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1.
Mol Cell Biochem ; 466(1-2): 83-89, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32016696

ABSTRACT

In the last decade, several reports highlight the importance of the low molecular weight protein tyrosine phosphatase (LMWPTP) in cancer aggressiveness and resistance. Specifically, in chronic myeloid leukemia, we have reported that high expression of the LMWPTP maintains Src and Bcr-Abl kinases in an activated status and the glucose metabolism is directed to lactate production and, in turn, favor the pentoses pathway (one of the key process for antioxidant and protective responses). In this present study, we investigated the possible correlation between the LMWPTP and autophagy. In resistant chronic myeloid leukemia cells, the antioxidant response is supported by the glycolytic metabolism and antioxidant enzymes such as SOD and catalase, both favored by the LMWPTP. Therefore, when the cells were challenged by hydrogen peroxide treatment, the LMWPTP level goes down as well as SOD, and in turn, autophagy process was stimulated. The findings presented here reveal a novel aspect by which LMWPTP cooperates for the resistance of CML towards stressor stimuli.


Subject(s)
Antioxidants/metabolism , Autophagy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/enzymology , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins/metabolism , Humans , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology
2.
Cell Metab ; 28(6): 866-880.e15, 2018 12 04.
Article in English | MEDLINE | ID: mdl-30146486

ABSTRACT

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASNKD) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASNKD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASNKD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade.


Subject(s)
Fatty Acid Synthase, Type I/physiology , Human Umbilical Vein Endothelial Cells/metabolism , Malonyl Coenzyme A/metabolism , Retinal Neovascularization/pathology , TOR Serine-Threonine Kinases/metabolism , Acetyl-CoA Carboxylase/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation , Fatty Acid Synthase, Type I/antagonists & inhibitors , Fatty Acid Synthase, Type I/genetics , Human Umbilical Vein Endothelial Cells/cytology , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Orlistat/therapeutic use , Protein Processing, Post-Translational , Retinal Neovascularization/drug therapy
3.
J Cell Biochem ; 118(11): 3846-3854, 2017 11.
Article in English | MEDLINE | ID: mdl-28387439

ABSTRACT

In chemoresistant leukemia cells (Lucena-1), the low molecular weight protein tyrosine phosphatase (LMWPTP) is about 20-fold more active than in their susceptible counterpart (K562). We found this phosphatase ensures the activated statuses of Src and Bcr-Abl. Since, phosphorylation and dephosphorylation of proteins represent a key post-translational regulation of several enzymes, we also explored the kinome. We hereby show that LMWPTP superactivation, together with kinome reprogramming, cooperate towards glucose addiction. Resistant leukemia cells present lower levels of oxidative metabolism, in part due to downexpression of the following mitochondrial proteins: pyruvate dehydrogenase subunit alpha 1, succinate dehydrogenase, and voltage-dependent anion channel. Those cells displayed higher expression levels of glucose transporter 1 and higher production of lactate. In addition, Lucena-1 siRNA LMWPTP cells showed lower expression levels of glucose transporter 1 and lower activity of lactate dehydrogenase. On the other hand, K562 cells overexpressing LMWPTP presented higher expression/activity of both proteins. In this study, we show that LMWPTP is a pivotal mediator of metabolic reprogramming that confers survival advantages to leukemia cells against death stimuli. J. Cell. Biochem. 118: 3846-3854, 2017. © 2017 Wiley Periodicals, Inc.


Subject(s)
Drug Resistance, Neoplasm , Glycolysis , Leukemia/metabolism , Neoplasm Proteins/metabolism , Protein Tyrosine Phosphatases/metabolism , Signal Transduction , Acute Disease , Humans , K562 Cells , Leukemia/pathology , Phosphorylation
4.
Sci Rep ; 5: 14534, 2015 Sep 28.
Article in English | MEDLINE | ID: mdl-26412398

ABSTRACT

Tuberous sclerosis complex (TSC) is caused by inactivating mutations in either TSC1 or TSC2 and is characterized by uncontrolled mTORC1 activation. Drugs that reduce mTOR activity are only partially successful in the treatment of TSC, suggesting that mTOR-independent pathways play a role in disease development. Here, kinome profiles of wild-type and Tsc2(-/-) mouse embryonic fibroblasts (MEFs) were generated, revealing a prominent role for PAK2 in signal transduction downstream of TSC1/2. Further investigation showed that the effect of the TSC1/2 complex on PAK2 is mediated through RHEB, but is independent of mTOR and p21RAC. We also demonstrated that PAK2 over-activation is likely responsible for the migratory and cell cycle abnormalities observed in Tsc2(-/-) MEFs. Finally, we detected high levels of PAK2 activation in giant cells in the brains of TSC patients. These results show that PAK2 is a direct effector of TSC1-TSC2-RHEB signaling and a new target for rational drug therapy in TSC.


Subject(s)
Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Tuberous Sclerosis/metabolism , Tumor Suppressor Proteins/metabolism , p21-Activated Kinases/metabolism , Animals , Cell Line , Cell Movement/genetics , Enzyme Activation , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Deletion , Gene Knockout Techniques , Humans , Mice , Molecular Targeted Therapy , Phosphorylation , Protein Binding , Signal Transduction/drug effects , Tuberous Sclerosis/drug therapy , Tuberous Sclerosis/genetics , Tuberous Sclerosis/pathology , Tuberous Sclerosis Complex 1 Protein , Tuberous Sclerosis Complex 2 Protein , Tumor Suppressor Proteins/genetics , rac GTP-Binding Proteins/metabolism
6.
J Pathol ; 234(3): 398-409, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25065357

ABSTRACT

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR-2 and activation of PAR-2 may induce their proliferation and migration. Interestingly, however, PAR-2 expression is increased in stroma-rich pancreatic cancer regions, suggesting a potential role of PAR-2 in the tumour microenvironment. Here, we assessed the importance of PAR-2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR-2-positive, whereas stromal cells are PAR-2-negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR-2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR-2-deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube-formation assays show that PAR-2 does not inhibit the intrinsic tube-forming capacity of lymphatic endothelial cells, but that PAR-2 actually inhibits cancer cell-induced tube formation. Overall, stromal PAR-2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR-2 in the tumour microenvironment and pinpoint PAR-2 as a negative regulator of lymphangiogenesis.


Subject(s)
Lymphangiogenesis/physiology , Lymphatic Metastasis/pathology , Pancreatic Neoplasms/pathology , Receptor, PAR-2/metabolism , Tumor Microenvironment/physiology , Animals , Blotting, Western , Disease Models, Animal , Humans , Immunohistochemistry , Mice, Inbred C57BL , Pancreatic Neoplasms/metabolism , Tissue Array Analysis
7.
Int J Cancer ; 135(10): 2294-304, 2014 Nov 15.
Article in English | MEDLINE | ID: mdl-24436106

ABSTRACT

Protease activated receptor (PAR)-1 expression in tumor cells is associated with disease progression and overall survival in a variety of cancers of epithelial origin; however, the importance of PAR-1 in the tumor microenvironment remains unexplored. Utilizing an orthotopic pancreatic cancer model in which tumor cells are PAR-1 positive whereas stromal cells are PAR-1 negative, we show that PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer. PAR-1 enhances monocyte recruitment into the tumor microenvironment by regulating monocyte migration and fibroblast dependent chemokine production thereby inducing chemoresistance. Overall, our data identify a novel role of PAR-1 in the pancreatic tumor microenvironment and suggest that PAR-1 may be an attractive target to reduce drug resistance in pancreatic cancer.


Subject(s)
Deoxycytidine/analogs & derivatives , Drug Resistance, Neoplasm , Liver Neoplasms/secondary , Pancreatic Neoplasms/pathology , Receptor, PAR-1/physiology , Stromal Cells/pathology , Animals , Antimetabolites, Antineoplastic/pharmacology , Cell Movement , Cell Proliferation , Cell Survival , Deoxycytidine/pharmacology , Disease Models, Animal , Disease Progression , Humans , Immunoenzyme Techniques , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Neovascularization, Pathologic , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Prognosis , Signal Transduction , Stromal Cells/metabolism , Tumor Cells, Cultured , Gemcitabine
8.
PLoS One ; 8(1): e54269, 2013.
Article in English | MEDLINE | ID: mdl-23342114

ABSTRACT

Melanoma is one of the most aggressive skin cancers due to its high capacity to metastasize. Treatment of metastatic melanomas is challenging for clinicians, as most therapeutic agents have failed to demonstrate improved survival. Thus, new candidates with antimetastatic activity are much needed. Riboavin (RF) is a component of the vitamin B complex and a potent photosensitizer. Previously, our group showed that the RF photoproducts (iRF) have potential as an antitumoral agent. Hence, we investigated the capacity of iRF on modulating melanoma B16F10 cells aggressiveness in vitro and in vivo. iRF decreases B16F10 cells survival by inhibiting mTOR as well as Src kinase. Moreover, melanoma cell migration was disrupted after treatment with iRF, mainly by inhibition of metalloproteinase (MMP) activity and expression, and by increasing TIMP expression. Interestingly, we observed that the Hedgehog (HH) pathway was inhibited by iRF. Two mediators of HH signaling, GLI1 and PTCH, were downregulated, while SUFU expression (an inhibitor of this cascade) was enhanced. Furthermore, inhibition of HH pathway signaling by cyclopamine and Gant 61 potentiated the antiproliferative action of RF. Accordingly, when a HH ligand was applied, the effect of iRF was almost completely abrogated. Our findings indicate that Hedgehog pathway is involved on the modulation of melanoma cell aggressiveness by iRF. Moreover, iRF treatment decreased pulmonary tumor formation in a murine experimental metastasis model. Research to clarify the molecular action of flavins, in vivo, is currently in progress. Taken together, the present data provides evidence that riboflavin photoproducts may provide potential candidates for improving the efficiency of melanoma treatment.


Subject(s)
Melanoma/drug therapy , Riboflavin/pharmacology , Riboflavin/therapeutic use , Animals , Blotting, Western , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Flavins/therapeutic use , Humans , Keratinocytes/cytology , Keratinocytes/drug effects , Male , Mice , Mice, Inbred C57BL , Pyridines/pharmacology , Pyrimidines/pharmacology , Veratrum Alkaloids/pharmacology
9.
PLoS One ; 7(10): e45362, 2012.
Article in English | MEDLINE | ID: mdl-23071514

ABSTRACT

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+)/c-Kit(+)/P-glycoprotein(+)/MRP1(+) TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.


Subject(s)
Cell Death/drug effects , Golgi Apparatus/drug effects , Indoles/therapeutic use , Leukemia/drug therapy , Apoptosis/drug effects , Apoptosis Regulatory Proteins/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calpain/antagonists & inhibitors , Cell Line, Tumor , Cell Survival/drug effects , Cyclic AMP-Dependent Protein Kinases/metabolism , Death-Associated Protein Kinases , Endoplasmic Reticulum Stress , Humans , Proto-Oncogene Proteins c-akt/metabolism
10.
PLoS One ; 7(9): e44312, 2012.
Article in English | MEDLINE | ID: mdl-22957062

ABSTRACT

The development of multidrug resistance (MDR) limits the efficacy of continuous chemotherapeutic treatment in chronic myelogenous leukemia (CML). Low molecular weight protein tyrosine phosphatase (LMW-PTP) is up-regulated in several cancers and has been associated to poor prognosis. This prompted us to investigate the involvement of LMW-PTP in MDR. In this study, we investigated the role of LMW-PTP in a chemoresistant CML cell line, Lucena-1. Our results showed that LMW-PTP is highly expressed and 7-fold more active in Lucena-1 cells compared to K562 cells, the non-resistant cell line. Knocking down LMW-PTP in Lucena-1 cells reverted chemoresistance to vincristine and imatinib mesylate, followed by a decrease of Src and Bcr-Abl phosphorylation at the activating sites, inactivating both kinases. On the other hand, overexpression of LMW-PTP in K562 cells led to chemoresistance to vincristine. Our findings describe, for the first time, that LMW-PTP cooperates with MDR phenotype, at least in part, through maintaining Src and Bcr-Abl kinases in more active statuses. These findings suggest that inhibition of LMW-PTP may be a useful strategy for the development of therapies for multidrug resistant CML.


Subject(s)
Drug Resistance, Neoplasm , Protein Tyrosine Phosphatases/antagonists & inhibitors , src-Family Kinases/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antineoplastic Agents/pharmacology , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Fusion Proteins, bcr-abl/physiology , Gene Expression Regulation, Leukemic , Humans , K562 Cells , Molecular Weight , Phosphorylation , Protein Tyrosine Phosphatases/genetics
11.
Drug Resist Updat ; 15(4): 211-22, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22910179

ABSTRACT

Hedgehog (Hh) signaling is a principal component of the morphogenetic code best known to direct pattern formation during embryogenesis. The Hh pathway remains active in adulthood however where it guides tissue regeneration and remodeling and Hh production in the niche plays an important role in maintaining stem cell compartment size. Deregulated Hh signaling activity is associated, depending on the context, with both cancer initiation and progression. Interestingly, the Hh pathway is remarkably druggable, raising hopes that inhibition of the pathway could support anticancer therapy. Indeed, a large body of preclinical data supports such an action, but promising clinical data are still limited to basal cell carcinoma (BSC) and medulloblastoma. Nevertheless cancer resistance against Hh targeting has already emerged as a major problem. Here we shall review the current situation with respect to targeting the Hh pathway in cancer in general and in chemotolerance in particular with a focus on the problems associated with the emergence of tumors resistant to treatment with inhibitors targeting the Hh receptor Smoothened (SMO).


Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Hedgehog Proteins/antagonists & inhibitors , Neoplasms/drug therapy , Signal Transduction/drug effects , Animals , Clinical Trials as Topic , Hedgehog Proteins/metabolism , Humans , Neoplasms/metabolism , Randomized Controlled Trials as Topic
12.
Mol Med ; 18: 1122-7, 2012 Sep 25.
Article in English | MEDLINE | ID: mdl-22371306

ABSTRACT

The major cause for plaque instability in atherosclerotic disease is neoangiogenic revascularization, but the factors controlling this process remain only partly understood. Hedgehog (HH) is a morphogen with important functions in revascularization, but its function in human healthy vessel biology as well as in atherosclerotic plaques has not been well investigated. Hence, we determined the status of HH pathway activity both in healthy vessels and atherosclerotic plaques. A series of 10 healthy organ donor-derived human vessels, 17 coronary atherosclerotic plaques and 24 atherosclerotic carotid plaques were investigated for HH pathway activity. We show that a healthy vessel is characterized by a high level of HH pathway activity but that atherosclerotic plaques are devoid of HH signaling despite the presence of HH ligand in these pathological structures. Thus, a dichotomy between healthy vessels and atherosclerotic plaques with respect to the activation status of the HH pathway exists, and it is tempting to suggest that downregulation of HH signaling contributes to long-term plaque stability.


Subject(s)
Blood Vessels/metabolism , Blood Vessels/pathology , Hedgehog Proteins/metabolism , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology , Signal Transduction , Hedgehog Proteins/genetics , Humans , Ligands , Plaque, Atherosclerotic/genetics , Signal Transduction/genetics
13.
Mol Med ; 17(9-10): 1119-26, 2011.
Article in English | MEDLINE | ID: mdl-21717035

ABSTRACT

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that TF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage.


Subject(s)
Chemokines/metabolism , Colitis/metabolism , Colon/metabolism , Thromboplastin/physiology , Animals , Cell Movement , Colitis/chemically induced , Colitis/genetics , Colon/pathology , Dextran Sulfate , Edema/metabolism , Edema/pathology , Enzyme-Linked Immunosorbent Assay , Female , Granulocytes/metabolism , Granulocytes/pathology , Humans , Immunohistochemistry , Inflammation Mediators/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Severity of Illness Index , Thromboplastin/deficiency , Thromboplastin/genetics , Tissue Culture Techniques
14.
Blood ; 118(10): 2889-95, 2011 Sep 08.
Article in English | MEDLINE | ID: mdl-21788337

ABSTRACT

Thrombomodulin (TM) is a predominantly endothelial transmembrane glycoprotein that modulates hemostatic function through a domain that controls thrombin-mediated proteolysis and an N-terminal lectin-like domain that controls inflammatory processes. To test the hypothesis that TM is a determinant of malignancy and dissect the importance of these functional domains in cancer biology, metastatic potential was evaluated in TM(Pro) mice expressing a mutant form of TM with reduced thrombin affinity and TM(LeD) mice lacking the N-terminal lectin-like domain. Studies of TM(Pro) mice revealed that TM is a powerful determinant of hematogenous metastasis. TM(Pro) mice exhibited a strongly prometastatic phenotype relative to control mice that was found to result from increased survival of tumor cells newly localized to the lung rather than any alteration in tumor growth. The impact of the TM(Pro) mutation on metastasis was dependent on both tumor cell-associated tissue factor and thrombin procoagulant function. In contrast, expression of a mutant form of TM lacking the lectin-like domain had no significant impact on metastasis. These studies directly demonstrate for the first time that TM-mediated regulation of tumor cell-driven procoagulant function strongly influences metastatic potential and suggest that endothelial cell-associated modulators of hemostasis may represent novel therapeutic targets in limiting tumor dissemination.


Subject(s)
Lectins/metabolism , Liver Neoplasms/secondary , Lung Neoplasms/secondary , Mutation/genetics , Thrombin/metabolism , Thrombomodulin/physiology , Animals , Carcinoma, Lewis Lung/metabolism , Carcinoma, Lewis Lung/pathology , Female , Hirudins/metabolism , Humans , Liver Neoplasms/metabolism , Lung Neoplasms/metabolism , Lymphatic Metastasis , Male , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Neoplastic Cells, Circulating , Oligonucleotides, Antisense/pharmacology , Platelet Count , Prothrombin/antagonists & inhibitors , Prothrombin/genetics , Recombinant Proteins/metabolism , Sarcoma, Experimental/metabolism , Sarcoma, Experimental/pathology
15.
Mol Med ; 17(3-4): 180-8, 2011.
Article in English | MEDLINE | ID: mdl-21203694

ABSTRACT

Although a relation between diminished human immunity and stress is well recognized both within the general public and the scientific literature, the molecular mechanisms by which stress alters immunity remain poorly understood. We explored a novel model for acute human stress involving volunteers performing a first-time bungee jump from an altitude of 60 m and exploited this model to characterize the effects of acute stress in the peripheral blood compartment. Twenty volunteers were included in the study; half of this group was pretreated for 3 d with the ß-receptor blocking agent propranolol. Blood was drawn 2 h before, right before, immediately after and 2 h after the jump. Plasma catecholamine and cortisol levels increased significantly during jumping, which was accompanied by significantly reduced ex vivo inducibility of proinflammatory cytokines as well as activation of coagulation and vascular endothelium. Kinome profiles obtained from the peripheral blood leukocyte fraction contained a strong noncanonical glucocorticoid receptor signal transduction signature after jumping. In apparent agreement, jumping down-regulated Lck/Fyn and cellular innate immune effector function (phagocytosis). Pretreatment of volunteers with propranolol abolished the effects of jumping on coagulation and endothelial activation but left the inhibitory effects on innate immune function intact. Taken together, these results indicate that bungee jumping leads to a catecholamine-independent immune suppressive phenotype and implicate noncanonical glucocorticoid receptor signal transduction as a major pathway linking human stress to impaired functioning of the human innate immune system.


Subject(s)
Altitude , Immunity, Innate/physiology , Sports , Stress, Physiological/physiology , Adolescent , Adrenergic beta-Antagonists/pharmacology , Adult , Blood Coagulation/drug effects , Blood Pressure/drug effects , Catecholamines/blood , Cytokines/metabolism , Heart Rate/drug effects , Humans , Hydrocortisone/blood , Immunity, Innate/drug effects , Leukocyte Count , Leukocytes/cytology , Leukocytes/drug effects , Leukocytes/metabolism , Lipopolysaccharides/pharmacology , Male , Phosphotransferases/metabolism , Propranolol/pharmacology , Prospective Studies , Signal Transduction/drug effects , Young Adult
17.
J Proteome Res ; 9(11): 6052-9, 2010 Nov 05.
Article in English | MEDLINE | ID: mdl-20839884

ABSTRACT

Hedgehog is one of the major morphogens and fulfils critical functions in both the development and maintenance of the vasculature. Hedgehog is highly hydrophobic and its diffusion toward target tissues remains only partly understood. In Drosophila, hedgehog transport via lipophorins is relevant for development, but neither the presence nor a function for a mammalian Hedgehog carried by human plasma lipoproteins has been established. We investigated the presence of Hedgehog on lipoprotein particles and determined its importance for maintaining the endothelium. LTQ-Orbitrap XL analysis of defined plasma lipoproteins revealed that Indian Hedgehog (Ihh) is present in the human very low density lipoprotein (VLDL) fraction but not in other plasma lipoprotein fractions (low density lipoprotein (LDL) and high density lipoprotein (HDL)). Using the same approach, neither Sonic Hedgehog nor Desert Hedgehog could be detected in plasma lipoprotein fractions. Most likely, primary white adipocytes are the source of Ihh loading on VLDL as both transcriptome as well as immunofluorescence analysis showed high expression of Ihh in these cells. Additionally, we show that the endothelial compartment is most likely to be affected by the presence of Ihh on VLDL. Indeed, VLDL increased survival of primary endothelial cells, suggesting that Ihh transport by VLDL is important for maintaining the human endothelium. In conclusion, our study shows that VLDL carries Ihh throughout the body in mammals and Hedgehog signaling by human plasma VLDL particles may affect blood vessel pathophysiology. A combination of three state-of-the-art technologies, proteomics, genomics, and confocal microscopy, appeared to be a powerful tool for analyzing plasma lipoprotein-associated proteins.


Subject(s)
Hedgehog Proteins/metabolism , Lipoproteins, VLDL/metabolism , Adipocytes , Humans , Lipoproteins , Lipoproteins, VLDL/blood , Protein Binding , Protein Transport
18.
Am J Pathol ; 177(5): 2483-94, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20864681

ABSTRACT

Klebsiella pneumoniae is a frequently isolated causative pathogen in respiratory tract infections. CD44 is a transmembrane adhesion molecule that has been implicated in several immunological processes. To determine the role of CD44 during Klebsiella pneumonia, we intranasally infected wild-type and CD44 knockout (KO) mice with 10(2) to 10(4) colony-forming units of K. pneumoniae or administered Klebsiella lipopolysaccharide. During lethal infection, CD44 deficiency was associated with reduced bacterial growth and dissemination accompanied by enhanced pulmonary inflammation. After infection with lower Klebsiella doses, CD44 KO mice but not wild-type mice demonstrated mortality. After infection with even lower bacterial doses, which were cleared by most mice of both strains, CD44 KO mice displayed enhanced lung inflammation 4 and 10 days postinfection, indicating that CD44 is important for the resolution of pulmonary inflammation after nonlethal pneumonia. In accordance, CD44 KO mice showed a diminished resolution of lung inflammation 4 days after intrapulmonary delivery of lipopolysaccharide. CD44 deficiency was associated with the accumulation of hyaluronan together with reduced gene expression levels of the negative regulators of Toll-like receptor signaling, interleukin-1R-associated kinase M, A20, and suppressor of cytokine signaling 3. In conclusion, the absence of CD44 affects various components and phases of the host response during Klebsiella pneumonia, reducing bacterial outgrowth and dissemination and enhancing pulmonary pathology during lethal infection, and diminishing the resolution of lung inflammation during sublethal infection.


Subject(s)
Hyaluronan Receptors/immunology , Klebsiella Infections/immunology , Klebsiella pneumoniae/metabolism , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/microbiology , Pneumonia/immunology , Pneumonia/microbiology , Animals , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Humans , Hyaluronan Receptors/genetics , Hyaluronic Acid/chemistry , Hyaluronic Acid/metabolism , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-1 Receptor-Associated Kinases/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Klebsiella Infections/pathology , Klebsiella pneumoniae/pathogenicity , Lipopolysaccharides/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Pneumonia/pathology , Pneumonia, Bacterial/pathology , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Survival Rate , Tumor Necrosis Factor alpha-Induced Protein 3
19.
Biochim Biophys Acta ; 1806(2): 287-303, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20659529

ABSTRACT

Most aspects of leukocyte physiology are under the control of reversible tyrosine phosphorylation. It is clear that excessive phosphorylation of signal transduction elements is a pivotal element of many different pathologies including haematological malignancies and accordingly, strategies that target such phosphorylation have clinically been proven highly successful for treatment of multiple types of leukemias and lymphomas. Cellular phosphorylation status is dependent on the resultant activity of kinases and phosphatases. The cell biology of the former is now well understood; for most cellular phosphoproteins we now know the kinases responsible for their phosphorylation and we understand the principles of their aberrant activity in disease. With respect to phosphatases, however, our knowledge is much patchier. Although the sequences of whole genomes allow us to identify phosphatases using in silico methodology, whereas transcription profiling allows us to understand how phosphatase expression is regulated during disease, most functional questions as to substrate specificity, dynamic regulation of phosphatase activity and potential for therapeutic intervention are still to a large degree open. Nevertheless, recent studies have allowed us to make meaningful statements on the role of tyrosine phosphatase activity in the three major signaling pathways that are commonly affected in leukemias, i.e. the Ras-Raf-ERK1/2, the Jak-STAT and the PI3K-PKB-mTOR pathways. Lessons learned from these pathways may well be applicable elsewhere in leukocyte biology as well.


Subject(s)
Hematologic Neoplasms/drug therapy , Protein Tyrosine Phosphatases/physiology , Animals , Hematologic Neoplasms/etiology , Hematologic Neoplasms/metabolism , Humans , Leukocyte Common Antigens/physiology , PTEN Phosphohydrolase/physiology , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 1/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/physiology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/physiology , Protein Tyrosine Phosphatases/antagonists & inhibitors , Signal Transduction
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