RESUMO
The proteasome inhibiter bortezomib has been successfully used to treat patients with relapsed multiple myeloma; however, many of these patients become thrombocytopenic, and it is not clear how the proteasome influences platelet production. Here we determined that pharmacologic inhibition of proteasome activity blocks proplatelet formation in human and mouse megakaryocytes. We also found that megakaryocytes isolated from mice deficient for PSMC1, an essential subunit of the 26S proteasome, fail to produce proplatelets. Consistent with decreased proplatelet formation, mice lacking PSMC1 in platelets (Psmc1(fl/fl) Pf4-Cre mice) exhibited severe thrombocytopenia and died shortly after birth. The failure to produce proplatelets in proteasome-inhibited megakaryocytes was due to upregulation and hyperactivation of the small GTPase, RhoA, rather than NF-κB, as has been previously suggested. Inhibition of RhoA or its downstream target, Rho-associated protein kinase (ROCK), restored megakaryocyte proplatelet formation in the setting of proteasome inhibition in vitro. Similarly, fasudil, a ROCK inhibitor used clinically to treat cerebral vasospasm, restored platelet counts in adult mice that were made thrombocytopenic by tamoxifen-induced suppression of proteasome activity in megakaryocytes and platelets (Psmc1(fl/fl) Pdgf-Cre-ER mice). These results indicate that proteasome function is critical for thrombopoiesis, and suggest inhibition of RhoA signaling as a potential strategy to treat thrombocytopenia in bortezomib-treated multiple myeloma patients.
Assuntos
Complexo de Endopeptidases do Proteassoma/fisiologia , Trombopoese , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Fator Plaquetário 4/fisiologia , Glicoproteína IIb da Membrana de Plaquetas/fisiologia , Inibidores de Proteassoma/farmacologia , Trombopoese/efeitos dos fármacos , Proteínas rho de Ligação ao GTP/fisiologia , Proteína rhoA de Ligação ao GTPRESUMO
Vascular malformations are a disruption of the normal vascular pattern in which it is expected that a capillary network of microscopic vessels lies interposed between high-pressure arteries that deliver blood and thin-walled veins that collect low-pressure blood for return to the heart. In the case of arteriovenous malformations, arteries or arterioles connect directly to the venous collection system, bypassing any capillary bed. Clinical consequences result from rupture and hemorrhage, from dramatically increased blood flow, or from the loss of capillary functions such as nutrient exchange and filtering function. These malformations can occur sporadically or as a component of inherited vascular malformation syndromes. In these and other hereditary vascular malformation syndromes, genetic studies have identified proteins and pathways involved in vascular morphogenesis and development. A common theme observed is that vascular malformations result from disruption in pathways involved in vascular stability. Here we review the vascular malformations and pathways involved in hereditary hemorrhagic telangiectasia, capillary malformation-arteriovenous malformation, cerebral cavernous malformations, and mucocutaneous venous malformations.
Assuntos
Malformações Arteriovenosas/genética , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Mutação/genética , Dermatopatias Vasculares/genética , Telangiectasia Hemorrágica Hereditária/genética , Capilares/anormalidades , Humanos , Mucosa/irrigação sanguínea , Pele/irrigação sanguíneaRESUMO
The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1ß (IL-1ß), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1ß on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1ß signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina/metabolismo , Fator 6 de Ribosilação do ADP , Adjuvantes Imunológicos/farmacologia , Animais , Artrite/patologia , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Purinas/farmacologia , Transdução de Sinais , Tiofenos/farmacologiaRESUMO
The innate immune system provides a first line of defense against invading pathogens by releasing multiple inflammatory cytokines, such as interleukin-1beta and tumor necrosis factor-alpha, which directly combat the infectious agent and recruit additional immune responses. This exuberant cytokine release paradoxically injures the host by triggering leakage from capillaries, tissue edema, organ failure, and shock. Current medical therapies target individual pathogens with antimicrobial agents or directly either blunt or boost the host's immune system. We explored a third approach: activating with the soluble ligand Slit an endothelium-specific, Robo4-dependent signaling pathway that strengthens the vascular barrier, diminishing deleterious aspects of the host's response to the pathogen-induced cytokine storm. This approach reduced vascular permeability in the lung and other organs and increased survival in animal models of bacterial endotoxin exposure, polymicrobial sepsis, and H5N1 influenza. Thus, enhancing the resilience of the host vascular system to the host's innate immune response may provide a therapeutic strategy for treating multiple infectious agents.
Assuntos
Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Infecções por Orthomyxoviridae/imunologia , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Sepse/imunologia , Transdução de Sinais , Animais , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Cateninas/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/fisiologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/complicações , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Sepse/complicações , Sepse/patologia , Transdução de Sinais/efeitos dos fármacos , delta CateninaRESUMO
PURPOSE OF REVIEW: The genetic basis for a variety of vascular malformation syndromes have been described, with an increasing functional understanding of the associated genes. RECENT FINDINGS: Genes responsible for familial vascular malformation syndromes have increasingly been shown to be involved in the control of vascular stability. SUMMARY: Genes involved in vascular stability pathways are good candidates for causing vascular malformation syndromes. Although these findings confirm the biologic importance of the involved pathways, further explanations are required to describe the focal nature of disease.
Assuntos
Vasos Sanguíneos/fisiologia , Doenças Vasculares/etiologia , Animais , Vasos Sanguíneos/citologia , Humanos , Doenças Vasculares/metabolismoRESUMO
PURPOSE: We demonstrate the establishment and characterization of a novel virus infection-induced seizure model in C57BL/6 mice. METHODS: C57BL/6 mice were infected with Theiler's murine encephalomyelitis virus (TMEV) or mock infected. Mice were followed for seizures, weight change, body temperature, motor function (righting reflex, rotorod) and neurological manifestations (inflammation [perivascular cuffing], pyknotic neurons, transforming growth factor [TGF]-beta expression). RESULTS: C57BL/6 mice are susceptible to seizures induced by TMEV infection. Approximately 50% of C57BL/6 mice develop transient afebrile seizures. Motor function and coordination are impaired in seized mice. Pyramidal neuron pyknosis and TGF-beta expression correlate with seizure activity in the hippocampus. DISCUSSION: The characterization of this model will enable the investigation of viral and immune contributions in the central nervous system to the development of seizure disorders in humans.
Assuntos
Infecções por Cardiovirus/complicações , Epilepsia Tônico-Clônica/etiologia , Convulsões/etiologia , Theilovirus , Animais , Encéfalo/patologia , Infecções por Cardiovirus/patologia , Epilepsia Tônico-Clônica/patologia , Feminino , Hipocampo/patologia , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Destreza Motora/fisiologia , Neurônios/patologia , Equilíbrio Postural/fisiologia , Reflexo/fisiologia , Convulsões/patologia , Fator de Crescimento Transformador beta/análiseRESUMO
The chemokine receptor CXCR4 and its cognate ligand CXCL12 recently have been proposed to regulate the directional trafficking and invasion of breast cancer cells to sites of metastases. However, effects of CXCR4 on the growth of primary breast cancer tumors and established metastases and survival have not been determined. We used stable RNAi to reduce expression of CXCR4 in murine 4T1 cells, a highly metastatic mammary cancer cell line that is a model for stage IV human breast cancer. Using noninvasive bioluminescence and magnetic resonance imaging, we showed that knockdown of CXCR4 significantly limited the growth of orthotopically transplanted breast cancer cells. Mice in which parental 4T1 cells were implanted had progressively enlarging tumors that spontaneously metastasized, and these animals all died from metastatic disease. Remarkably, RNAi of CXCR4 prevented primary tumor formation in some mice, and all mice transplanted with CXCR RNAi cells survived without developing macroscopic metastases. To analyze effects of CXCR4 on metastases to the lung, an organ commonly affected by metastatic breast cancer, we injected tumor cells intravenously and monitored cell growth with bioluminescence imaging. Inhibiting CXCR4 with RNAi, or the specific antagonist AMD3100, substantially delayed the growth of 4T1 cells in the lung, although neither RNAi nor AMD3100 prolonged overall survival in mice with experimental lung metastases. These data indicate that CXCR4 is required to initiate proliferation and/or promote survival of breast cancer cells in vivo and suggest that CXCR4 inhibitors will improve treatment of patients with primary and metastatic breast cancer.
Assuntos
Neoplasias Mamárias Experimentais/patologia , Receptores CXCR4/fisiologia , Animais , Benzilaminas , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Ciclamos , Modelos Animais de Doenças , Feminino , Compostos Heterocíclicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Imageamento por Ressonância Magnética , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Metástase Neoplásica , Transplante de Neoplasias , Interferência de RNA , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , TransfecçãoRESUMO
Signaling pathways regulating proliferation, differentiation, and apoptosis are commonly mediated through protein-protein interactions as well as reversible phosphorylation of proteins. To facilitate the study of regulated protein-protein interactions in cells and living animals, we optimized firefly luciferase protein fragment complementation by screening incremental truncation libraries of N- and C-terminal fragments of luciferase. Fused to the rapamycin-binding domain (FRB) of the kinase mammalian target of rapamycin and FK506-binding protein 12 (FKBP), respectively, the optimized FRB-N-terminal luciferase fragment (NLuc)/C-terminal luciferase fragment (CLuc)-FKBP luciferase complementation imaging (LCI) pair reconstituted luciferase activity in cells upon single-site binding of rapamycin in an FK506-competitive manner. LCI was used in three independent applications. In mice bearing implants of cells expressing the FRB-NLuc/CLuc-FKBP LCI pair, dose- and time-dependent luciferase activity allowed target-specific pharmacodynamic analysis of rapamycin-induced protein-protein interactions in vivo. In cells expressing a Cdc25C-NLuc/CLuc-14-3-3epsilon LCI pair, drug-mediated disruption of cell cycle regulated protein-protein interactions was demonstrated with the protein kinase inhibitor UCN-01 in a phosphoserine-dependent manner. When applied to IFN-gamma-dependent activation of Janus kinase/signal transducer and activator of transcription 1 (STAT1), LCI revealed, in the absence of ligand-induced phosphorylation, STAT1 proteins existing in live cells as preformed dimers. Thus, optimized LCI provides a platform for near real-time detection and characterization of regulated and small molecule-induced protein-protein interactions in intact cells and living animals and should enable a wide range of novel applications in drug discovery, chemical genetics, and proteomics research.