (0.0017-0.0030) ), significances were found in the mild groups (Z = -3.72, P < 0.01). CONCLUSIONS: Arsenic could induce the changes of ERK2 and JNK1mRNA expression in the MAPK path way in arseniasis patients.It suggests that the MAPK signaling pathway take part in the occurrence and development process of arseniasis caused by burning coal.


Subject(s)
Arsenic Poisoning/blood , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 8/blood , Adult , Air Pollution, Indoor , Arsenic Poisoning/etiology , Case-Control Studies , Coal , Female , Humans , Male , Middle Aged , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 8/genetics , RNA, Messenger/genetics , Transcription, Genetic
5.
J Strength Cond Res ; 26(3): 599-607, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22343976

ABSTRACT

Extracellular signal-regulated kinase (ERK) 1/2 signaling has been shown to be increased after heavy resistance exercise and suggested to play a role in the hypertrophic adaptations that are known to occur with training. However, the role that ERK1/2 may play in response to lower intensities of resistance exercise is unknown. Therefore, the purpose of this study was to determine the effects of resistance exercise intensity on ERK1/2 activity in human skeletal muscle. Twelve recreationally active men completed separate bouts of single-legged resistance exercise with 8-10 repetitions (reps) at 80-85% 1 repetition maximum (1RM) (85%) and 18-20 reps at 60-65% 1RM (65%) in a randomized crossover fashion. For both resistance exercise sessions, vastus lateralis biopsies and blood draws were taken immediately before exercise (PRE) and at 30 minutes (30MPST), 2 hours (2HRPST), and 6 hours (6HRPST) post exercise, with an additional blood draw occurring immediately after exercise (POST). The phosphorylated levels of pIGF-1R, pMEK1, pERK1/2, and activated Elk-1 were assessed by phosphoELISA, and serum insulin-like growth factor 1 (IGF-1) was assessed via enzyme-linked immunosorbent assay. Statistical analyses used a 2 × 4 (muscle responses) and 2 × 5 (serum responses) multivariate analysis of variance on delta values from baseline (p < 0.05). Both exercise intensities significantly increased the activity of insulin-like growth factor 1 receptor (IGF-1R), mitogen-activated protein kinase 1, ERK1/2, and Elk-1, with peak activity occurring at 2HRPST (p < 0.001). However, 65% resulted in a preferential increase in IGF-1R and Elk-1 activation when compared with 85% (p < 0.05). No differences were observed for serum IGF-1 levels regardless of intensity and time. These findings demonstrate that resistance exercise upregulates ERK1/2 signaling in a manner that does not appear to be preferentially dependent on exercise intensity.


Subject(s)
Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinases/metabolism , Physical Exertion/physiology , Resistance Training , Cross-Over Studies , Enzyme Activation/physiology , Humans , Insulin-Like Growth Factor I/analysis , Male , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3/blood , Mitogen-Activated Protein Kinases/blood , Muscle Strength/physiology , Muscle, Skeletal/enzymology , Muscle, Skeletal/physiology , Young Adult , ets-Domain Protein Elk-1/blood
6.
Cell Death Dis ; 2: e150, 2011 Apr 28.
Article in English | MEDLINE | ID: mdl-21525937

ABSTRACT

We reported a relevant activity of the combination between sorafenib and octreotide long-acting release (LAR) in advanced hepatocellular carcinoma (HCC) patients. In this work, we have studied if oxidative stress in both serum and peripheral blood mononuclear cells (PBMC) and pERK activation status in PBMC could be predictive of response. In the 20 responsive patients, the decrease of reactive oxygen species levels was already detectable after 10 days (T10) from the beginning of sorafenib administration, and this effect was enhanced by the combined treatment with sorafenib+octreotide LAR (T21). This effect correlated with the modulation of superoxide dismutase (SOD) activity (physiological scavenger of O(2-)) and of serum nitric oxide (NO) levels. Sorafenib alone induced an increase of about 40% of NO levels and of about two-fold of SOD activity in responsive patients, and both effects were significantly potentiated by the combined treatment. We found a gradual reduction of Erk1/2 activity, as evaluated by cytofluorimetric analysis, in 15 responsive patients reaching about 50% maximal decrease at T21. On the other hand, in 17 resistant patients, Erk1/2 activity was about 80% increased at T21. The determination of both the oxidative stress status and pERK activity in PBMC has high value in the prediction of response to sorafenib+octreotide therapy in HCC patients.


Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Biomarkers, Tumor/blood , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3/blood , Oxidative Stress , Benzenesulfonates/administration & dosage , Carcinoma, Hepatocellular/metabolism , Delayed-Action Preparations/administration & dosage , Drug Resistance, Neoplasm , Female , Humans , Leukocytes, Mononuclear/metabolism , Liver Neoplasms/metabolism , Male , Niacinamide/analogs & derivatives , Nitric Oxide/blood , Octreotide/administration & dosage , Phenylurea Compounds , Phosphorylation , Pyridines/administration & dosage , Reactive Oxygen Species/blood , Sorafenib , Superoxide Dismutase/blood , Treatment Outcome
7.
Hypertension ; 55(1): 137-43, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19901160

ABSTRACT

Pressure overload has been shown to induce mitogen activated protein kinases (MAPKs) and reactivate the atrial natriuretic factor in the heart. To test the sensitivity of these signals to pressure overload, we assayed the activity of MAPKs extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 in protein lysates from the left ventricle (LV) or white blood cells (WBC) isolated from aortic banded mice with varying levels of pressure overload. In separated mice we measured atrial natriuretic factor mRNA levels by Northern blotting. As expected, a significant induction of atrial natriuretic factor mRNA levels was observed after aortic banding, and it significantly correlated with the trans-stenotic systolic pressure gradient but not with the LV weight:body weight ratio. In contrast, a significant correlation with systolic pressure gradient or LV weight:body weight ratio was observed for all of the MAPK activity detected in LV samples or WBCs. Importantly, LV activation of MAPKs significantly correlated with their activation in WBCs from the same animal. To test whether MAPK activation in WBCs might reflect uncontrolled blood pressure levels in humans, we assayed extracellular signal-regulated kinase, c-Jun N-terminal kinase 1, and p38 activation in WBCs isolated from normotensive volunteers, hypertensive patients with controlled blood pressure values, or hypertensive patients with uncontrolled blood pressure values. Interestingly, in hypertensive patients with controlled blood pressure values, LV mass and extracellular signal-regulated kinase phosphorylation were significantly reduced compared with those in hypertensive patients with uncontrolled blood pressure values. These results suggest that MAPKs are sensors of pressure overload and that extracellular signal-regulated kinase activation in WBCs might be used as a novel surrogate biomarker of uncontrolled human hypertension.


Subject(s)
Blood Pressure/physiology , Hypertension/physiopathology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/metabolism , Animals , Aorta/physiopathology , Atrial Natriuretic Factor/genetics , Blotting, Northern , Blotting, Western , Constriction, Pathologic , Enzyme Activation , Female , Gene Expression , Humans , Hypertension/enzymology , Hypertension/genetics , Hypertrophy , Leukocytes/enzymology , Male , Mice , Middle Aged , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 8/blood , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinases/blood , Myocardium/metabolism , Myocardium/pathology , Phosphorylation , Pressure , p38 Mitogen-Activated Protein Kinases/blood , p38 Mitogen-Activated Protein Kinases/metabolism
8.
Osteoporos Int ; 21(1): 189-93, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19350338

ABSTRACT

UNLABELLED: A 60-year-old postmenopausal woman diagnosed as primary osteoporosis began to take raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated after the treatment, concomitant with the significant upregulation of p44/p42 mitogen-activated protein (MAP) kinase induced by adenosine diphosphate (ADP). After the cessation of raloxifene, the spontaneous microaggregates of platelets and the acceleration of ADP-induced p44/p42 MAP kinase phosphorylation was diminished. We concluded that raloxifene caused platelet hyperaggregability to shear stress and p44/p42 MAP kinase was involved in the pathological state. INTRODUCTION: A 60-year-old postmenopausal woman suffering from severe lumbago was diagnosed as primary osteoporosis with combined vertebral fractures. After the acute phase, she began to take 60 mg daily of oral raloxifene. The spontaneous microaggregates of platelets induced by shear stress were accelerated significantly after 8 weeks from the beginning of raloxifene treatment and observed at 12 weeks. RESULTS: The platelet aggregation induced by ADP was little changed; however, low doses (0.3 and 1 microM) of ADP significantly induced the phosphorylation of p44/p42 MAP kinase in the platelets obtained at 12 weeks. Although there were few subjective complaints except for paroxysmal headache, the medication was stopped with her consent to avoid any adverse effects. The spontaneous microaggregates of platelets gradually decreased after the cessation of medication. At 12 weeks after the cessation, the phosphorylation of p44/p42 MAP kinase induced by low doses of ADP was no more observed. CONCLUSION: These results strongly suggest that raloxifene caused platelet hyperaggregability to shear stress and subclinical thrombus formation in this case and that p44/p42 MAP kinase was involved in the pathological state.


Subject(s)
Bone Density Conservation Agents/pharmacology , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3/blood , Platelet Aggregation/drug effects , Raloxifene Hydrochloride/pharmacology , Blood Platelets/enzymology , Cells, Cultured , Female , Humans , Middle Aged , Osteoporosis, Postmenopausal/blood , Osteoporosis, Postmenopausal/drug therapy , Platelet Aggregation/physiology , Up-Regulation/drug effects
9.
J Leukoc Biol ; 86(5): 1179-89, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19641039

ABSTRACT

During neutrophil activation and degranulation, MMP-9 and MPO are released into the extracellular space to propagate inflammatory disorders. As kinin peptides are major participants in acute inflammatory responses, and the G-protein-coupled B(1)R mediates the chemotaxis of human neutrophils, we examined the release of the neutrophil enzymes MMP-9 and MPO by the B(1)R agonist LDBK and determined the signaling pathways that may regulate this cellular effect. Cytochalasin-treated and -untreated neutrophils were suspended in HBSS and stimulated with a range concentration of LDBK for 5 min. Zymography and Western blotting revealed that LDBK induced the release of MMP-9 and MPO. The use of specific signaling transduction inhibitors showed that release of MMP-9 depended on ERK1/2 and p38 MAPKs, whereas release of MPO involved only the p38 cascade. Inhibition of the key steps in these pathways showed that the release of both enzymes depended on PKC and PI3K. Stimulation of neutrophils with LDBK produced phosphorylation of ERK1/2 and p38 MAPK, which was inhibited by B(1)R antagonists. The phosphorylated ERK1/2 MAPK translocated to the neutrophil nucleus, suggesting that transcription of new genes may follow activation of B(1)R. Our results demonstrate that in human neutrophils, activation of kinin B(1)R by LDBK initiates separate signaling cascades that trigger the release of MMP-9 and MPO from tertiary and primary granules, respectively, suggesting that the B(1)R plays a pivotal role in inflammatory disorders.


Subject(s)
Matrix Metalloproteinase 9/blood , Mitogen-Activated Protein Kinases/blood , Neutrophils/enzymology , Peroxidase/blood , Receptor, Bradykinin B1/physiology , Receptors, G-Protein-Coupled/blood , Cytochalasins/pharmacology , Exocytosis , Humans , Inflammation/blood , Inflammation/enzymology , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3/blood , Neutrophils/drug effects , Neutrophils/physiology , Phosphorylation , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/physiology , Reference Values , Signal Transduction/drug effects , Signal Transduction/physiology
10.
J Thromb Haemost ; 4(2): 443-52, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16420578

ABSTRACT

The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). In SIPA, ERK2 was rapidly phosphorylated during GPIb stimulation, its activation contributing to SIPA for 50%, independently of P2X(1) regulation. Thrombotic occlusion of injured arterioles occurred considerably faster in TG (4.3 +/- 2.3 min) than in WT (38 +/- 8 min) arterioles, but occlusion times in TG (19 +/- 12) and WT (48 +/- 4.5 min) venules differed less. Both the alphabeta-meATP triggered desensitization of platelet P2X(1), as well as P2X(1) antagonism by NF279 or NF449 prolonged mean occlusion to about 75 min in WT and 65 min in TG arterioles, but venular occlusion times were less affected. Preventing ERK2 activation by U0126 prolonged occlusion times in TG (41 +/- 10 min) and WT (51 +/- 17) arterioles more than in TG (46 +/- 5 min) and WT (56 +/- 6 min) venules, uncovering a role for ERK2 in shear controlled thrombosis. Antagonism of GPIb by a recombinant murine von Willebrand factor (VWF)-A1 fragment prolonged occlusion times to comparable values, ranging from 55 to 58 min, both in TG and WT arterioles and venules. Further inhibition strategies, combining VWF-A1, U0126 and NF449 in WT and TG mice and resulting in occlusion in various time windows, identified that inhibition by VWF-A1 largely abrogated the ERK2 contribution to thrombosis. In conclusion, P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions.


Subject(s)
Mitogen-Activated Protein Kinase 1/blood , Platelet Glycoprotein GPIb-IX Complex/metabolism , Receptors, Purinergic P2/blood , Thrombosis/blood , Thrombosis/enzymology , Animals , Arterioles , Enzyme Activation , Humans , In Vitro Techniques , Mice , Mice, Transgenic , Phosphorylation , Platelet Aggregation , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X , Recombinant Proteins/blood , Recombinant Proteins/genetics , Stress, Mechanical , Thrombosis/etiology , Venous Thrombosis/blood , Venous Thrombosis/enzymology , Venous Thrombosis/etiology , von Willebrand Factor/metabolism
11.
J Hepatol ; 43(2): 317-23, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15964094

ABSTRACT

BACKGROUND/AIMS: Pharmacological blockade of the renin-angiotensin system (RAS) attenuates liver fibrogenesis in rats. Here, we provide genetic evidence implicating angiotensin type 1 (AT1) receptors in liver fibrogenesis. METHODS: Wild type (WT) and AT1a knockout [AT1a (-/-)] mice were subjected to either sham operation or bile-duct ligation. Fibrosis was assessed by Sirius Red staining and hydroxyproline hepatic content. Fibrogenic and inflammatory cytokines were measured by ELISA. RESULTS: Bile duct ligation-induced elevation of serum liver enzymes was similar in WT and AT1a (-/-) mice. Bile duct ligated WT mice showed inflammatory changes and severe septal fibrosis. In contrast, AT1a (-/-) mice showed minor fibrotic lesions. Collagen accumulation was lower in AT1a (-/-) mice compared to WT mice. The increase in hepatic concentration of TGFbeta1 and pro-inflammatory cytokines was attenuated in AT1a (-/-) mice compared to WT mice. Immunohistochemistry analysis revealed decreased infiltration by inflammatory cells, lipid peroxidation products as well as decreased phosphorylation of c-Jun and p42/44 MAPK in AT1a (-/-) mice compared to AT1 (+/+) mice. CONCLUSIONS: AT1 receptors play an important role in the development of fibrosis. Pharmacological blockade of AT1 receptors appears to be a promising approach to treat liver fibrosis.


Subject(s)
Hepatitis/pathology , Liver Cirrhosis/pathology , Receptor, Angiotensin, Type 1/deficiency , Animals , Biomarkers/blood , Cell Proliferation , Disease Models, Animal , Disease Progression , Enzyme-Linked Immunosorbent Assay , Gene Expression , Hepatitis/blood , Immunohistochemistry , JNK Mitogen-Activated Protein Kinases/blood , Liver Cirrhosis/blood , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 1/blood , Phosphorylation , RNA/genetics , Receptor, Angiotensin, Type 1/blood , Receptor, Angiotensin, Type 1/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1
12.
Thromb Haemost ; 92(1): 114-23, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15213852

ABSTRACT

Stimulating human platelets with thrombin induces the activation of the extracellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 microM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 microM) and MRS2179 (100 microM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 microM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 microM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via alpha(2A)-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthio-ADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2 -receptor with U46619 (10 microM), which leads to ADP secretion and P2Y12 receptor-dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.


Subject(s)
Adenosine Diphosphate/metabolism , Blood Platelets/physiology , Membrane Proteins/blood , Mitogen-Activated Protein Kinase 1/blood , Receptors, Purinergic P2/blood , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adenosine Diphosphate/blood , Adenosine Diphosphate/pharmacology , Blood Platelets/drug effects , Enzyme Activation/drug effects , Epinephrine/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/blood , Humans , In Vitro Techniques , MAP Kinase Signaling System/drug effects , Receptors, Purinergic P2Y12 , Signal Transduction , Thrombin/pharmacology , Thromboxane A2/blood
13.
J Immunol ; 172(6): 3758-65, 2004 Mar 15.
Article in English | MEDLINE | ID: mdl-15004180

ABSTRACT

LL-37 is a cationic peptide that is found in the granules of neutrophils and is secreted by epithelial cells from a variety of tissues. Levels of LL-37 in vivo increase upon infection, and its production and secretion are increased upon stimulation with proinflammatory mediators. It has been postulated that LL-37 modulates the immune response by interacting with the effector cells of innate immunity; however, the mechanism of this interaction is unknown. LL-37 induced phosphorylation and activation of the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) and p38, in human peripheral blood-derived monocytes and a human bronchial epithelial cell line, but not in B or T lymphocytes. Phosphorylation was not dependent on the G protein-coupled formyl peptide-like receptor 1, which was previously proposed to be the receptor for LL-37-induced chemotaxis on human monocytes and T cells. Activation of ERK1/2 and p38 was markedly increased by the presence of GM-CSF, but not M-CSF. Exposure to LL-37 also led to the activation of Elk-1, a transcription factor that is downstream of and activated by phosphorylated ERK1/2, the up-regulation of various Elk-1-controlled genes, and the transcription and secretion of IL-8. Inhibition of either p38 or ERK1/2 kinases led to a reduction in LL-37-induced IL-8 secretion and inhibition of the transcription of various chemokine genes. The ability of LL-37 to signal through these pathways has broad implications in immunity, monocyte activation, proliferation, and differentiation.


Subject(s)
Antimicrobial Cationic Peptides/physiology , MAP Kinase Signaling System/immunology , Mitogen-Activated Protein Kinases/metabolism , Monocytes/enzymology , Antimicrobial Cationic Peptides/blood , Cathelicidins , Cells, Cultured , Chemokines/genetics , Cytotoxicity, Immunologic , Dose-Response Relationship, Immunologic , Drug Synergism , Enzyme Activation/immunology , Epithelial Cells/enzymology , GTP-Binding Proteins/physiology , Gene Expression Regulation/immunology , Granulocyte-Macrophage Colony-Stimulating Factor/physiology , Humans , Interleukin-8/metabolism , Macrophage Activation/immunology , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/blood , Mitogen-Activated Protein Kinases/physiology , Monocytes/immunology , Receptor, EphA8/genetics , Transcription, Genetic/immunology , p38 Mitogen-Activated Protein Kinases
14.
Diabetes ; 52(3): 621-33, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12606501

ABSTRACT

Twenty-three nondiabetic volunteers were divided into three groups. In group A (n = 9), the glucose infusion was adjusted to maintain blood glucose at 5 mmol/l (euglycemic clamp). In group B (n = 9), the glucose infusion was adjusted to maintain blood glucose at 10 mmol/l (hyperglycemic clamp) over 2 h. Group C consisted of five volunteers who were studied as the control group. Peripheral blood mononuclear cells (PBMCs) were isolated before and at the end of a 2-h clamp. In group C, PBMCs were isolated before and after 2 h without performing a clamp. The euglycemic clamp as well as "no clamp" had no effects on all parameters studied. In contrast, a significant increase in carboxymethyllysine (CML) content and p21(ras) and p42/44 mitogen-activated protein kinase (MAPK) phosphorylation was observed at the end of a 2-h hyperglycemic clamp. The nuclear factor (NF)-kappaB (but not Oct-1) binding activity increased significantly in the hyperglycemic clamp. Western blots confirmed NF-kappaB-p65-antigen translocation into the nucleus. IkappaBalpha did not change significantly in both groups. Hyperglycemia-mediated NF-kappaB activation and increase of CML content, p21(ras), and p42/44 MAPK phosphorylation was also seen in ex vivo-isolated PBMCs stimulated with 5 or 10 mmol/l glucose. Addition of insulin did not influence the results. Inhibition of activation of ras, MAPK, or protein kinase C blocked hyperglycemia-mediated NF-kappaB activation in ex vivo-isolated PBMCs stimulated with 10 mmol/l glucose. Similar data were obtained using an NF-kappaB-luciferase reporter plasmid. Therefore, we can conclude that an acute hyperglycemia-mediated mononuclear cell activation is dependent on activation of ras, p42/p44 MAPK phosphorylation, and subsequent NF-kappaB activation and results in transcriptional activity in PBMCs.


Subject(s)
Hyperglycemia/blood , Leukocytes, Mononuclear/metabolism , Lysine/analogs & derivatives , Lysine/blood , Mitogen-Activated Protein Kinases/blood , NF-kappa B/blood , Proto-Oncogene Proteins p21(ras)/blood , Animals , Blotting, Western , Cattle , Electrophoresis, Polyacrylamide Gel , Endothelium, Vascular/metabolism , Glucose Clamp Technique , Humans , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3 , Nuclear Proteins/blood , Phosphorylation , Signal Transduction , Transfection
15.
J Biol Chem ; 276(19): 15666-75, 2001 May 11.
Article in English | MEDLINE | ID: mdl-11278478

ABSTRACT

Agonists elevate the cytosolic calcium concentration in human platelets via a receptor-operated mechanism, involving both Ca(2+) release from intracellular stores and subsequent Ca(2+) entry, which can be inhibited by platelet inhibitors, such as prostaglandin E(1) and nitroprusside which elevate cAMP and cGMP, respectively. In the present study we investigated the mechanisms by which cAMP and cGMP modulate store-mediated Ca(2+) entry. Both prostaglandin E(1) and sodium nitroprusside inhibited thapsigargin-evoked store-mediated Ca(2+) entry and actin polymerization. However, addition of these agents after induction of store-mediated Ca(2+) entry did not affect either Ca(2+) entry or actin polymerization. Furthermore, prostaglandin E(1) and sodium nitroprusside dramatically inhibited the tyrosine phosphorylation induced by depletion of the internal Ca(2+) stores or agonist stimulation without affecting the activation of Ras or the Ras-activated phosphatidylinositol 3-kinase or extracellular signal-related kinase (ERK) pathways. Inhibition of cyclic nucleotide-dependent protein kinases prevented inhibition of agonist-evoked Ca(2+) release but it did not have any effect on the inhibition of Ca(2+) entry or actin polymerization. Phenylarsine oxide and vanadate, inhibitors of protein-tyrosine phosphatases prevented the inhibitory effects of the cGMP and cAMP elevating agents on Ca(2+) entry and actin polymerization. These results suggest that Ca(2+) entry in human platelets is directly down-regulated by cGMP and cAMP by a mechanism involving the inhibition of cytoskeletal reorganization via the activation of protein tyrosine phosphatases.


Subject(s)
Actins/blood , Blood Platelets/physiology , Calcium/blood , Carbazoles , Cyclic AMP/blood , Cyclic GMP/blood , Mitogen-Activated Protein Kinases/blood , Platelet Aggregation Inhibitors/pharmacology , Protein Tyrosine Phosphatases/blood , Actins/chemistry , Adenosine Diphosphate/pharmacology , Alprostadil/pharmacology , Arsenicals/pharmacology , Blood Platelets/drug effects , Egtazic Acid/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , In Vitro Techniques , Indoles/pharmacology , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3 , Nitroprusside/pharmacology , Phosphorylation , Platelet Activation/drug effects , Platelet Activation/physiology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Pyrroles/pharmacology , Thapsigargin/pharmacology , Thrombin/pharmacology , Vanadates/pharmacology
16.
J Biol Chem ; 276(19): 15659-65, 2001 May 11.
Article in English | MEDLINE | ID: mdl-11278479

ABSTRACT

Extracellular signal-regulated kinases (ERKs), are common participants in a broad variety of signal transduction pathways. Several studies have demonstrated the presence of ERKs in human platelets and their activation by the physiological agonist thrombin. Here we report the involvement of the ERK cascade in store-mediated Ca(2+) entry in human platelets. Treatment of dimethyl-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-loaded platelets with thapsigargin to deplete the intracellular Ca(2+) stores resulted in a time- and concentration-dependent activation of ERK1 and ERK2. Incubation with either U0126 or PD 184352, specific inhibitors of mitogen-activated protein kinase kinase (MEK), prevented thapsigargin-induced ERK activation. Furthermore, U0126 and PD 184352 reduced Ca(2+) entry stimulated by thapsigargin or thrombin, in a concentration-dependent manner. The role of ERK in store-mediated Ca(2+) entry was found to be independent of phosphatidylinositol 3- and 4-kinases, the tyrosine kinase pathway, and actin polymerization but sensitive to treatment with inhibitors of Ras, suggesting that the ERK pathway might be a downstream effector of Ras in mediating store-mediated Ca(2+) entry in human platelets. In addition, we have found that store depletion stimulated ERK activation does not require PKC activity. This study demonstrates for the first time a novel mechanism for regulation of store-mediated Ca(2+) entry in human platelets involving the ERK cascade.


Subject(s)
Blood Platelets/physiology , Calcium/blood , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinases/blood , Platelet Activation , Actins/blood , Benzamides/pharmacology , Blood Platelets/drug effects , Butadienes/pharmacology , Cytochalasin D/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Farnesol/analogs & derivatives , Farnesol/pharmacology , Humans , In Vitro Techniques , Kinetics , MAP Kinase Signaling System/drug effects , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinase 3 , Nitriles/pharmacology , Thapsigargin/pharmacology , Thrombin/pharmacology
17.
J Clin Endocrinol Metab ; 86(3): 1301-5, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11238524

ABSTRACT

Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 +/- 5 pmol/min.mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 +/- 11 pmol/min.mg protein; P < 0.05) and phosphorylation significantly increased (P < 0.01). MEK activity was significantly increased by hyperglycemia (1251 +/- 136 vs. 2000 +/- 42 cpm; P = 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.


Subject(s)
Cell Cycle Proteins , Hyperglycemia/enzymology , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinases/blood , Monocytes/enzymology , Phosphoprotein Phosphatases , Adult , Dual Specificity Phosphatase 1 , Female , Glucose/administration & dosage , Humans , Immediate-Early Proteins/blood , Immunoblotting , MAP Kinase Signaling System , Male , Mitogen-Activated Protein Kinase 3 , Phosphorylation , Protein Phosphatase 1 , Protein Tyrosine Phosphatases/blood , Somatostatin/administration & dosage
18.
Mol Med ; 7(11): 755-60, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11788789

ABSTRACT

BACKGROUND: All three major members of the MAPK family (i.e., p38 MAPK, p42/p44 MAPK, and c-Jun N terminal kinase (JNK)) have been shown to control cellular responses to inflammation in vitro. Therefore these kinases have been designated suitable targets for anti-inflammatory therapy. However, the extent to which these kinases are actually activated during inflammation in humans in vivo has not been investigated. We employed experimental human endotoxemia, a model of systemic inflammation, to address this question. MATERIALS AND METHODS: Male volunteers were intravenously infused with 4 ng/kg bw lipopolysaccharide (LPS). Directly before LPS infusion and up to 24 h thereafter, activation of p38 MAPK, p42/p44 MAPK and JNK was assessed in peripheral blood, using Western blot and in vitro kinase assays. RESULTS: We observed that LPS induced a strong but transient phosphorylation and activation of p38 MAPK and p42/p44 MAPK, maximal activity being reached after 1 hr of LPS infusion. Strikingly, no JNK phosphorylation or activation was detected under these circumstances. CONCLUSIONS: These results suggest that both inhibitors of p38 MAPK and p42/p44 MAPK but not JNK are potentially useful for anti-inflammatory therapy.


Subject(s)
Endotoxemia/blood , Mitogen-Activated Protein Kinase 1/blood , Mitogen-Activated Protein Kinases/blood , Signal Transduction , Adult , Blotting, Western , Enzyme Activation , Humans , JNK Mitogen-Activated Protein Kinases , Leukocytes/metabolism , Lipopolysaccharides/administration & dosage , Mitogen-Activated Protein Kinase 3 , Phosphorylation , p38 Mitogen-Activated Protein Kinases
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