ABSTRACT
Osteopontin (OPN) is a phosphoglycoprotein secreted into the extracellular matrix upon liver injury, acting as a cytokine stimulates the deposition of fibrillary collagen in liver fibrogenesis. In livers of mice subjected to bile duct ligation (BDL) and in cultured activated hepatic stellate cells (HSCs), we show that OPN, besides being overexpressed, is substantially phosphorylated by family with sequence similarity 20, member C (Fam20C), formerly known as Golgi casein kinase (G-CK), which is exclusively resident in the Golgi apparatus. In both experimental models, Fam20C becomes overactive when associated with a 500-kDa multiprotein complex, as compared with the negligible activity in livers of sham-operated rats and in quiescent HSCs. Fam20C knockdown not only confirmed the role of Fam20C itself in OPN phosphorylation, but also revealed that phosphorylation was essential for OPN secretion. However, OPN acts as a fibrogenic factor independently of its phosphorylation state, as demonstrated by the increased expression of Collagen-I by HSCs incubated with either a phosphorylated or nonphosphorylated form of recombinant OPN. Collectively, our results confirm that OPN promotes liver fibrosis and highlight Fam20C as a novel factor driving this process by favoring OPN secretion from HSCs, opening new avenues for deciphering yet unidentified mechanisms underlying liver fibrogenesis.
Subject(s)
Calcium-Binding Proteins/metabolism , Extracellular Matrix Proteins/metabolism , Hepatic Stellate Cells/metabolism , Liver/pathology , Osteopontin/metabolism , Protein Serine-Threonine Kinases/metabolism , Animals , Cytokines/metabolism , Liver/metabolism , Liver Cirrhosis/metabolism , Male , Mice , Mice, Knockout , Phosphorylation , Rats , Rats, Wistar , Signal TransductionABSTRACT
Endometriosis, an estrogen-dependent chronic gynecological disease, is characterized by a systemic inflammation that affects circulating red blood cells (RBC), by reducing anti-oxidant defenses. The aim of this study was to investigate the potential beneficial effects of licorice intake to protect RBCs from dapsone hydroxylamine (DDS-NHOH), a harmful metabolite of dapsone, commonly used in the treatment of many diseases. A control group (CG, n = 12) and a patient group (PG, n = 18) were treated with licorice extract (25 mg/day), for a week. Blood samples before (T0) and after (T1) treatment were analyzed for: i) band 3 tyrosine phosphorylation and high molecular weight aggregates; and ii) glutathionylation and carbonic anhydrase activity, in the presence or absence of adjunctive oxidative stress induced by DDS-NHOH. Results were correlated with plasma glycyrrhetinic acid (GA) concentrations, measured by HPLC-MS. Results showed that licorice intake decreased the level of DDS-NHOH-related oxidative alterations in RBCs, and the reduction was directly correlated with plasma GA concentration. In conclusion, in PG, the inability to counteract oxidative stress is a serious concern in the evaluation of therapeutic approaches. GA, by protecting RBC from oxidative assault, as in dapsone therapy, might be considered as a new potential tool for preventing further switching into severe endometriosis.
Subject(s)
Anti-Infective Agents/adverse effects , Dapsone/adverse effects , Endometriosis/chemically induced , Glycyrrhiza , Plant Extracts/therapeutic use , Protective Agents/therapeutic use , Adult , Antioxidants/therapeutic use , Endometriosis/prevention & control , Erythrocytes/drug effects , Female , Glycyrrhiza/chemistry , Humans , Oxidative Stress/drug effects , Young AdultABSTRACT
Bicarbonate uptake is one of the early steps of capacitation, but the identification of proteins regulating anion fluxes remains elusive. The aim of this study is to investigate the role of sperm solute carrier 4 (SLC4) A1 (spAE1) in the capacitation process. The expression, location, and tyrosine-phosphorylation (Tyr-P) level of spAE1 were assessed. Thereby, it was found that 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), an SLC4 family channel blocker, inhibited capacitation in a dose-dependent manner by decreasing acrosome reaction (ARC% 24.5 ± 3.3 vs 64.9 ± 4.3, p < 0.05) and increasing the percentage of not viable cells (NVC%), comparable to the inhibition by I-172, a cystic fibrosis transmembrane conductance regulator (CFTR) blocker (AR% 30.5 ± 4.4 and NVC% 18.6 ± 2.2). When used in combination, a synergistic inhibitory effect was observed with a remarkable increase of the percentage of NVC (45.3 ± 4.1, p < 0.001). spAE1 was identified in sperm membrane as a substrate for Tyr-protein kinases Lyn and Syk, which were identified as both soluble and membrane-bound pools. spAE1-Tyr-P level increased in the apical region of sperm under capacitating conditions and was negatively affected by I-172 or DIDS, and, to a far greater extent, by a combination of both. In conclusion, we demonstrated that spAE1 is expressed in sperm membranes and it is phosphorylated by Syk, but above all by Lyn on Tyr359, which are involved in sperm viability and capacitation.
Subject(s)
SLC4A Proteins/metabolism , Sperm Capacitation/physiology , Spermatozoa/physiology , Tyrosine/metabolism , Acrosome Reaction , Cell Membrane , Cell Survival , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Male , Phosphorylation , SLC4A Proteins/geneticsABSTRACT
Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyn-containing high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD34+-derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in chorea-acanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation.
Subject(s)
Autophagy , Erythroid Cells/pathology , Neuroacanthocytosis/pathology , Adult , Anion Exchange Protein 1, Erythrocyte/metabolism , Autophagy/drug effects , Autophagy-Related Protein 7/metabolism , Autophagy-Related Protein-1 Homolog/metabolism , Benzoquinones/pharmacology , Bortezomib/pharmacology , Cell Differentiation/drug effects , Cytosol/drug effects , Cytosol/metabolism , Demography , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythrocytes/pathology , Erythrocytes/ultrastructure , Erythroid Cells/drug effects , Erythroid Cells/ultrastructure , Erythropoiesis/drug effects , Female , Heat-Shock Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Lactams, Macrocyclic/pharmacology , Lysosomes/drug effects , Lysosomes/metabolism , Male , Middle Aged , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Weight , Multivesicular Bodies/drug effects , Multivesicular Bodies/metabolism , Phosphorylation/drug effects , Proteasome Endopeptidase Complex/metabolism , Proteolysis/drug effects , src-Family Kinases/metabolismABSTRACT
Polycystic ovary syndrome (PCOS)is a gynecological endocrine disorder which is associated with systemic inflammatory status inducing red blood cells (RBC) membrane alterations related to insulin resistance and testosterone levels which could be greatly improved by myo-inositol (MYO) uptake. In this study we aim to evaluate the effect of MYO in reducing oxidative-related alterations through in vitro study on PCOS RBC. Blood samples from two groups of volunteers, control group (CG, n = 12) and PCOS patient group (PG, n = 12), were analyzed for band 3 tyrosine phosphorylation (Tyr-P), high molecular weight aggregate (HMWA), IgG in RBC membranes, and glutathione (GSH) in cytosol, following O/N incubation in the presence or absence of MYO. PCOS RBC underwent oxidative stress as indicated by higher band 3 Tyr-P and HMWA and increased membrane bound autologous IgG. Twenty four hours (but not shorter time) MYO incubation, significantly improved both Tyr-P level and HMWA formation and concomitant membrane IgG binding. However, no relevant modification of GSH content was detected. PCOS RBC membranes are characterized by increased oxidized level and enhanced sensitivity to oxidative injuries leading to potential premature RBC removal. MYO treatment is effective in reducing oxidative related abnormalities in PCOS patients probably restoring the inositol phospholipid pools of the membranes.
Subject(s)
Erythrocytes/drug effects , Inositol/pharmacology , Polycystic Ovary Syndrome/blood , Adult , Erythrocytes/metabolism , Female , Glutathione/metabolism , Humans , Immunoglobulin G/metabolism , Phosphorylation/drug effects , Young AdultABSTRACT
Astaxanthin (Asta), red pigment of the carotenoid family, is known for its anti-oxidant, anti-cancer, anti-diabetic, and anti-inflammatory properties. In this study, we evaluated the effects of Asta on isolated human sperm in the presence of human papillomavirus (HPV) 16 capsid protein, L1. Sperm, purified by gradient separation, were treated with HPV16-L1 in both a dose and time-dependent manner in the absence or presence of 30 min-Asta pre-incubation. Effects of HPV16-L1 alone after Asta pre-incubation were evaluated by rafts (CTB) and Lyn dislocation, Tyr-phosphorylation (Tyr-P) of the head, percentages of acrosome-reacted cells (ARC) and endogenous reactive oxygen species (ROS) generation. Sperm membranes were also analyzed for the HPV16-L1 content. Results show that HPV16-L1 drastically reduced membrane rearrangement with percentage of sperm showing head CTB and Lyn displacement decreasing from 72% to 15.8%, and from 63.1% to 13.9%, respectively. Accordingly, both Tyr-P of the head and ARC decreased from 68.4% to 10.2%, and from 65.7% to 14.6%, respectively. Asta pre-incubation prevented this drop and restored values of the percentage of ARC up to 40.8%. No alteration was found in either the ROS generation curve or sperm motility. In conclusion, Asta is able to preserve sperm by reducing the amount of HPV16-L1 bound onto membranes.
Subject(s)
Acrosome Reaction/drug effects , Capsid Proteins/metabolism , Human papillomavirus 16/pathogenicity , Oncogene Proteins, Viral/metabolism , Spermatozoa/drug effects , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane/virology , Chlorophyceae/chemistry , Drug Evaluation, Preclinical , Humans , Male , Papillomavirus Infections/prevention & control , Papillomavirus Infections/virology , Protein Binding/drug effects , Reactive Oxygen Species , Sperm Capacitation/drug effects , Sperm Motility/drug effects , Spermatozoa/virology , Xanthophylls/pharmacology , Xanthophylls/therapeutic useABSTRACT
Aberrant protein kinase activities, and the consequent dramatic increase of Ser/Thr and -Tyr phosphorylation, promote the deregulation of the survival pathways in chronic lymphocytic leukemia (CLL), which is crucial to the pathogenesis and progression of the disease. In this study, we show that the tumor suppressor protein phosphatase 2A (PP2A), one of the major Ser/Thr phosphatases, is in an inhibited form because of the synergistic contribution of 2 events, the interaction with its physiologic inhibitor SET and the phosphorylation of Y307 of the catalytic subunit of PP2A. The latter event is mediated by Lyn, a Src family kinase previously found to be overexpressed, delocalized, and constitutively active in CLL cells. This Lyn/PP2A axis accounts for the persistent high level of phosphorylation of the phosphatase's targets and represents a key connection linking phosphotyrosine- and phosphoserine/threonine-mediated oncogenic signals. The data herein presented show that the disruption of the SET/PP2A complex by a novel FTY720-analog (MP07-66) devoid of immunosuppressive effects leads to the reactivation of PP2A, which in turn triggers apoptosis of CLL cells. When used in combination with SFK inhibitors, the action of MP07-66 is synergistically amplified, providing a new option in the therapeutic strategy for CLL patients.
Subject(s)
Histone Chaperones/metabolism , Immunosuppressive Agents/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Propylene Glycols/pharmacology , Protein Phosphatase 2/metabolism , Sphingosine/analogs & derivatives , Transcription Factors/metabolism , src-Family Kinases/metabolism , Apoptosis/drug effects , DNA-Binding Proteins , Fingolimod Hydrochloride , HSP90 Heat-Shock Proteins/metabolism , Humans , Immunosuppressive Agents/chemistry , Phosphorylation/drug effects , Phosphotyrosine/metabolism , Propylene Glycols/chemistry , Protein Interaction Maps/drug effects , Signal Transduction/drug effects , Sphingosine/chemistry , Sphingosine/pharmacology , Tumor Cells, CulturedABSTRACT
Lyn, a member of the Src family of kinases, is a key factor in the dysregulation of survival and apoptotic pathways of malignant B cells in chronic lymphocytic leukemia. One of the effects of Lyn's action is spatial and functional segregation of the tyrosine phosphatase SHP-1 into two pools, one beneath the plasma membrane in an active state promoting pro-survival signals, the other in the cytosol in an inhibited conformation and unable to counter the elevated level of cytosolic tyrosine phosphorylation. We herein show that SHP-1 activity can be elicited directly by nintedanib, an agent also known as a triple angiokinase inhibitor, circumventing the phospho-S591-dependent inhibition of the phosphatase, leading to the dephosphorylation of pro-apoptotic players such as procaspase-8 and serine/threonine phosphatase 2A, eventually triggering apoptosis. Furthermore, the activation of PP2A by using MP07-66, a novel FTY720 analog, stimulated SHP-1 activity via dephosphorylation of phospho-S591, which unveiled the existence of a positive feedback signaling loop involving the two phosphatases. In addition to providing further insights into the molecular basis of this disease, our findings indicate that the PP2A/SHP-1 axis may emerge as an attractive, novel target for the development of alternative strategies in the treatment of chronic lymphocytic leukemia.
Subject(s)
Apoptosis/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Protein Phosphatase 2/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Signal Transduction/drug effects , Feedback, Physiological , Humans , Indoles/pharmacology , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Molecular Targeted Therapy/methods , Tumor Cells, CulturedABSTRACT
Endometriosis, an estrogen-dependent chronic gynecological disease in women of reproductive age, is characterized by a systemic inflammation status involving also red blood cells (RBCs). In this study, we evaluated how the protein oxidative status could be involved in the worsening of RBC conditions due to dapsone intake in endometriotic women in potential treatment for skin or infection diseases. Blood samples from two groups of volunteers, control group (CG) and endometriosis patient group (PG), were analyzed for their content of band 3 tyrosine phosphorylation (Tyr-P) and high molecular weight aggregate (HMWA) in membranes, and glutathione (GSH) content and carbonic anhydrase (CA) activity in cytosol. In endometriotic patients, RBC showed the highest level of oxidative-related alterations both in membrane and cytosol. More interestingly, the addition of dapsone hydroxylamine (DDS-NHOH) could induce further increase of both membranes and cytosol markers, with an enhancement of CA activity reaching about 66% of the total cell enzyme amount. In conclusion, in PG the systemic inflammatory status leads to the inability of counteracting adjunctive oxidative stress, with a potential involvement of CA-related pathologies, such as glaucoma. Hence, the importance of the evaluation of therapeutic approaches worsening oxidative imbalance present in PG RBC is underlined.
Subject(s)
Dapsone/analogs & derivatives , Endometriosis/blood , Erythrocytes/drug effects , Oxidative Stress/drug effects , Adult , Carbonic Anhydrases/metabolism , Dapsone/pharmacology , Erythrocytes/enzymology , Female , Humans , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Young AdultABSTRACT
Fam20C is an atypical kinase implicated in bio-mineralization and phosphate homeostasis disorders, and has recently been shown to account for the activity of an orphan enzyme ("genuine casein kinase", G-CK) previously characterized for its ability to phosphorylate casein and a plethora of secreted proteins at serine residues specified by the S-x-E/pS motif. Fam20C/G-CK activity is only appreciable in the presence of high Mn2+ concentration (>1 mM), and is negligible if Mn2+ is replaced by physiological Mg2+ concentrations. Here we show that sphingosine (but not its biological precursor ceramide) not only stimulates several-fold Fam20C activity in the presence of Mn2+, but also confers a comparable activity to Fam20C assayed with Mg2+. Activation by sphingosine is evident using a variety of substrates, and is accounted for by both higher Vmax and decreased KmATP, as judged from kinetics run with the ß(28-40) substrate peptide and a physiological substrate, BMP-15. Sphingosine also protects Fam20C from thermal inactivation. Consistent with the in vitro results, by treating Fam20C expressing HEK293T cells with myriocin, a potent inhibitor of the sphingosine biosynthetic pathway, the activity of Fam20C released into the conditioned medium is substantially decreased corroborating the concept that sphingosine (or related metabolite(s)) is a co-factor required by Fam20C to optimally display its biological functions. None of the small molecule kinase inhibitors tested so far were able to inhibit Fam20C. Interestingly however fingolimod, an immunosuppressive drug structurally related to sphingosine, used for the treatment of multiple sclerosis, is a powerful activator of Fam20C, both wild type and its pathogenic, loss of function, T268M mutant. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Subject(s)
Casein Kinase I/genetics , Extracellular Matrix Proteins/genetics , Multiple Sclerosis/genetics , Sphingosine/biosynthesis , Amino Acid Sequence , Casein Kinase I/chemistry , Extracellular Matrix Proteins/chemistry , Fatty Acids, Monounsaturated/pharmacology , Gene Expression Regulation/drug effects , HEK293 Cells , Humans , Multiple Sclerosis/enzymology , Phosphorylation , Sphingosine/antagonists & inhibitors , Sphingosine/metabolism , Transcriptional Activation/drug effectsABSTRACT
Lyn, a member of the group of tyrosine kinases named the Src family kinases (SFKs), is overexpressed, associated with an aberrant multiprotein complex and constitutively active in B-cell chronic lymphocytic leukemia (B-CLL) cells, resulting in a high level of tyrosine phosphorylation and contributing to their resistance to apoptosis. By using biochemical and bioinformatics tools, we identified procaspase-8 (procasp8), the caspase-8 zymogen, as a cytosolic target for Lyn in B-CLL cells, the phosphorylation of which at Tyr380 promotes the formation of an inactive procasp8 homodimer. This complex remains segregated in the cytosol and appears to be crucial in mediating the antiapoptotic function of Lyn in this disease. The significance of the Lyn-procasp8 axis in impairing apoptosis in B-CLL cells was further confirmed by pharmacological and genetic inhibition of procasp8, which drastically reduced the apoptosis induced by the SFK inhibitors PP2 and dasatinib. Our data highlight that Lyn's dysregulated expression, activity, and localization in B-CLLs support resistance to cell demise by inhibiting an early player of apoptotic signaling, and potentially broaden the perspectives of developing new strategies for the treatment of this disease.
Subject(s)
Apoptosis , Caspase 8/chemistry , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , src-Family Kinases/metabolism , Blotting, Western , Caspase 8/metabolism , Cell Proliferation , Computational Biology , Cytosol/metabolism , Electrophoresis, Gel, Two-Dimensional , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Phosphorylation , Protein Multimerization , Proteome/analysis , Tumor Cells, Cultured , Tyrosine/metabolismABSTRACT
The dimerization and auto-transphosphorylation of platelet-derived growth factor receptor (PDGFR) upon engagement by platelet-derived growth factor (PDGF) activates signals promoting the mitogenic response of hepatic stellate cells (HSCs) due to liver injury, thus contributing to the development of hepatic fibrosis. We demonstrate that the tyrosine phosphatases Src homology 2 domain-containing phosphatase 1 and 2 (SHP-1 and SHP-2) act as crucial regulators of a complex signaling network orchestrated by PDGFR activation in a spatio-temporal manner with diverse and opposing functions in HSCs. In fact, silencing of either phosphatase shows that SHP-2 is committed to PDGFR-mediated cell proliferation, whereas SHP-1 dephosphorylates PDGFR hence abrogating the downstream signaling pathways that result in HSC activation. In this regard, SHP-1 as an off-switch of PDGFR signaling appears to emerge as a valuable molecular target to trigger as to prevent HSC proliferation and the fibrogenic effects of HSC activation. We show that boswellic acid, a multitarget compound with potent anti-inflammatory action, exerts an anti-proliferative effect on HSCs, as in other cell models, by upregulating SHP-1 with subsequent dephosphorylation of PDGFR-ß and downregulation of PDGF-dependent signaling after PDGF stimulation. Moreover, the synergism resulting from the combined use of boswellic acid and imatinib, which directly inhibits PDGFR-ß activity, on activated HSCs offers new perspectives for the development of therapeutic strategies that could implement molecules affecting diverse players of this molecular circuit, thus paving the way to multi-drug low-dose regimens for liver fibrosis.
Subject(s)
Hepatic Stellate Cells/cytology , Hepatic Stellate Cells/enzymology , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Signal Transduction , Animals , Becaplermin , Benzamides/pharmacology , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Proliferation/drug effects , Down-Regulation/drug effects , Enzyme Activation/drug effects , Hepatic Stellate Cells/drug effects , Imatinib Mesylate , Male , Piperazines/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/antagonists & inhibitors , Proto-Oncogene Proteins c-sis/pharmacology , Pyrimidines/pharmacology , Rats , Rats, Wistar , Signal Transduction/drug effects , Triterpenes/pharmacologyABSTRACT
Astaxanthin (Asta), a photo-protective red pigment of the carotenoid family, is known for its multiple beneficial properties. In this study, the effects of Asta on isolated human sperm were evaluated. Capacitation involves a series of transformations to let sperm acquire the correct features for potential oocyte fertilization, including the generation of a controlled amount of reactive oxygen species (ROS), cholesterol depletion of the sperm outer membrane, and protein tyrosine phosphorylation (Tyr-P) process in the head region. Volunteers, with normal spermiogram values, were divided in two separate groups on the basis of their ability to generate the correct content of endogenous ROS. Both patient group (PG) and control group (CG) were analysed for Tyr-phosphorylation (Tyr-P) pattern and percentages of acrosome-reacted cells (ARC) and non-viable cells (NVC), in the presence or absence of Asta. In addition, the involvement of ROS on membrane reorganization and the presence of Lyn, a Src family kinase associated with lipid rafts, were investigated. Results show that Lyn is present in the membranes of human sperm, mainly confined in midpiece in resting conditions. Following capacitation, Lyn translocated to the head concomitantly with raft relocation, thus allowing the Tyr-P of head proteins. Asta succeeded to trigger Lyn translocation in PG sperm thus bypassing the impaired ROS-related mechanism for rafts and Lyn translocation. In this study, we showed an interdependence between ROS generation and lipid rafts and Lyn relocation leading the cells to undergo the successive acrosome reaction (AR). Asta, by ameliorating PG sperm functioning, may be utilised to decrease male idiopathic infertility.
Subject(s)
Membrane Microdomains/drug effects , Sperm Capacitation/drug effects , Spermatozoa/drug effects , src-Family Kinases/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Humans , Male , Membrane Microdomains/physiology , Protein Transport/drug effects , Protein Transport/physiology , Reactive Oxygen Species/metabolism , Spermatozoa/physiology , Xanthophylls/pharmacology , src-Family Kinases/geneticsABSTRACT
Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.
Subject(s)
Alternative Splicing , Cortactin/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Signal Transduction , src-Family Kinases/metabolism , Adult , Aged , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Case-Control Studies , Cortactin/metabolism , Drug Resistance, Neoplasm/genetics , Female , Gene Expression , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Male , Middle Aged , Protein Isoforms , Protein Processing, Post-TranslationalABSTRACT
OBJECTIVES: Neural stem/progenitor cells derived from olfactory neuroepithelium (hereafter olfactory neural stem/progenitor cells, ONSPCs) are emerging as a potential tool in the exploration of psychiatric disorders. The present study intended to assess whether ONSPCs could help discern individuals with schizophrenia (SZ) from non-schizophrenic (NS) subjects by exploring specific cellular and molecular features. METHODS: ONSPCs were collected from 19 in-patients diagnosed with SZ and 31 NS individuals and propagated in basal medium. Mitochondrial ATP production, expression of ß-catenin and cell proliferation, which are described to be altered in SZ, were examined in freshly isolated or newly thawed ONSPCs after a few culture passages. RESULTS: SZ-ONSPCs exhibited a lower mitochondrial ATP production and insensitivity to agents capable of positively or negatively affecting ß-catenin expression with respect to NS-ONSPCs. As to proliferation, it declined in SZ-ONSPCs as the number of culture passages increased compared to a steady level of growth shown by NS-ONSPCs. CONCLUSIONS: The ease and safety of sample collection as well as the differences observed between NS- and SZ-ONSPCs, may lay the groundwork for a new approach to obtain biological material from a large number of living individuals and gain a better understanding of the mechanisms underlying SZ pathophysiology.
Subject(s)
Cell Proliferation , Neural Stem Cells , Olfactory Mucosa , Schizophrenia , beta Catenin , Schizophrenia/metabolism , Schizophrenia/pathology , Humans , Adult , Male , Female , beta Catenin/metabolism , Olfactory Mucosa/cytology , Olfactory Mucosa/metabolism , Olfactory Mucosa/pathology , Adenosine Triphosphate/metabolism , Middle Aged , Cells, Cultured , Mitochondria/metabolism , Neuroepithelial Cells/metabolismABSTRACT
The epithelial-mesenchymal transition (EMT) of proximal tubular epithelial cells (PTECs) into myofibroblasts contributes to the establishment of fibrosis that leads to end stage renal disease. FGF-2 induces EMT in PTECs. Because the interaction between FGF-2 and its receptor is mediated by heparan sulfate (HS) and syndecans, we speculated that a deranged HS/syndecans regulation impairs FGF-2 activity. Heparanase is crucial for the correct turnover of HS/syndecans. The aim of the present study was to assess the role of heparanase on epithelial-mesenchymal transition induced by FGF-2 in renal tubular cells. In human kidney 2 (HK2) PTEC cultures, although FGF-2 induces EMT in the wild-type clone, it is ineffective in heparanase-silenced cells. The FGF-2 induced EMT is through a stable activation of PI3K/AKT which is only transient in heparanase-silenced cells. In PTECs, FGF-2 induces an autocrine loop which sustains its signal through multiple mechanisms (reduction in syndecan-1, increase in heparanase, and matrix metalloproteinase 9). Thus, heparanase is necessary for FGF-2 to produce EMT in PTECs and to sustain FGF-2 intracellular signaling. Heparanase contributes to a synergistic loop for handling syndecan-1, facilitating FGF-2 induced-EMT. In conclusion, heparanase plays a role in the tubular-interstitial compartment favoring the FGF-2-dependent EMT of tubular cells. Hence, heparanase is an interesting pharmacological target for the prevention of renal fibrosis.
Subject(s)
Epithelial-Mesenchymal Transition , Fibroblast Growth Factor 2/metabolism , Glucuronidase/metabolism , Kidney Failure, Chronic/metabolism , Kidney Tubules/metabolism , Syndecan-1/metabolism , Autocrine Communication/genetics , Cell Line , Enzyme Activation/genetics , Fibroblast Growth Factor 2/genetics , Fibrosis , Glucuronidase/genetics , Humans , Kidney Failure, Chronic/pathology , Kidney Tubules/pathology , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Receptor, Fibroblast Growth Factor, Type 2/genetics , Receptor, Fibroblast Growth Factor, Type 2/metabolism , Signal Transduction/genetics , Syndecan-1/geneticsABSTRACT
Acanthocytic RBCs are a peculiar diagnostic feature of chorea-acanthocytosis (ChAc), a rare autosomal recessive neurodegenerative disorder. Although recent years have witnessed some progress in the molecular characterization of ChAc, the mechanism(s) responsible for generation of acanthocytes in ChAc is largely unknown. As the membrane protein composition of ChAc RBCs is similar to that of normal RBCs, we evaluated the tyrosine (Tyr)-phosphorylation profile of RBCs using comparative proteomics. Increased Tyr phosphorylation state of several membrane proteins, including band 3, ß-spectrin, and adducin, was noted in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs, band 3 Tyr phosphorylation by Lyn was independent of the canonical Syk-mediated pathway. The ChAc-associated alterations in RBC membrane protein organization appear to be the result of increased Tyr phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton as supported by coimmunoprecipitation of ß-adducin with band 3 only in ChAc RBC-membrane treated with the Lyn-inhibitor PP2. We propose this altered association between membrane skeleton and membrane proteins as novel mechanism in the generation of acanthocytes in ChAc.
Subject(s)
Acanthocytes/enzymology , Erythrocyte Membrane/enzymology , Neuroacanthocytosis/metabolism , src-Family Kinases/metabolism , Acanthocytes/pathology , Adult , Anion Exchange Protein 1, Erythrocyte/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/metabolism , Enzyme Activation/physiology , Erythrocyte Membrane/pathology , Female , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Male , Middle Aged , Neuroacanthocytosis/pathology , Phosphorylation/physiology , Protein-Tyrosine Kinases/metabolism , Proteomics , Syk Kinase , Tyrosine/metabolismABSTRACT
Sixteen flavonoids and related compounds have been tested for their ability to inhibit three acidophilic Ser/Thr protein kinases: the Golgi apparatus casein kinase (G-CK) recently identified with protein FAM20C, protein kinase CK1, and protein kinase CK2. While G-CK is entirely insensitive to all compounds up to 40 µM concentration, consistent with the view that it is not a member of the kinome, and CK1 is variably inhibited in an isoform-dependent manner by fisetin and luteolin, and to a lesser extent by myricetin and quercetin, CK2 is susceptible to drastic inhibition by many flavonoids, displaying with six of them IC(50) values < 1 µM. A common denominator of these compounds (myricetin, quercetin, fisetin, kaempferol, luteolin, and apigenin) is a flavone scaffold with at least two hydroxyl groups at positions 7 and 4'. Inhibition is competitive with respect to the phospho-donor substrate ATP. The crystal structure of apigenin and luteolin in complex with the catalytic subunit of Zea mays CK2 has been solved, revealing their ability to interact with both the hinge region (Val116) and the positive area near Lys68 and the conserved water W1, the two main polar ligand anchoring points in the CK2 active site. Modeling experiments account for the observation that luteolin but not apigenin inhibits also CK1. The observation that luteolin shares its pyrocatechol moiety with tyrphostin AG99 prompted us to solve also the structure of this compound in complex with CK2. AG99 was found inside the ATP pocket, consistent with its mode of inhibition competitive with respect to ATP. As in the case of luteolin, the pyrocatechol group of AG99 is critical for binding, interacting with the positive area in the deepest part of the CK2 active site.
Subject(s)
Casein Kinase II/antagonists & inhibitors , Casein Kinase II/chemistry , Flavonoids/pharmacology , Protein Kinase Inhibitors/pharmacology , Tyrphostins/pharmacology , Adenosine Triphosphate/metabolism , Binding, Competitive , Casein Kinase II/metabolism , Catalytic Domain , Flavonoids/metabolism , Humans , Inhibitory Concentration 50 , Models, Molecular , Protein Kinase Inhibitors/metabolism , Tyrphostins/metabolism , Zea mays/enzymologyABSTRACT
Diabetic nephropathy is one of the main causes of end-stage renal disease, in which the development of tubular damage depends on factors such as high glucose levels, albuminuria and advanced glycation end-product. In this study, we analyzed the involvement of heparanase, a heparan sulfate glycosidase, in the homeostasis of proximal tubular epithelial cells in the diabetic milieu. In vitro studies were performed on a wild-type and stably heparanase-silenced adult tubular line (HK2) and HEK293. Gene and protein expression analyses were performed in the presence and absence of diabetic mediators. Albumin and advanced glycation end-product, but not high glucose levels, increased heparanase expression in adult tubular cells via the AKT/PI3K signaling pathway. This over-expression of heparanase is then responsible for heparan sulfate reduction via its endoglycosidase activity and its capacity to regulate the heparan sulfate-proteoglycans core protein. In fact, heparanase regulates the gene expression of syndecan-1, the most abundant heparan sulfate-proteoglycans in tubular cells. We showed that heparanase is a target gene of the diabetic nephropathy mediators albumin and advanced glycation end-product, so it may be relevant to the progression of diabetic nephropathy. It could take part in several processes, e.g. extracellular-matrix remodeling and cell-cell crosstalk, via its heparan sulfate endoglycosidase activity and capacity to regulate the expression of the heparan sulfate-proteoglycan syndecan-1.
Subject(s)
Albumins/metabolism , Glucuronidase/metabolism , Glycation End Products, Advanced/metabolism , Kidney Tubules, Proximal/metabolism , Base Sequence , Cell Line , Diabetic Nephropathies/complications , Diabetic Nephropathies/genetics , Diabetic Nephropathies/metabolism , Glucose/metabolism , Glucuronidase/antagonists & inhibitors , Glucuronidase/genetics , HEK293 Cells , Heparitin Sulfate/metabolism , Homeostasis , Humans , Kidney Failure, Chronic/etiology , Kidney Failure, Chronic/genetics , Kidney Failure, Chronic/metabolism , Kidney Tubules, Proximal/cytology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Serum Albumin/metabolism , Serum Albumin, Bovine/metabolism , Syndecan-1/genetics , Syndecan-1/metabolism , Transcriptional Activation , Glycated Serum AlbuminABSTRACT
Liver regeneration (LR) is a compensatory growth that occurs in response to resection or injury of the liver aimed at restoring the liver mass and maintaining body homeostasis. The activation of intracellular signaling pathways due to extracellular stimuli mainly reflects a highly coordinated spatial and temporal organization of phosphotyrosine-based signals generated by the concerted action of three basic functional modules, namely protein tyrosine kinases, protein tyrosine phosphatases, and the Src homology 2 (SH2) domain. In this review, we have selected a set of signaling proteins downstream of activated cytokine and growth factor receptors that highlight the multifaceted aspects of tyrosine phosphorylation with their impact on the course of LR. Besides being a process of remarkable biological interest, LR has recently emerged as a model for dissecting molecular mechanisms underlying diverse pathophysiological states, offering new perspectives in primarily, but not only, managing life-threatening liver diseases.