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1.
Blood ; 134(9): 776-781, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31243040

RESUMO

Soluble thrombomodulin plasma concentrations are elevated in steroid-resistant graft-versus-host disease (GVHD), implying endothelial hypofunctioning for thrombomodulin-dependent generation of activated protein C's (APC) anticoagulant, anti-inflammatory, and antiapoptotic functions. Recombinant thrombomodulin or APC administration decreases acute GVHD, manifested by intense inflammation and tissue destruction. Here, we administered recombinant murine wild-type (WT) APC to mice with established chronic GVHD (cGVHD), a less-inflammatory autoimmune-like disease. WT APC normalized bronchiolitis obliterans-induced pulmonary dysfunction. Signaling-selective APC variants (3A-APC [APC with lysine 191-193 replaced with 3 alanines] or 5A-APC [APC with lysine 191-193 replaced with 3 alanines and arginine 229/230 replaced with 2 alanines]) with normal cytoprotective properties, but greatly reduced anticoagulant activity, provided similar results. Mechanistically, WT APC and signaling-selective variants reduced T follicular helper cells, germinal center formation, immunoglobulin, and collagen deposition. WT APC can potentially cleave protease-activated receptor 1 (PAR1) at Arg41 or Arg46, the latter causing anti-inflammatory signaling. cGVHD was reduced in recipients of T cells from WT PAR1 or mutated Gln41-PAR1 donors but not from mutated Gln46-PAR1 donors. These data implicate donor T-cell APC-induced noncanonical cleavage at Arg46-PAR1, which is known to confer cytoprotective and anti-inflammatory activities. Together, these data indicate that APC anticoagulant activity is dispensable, whereas anti-inflammatory signaling and cytoprotective cell signaling by APC are essential. Because a phase 2 ischemic stroke clinical trial did not raise any safety issues for 3A-APC treatment, our studies provide a foundational platform for testing in clinical cGVHD therapy.


Assuntos
Doença Enxerto-Hospedeiro/tratamento farmacológico , Proteína C/uso terapêutico , Receptor PAR-1/metabolismo , Linfócitos T/efeitos dos fármacos , Animais , Doença Crônica , Doença Enxerto-Hospedeiro/metabolismo , Doença Enxerto-Hospedeiro/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Recombinantes/uso terapêutico , Linfócitos T/metabolismo , Linfócitos T/patologia
2.
Blood ; 131(11): 1163-1171, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29343482

RESUMO

Activated protein C (APC) cleaves protease-activated receptor 1 (PAR1) in vitro at R46 to initiate beneficial cell signaling; however, thrombin and APC can cleave at R41. To elucidate PAR1-dependent aspects of the pharmacologic in vivo mechanisms of APC, we generated C57BL/6 mouse strains carrying QQ41 or QQ46 point mutations in PAR1 (F2r gene). Using these strains, we determined whether or not recombinant murine signaling-selective APC mutants would reduce septic death or provide neuroprotection against ischemic stroke when mice carried PAR1-homozygous mutations that prevent cleavage at either R41 or R46. Intercrossing PAR1+/R46Q mice generated expected numbers of PAR1+/+, PAR1+/R46Q, and R46Q/R46Q offspring whereas intercrossing PAR1+/R41Q mice gave decreased R41Q/R41Q homozygotes (resembling intercrossing PAR1+/PAR1-knockout mice). QQ41-PAR1 and QQ46-PAR1 brain endothelial cells showed the predicted retention or loss of cellular responses to thrombin receptor-activating peptide, thrombin, or APC for each PAR1 mutation. In sepsis studies, exogenous APC reduced mortality from 50% to 10% in Escherichia coli-induced pneumonia for wild-type (Wt) PAR1 and QQ41-PAR1 mice (P < .01) but had no benefit for QQ46-PAR1 mice. In transient distal middle cerebral artery occlusion stroke studies, exogenous APC significantly reduced infarct size, edema, and neuronal apoptosis for Wt mice and QQ41-PAR1 mice but had no detectable benefits for mice carrying QQ46-PAR1. In functional studies of forelimb-asymmetry and foot-fault tests at 24 hours after stroke induction, signaling-selective APC was beneficial for Wt and QQ41-PAR1 mice but not QQ46-PAR1 mice. These results support the concept that APC-induced, PAR1-dependent biased signaling following R46 cleavage is central to the in vivo benefits of APC.


Assuntos
Mutação Puntual , Proteína C/metabolismo , Proteólise , Receptor PAR-1/metabolismo , Sepse/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/metabolismo , Substituição de Aminoácidos , Animais , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Camundongos , Camundongos Mutantes , Proteína C/genética , Receptor PAR-1/genética , Sepse/genética , Sepse/patologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/patologia
3.
Blood ; 128(14): 1870-1878, 2016 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-27421960

RESUMO

To test the hypothesis that skeletal muscle myosins can directly influence blood coagulation and thrombosis, ex vivo studies of the effects of myosin on thrombogenesis in fresh human blood were conducted. Addition of myosin to blood augmented the thrombotic responses of human blood flowing over collagen-coated surfaces (300 s-1 shear rate). Perfusion of human blood over myosin-coated surfaces also caused fibrin and platelet deposition, evidencing myosin's thrombogenicity. Myosin markedly enhanced thrombin generation in both platelet-rich plasma and platelet-poor plasma, indicating that myosin promoted thrombin generation in plasma primarily independent of platelets. In purified reaction mixtures composed only of factor Xa, factor Va, prothrombin, and calcium ions, myosin greatly enhanced prothrombinase activity. The Gla domain of factor Xa was not required for myosin's prothrombinase enhancement. When binding of purified clotting factors to immobilized myosin was monitored using biolayer interferometry, factors Xa and Va each showed favorable binding interactions. Factor Va reduced by 100-fold the apparent Kd of myosin for factor Xa (Kd ∼0.48 nM), primarily by reducing koff, indicating formation of a stable ternary complex of myosin:Xa:Va. In studies to assess possible clinical relevance for this discovery, we found that antimyosin antibodies inhibited thrombin generation in acute trauma patient plasmas more than in control plasmas (P = .0004), implying myosin might contribute to acute trauma coagulopathy. We posit that myosin enhancement of thrombin generation could contribute either to promote hemostasis or to augment thrombosis risk with consequent implications for myosin's possible contributions to pathophysiology in the setting of acute injuries.


Assuntos
Fator Va/metabolismo , Fator Xa/metabolismo , Protrombina/metabolismo , Miosinas de Músculo Esquelético/farmacologia , Trombose/patologia , Doença Aguda , Animais , Circulação Sanguínea/efeitos dos fármacos , Estudos de Casos e Controles , Humanos , Proteínas Imobilizadas/farmacologia , Interferometria , Modelos Biológicos , Plasma Rico em Plaquetas/metabolismo , Ligação Proteica/efeitos dos fármacos , Coelhos , Trombose/metabolismo , Ferimentos e Lesões/sangue , Ferimentos e Lesões/patologia
4.
Arterioscler Thromb Vasc Biol ; 36(3): 518-24, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26800564

RESUMO

OBJECTIVE: Activated protein C (APC), a plasma serine protease, initiates cell signaling that protects endothelial cells from apoptosis and endothelial barrier disruption. Apolipoprotein E receptor 2 (ApoER2; LRP8) is a receptor known for mediating signaling initiated by reelin in neurons. ApoER2 contributes to APC-initiated signaling in monocytic U937 cells. The objective was to determine whether ApoER2 is required for APC's beneficial signaling in the endothelial cell surrogate EA.hy926 line. APPROACH AND RESULTS: We used small interfering RNA and inhibitors to probe requirements for specific receptors for APC's antiapoptotic activity and for phosphorylation of disabled-1 by Src family kinases and of Akt. When small interfering RNA for ApoER2 or endothelial cell protein C receptor or protease activated receptor 1 was used, APC's antiapoptotic activity was ablated, indicating that each of these receptors was required. In EA.hy926 cells, APC induced a 2- to 3-fold increased phosphorylation of Ser473-Akt and Tyr232-disabled-1, a phosphorylation known to trigger disabled-1-mediated signaling in other cell types. Ser473-Akt phosphorylation was inhibited by ApoER2 small interfering RNA or by inhibitors of Src (PP2), phosphatidylinositol-3 kinase (LY303511), and protease activated receptor 1 (SCH79797). ApoER2 small interfering RNA blocked the ability of APC to prevent thrombin-induced endothelial barrier disruption in TransEndothelial Resistance assays. Binding studies using purified APC and purified immobilized wild-type and mutated ApoER2 ectodomains suggested that APC binding involves Lys49, Asp50, and Trp64 on the surface of the N-terminal LA1 domain of ApoER2. CONCLUSIONS: ApoER2 contributes cooperatively with endothelial cell protein C receptor and protease activated receptor 1 to APC-initiated endothelial antiapoptotic and barrier protective signaling.


Assuntos
Permeabilidade da Membrana Celular , Células Endoteliais/enzimologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Apoptose , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Impedância Elétrica , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática , Proteínas da Matriz Extracelular/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/genética , Proteínas do Tecido Nervoso/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteína C/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/genética , Receptor PAR-1/metabolismo , Proteína Reelina , Serina Endopeptidases/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismo
5.
Carcinogenesis ; 36 Suppl 1: S89-110, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26106145

RESUMO

Cell death is a process of dying within biological cells that are ceasing to function. This process is essential in regulating organism development, tissue homeostasis, and to eliminate cells in the body that are irreparably damaged. In general, dysfunction in normal cellular death is tightly linked to cancer progression. Specifically, the up-regulation of pro-survival factors, including oncogenic factors and antiapoptotic signaling pathways, and the down-regulation of pro-apoptotic factors, including tumor suppressive factors, confers resistance to cell death in tumor cells, which supports the emergence of a fully immortalized cellular phenotype. This review considers the potential relevance of ubiquitous environmental chemical exposures that have been shown to disrupt key pathways and mechanisms associated with this sort of dysfunction. Specifically, bisphenol A, chlorothalonil, dibutyl phthalate, dichlorvos, lindane, linuron, methoxychlor and oxyfluorfen are discussed as prototypical chemical disruptors; as their effects relate to resistance to cell death, as constituents within environmental mixtures and as potential contributors to environmental carcinogenesis.


Assuntos
Carcinogênese/induzido quimicamente , Carcinógenos Ambientais/efeitos adversos , Morte Celular/efeitos dos fármacos , Exposição Ambiental/efeitos adversos , Substâncias Perigosas/efeitos adversos , Neoplasias/induzido quimicamente , Neoplasias/etiologia , Animais , Homeostase/efeitos dos fármacos , Humanos
6.
Blood ; 120(26): 5237-46, 2012 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-23149848

RESUMO

Activated protein C (APC) exerts endothelial cytoprotective actions that require protease-activated receptor 1 (PAR1), whereas thrombin acting via PAR1 causes endothelial disruptive, proinflammatory actions. APC's activities, but not thrombin's, require PAR1 located in caveolae. PAR1 is a biased 7-transmembrane receptor because G proteins mediate thrombin's signaling, whereas ß-arrestin 2 mediates APC's signaling. Here we elucidate novel mechanisms for APC's initiation of signaling. Biochemical studies of APC's protease specificity showed that APC cleaved PAR1 sequences at both Arg41 and Arg46. That PAR1 cleavage at Arg46 can occur on cells was supported by APC's cleavage of N-terminal-SEAP-tagged R41Q-PAR1 but not R41Q/R46Q-PAR1 mutants transfected into cells and by anti-PAR1 epitope mapping of APC-treated endothelial cells. A synthetic peptide composing PAR1 residues 47-66, TR47, stimulated protective signaling in endothelial cells as reflected in Akt and glycogen synthase kinase 3ß phosphorylation, Ras-related C3 botulinum toxin substrate 1 activation, and barrier stabilization effects. In mice, the TR47 peptide reduced VEGF-induced vascular leakage. These in vitro and in vivo data imply that the novel PAR1 N-terminus beginning at residue Asn47, which is generated by APC cleavage at Arg46, mediates APC's cytoprotective signaling and that this unique APC-generated N-terminal peptide tail is a novel biased agonist for PAR1.


Assuntos
Proteína C/farmacologia , Proteólise , Receptor PAR-1/agonistas , Receptor PAR-1/química , Receptor PAR-1/metabolismo , Animais , Arginina/química , Permeabilidade Capilar/efeitos dos fármacos , Domínio Catalítico , Células Cultivadas , Agonismo de Drogas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Biológicos , Modelos Moleculares , Proteína C/metabolismo , Proteólise/efeitos dos fármacos , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores
7.
Indian J Ophthalmol ; 71(5): 2193-2198, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37202947

RESUMO

Purpose: The second wave of coronavirus disease 2019 (COVID-19) pandemic triggered a mucormycosis epidemic in India. Diabetes mellitus and dysregulated immune response were contributors, and rhino-orbital-cerebral mucormycosis (ROCM) was the most common presentation. It is however not known whether bio-chemical parameters at presentation correlate with stage of ROCM or final outcome in terms of vision or mortality. Methods: This retrospective, hospital-based study included all in-patients of mucormycosis with ophthalmic manifestations at presentation admitted during June 1, 2021 to August 31, 2021. It aimed to evaluate the association between severity of infection, serum levels of HbA1c, ferritin, interleukin-6 (IL-6), C-reactive protein (CRP), and D-dimer levels at presentation and outcome. Results: There were altogether 47 eligible cases having a mean age of 48.8 ± 10.9 years with a male:female ratio of 2.6:1; forty-two (89.4%) had pre-existing diabetes, and five (10.6%) had steroid-induced hyperglycemia. The mean HbA1c among diabetics was 9.7 ± 2.1. HbA1c and serum CRP showed an increase over subsequent stages, which was not statistically significant (P = 0.31). IL-6 values for all stages were similar (P = 0.97). Only serum ferritin levels showed a statistically significant increase over stages (P = 0.04). IL-6 was significantly lower (P = 0.03) in patients who survived, whereas CRP levels were significantly lower in patients who had final visual acuity (VA) better than only perception of light (P = 0.03). Conclusion: Uncontrolled diabetes mellitus is a significant association of ROCM. Serum ferritin levels at presentation best correlate with extent of the disease. CRP levels are best to prognosticate cases that will have sufficient VA to carry on activities of daily living, whereas IL-6 levels are best associated with survival.


Assuntos
COVID-19 , Oftalmopatias , Mucormicose , Doenças Orbitárias , Humanos , Feminino , Masculino , Adulto , Pessoa de Meia-Idade , Mucormicose/diagnóstico , Mucormicose/epidemiologia , Centros de Atenção Terciária , Estudos Transversais , Atividades Cotidianas , Hemoglobinas Glicadas , Interleucina-6 , Estudos Retrospectivos , COVID-19/complicações , COVID-19/epidemiologia , Proteína C-Reativa , Ferritinas , Doenças Orbitárias/diagnóstico
8.
Arch Biochem Biophys ; 516(1): 45-51, 2011 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-21964243

RESUMO

Type II phosphatidylinositol (PtdIns) 4-kinases produce PtdIns 4-phosphate, an early key signaling molecule in phosphatidylinositol cycle, which is indispensable for T cell activation. Type II PtdIns 4-kinase alpha and beta have similar biochemical properties. To distinguish these isoforms Epigallocatechin gallate (EGCG) has been evaluated as a specific inhibitor. EGCG is the major active catechin in green tea having anti-inflammatory, antiatherogenic and cancer chemopreventive properties. The precise mechanism of actions and molecular targets of EGCG in early signaling cascades are not well understood. In the present study, we have shown that EGCG inhibits type II PtdIns 4-kinases (α and ß isoforms) and PtdIns 3-kinase activity in vitro. EGCG directly bind to both alpha and beta isoforms of type II PtdIns 4-kinases with a Kd of 2.62 µM and 1.02 µM, respectively. Type II PtdIns 4-kinase-EGCG complex have different binding pattern at its excited state. Both isoforms showed significant change in helicity upon binding with EGCG. EGCG modulates its effect by interacting with ATP binding pocket; the residues likely to be involved in EGCG binding were predicted by Autodock. Our findings suggest that EGCG inhibits two isoforms and could be a key to regulate T cell activation.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , 1-Fosfatidilinositol 4-Quinase/metabolismo , Anticarcinógenos/farmacologia , Catequina/análogos & derivados , Inibidores Enzimáticos/farmacologia , Fosfatidilinositóis/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , Sequência de Aminoácidos , Sítios de Ligação , Camellia sinensis/química , Catequina/farmacologia , Humanos , Células Jurkat , Modelos Moleculares , Dados de Sequência Molecular , Neoplasias/prevenção & controle , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência
9.
J Family Med Prim Care ; 10(6): 2195-2201, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34322412

RESUMO

CONTEXT: There is a global need for quality eye banking practices and sensitization of primary care physicians toward corneal donation. AIMS: To evaluate performance of a recently established eye bank (EB) and quality of corneas obtained, and identify areas of improvement during procurement and utilization of donor corneas. SETTINGS AND DESIGN: This retrospective observational study is based on records of corneas collected through hospital cornea retrieval programme (HRCP) in the EB of a tertiary care institution during the first 2 years of its establishment. METHODS AND MATERIAL: Data on demographic characteristics of donors, death-preservation interval, specular microscopy parameters of corneas, indications for utilization, and reasons for non-utilization of corneas were collected. STATISTICAL ANALYSIS USED: Means, standard deviation, range, frequencies, and proportions were analyzed. Spearman's correlation coefficient and Kruskal-Wallis test were applied taking P < 0.05 as significant. RESULTS: The EB retrieved 54 corneas from 27 donors with mean age 42.3 ± 24.2 years. All tissues were preserved in Cornisol®. Majority (50%) of transplantable tissues had an endothelial cell density (ECD) between 2,000 and 2,500 cells/mm2. ECD decreased significantly with increasing age (Spearman's ρ -0.747, P < 0.001; Kruskal-Wallis P < 0.001). Overall utilization rate of tissues was 87.04% (47/54), and utilizable corneas (50/54, 92.6%) were mainly used for optical purposes (34/50, 68%). CONCLUSIONS: Successful HCRP of the recently established EB has shown considerable promise in terms of quality and utilisation of corneas. There is need for active involvement of primary care physicians in contributing to increasing voluntary eye donation through awareness, advocacy, and social mobilization.

10.
11.
Mol Immunol ; 43(5): 454-63, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16337488

RESUMO

Phosphatidylinositol lipid signaling cascades are integral part of TCR-CD3 signaling. The mechanisms by which phosphatidylinositol kinases are coupled to TCR-CD3 complex remain elusive. Here we report an association of type II PtdIns 4-kinase with TCR-CD3 zeta chain upon cross-linking. Mapping studies have revealed that the C-terminal ITAM is critical for docking of the enzyme on the zeta chain. The association is shown to be tyrosyl phosphorylation dependent as mutation of Y-151 and Y-142 on the C-terminal ITAM disrupts interaction of the two proteins. Identification of the associated type II PtdIns 4-kinase revealed that the beta isoform of the enzyme interacts with the zeta chain in vivo.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , Motivos de Aminoácidos , Anticorpos Monoclonais/imunologia , Ativação Enzimática , Humanos , Capeamento Imunológico , Células Jurkat , Linfócitos/metabolismo , Proteínas de Membrana/química , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/química , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Receptores de Antígenos de Linfócitos T/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
12.
Blood Adv ; 1(18): 1423-1426, 2017 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-29296783

RESUMO

Protein S anticoagulant cofactor sensitivity and PAR1 cleavage activity were assayed for 9 recombinant APC mutants.Residues L38, K43, I73, F95, and W115 on one face of the APC light chain define an extended surface containing the protein S binding site.

13.
Mol Immunol ; 42(5): 561-8, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15607814

RESUMO

T cells show rapid reorganization of cytoskeleton in response to antigenic stimulation. The molecular mechanisms by which TCR-CD3 regulates actin cytoskeleton are not well defined. Here we show that a type II PtdIns 4-kinase associates with cytoskeletal fraction in splenic lymphocytes in response to Con A. Protein tyrosyl phosphorylation of type II PtdIns 4-kinase appears to be the mechanism for its association with cytoskeleton. Over-lay blots suggest that the enzyme binds to TCR-CD3 zeta chain in the cytoskeletal fraction. Anti-TCR-CD3 zeta antibodies competitively inhibit PtdIns 4-kinase association with TCR-CD3 zeta chain. Immunodepletion of TCR-CD3 zeta decreases PtdIns 4-kinase activity in the cytoskeletal fraction with a concomitant increase in PtdIns 4-kinase activity in anti-TCR-CD3 zeta immunoprecipitates. We propose that the association of type II PtdIns 4-kinase with TCR-CD3 zeta chain may bring the enzyme into close proximity of actin and a possible regulation of actin polymerization through localized production of PtdIns4P and PtdIns(4,5)P2.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Actinas/metabolismo , Animais , Concanavalina A/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Técnicas In Vitro , Proteínas de Membrana/química , Fosfatidilinositol 4,5-Difosfato , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilação , Ratos , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/efeitos dos fármacos , Tirosina/química
14.
Biochimie ; 95(8): 1560-6, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23619705

RESUMO

The early signaling events in T cell activation through CD3 receptor include a rapid change in intra cellular free calcium concentration and reorganization of actin cytoskeleton. Phosphatidylinositol 4-kinases (PtdIns 4-kinases) are implicated as key components in these early signaling events. The role of type II PtdIns 4-kinase ß in CD3 receptor signaling was investigated with the help of short hairpin RNA sequences. Cross-linking of CD3 receptors on Jurkat T Cells with monoclonal antibodies showed an early increase in type II PtdIns 4-kinase activity and co-localization of type II PtdIns 4-kinase ß with CD3 ζ. Transfection of Jurkat T Cells with shRNAs inhibited CD3 receptor mediated type II PtdIns 4-kinase activation with a concomitant reduction in intra cellular calcium release, suggesting a role for type II PtdIns 4-kinase ß in CD3 receptor signal transduction. Knock-down of type II PtdIns 4-kinase ß with shRNAs also correlated with a decrease in PtdIns 4-kinase activity in cytoskeleton fractions and reduced adhesion to matrigel surfaces. These results indicate that type II PtdIns 4-kinase ß is a key component in early T cell activation signaling cascades.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Ativação Linfocitária/fisiologia , Linfócitos T/enzimologia , Complexo CD3/metabolismo , Adesão Celular , Humanos , Células Jurkat , Microscopia Confocal , Linfócitos T/imunologia
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