RESUMO
There are no non-hormonal male contraceptives currently on the market despite decades of efforts toward the development of "male pills". Here, we report that triptonide, a natural compound purified from the Chinese herb Tripterygium Wilfordii Hook F displays reversible male contraceptive effects in both mice and monkeys. Single daily oral doses of triptonide induces deformed sperm with minimal or no forward motility (close to 100% penetrance) and consequently male infertility in 3-4 and 5-6 weeks in mice and cynomolgus monkeys, respectively. Male fertility is regained in ~4-6 weeks after cessation of triptonide intake in both species. Either short- or long-term triptonide treatment causes no discernable systematic toxic side effects based on histological examination of vital organs in mice and hematological and serum biochemical analyses in monkeys. Triptonide appears to target junction plakoglobin and disrupts its interactions with SPEM1 during spermiogenesis. Our data further prove that targeting late spermiogenesis represents an effective strategy for developing non-hormonal male contraceptives.
Assuntos
Anticoncepcionais Masculinos/farmacologia , Triterpenos/farmacologia , Administração Oral , Animais , Anticoncepcionais Masculinos/administração & dosagem , Quebras de DNA de Cadeia Dupla , Infertilidade Masculina/patologia , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Motilidade dos Espermatozoides/efeitos dos fármacos , Testículo/metabolismo , Triterpenos/administração & dosagem , gama Catenina/metabolismoRESUMO
Monocytes/macrophages are critical for both immunity and homoeostasis. They are the outposts of the immune system in detecting invading pathogens or foreign antigens for homoeostatic clearance and antigen processing for the initiation and effector stages of both innate and adaptive immunity. In addition, monocytes/macrophages often function as control switches for immune system balance between pro- and anti-inflammatory reactions. In the beginning of this article, I would like to briefly introduce the achievements of Metchnikoff and Ehrlich in immunology, including Metchnikoff's cell theory, since they have both greatly influenced the advancement of modern immunology. Additionally, I will honour the 100th anniversary of the 1908 Nobel Prize in Physiology or Medicine. Next, I would like to emphasize the concept of the MPS (mononuclear phagocyte system) by examining recent developments regarding the MPS. Thus the article consists of three parts. The first part describes the regulation of growth and differentiation in the MPS. The second part addresses how the key macrophage transcription factor gene PU.1 and the csf1r (colony-stimulating factor-1 receptor gene) play a critical role in haematopoietic myelopoiesis, or the generation of the cells of the MPS. The third part describes PMA-induced monocyte/macrophage differentiation and immune modulation of suppressor macrophages. Finally, this review discusses the latest findings and implications regarding the MPS and macrophages.
Assuntos
Alergia e Imunologia/história , Sistema Fagocitário Mononuclear/imunologia , Prêmio Nobel , Alemanha , História do Século XIX , História do Século XX , Macrófagos/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Federação Russa , Transativadores/genética , Transativadores/imunologiaRESUMO
BACKGROUND INFORMATION: MD-2 is associated with the extracellular domain of TLR4 (Toll-like receptor 4) and augments TLR4-dependent LPS (lipopolysaccharide) responses in vitro. Our previous investigation found that PMA-induced HL-60 cell differentiation to macrophages is associated largely with TLR2 and CD14 and, to a much lesser extent, with TLR4. RESULTS: We studied the MD-2 expression during differentiation of HL-60 cells induced by PMA. The results showed that PMA, but not VitD(3) (1alpha,25-dihydroxy-vitamin D(3)), strongly induces MD-2 gene expression by HL-60 cells in a time- and dose-dependent manner. Treatment with an MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] inhibitor (PD98059) and a JNK (c-Jun N-terminal kinase) inhibitor (SP600125) suppresses PMA-induced MD-2 gene expression, whereas impairment of p38 function by treatment with the inhibitor SB203580 has no effect on MD-2 mRNA. In order to reveal the possible molecular mechanism for such a regulation of MD-2 gene expression, we cloned and analysed the putative MD-2 gene promoter. Transient transfection of different deletion mutants demonstrated that the region -185/-171 (5'-TCCTTTACAGGAAGT-3') of the MD-2 gene promoter is closely related to gene transcription in response to PMA. Additionally, the transcription factor Elk-1 has been found to bind this specific motif. CONCLUSIONS: These results suggest that ERK and JNK pathways are involved in PMA-mediated MD-2 gene expression during HL-60 cell differentiation, and the activation of the MEK/possible ERK/Elk signal pathway is the mechanism responsible for PMA-induced MD-2 gene expression in differentiated HL-60 cells.
Assuntos
Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Antígeno 96 de Linfócito/genética , Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Antígeno 96 de Linfócito/metabolismo , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
BACKGROUND INFORMATION: The heat-shock response is a self-defence mechanism that protects cells and organisms from a wide range of harmful stresses. Recent studies revealed that it involved the regulation of cytokine expression. Interleukin-18 (IL-18) is an important cytokine in mediating immune response. RESULTS: We studied interferon-gamma (IFN-gamma)-induced IL-18 expression in heat-shock-treated murine peritoneal macrophages. Our results showed that the heat-shock response significantly inhibited the expression of IFN-gamma-induced pro-inflammatory cytokine IL-18. Interferon consensus sequence binding protein (ICSBP) is a transcription factor that binds to the promoter of IL-18 and regulates the transcription of IL-18. Further research on the down-regulation mechanism showed that the DNA-binding activity of ICSBP was greatly reduced by the heat shock response. CONCLUSIONS: These results suggest that the inhibitory effect of heat-shock response on IL-18 production in IFN-gamma-stimulated macrophages is related to the suppression of the binding activity of ICSBP.
Assuntos
Resposta ao Choque Térmico , Interleucina-18/metabolismo , Macrófagos Peritoneais/metabolismo , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Fatores Reguladores de Interferon , Interferon gama/farmacologia , Interleucina-18/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Interferon-gamma (IFN-gamma) is a macrophage-activating cytokine that serves critical functions in innate and adaptive immunity and is thought to be mediated by the Jak-Stat signaling pathway. The present study establishes for the first time that cyclic adenosine monophosphate, protein kinase A, and cAMP response element-binding protein (cAMP/PKA/CREB) are coregulators of the IFN-gamma signaling pathway. Experimental data indicate that exogenous IFN-gamma stimulated cAMP accumulation and PKA activation in time-dependent and dose-dependent manners in murine peritoneal macrophages. Moreover, IFN-gamma stimulated CREB phosphorylation and CREB DNA binding, which could be significantly attenuated by PKA inhibition with H89. It appears that a novel cAMP/PKA/CREB signaling pathway is activated by IFN-gamma in macrophages, suggesting that an alternate signaling pathway exists in macrophages in response to IFN-gamma.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Interferon gama/fisiologia , Macrófagos Peritoneais/metabolismo , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo II Dependente de AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Ativação Enzimática , Feminino , Interferon gama/farmacologia , Isoquinolinas/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Sulfonamidas/farmacologiaRESUMO
BACKGROUND: Erianin is a natural product derived from Dendrobium chrysotoxum, with promising antitumor activity. MATERIALS AND METHODS: To evaluate the metabolic effect of erianin, a cytosensor assay for acidification rate, MTT assay, measurement of lactate, glucose and ATP were performed in human umbilical vein endothelial cells (HUVECs) exposed to 1-100 nM erianin. JNK/SAPK activity was detected by Western blot. RESULTS: Twelve- or 24- hour incubation with erianin induced a dose-dependent metabolic inhibition, as indicated by reduced acidification rate and cell viability, with an endothelium-selectivity. Erianin caused decreases in lactate production, glucose consumption and intracellular ATP level. Pretreatment with the JNK/SAPK inhibitor SP600125 significantly abolished these inhibitory responses, and especially restored the erianin-induced decreases in ATP and the erianin-induced phosphorylation of JNK/SAPK with dose- and time- dependence. CONCLUSION: Erianin inhibited endothelial metabolism in a JNK/SAPK-dependent manner. This mechanism may be involved in the potential antitumnor and antiangiogenic actions of erianin.
Assuntos
Inibidores da Angiogênese/farmacologia , Bibenzilas/farmacologia , Endotélio Vascular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Antracenos/farmacologia , Técnicas Biossensoriais , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Formazans/metabolismo , Glucose/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Proteína Quinase 10 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fenol , Proteínas Tirosina Quinases/antagonistas & inibidores , Sais de Tetrazólio/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
To investigate the possible mechanism of gene transcription changes induced by magnetic field (MF), we examined the DNA binding behavior of the transcription factor cyclic-AMP responsive element binding protein (CREB) in HL60 cells after exposure to a 0.1mT 50-Hz extremely low frequency (ELF) sinusoidal MF by a gel shift assay. Magnetic field induced a time-dependent activation of CREB binding. The complex formation increased shortly after MF exposure for 10min, reaching a peak level after 1h, and then recovered to basal level at 4h after exposure. A novel MF-induced ATF2/ATF2 homodimer formation was observed after MF exposure for 30min, 1, and 2h. Furthermore, We found that the MF-induced increase of CREB DNA binding in HL60 cells was dependent on both extracellular and intracellular Ca(2+) but not PKA, PKC, ERK, or p38 MAPK by using various pathway inhibitors. These data indicate that MF exposure activates CREB DNA binding through calcium-related signal transduction pathways under our experimental conditions.
Assuntos
Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , DNA/metabolismo , Campos Eletromagnéticos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Núcleo Celular/metabolismo , Células HL-60 , Humanos , Ligação Proteica , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Heat shock response has been implicated in cytoprotective effects from cellular damage and in the regulation of cytokine expression. We report the effect of heat shock on LPS-induced expression of IL-18, an important cytokine that has diverse immune regulatory effects on T cells, B cells, NK cells, and nonimmune cells. The augmentation of LPS-induced IL-18 mRNA and protein was significantly suppressed in murine peritoneal macrophages after 43 degrees C heat shock treatment. In addition, the JNK MAPK inhibitor SP600125 inhibited IL-18 mRNA transcription in a dose-dependent manner. To examine the possibility that the inhibition of IL-18 may be mediated through the inactivation of JNK, the activity of JNK was measured by using Western blot and kinase assays. Our data show that heat shock response decreased LPS-induced phosphorylation of JNK and its downstream substrate c-Jun. AP-1, a transcriptional factor composed of c-Jun, could regulate the expression of IL-18. Also, its DNA-binding activity was reduced by the heat shock response. These findings suggest that treatment of heat stress results in inhibition of IL-18 production in macrophages mainly through the JNK/AP-1 signaling pathway.
Assuntos
Resposta ao Choque Térmico , Interleucina-18/biossíntese , Sistema de Sinalização das MAP Quinases , Macrófagos Peritoneais/imunologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/genética , Interleucina-18/genética , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Lipopolissacarídeos/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/enzimologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fosforilação , RNA Mensageiro/biossíntese , Fator de Transcrição AP-1/metabolismoRESUMO
The effects of a 50 Hz extremely low frequency (ELF) sinusoidal magnetic field (MF) on the expression of genes relating to cytokine receptors were studied in HL60 cells. Transcription levels of tumor necrosis factor receptor (TNFR) p55 and p75, interleukin-6 receptor-alpha (IL-6Ralpha) and transforming growth factor-beta receptor 1 (TGFbetaR1) were quantified in cells exposed to an intensity of 0.1 or 0.8 mT for periods ranging from 30 min to 72 h. Cells treated with 10 nM of phorbol 12-myristate 13-acetate (PMA) for 8 h served as a positive control. Gene expression values were assessed by the ribonuclease protection assay (RPA) and normalized to those of the noninducible gene GAPDH. The results showed that MF exposure at 0.1 and 0.8 mT for 72 h increased TNFR p75 and IL-6Ralpha mRNA expression in HL60 cells. No significant change in gene expression levels of TNFR p55 and TGFbetaR1 was observed under any of the exposure conditions. In addition, we report here for the first time that IL-6Ralpha mRNA expression can be suppressed by PMA in HL60 cells.
Assuntos
Expressão Gênica , Magnetismo/efeitos adversos , Receptores de Citocinas/genética , Receptores de Ativinas Tipo I/genética , Antígenos CD/genética , Expressão Gênica/efeitos dos fármacos , Células HL-60 , Humanos , Proteínas Serina-Treonina Quinases , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Interleucina-6/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Macrophages form a crucial bridge between the innate and adaptive immune response. One of their most important functions is to recognize infectious microorganisms. Toll-like receptors (TLRs) are key elements in pathogen recognition, and among them, TLR2 and TLR4 are most discussed. However, expression patterns of TLRs during myeloid cell differentiation to macrophage are unknown. In this study, we examined differentiation in the model human myeloid cell line, HL-60, treated with phorbol 12-myristate 13-acetate (PMA) or VitD(3). Expression of TLR2, TLR4, and CD14 were measured by reverse transcription-PCR, RNase protection assay, and fluorescence-activated cell sorter assays. After treatment by PMA (1, 10, and 100 nM) for 12, 24, and 48 h, expression of TLR2 and CD14 mRNA was increased in a time- and dose-dependent manner. However, VitD(3) only induced expression of CD14 but not TLR2 in HL-60 cells. TLR4 was expressed constitutively before differentiation and increased slightly after that. Thus, PMA-mediated differentiation of HL-60 cells to macrophages is associated largely with TLR2 expression and, to a much lesser extent, with TLR4. Furthermore, up-regulation of TLR2 and CD14 mRNA expression by PMA was abrogated by a protein kinase C inhibitor, Calphostine C, suggesting the up-regulation of TLR2 and CD14 mRNA is dependent on the activation of protein kinase C. Coexpression of CD14/TLR2 and/or CD14/TLR4 may be essential but not sufficient for the production of tumor necrosis factor-alpha in response to lipopolysaccharide in our system.
Assuntos
Calcitriol/farmacologia , Proteínas de Drosophila , Receptores de Lipopolissacarídeos/biossíntese , Glicoproteínas de Membrana/biossíntese , Receptores de Superfície Celular/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Células HL-60 , Humanos , Receptores de Lipopolissacarídeos/genética , Macrófagos/fisiologia , Glicoproteínas de Membrana/genética , Naftalenos/farmacologia , Fagocitose/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Receptores de Superfície Celular/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Ativação Transcricional , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Previous experimental investigation indicated that immuno-suppressor activities of suppressor macrophages on T B lymphocytes and NK cells could be prevented by treatment with LPS but the tumoricidal activities of those macrophages could be kept or even enhanced after the same treatment. During this complicated course LPS-mediated immuno-modulation was accompanied by activation of PKC and MAPK signal pathways. In order to explore the effect of another signal on MAPK pathway this model of immuno-modulated macrophage was utilized to study the regulatory effect on the activation of three family members of MAPK (ERK1/2 JNK and p38) by cAMP/PKA and PMA/PKC. The results showed that 1) LPS-mediated immuno-modulation was accompanied by dynamic changes of intracellular cAMP amount and PKA activity. 2) A specific PKC activator PMA induced strongly the activation of ERK1/2 JNK and p38 MAPK. 3) In contrast the activation of cAMP/PKA mediated a significant inhibiton of the phosphorylation of ERK1/2 JNK and p38 MAPK and ATF-2 but it enhanced the phosphorylation of CREB. These results suggest that a complicated "cross-talk" may exist among PKC and PKA and MAPK signaling pathways in the regulation of murine peritoneal suppressor macrophages by LPS.