RESUMO
After a primary immune response, T cell memory occurs when a subset of Ag-specific T cells resists peripheral selection by acquiring resistance to TCR-induced death. Recent data have implicated Bcl-2 interacting mediator of death (Bim) as an essential mediator of the contraction phase of T cell immunity. In this article, we describe that stromal-derived factor-1α (SDF-1α) ligation of CXCR4 on activated T cells promotes two parallel processes that favor survival, phospho-inactivation of Foxo3A, as well as Bim extralong isoform (Bim(EL)) degradation, both in an Akt- and Erk-dependent manner. Activated primary CD4 T cells treated with SDF-1α therefore become resistant to the proapoptotic effects of TCR ligation or IL-2 deprivation and accumulate cells of a memory phenotype. Unlike SDF-1α, gp120 ligation of CXCR4 has the opposite effect because it causes p38-dependent Bim(EL) upregulation. However, when activated CD4 T cells are treated with both gp120 and SDF-1α, the SDF-1α-driven effects of Bim(EL) degradation and acquired resistance to TCR-induced death predominate. These results provide a novel causal link between SDF-1α-induced chemotaxis, degradation of Bim(EL), and the development of CD4 T cell memory.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL12/imunologia , Memória Imunológica/imunologia , Proteínas de Membrana/imunologia , Proteínas Proto-Oncogênicas/imunologia , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxia de Leucócito/imunologia , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Ativação Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Receptores CXCR4/imunologia , Receptores CXCR4/metabolismo , Transdução de Sinais/imunologia , Transfecção , Regulação para CimaRESUMO
Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Receptor fas/metabolismo , Carcinógenos/farmacologia , Ativadores de Enzimas/farmacologia , Proteína Ligante Fas/farmacologia , Células HL-60 , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células Jurkat , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
NF-kappaB is an ubiquitous transcription factor that is a key in the regulation of the immune response and inflammation. T-cell receptor (TCR) cross-linking leads to NF-kappaB activation, an IkappaB kinase (IKK)-dependent process. However, the upstream kinases that regulate IKK activity following TCR activation remain to be fully characterized. Herein, we demonstrate using genetic analysis, pharmacological inhibition, and RNA interference (RNAi) that the conventional protein kinase C (PKC) isoform PKCalpha, but not PKCbeta1, is required for the activation of the IKK complex following T-cell activation triggered by CD3/CD28 cross-linking. We find that in the presence of Ca(2+) influx, the catalytically active PKCalphaA25E induces IKK activity and NF-kappaB-dependent transcription; which is abrogated following the mutations of two aspartates at positions 246 and 248, which are required for Ca(2+) binding to PKCalpha and cell membrane recruitment. Kinetic studies reveal that an early phase (1 to 5 min) of IKK activation following TCR/CD28 cross-linking is PKCalpha dependent and that a later phase (5 to 25 min) of IKK activation is PKCtheta dependent. Activation of IKK- and NF-kappaB-dependent transcription by PKCalphaA25E is abrogated by the PKCtheta inhibitor rottlerin or the expression of the kinase-inactive form of PKCtheta. Taken together, our results suggest that PKCalpha acts upstream of PKCtheta to activate the IKK complex and NF-kappaB in T lymphocytes following TCR activation.
Assuntos
Isoenzimas/metabolismo , NF-kappa B/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/metabolismo , Acetofenonas/farmacologia , Ácido Aspártico/genética , Benzopiranos/farmacologia , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Cálcio/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica , Humanos , Quinase I-kappa B , Interleucina-2/genética , Isoenzimas/efeitos dos fármacos , Células Jurkat , Ativação Linfocitária , Mutação Puntual , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C-alfa , Proteína Quinase C-theta , Linfócitos T/enzimologia , Transcrição Gênica , Dedos de ZincoRESUMO
Available data associate lipids concentrations in men with body mass index, anabolic steroids, age, and certain cytokines. Data were less clear in women, especially across the full adult lifespan, and when segmented by premenopausal and postmenopausal status. SUBJECTS: 120 healthy women (60 premenopausal and 60 postmenopausal) in Olmsted County, MN, USA, a stable well studied clinical population. Dependent variables: measurements of 10 h fasting high-density lipoprotein cholesterol, total cholesterol, low-density lipoprotein cholesterol, and triglycerides. INDEPENDENT VARIABLES: testosterone, estrone, estradiol, 5-alpha-dihydrotestosterone, and sex-hormone binding globulin (by mass spectrometry); insulin, glucose, and albumin; abdominal visceral, subcutaneous, and total abdominal fat [abdominal visceral fat, subcutaneous fat, total abdominal fat by computerized tomography scan]; and a panel of cytokines (by enzyme-linked immunosorbent assay). Multivariate forward-selection linear-regression analysis was applied constrained to P < 0.01. Lifetime data: High-density lipoprotein cholesterol was correlated jointly with age (P < 0.0001, positively), abdominal visceral fat (P < 0.0001, negatively), and interleukin-6 (0.0063, negatively), together explaining 28.1 % of its variance (P = 2.3 × 10-8). Total cholesterol was associated positively with multivariate age only (P = 6.9 × 10-4, 9.3 % of variance). Triglycerides correlated weakly with sex-hormone binding globulin (P = 0.0115), and strongly with abdominal visceral fat (P < 0.0001), and interleukin-6 (P = 0.0016) all positively (P = 1.6 × 10-12, 38.9 % of variance). Non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol correlated positively with both total abdominal fat and interleukin-8 (P = 2.0 × 10-5, 16.9 % of variance; and P = 0.0031, 9.4 % of variance, respectively). Premenopausal vs. postmenopausal comparisons identified specific relationships that were stronger in premenopausal than postmenopausal individuals, and vice versa. Age was a stronger correlate of low-density lipoprotein cholesterol; interleukin-6 of triglycerides and high-density lipoprotein; and both sex-hormone binding globulin and total abdominal fat of non high-density lipoprotein cholesterol in premenopausal than postmenopausal women. Conversely, sex-hormone binding globulin, abdominal visceral fat, interleukin-8, adiponectin were stronger correlates of triglycerides; abdominal visceral fat, and testosterone of high-density lipoprotein cholesterol; and age of both non high-density lipoprotein and low-density lipoprotein in postmenopausal than premenopausal women. Our data delineate correlations of total abdominal fat and interleukin-8 (both positively) with non high-density lipoprotein cholesterol and low-density lipoprotein cholesterol in healthy women across the full age range of 21-79 years along with even more specific associations in premenopausal and postmenopausal individuals. Whether some of these outcomes reflect causal relationships would require longitudinal and interventional or genetic studies.
Assuntos
Gordura Abdominal/metabolismo , Lipídeos/sangue , Pós-Menopausa/sangue , Pré-Menopausa/sangue , Gordura Abdominal/diagnóstico por imagem , Adulto , Idoso , Feminino , Hormônios Esteroides Gonadais/sangue , Humanos , Interleucina-6/sangue , Interleucina-8/sangue , Pessoa de Meia-Idade , Globulina de Ligação a Hormônio Sexual/metabolismo , Tomografia Computadorizada por Raios X , Adulto JovemRESUMO
CONTEXT: SHBG concentrations correlate inconsistently with metabolic parameters. HYPOTHESIS: SHBG assay platforms contribute to nonuniformities according to the literature. DESIGN: The design of the study was a noninterventional quantification of SHBG by two immuno- and two mass spectrometric assays and abdominal visceral fat by computed tomography scan. SETTING: The study was conducted at the Center for Translational Science Activities. PARTICIPANTS: Healthy men (n=120) aged 18-80 years with a body mass index of 20-43 kg/m2 participated I the study. OUTCOMES: Outcomes of the study included a correlation of log SHBG with age, metabolic surrogates [body mass index, albumin, glucose, insulin, abdominal (total and visceral) fat, homeostasis model assessment insulin resistance index], sex steroids (estrone, 17ß-estradiol, T, and dihydrotestosterone by mass spectrometry), and adipocytokines (IL-1ß, IL-6, IL-8, IL-10 and IL-12, TNF-α, and adiponectin). RESULTS: By univariate regression, age (P<10(-4)), dihydrotestosterone (P<10(-4)), T (P≤.00022), and adiponectin (P≤.0084) were positive correlates, and insulin and homeostasis model assessment insulin resistance index were negative correlates (P≤.0060) of SHBG in all four assays. Stepwise multivariate analysis unveiled that age and T together could explain 38.1%-52.5% of the statistical variance in SHBG in all assays (P<10(-11)). Multivariate regression without sex steroids unveiled that age (P<10(-5)) and insulin (P<10(-3)) are jointly associated with SHBG levels in the four assays with overall R2=0.215-0.293 and P<10(-6). In one immunological SHBG assay each, abdominal visceral fat and adiponectin were weak multivariates also. CONCLUSION: Immunological and mass spectrometric SHBG assays yield both consistent and inconsistent correlations with key metabolic variables in healthy men, thereby potentially explaining earlier inconsistencies in the literature.
Assuntos
Doenças Metabólicas/sangue , Globulina de Ligação a Hormônio Sexual/análise , Globulina de Ligação a Hormônio Sexual/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Saúde , Humanos , Imunoensaio/métodos , Masculino , Espectrometria de Massas/métodos , Doenças Metabólicas/epidemiologia , Doenças Metabólicas/etiologia , Pessoa de Meia-Idade , Fatores Sexuais , Adulto JovemRESUMO
OBJECTIVE: Sex-hormone binding globulin (SHBG) concentrations across the adult female lifespan are not well defined. To address this knowledge gap, SHBG was quantified by both immunological and criterion methods, viz, mass spectrometry (MS). SETTING: Center for Translational Science Activities (CTSA). PARTICIPANTS: Healthy nonpregnant women (N=120) ages 21 to 79 years. OUTCOMES: SHBG, testosterone (T), estradiol (E2) and estrone (E1) each determined by MS. Uni- and multivariate regression of SHBG concentrations on age, body mass index (BMI), total and visceral abdominal fat (TAF, AVF), albumin, glucose, insulin, sex steroids, selected cytokines, blood pressure, and lipids. RESULTS: By univariate regression, MS-estimated SHBG correlated negatively with BMI, TAF, AVF, insulin, free T and bioavailable T (bio T) (each P≤10(-4)), but not with blood pressure or lipids. By stepwise multivariate regression analysis, free and total T (both positive) and bio T (negative) were correlated with SHBG in all 4 assays (each P<10(-15), R(2)≥0.481). In addition, TAF and BMI were negatively associated with SHBG (P≤0.0066) in 2 SHBG assays, and estrone and IL-8 with SHBG weakly (P≤0.035) in one SHBG assay each. When nonsignificant cytokines were excluded, SHBG was jointly associated with AVF, total T and HDL (P<10(-9), R(2)=0.358). CONCLUSION: According to MS, three metabolic factors, T, AVF and HDL, together explain more than one-third of the interindividual variation in SHBG levels. We speculate that these measures reflect insulin action.
Assuntos
Gordura Intra-Abdominal/metabolismo , Espectrometria de Massas , Globulina de Ligação a Hormônio Sexual/metabolismo , Testosterona/sangue , Adulto , Idoso , Glicemia/metabolismo , Índice de Massa Corporal , HDL-Colesterol/sangue , Estradiol/sangue , Estrona/sangue , Feminino , Humanos , Insulina/sangue , Interleucinas/sangue , Menopausa , Pessoa de Meia-Idade , Albumina Sérica/metabolismo , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
CONTEXT: Prostate-specific antigen (PSA) is a 34-kDa glycoprotein with chymotrypsin-like enzyme activity that circulates both in free forms and complexed to various enzyme inhibitors including antichymotrypsin and α2-macroglobulin. Prostate-specific antigen bound to α2-macroglobulin is not detected by commercial PSA immunoassays. OBJECTIVE: To develop a mass spectrometry assay that detects the same forms of PSA as the immunoassays, which could serve as a reference for harmonizing PSA immunoassays. DESIGN: Prostate-specific antigen was immune extracted from serum, trypsin was digested, and the LSEPAELTDAVK peptide was quantitated on an API 5000 spectrometer. Calibrators were made by adding 10% free and 90% antichymotrypsin-bound PSA to female sera. The assay was standardized to the World Health Organization 96/670 reference standard. Validation of clinical utility and comparisons with 2 immunoassays (Roche cobas and Beckman Access) were performed using frozen sera aliquots from 100 men undergoing prostate biopsy (50 negative, 50 with cancer) and 5 serial samples collected over time from 5 men with advanced prostate cancer. RESULTS: The antibody extraction efficiency was greater than 99%. The assay has an analytic range from 1.2 to 76 ng/mL, with precision ranging from 8.6% at 1.5 ng/mL to 5.4% at 27 ng/mL. The mass spectrometry assay correlated well with 2 immunoassays. All 3 assays showed statistically equivalent separation of prostate cancer from benign disease using receiver operating characteristic curve analysis. CONCLUSIONS: This mass spectrometry assay can reliably measure PSA concentrations in human serum and could serve as a reference standard for harmonizing PSA immunoassays.
Assuntos
Fragmentos de Peptídeos/análise , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/sangue , Métodos Analíticos de Preparação de Amostras , Anticorpos Monoclonais/química , Calibragem , Humanos , Indicadores e Reagentes/química , Masculino , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Antígeno Prostático Específico/química , Antígeno Prostático Específico/isolamento & purificação , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/diagnóstico , Proteólise , Curva ROC , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem , Tripsina/metabolismoRESUMO
OBJECTIVES: Harmonization of prostate-specific antigen (PSA) immunoassays is important for good patient care. The specificity of the antibodies used to detect circulating PSA could cause differences in the PSA measurements. METHODS: We used mass spectrometry (MS) to quantitate the concentration of five peptides cleaved from trypsin digestion of PSA and compared these measurements with six automated immunoassays. Linear regression and a mixed-effects model were used to analyze the results. RESULTS: PSA measurements from the immunoassays and the five MS peptide assays were highly correlated (R(2) > 0.99), but the recovery of the World Health Organization standard and the regression slopes differed across assays. The same relative patterns of immunoassay differences were seen in comparing their results with each of the five MS peptide measurements from different parts of the circulating PSA molecules. CONCLUSIONS: Mass spectrometry quantitation of peptides derived from trypsin digestion of immune-extracted PSA could be used to harmonize PSA immunoassays.
Assuntos
Fragmentos de Peptídeos/sangue , Antígeno Prostático Específico/química , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Epitopos , Feminino , Humanos , Imunoensaio/normas , Masculino , Antígeno Prostático Específico/imunologia , Antígeno Prostático Específico/metabolismo , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
OBJECTIVE: HIV gp120 is a pleiotropic protein present in the plasma and tissues of HIV-infected patients, which affects a variety of homeostatic functions. In this report, we examine the mechanism of how gp120 blocks CD4 T cells from migrating towards SDF-1α. METHODS: In vitro treatment of primary CD4 T cells with CXCR4 tropic gp120, SDF, and measurement of chemotaxis and cell signaling. RESULTS: gp120 signaling through CD4 receptor and Lck are required for its ability to inhibit chemotaxis induced by SDF, as demonstrated by CD4 receptor decoys, Lck inhibitors, as well as cells deficient in Lck, in which Lck expression is restored. Blocking Lck abrogates the ability of CXCR4 tropic gp120 to antagonize SDF-1α-induced chemotaxis. This inhibition is associated with cofilin phosphorylation, thereby providing a potential mechanism. CONCLUSION: We conclude that the ability of gp120 to inhibit SDF-1α-induced chemotaxis is mediated by the CD4 receptor and Lck signaling, potentially by promoting cofilin phosphorylation.
RESUMO
HIV-infected patients exhibit quantitative and qualitative defects in CD4 T cells, including having increased numbers of CD4+CD45R0+/CD45RA+ T cells, although it remains unclear how these cells arise. Here we demonstrate that gp120 treatment of activated but not resting primary human CD4 T cells decreases number of cells with single positive CD45R0+/CD45RA- effector memory phenotype while proportionally increasing the subset of cells with double positive CD45R0+/CD45RA+ mixed phenotype. We found that double positive CD45R0+/CD45RA+CD4 T cells preferentially undergo apoptosis while single positive CD45R0+/CD45RA- and CD45R0-/CD45RA+ do not. Blocking gp120-CD4 interaction with sCD4 or inhibition Lck activity reverses gp120 induced increase in double positive CD45R0+/CD45RA+CD4 T cells and subsequently diminishes the apoptosis of double positive CD45R0+/CD45RA+ cells. Altogether these data indicate that gp120 ligation of the CD4 receptor increases the number of double positive CD45R0+/CD45RA+ CD4 T cells which subsequently undergo apoptosis in a CD4 dependent manner.
RESUMO
BACKGROUND: HIV envelope glycoprotein gp120 causes cellular activation resulting in anergy, apoptosis, proinflammatory cytokine production, and through an unknown mechanism, enhanced HIV replication. METHODOLOGY/PRINCIPAL FINDINGS: We describe that the signals which promote apoptosis are also responsible for the enhanced HIV replication. Specifically, we demonstrate that the caspase 8 cleavage fragment Caspase8p43, activates p50/p65 Nuclear Factor kappaB (NF-kappaB), in a manner which is inhibited by dominant negative IkappaBalpha. This caspase 8 dependent NF-kappaB activation occurs following stimulation with gp120, TNF, or CD3/CD28 crosslinking, but these treatments do not activate NF-kappaB in cells deficient in caspase 8. The Casp8p43 cleavage fragment also transactivates the HIV LTR through NF-kappaB, and the absence of caspase 8 following HIV infection greatly inhibits HIV replication. CONCLUSION/SIGNIFICANCE: Gp120 induced caspase 8 dependent NF-kappaB activation is a novel pathway of HIV replication which increases understanding of the biology of T-cell death, as well as having implications for understanding treatment and prevention of HIV infection.
Assuntos
Caspase 8/metabolismo , Proteína gp120 do Envelope de HIV/fisiologia , HIV-1/fisiologia , NF-kappa B/fisiologia , Replicação Viral , Apoptose , Sequência de Bases , Linhagem Celular , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Repetição Terminal Longa de HIV , Humanos , HidróliseRESUMO
Acute HIV-1 infection of CD4 T cells often results in apoptotic death of infected cells, yet it is unclear what evolutionary advantage this offers to HIV-1. Given the independent observations that acute T cell HIV-1 infection results in (1) NF-kappaB activation, (2) caspase 8 dependent apoptosis, and that (3) caspase 8 directly activates NF-kappaB, we questioned whether these three events might be interrelated. We first show that HIV-1 infected T cell apoptosis, NF-kappaB activation, and caspase 8 cleavage by HIV-1 protease are coincident. Next we show that HIV-1 protease not only cleaves procaspase 8, producing Casp8p41, but also independently stimulates NF-kappaB activity. Finally, we demonstrate that the HIV protease cleavage of caspase 8 is necessary for optimal NF-kappaB activation and that the HIV-1 protease specific cleavage fragment Casp8p41 is sufficient to stimulate HIV-1 replication through NF-kappaB dependent HIV-LTR activation both in vitro as well as in cells from HIV infected donors. Consequently, the molecular events which promote death of HIV-1 infected T cells function dually to promote HIV-1 replication, thereby favoring the propagation and survival of HIV-1.
Assuntos
Sobrevivência Celular , Protease de HIV/metabolismo , HIV-1/fisiologia , NF-kappa B/fisiologia , Replicação Viral , Síndrome de Alstrom , Apoptose , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/virologia , Caspase 8/genética , Ativação Enzimática , Genes Reporter , Repetição Terminal Longa de HIV/genética , HIV-1/enzimologia , HIV-1/genética , Humanos , Células Jurkat , Plasmídeos , Linfócitos T/virologia , TransfecçãoRESUMO
HIV-1 infection causes the depletion of host CD4 T cells through direct and indirect (bystander) mechanisms. Although HIV Env has been implicated in apoptosis of uninfected CD4 T cells via gp120 binding to either CD4 and/or the chemokine receptor 4 (CXCR4), conflicting data exist concerning the molecular mechanisms involved. Using primary human CD4 T cells, we demonstrate that gp120 binding to CD4 T cells activates proapoptotic p38, but does not activate antiapoptotic Akt. Because ligation of the CD4 receptor alone or the CXCR4 receptor alone causes p38 activation and apoptosis, we used the soluble inhibitors, soluble CD4 (sCD4) or AMD3100, to delineate the role of CD4 and CXCR4 receptors, respectively, in gp120-induced p38 activation and death. sCD4 alone augments gp120-induced death, suggesting that CXCR4 signaling is principally responsible. Supporting that model, AMD3100 reduces death caused by gp120 or by gp120/sCD4. Finally, prevention of gp120-CXCR4 interaction with 12G5 Abs blocks p38 activation and apoptosis, whereas inhibition of CD4-gp120 interaction with Leu-3a has no effect. Consequently, we conclude that gp120 interaction with CXCR4 is required for gp120 apoptotic effects in primary human T cells.
Assuntos
Apoptose , Antígenos CD4/fisiologia , Proteína gp120 do Envelope de HIV/fisiologia , Receptores CXCR4/fisiologia , Linfócitos T/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Benzilaminas , Células Cultivadas , Ciclamos , Compostos Heterocíclicos/farmacologia , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die. We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4(+) T cells, predominantly in the CD27(+) memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.
Assuntos
Apoptose , Linfócitos T CD4-Positivos/enzimologia , Caspase 8/metabolismo , Infecções por HIV/enzimologia , Protease de HIV/metabolismo , HIV-1/enzimologia , Apoptose/genética , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/virologia , Caspase 8/genética , Infecções por HIV/genética , Protease de HIV/genética , HIV-1/genética , Humanos , Memória Imunológica/genética , Células Jurkat , Membro 7 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Latently human immunodeficiency virus (HIV)-infected memory CD4(+) T cells represent the major obstacle to eradicating HIV from infected patients. Antigens, T-cell receptor (TCR) ligation, and phorbol esters can reactivate HIV from latency in a protein kinase C (PKC)-dependent manner; however, it is unknown which specific PKC isoforms are required for this effect. We demonstrate that constitutively active (CA) forms of both PKCtheta, PKCthetaA148E, and PKCalpha, PKCalphaA25E, induce HIV long terminal repeat (LTR)-dependent transcription in Jurkat and primary human CD4(+) T cells and that both PKCthetaA148E and PKCalphaA25E cause HIV reactivation in J1.1 T cells. Suppression of both PKCalpha and PKCtheta with short hairpinned (sh) RNA inhibited CD3/CD28-induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells. Both prostratin and phorbol myristate 13-acetate induced HIV LTR-dependent transcription and HIV reactivation in J1.1 T cells that was blocked by shRNA against either PKCalpha or PKCtheta. Since suppression of PKCalpha and PKCtheta together has no greater inhibitory effect on HIV reactivation than inhibition of PKCalpha alone, our data confirm that PKCalpha and PKCtheta act in sequence. The requirement for PKCalpha and PKCtheta for prostratin-induced HIV reactivation and the ability of selective PKCalpha or PKCtheta agonists to induce HIV transcription indicate that these PKC isoforms are important targets for therapeutic drug design.