RESUMO
Previous studies suggest that thymus produces a homogenous population of natural regulatory T (Treg) cells that express a transcriptional factor FOXP3 and control autoimmunity through a cell-contact-dependent mechanism. We found two subsets of FOXP3+ natural Treg cells defined by the expression of the costimulatory molecule ICOS in the human thymus and periphery. Whereas the ICOS+FOXP3+ Treg cells used interleukin-10 to suppress dendritic cell function and transforming growth factor (TGF)-beta to suppress T cell function, the ICOS-FOXP3+ Treg cells used TGF-beta only. The survival and proliferation of the two subsets of Treg cells were differentially regulated by signaling through ICOS or CD28, respectively. We suggest that the selection of natural Treg cells in thymus is coupled with Treg cell differentiation into two subsets imprinted with different cytokine expression potentials and use both cell-contact-dependent and independent mechanisms for immunosuppression in periphery.
Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos CD28/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis , Interleucina-10/imunologia , Interleucina-10/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Timo/citologia , Timo/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Over the past 30 years, the therapeutic outcome for pancreatic ductal adenocarcinoma (PDAC) has remained stagnant due to the lack of effective treatments. We performed a genome-wide analysis to identify novel therapeutic targets for PDAC. Our analysis showed that Homo sapiens chromosome 16 open reading frame 74 (C16orf74) was upregulated in most patients with PDAC and associated with poor prognosis. Previously, we demonstrated that C16orf74 interacts with the catalytic subunit alpha of protein phosphatase 3 and plays an important role in PDAC invasion. However, the pathophysiologic function of C16orf74 is still unclear. In this study, through the analysis of C16orf74 interaction, we demonstrate a new strategy to inhibit the growth and invasion of PDAC. C16orf74 exists in the homodimer form under the cell membrane and binds integrin αVß3 and is also involved in invasion by activating Rho family (Rac1) and MMP2. Considering that this dimeric form was found to be involved in the function of C16orf74, we designed an 11R-DB (dimer block) cell-permeable dominant-negative peptide that inhibits the dimer form of C16orf74. 11R-DB suppressed invasion and proliferation of PDAC cell lines by inhibiting phosphorylation of Akt and mTOR and also by inactivation of MMP2. 11R-DB also showed antitumor effects in an orthotopic xenograft model and peritoneal metastasis model. Thus, this study demonstrates that dimerized C16orf74, present in the cell membrane, is involved in pancreatic cancer invasion and proliferation. In addition, the C16orf74 dimer block cell-permeable peptide (11R-DB) has a potent therapeutic effect on PDAC in vitro and in vivo.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Animais , Biomarcadores Tumorais/metabolismo , Células COS , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Chlorocebus aethiops , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Integrina alfaVbeta3/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Peptídeos/farmacologia , Multimerização Proteica , Transdução de Sinais , Regulação para Cima , Neoplasias PancreáticasRESUMO
Although a number of p53 target genes have been identified, the mechanisms of p53-dependent activities that determine cellular survival or death are still not fully understood. Here we report isolation of a novel p53 target gene, designated p53-inducible cell-survival factor (p53CSV). p53CSV contains a p53-binding site within its second exon and the reduction of expression by small interfering RNA enhanced apoptosis, whereas overexpression protected cells from apoptosis caused by DNA damage. p53CSV is induced significantly when cells have a low level of genotoxic stresses, but not when DNA damage is severe. p53CSV can modulate apoptotic pathways through interaction with Hsp70 that probably inhibits activity of apoptosis protease activating factor-1. Our results imply that under specific conditions of stress, p53 regulates transcription of p53CSV and that p53CSV is one of the important players in the p53-mediated cell survival.
Assuntos
Proteína Supressora de Tumor p53/fisiologia , Sequência de Aminoácidos , Fator Apoptótico 1 Ativador de Proteases , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Dano ao DNA/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Dados de Sequência Molecular , Proteínas/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
The chemokine receptor CXCR3 has been shown to play a key role in the recruitment of T cells to sites of inflammation such as allografts. Here, we investigated which signals and conditions areresponsible for CXCR3 induction. CXCR3 was induced on T cells that were stimulated with anti-CD3 plus anti-CD28 monoclonal antibodies and then recultured without any external stimuli. CXCR3 expression was inhibited when TCR stimulation was persistent in the reculture. CXCR3 induction also depended on the stimulation with IFN-gamma because CXCR3 expression was not induced in IFN-gamma-deficient T cells. The induction of another Th1 chemokine receptor CCR5 absolutely required IL-12 stimulation and STAT4 involvement. In contrast, CXCR3 was induced on STAT4-deficient T cells independently of IL-12 stimulation as long as IFN-gamma was produced as a result of potent TCR stimulation. These results show that CXCR3 induction on TCR-triggered T cells requires the release of these T cells from persistent TCR signaling and the stimulation with IFN-gamma and also indicate the differential regulatory mechanisms underlying the induction of two Th1 chemokine receptors.
Assuntos
Interferon gama/fisiologia , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Quimiocinas/biossíntese , Linfócitos T/metabolismo , Animais , Proteínas de Ligação a DNA/fisiologia , Feminino , Interleucina-10/farmacologia , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR5/biossíntese , Receptores CXCR3 , Fator de Transcrição STAT4 , Transativadores/fisiologiaRESUMO
IL-12 and IL-18 synergistically enhance IFN-gamma mRNA transcription by activating STAT4 and AP-1, respectively. However, it is still unknown how STAT4/AP-1 elicit IFN-gamma promoter activation. Using an IL-12/IL-18-responsive T cell clone, we investigated the mechanisms underlying synergistic enhancement of IFN-gamma mRNA expression induced by these two cytokines. Synergy was observed in a reporter gene assay using an IFN-gamma promoter fragment that binds AP-1, but not STAT4. An increase in c-Jun, a component of AP-1, in the nuclear compartment was elicited by stimulation with either IL-12 or IL-18, but accumulation of serine-phosphorylated c-Jun was induced only by IL-18 capable of activating c-Jun N-terminal kinase. The binding of AP-1 to the relevant promoter sequence depended on the presence of STAT4. STAT4 bound with c-Jun, and a phosphorylated c-Jun-STAT4 complex most efficiently interacted with the AP-1-relevant promoter sequence. Enhanced cobinding of STAT4 and c-Jun to the AP-1 sequence was also observed when activated lymph node T cells were exposed to IL-12 plus IL-18. These results show that STAT4 up-regulates AP-1-mediated IFN-gamma promoter activation without directly binding to the promoter sequence, providing a mechanistic explanation for IL-12/IL-18-induced synergistic enhancement of IFN-gamma gene expression.
Assuntos
Proteínas de Ligação a DNA/biossíntese , Interferon gama/genética , Interleucina-12/fisiologia , Interleucina-18/fisiologia , Regiões Promotoras Genéticas/imunologia , Transdução de Sinais/imunologia , Transativadores/biossíntese , Fator de Transcrição AP-1/metabolismo , Regulação para Cima/imunologia , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular , Células Clonais , Meios de Cultivo Condicionados , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Sinergismo Farmacológico , Interferon gama/biossíntese , Interleucina-12/metabolismo , Ativação Linfocitária , Camundongos , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Proteínas Proto-Oncogênicas c-jun/biossíntese , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição STAT4 , Serina/metabolismo , Transdução de Sinais/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transativadores/metabolismo , Transativadores/fisiologia , Fator de Transcrição AP-1/biossíntese , Regulação para Cima/genéticaRESUMO
T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [(3)H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [(3)H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-kappaB binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-kappaB to CD28-responsive element and the NF-kappaB binding site. Decreased NF-kappaB activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-kappaB member, c-Rel, to the nuclear compartment due to the lack of IkappaBbeta inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-kappaB.
Assuntos
Antígenos CD28/fisiologia , Regulação da Expressão Gênica/imunologia , Proteínas I-kappa B , Interleucina-2/genética , Interleucina-2/metabolismo , Proteínas Nucleares , Regiões Promotoras Genéticas/imunologia , Animais , Sítios de Ligação/imunologia , Antígenos CD5/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/metabolismo , Feminino , Interleucina-2/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes , NF-kappa B/análise , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , NF-kappa B/metabolismo , Fatores de Transcrição NFATC , Transporte Proteico/imunologia , Proteínas Proto-Oncogênicas c-rel/biossíntese , Elementos de Resposta/imunologia , Linfócitos T/imunologia , Timidina/metabolismo , Fator de Transcrição AP-1/biossíntese , Fatores de Transcrição/biossíntese , TrítioRESUMO
IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending on co-stimulation with IL-12. IL-12 stimulation greatly enhanced the promoter-binding activity of c-Jun/AP-1, a critical transcription factor for IFN-gamma gene expression in wild-type T cells, but not in STAT4-deficient (STAT4(-/-)) T cells. Comparable amounts of c-Jun were induced by TCR stimulation in both wild-type and STAT4(-/-) T cells irrespective of IL-12 co-stimulation. However, c-Jun bound to STAT4 in IL-12-co-stimulated wild-type T cells. c-Jun forming a complex with STAT4 efficiently interacted with the AP-1-related sequence of the IFN-gamma promoter. Such an enhanced c-Jun binding did not occur in STAT4(-/-) T cells. These results show that STAT4 contributes to enhancing IFN-gamma expression by up-regulating the binding of TCR signal-induced AP-1 to the relevant promoter sequence.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Interferon gama/biossíntese , Interleucina-12/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transativadores/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Proteínas de Ligação a DNA/genética , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Genes jun/genética , Genes jun/fisiologia , Interferon gama/genética , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Fator de Transcrição STAT4 , Células Th1/imunologia , Transativadores/genética , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismoRESUMO
T cells that are intrathymically lineage committed are believed to maintain their CD4 or CD8 co-receptor expression. Here, we investigated whether intrathymic lineage commitment involves irreversible genetic modification or whether co-receptor expression can be reprogrammed depending on external stimuli. The CD4(+) T(h)1 clone 2D6 established from splenic T cells as an IL-12-dependent line survived in culture with IL-2, IL-7 or IL-15 alone. Surprisingly, CD8 expression occurred in 2D6 cells upon replacement of IL-12 with any one of the three cytokines that stimulate the common cytokine receptor gamma chain, yielding CD4(+)CD8(+) 2D6 cells. CD8 expression declined when IL-2 was replaced with IL-12 and CD8 induction was inhibited when IL-12 was included in IL-2 or IL-7 culture. Our observations show that even a lineage-committed mature T cell can be reprogrammed for co-receptor expression in response to particular external stimuli.