RESUMO
SAF-1, a zinc finger transcription factor, is activated by a number of inflammatory agents, including interleukin-1 (IL-1) and IL-6. It is involved in the cytokine-mediated transcriptional induction of serum amyloid A, an acute-phase plasma protein that is associated with the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here, we show that the mitogen-activated protein (MAP) kinase signaling pathway regulates cytokine-mediated induction of the DNA-binding activity and transactivation potential of SAF-1. Phosphorylation of endogenous SAF-1 in response to IL-1 and IL-6 was markedly inhibited by the addition of MAP kinase inhibitors. Consistent with this finding, we show that a consensus MAP kinase phosphorylation site, PPTP, within SAF-1 could be phosphorylated by MAP kinase in vitro. To analyze the contribution of MAP kinase in the activation of SAF-1, we prepared two independent mutant proteins in which the threonine residue of the PPTP motif was altered to either valine or alanine. These mutant proteins lost the ability to be phosphorylated by MAP kinase both in vivo and in vitro and exhibited a significantly reduced ability to promote expression of the SAF-1-regulated promoter. While the DNA-binding activity of wild-type SAF-1 protein was markedly increased upon phosphorylation with MAP kinase, no such increase could be detected with the mutant SAF-1 proteins. Further analysis with the GAL-4 reporter system showed that mutation of the MAP kinase phosphorylation site considerably lowers the transactivation potential of SAF-1. Together, these results show that activation of SAF-1 in response to IL-1 and -6 is mediated via MAP kinase-regulated phosphorylation.
Assuntos
Citocinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Inibidores Enzimáticos/farmacologia , Genes Reporter , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Ativação Transcricional/fisiologia , Dedos de ZincoRESUMO
The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Dipeptídeos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Animais , Neoplasias da Mama/patologia , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Humanos , Proteínas Inibidoras de Apoptose/metabolismo , Camundongos Nus , Proteínas Mitocondriais/metabolismo , Receptores do Fator de Necrose Tumoral , Transdução de Sinais/efeitos dos fármacos , Fator 2 Associado a Receptor de TNF/metabolismoRESUMO
Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/química , Dipeptídeos/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Indóis/farmacologia , Proteínas Mitocondriais/química , Mimetismo Molecular , Neoplasias Experimentais/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Proteínas Reguladoras de Apoptose , Dipeptídeos/uso terapêutico , Descoberta de Drogas , Indóis/uso terapêutico , Camundongos , Modelos MolecularesRESUMO
The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4), a platelet-specific chemokine released on platelet activation, has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin RNA is time and dose dependent. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. PF4 induces E-selectin expression by activation of transcriptional activity. Activation of nuclear factor-kappaB is critical for PF4-induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. Further, we have identified the low-density lipoprotein receptor-related protein as the cell surface receptor mediating this effect. These results demonstrate that PF4 is able to increase expression of E-selectin by endothelial cells and represents another potential mechanism by which platelets may participate in atherosclerotic lesion progression.
Assuntos
Selectina E/genética , Endotélio Vascular/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , NF-kappa B/metabolismo , Fator Plaquetário 4/farmacologia , Arteriosclerose/etiologia , Plaquetas/patologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Humanos , Cinética , RNA/biossíntese , RNA/efeitos dos fármacos , Transcrição Gênica , Veias Umbilicais/citologia , Regulação para Cima/genéticaRESUMO
Megakaryocyte (MK)-specific transgene expression has proved valuable in studying thrombotic and hemostatic processes. Constitutive expression of genes, however, could result in altered phenotypes due to compensatory mechanisms or lethality. To circumvent these limitations, we used the tetracycline/doxycycline (Tet)-off system to conditionally over-express genes in megakaryocytes and platelets in vivo. We generated 3 transactivator transgenic lines expressing the Tet transactivator element (tTA), under the control of the MK-specific platelet factor 4 promoter (PF4-tTA-VP16). Responder lines were simultaneously generated, each with a bidirectional minimal cytomegalovirus (CMV)-tTA responsive promoter driving prokaryotic beta-galactosidase gene, as a cellular reporter, and a gene of interest (in this case, the mitotic regulator Aurora-B). A transactivator founder line that strongly expressed PF4-driven tTA-viral protein 16 (VP16) was crossbred to a responder line. The homozygous double-transgenic mouse line exhibited doxycycline-dependent transgene overexpression in MKs and platelets. Using this line, platelets were conveniently indicated at sites of induced stress by beta-galactosidase staining. In addition, we confirmed our earlier report on effects of constitutive expression of Aurora-B, indicating a tight regulation at protein level and a modest effect on MK ploidy. Hence, we generated a new line, PF4-tTA-VP16, that is available for conditionally overexpressing genes of interest in the MK/platelet lineage in vivo.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Transgenes/genética , Animais , Aurora Quinase B , Aurora Quinases , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Doxiciclina/administração & dosagem , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Imuno-Histoquímica , Megacariócitos/citologia , Megacariócitos/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Modelos Animais , Fenótipo , Contagem de Plaquetas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Tetraciclina/administração & dosagem , Transgenes/efeitos dos fármacos , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Fragmentation of polyploid megakaryocytes into platelets has great relevance for blood homeostasis. Apoptotic cell death is a highly regulated genetic program, which has been observed in mature megakaryocytes fragmenting into platelets. The antiapoptotic protein BclxL has been reported as up-regulated during megakaryocytic differentiation in vitro, but absent during late megakaryopoiesis. Our study focused on examining BclxL levels in megakaryocytes in vivo and in assessing the effect of its overexpression in transgenic mice (via the platelet factor 4 [PF4] promoter) on megakaryocyte development and platelet fragmentation. Interestingly, in the wild-type and less in PF4-driven transgenic mice, BclxL was not detected in a fraction of the large mature megakaryocytes, suggesting a regulation on the protein level. BclxL overexpression was associated with a moderate increase in megakaryocyte number, with no significant change in ploidy level or platelet counts. When the mice were challenged by induction of immune thrombocytopenia, the rate of platelet recovery was significantly slower in the transgenic mice as compared with controls. Moreover, proplatelet formation in vitro by transgenic megakaryocytes was limited. Transgenic megakaryocytes displayed poorly developed platelet demarcation membranes and cell margin extensions. Our study indicates that regulated expression of BclxL in megakaryocytes is important for the development of cells with a high potential to fragment into platelets.
Assuntos
Plaquetas/citologia , Diferenciação Celular/genética , Regulação da Expressão Gênica , Megacariócitos/citologia , Megacariócitos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Plaquetas/fisiologia , Camundongos , Camundongos Transgênicos , Trombocitopenia/genética , Trombocitopenia/imunologia , Proteína bcl-XRESUMO
Megakaryocytes skip late anaphase and cytokinesis during endomitosis. We found normal expression and localization of a fundamental regulator of mitosis, Aurora-B/AIM-1, during prophase in polyploidizing mouse bone marrow megakaryocytes. At late anaphase, however, Aurora-B/AIM-1 is absent or mislocalized. Megakaryocytes treated with a proteasome inhibitor display Aurora-B/AIM-1 properly expressed and localized to the midzone, suggesting that protein degradation contributes to this atypical appearance. In contrast, survivin, an Aurora-B/AIM-1 coregulator of mitosis, is not detected at any stage of the endomitotic cell cycle, and in most megakaryocytes proteasome inhibition does not rescue this phenotype. To further explore the importance of reduced Aurora-B/AIM-1 for polyploidization, it was overexpressed in megakaryocytes of transgenic mice. The phenotype includes increased transgenic mRNA, but not protein, in polyploidy megakaryocytes, further suggesting that Aurora-B/AIM-1 is regulated at the protein level. Aurora-B/AIM-1 protein is, however, elevated in diploid transgenic megakaryocytes. Transgenic mice also exhibit enhanced numbers of megakaryocytes with increased proliferative potential, and some mice exhibit mild decreases in ploidy level. Hence, the molecular programming involved in endomitosis is characterized by the mislocalization or absence of at least 2 critical mitotic regulators, Aurora-B/AIM-1 and survivin. Future studies will examine the impact of survivin restoration on mouse megakaryocyte polyploidization.