RESUMO
Endothelial nitric oxide (NO) synthase (eNOS) plays a critical role in the maintenance of blood vessel homeostasis. Recent findings suggest that cytoskeletal dynamics play an essential role in regulating eNOS expression and activation. Here, we sought to test whether modulation of cytoskeletal dynamics through pharmacological regulation of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation affects eNOS expression and endothelial function in vitro and in vivo We found that tubulin acetylation inducer (tubacin), a compound that appears to selectively inhibit HDAC6 activity, dramatically increased eNOS expression in several different endothelial cell lines, as determined by both immunoblotting and NO production assays. Mechanistically, we found that these effects were not mediated by tubacin's inhibitory effect on HDAC6 activity, but rather were due to its ability to stabilize eNOS mRNA transcripts. Consistent with these findings, tubacin also inhibited proinflammatory cytokine-induced degradation of eNOS transcripts and impairment of endothelium-dependent relaxation in the mouse aorta. Furthermore, we found that tubacin-induced up-regulation in eNOS expression in vivo is associated with improved endothelial function in diabetic db/db mice and with a marked attenuation of ischemic brain injury in a murine stroke model. Our findings indicate that tubacin exhibits potent eNOS-inducing effects and suggest that this compound might be useful for the prevention or management of endothelial dysfunction-associated cardiovascular diseases.
Assuntos
Anilidas/farmacologia , Endotélio Vascular/patologia , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/química , Acetilação , Animais , Aorta/metabolismo , Encéfalo/patologia , Isquemia Encefálica/patologia , Modelos Animais de Doenças , Feminino , Regulação Enzimológica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Acidente Vascular Cerebral/fisiopatologia , Tubulina (Proteína)/química , Regulação para CimaRESUMO
Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. We have previously shown that ASK1 is expressed in platelets and regulates agonist-induced platelet activation and thrombosis. However, the mechanism by which platelet agonists cause activation of ASK1 is unknown. Here, we show that in platelets agonist-induced activation of p38 is exclusively dependent on ASK1. Both thrombin and collagen were able to activate ASK1/p38. Activation of ASK1/p38 was strongly dependent on thromboxane A2 (TxA2) and ADP. Agonist-induced ASK1 activation is blocked by inhibition of phospholipase C (PLC) ß/γ activity or by chelating intracellular Ca2+. Furthermore, treatment of platelets with thapsigargin or Ca2+ ionophore robustly induced ASK1/p38 activation. In addition, calcium and integrin-binding protein 1 (CIB1), a Ca2+-dependent negative regulator of ASK1, associates with ASK1 in resting platelets and is dissociated upon platelet activation by thrombin. Dissociation of CIB1 corresponds with ASK1 binding to tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6) and the autophosphorylation of ASK1 Thr838 within the catalytic domain results in full activation of ASK1. Furthermore, genetic ablation of Cib1 in mice augments agonist-induced Ask1/p38 activation. Together our results suggest that in resting platelets ASK1 is bound to CIB1 at low Ca2+ concentrations. Agonist-induced platelet activation causes an increase in intracellular Ca2+ concentration that leads to the dissociation of CIB1 from ASK1, allowing for proper dimerization through ASK1 N-terminal coiled-coil (NCC) domains.
Assuntos
Plaquetas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , MAP Quinase Quinase Quinase 5/metabolismo , Ativação Plaquetária/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Plaquetas/citologia , Cálcio/metabolismo , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação ProteicaRESUMO
Ischemic stroke is a leading cause of morbidity and mortality worldwide and, despite reperfusion either via thrombolysis or thrombectomy, stroke patients often suffer from lifelong disabilities. These persistent neurological deficits may be improved by treating the ischemia/reperfusion (I/R) injury that occurs following ischemic stroke. There are currently no approved therapies to treat I/R injury, and thus it is imperative to find new targets to decrease the burden of ischemic stroke and related diseases. Platelets, cell fragments from megakaryocytes, are primarily known for their role in hemostasis. More recently, investigators have studied the nonhemostatic role of platelets in inflammatory pathologies, such as I/R injury after ischemic stroke. In this review, we seek to provide an overview of how I/R can lead to platelet activation and how activated platelets, in turn, can exacerbate I/R injury after stroke. We will also discuss potential mechanisms by which platelets may ameliorate I/R injury.
Assuntos
Traumatismo por Reperfusão , Acidente Vascular Cerebral , Plaquetas , Humanos , Isquemia , Ativação PlaquetáriaRESUMO
BACKGROUND: Immune complexes (ICs) bind to and activate platelets via FcγRIIA, causing patients to experience thrombocytopenia, as well as an increased risk of forming occlusive thrombi. Although platelets have been shown to mediate IC-induced pathologies, the mechanisms involved have yet to be fully elucidated. We identified that apoptosis signal-regulating kinase 1 (ASK1) is present in both human and mouse platelets and potentiates many platelet functions. OBJECTIVES: Here we set out to study ASK1's role in regulating IC-mediated platelet functions in vitro and IC-induced pathologies using an in vivo mouse model. METHODS: Using human platelets treated with an ASK1-specific inhibitor and platelets from FCGR2A/Ask1-/- transgenic mice, we examined various platelet functions induced by model ICs in vitro and in vivo. RESULTS: We found that ASK1 was activated in human platelets following cross-linking of FcγRIIA using either anti-hCD9 or IV.3 + goat-anti-mouse. Although genetic deletion or inhibition of ASK1 significantly attenuated anti-CD9-induced platelet aggregation, activation of the canonical FcγRIIA signaling targets Syk and PLCγ2 was unaffected. We further found that anti-mCD9-induced cPla2 phosphorylation and TxA2 generation is delayed in Ask1 null transgenic mouse platelets leading to diminished δ-granule secretion. In vivo, absence of Ask1 protected FCGR2A transgenic mice from thrombocytopenia, thrombosis, and systemic shock following injection of anti-mCD9. In whole blood microfluidics, platelet adhesion and thrombus formation on fibrinogen was enhanced by Ask1. CONCLUSIONS: These findings suggest that ASK1 inhibition may be a potential target for the treatment of IC-induced shock and other immune-mediated thrombotic disorders.
Assuntos
Trombocitopenia , Trombose , Animais , Apoptose , Plaquetas , Humanos , MAP Quinase Quinase Quinase 5/genética , Camundongos , Ativação Plaquetária , Agregação Plaquetária , Trombocitopenia/genética , Trombose/genéticaRESUMO
This cohort study characterizes US trends in traumatic brain injuryrelated mortality by age, sex, and race and ethnicity.
Assuntos
Lesões Encefálicas Traumáticas , Humanos , Estados Unidos/epidemiologia , EtnicidadeRESUMO
Convergent evolution is often due to selective pressures generating a similar phenotype. We observe relatively recent duplications in a spectrum of Saccharomycetaceae yeast species resulting in multiple phosphatases that are regulated by different nutrient conditions - thiamine and phosphate starvation. This specialization is both transcriptional and at the level of phosphatase substrate specificity. In Candida glabrata, loss of the ancestral phosphatase family was compensated by the co-option of a different histidine phosphatase family with three paralogs. Using RNA-seq and functional assays, we identify one of these paralogs, CgPMU3, as a thiamine phosphatase. We further determine that the 81% identical paralog CgPMU2 does not encode thiamine phosphatase activity; however, both are capable of cleaving the phosphatase substrate, 1-napthyl-phosphate. We functionally demonstrate that members of this family evolved novel enzymatic functions for phosphate and thiamine starvation, and are regulated transcriptionally by either nutrient condition, and observe similar trends in other yeast species. This independent, parallel evolution involving two different families of histidine phosphatases suggests that there were likely similar selective pressures on multiple yeast species to recycle thiamine and phosphate. In this work, we focused on duplication and specialization, but there is also repeated loss of phosphatases, indicating that the expansion and contraction of the phosphatase family is dynamic in many Ascomycetes. The dynamic evolution of the phosphatase gene families is perhaps just one example of how gene duplication, co-option, and transcriptional and functional specialization together allow species to adapt to their environment with existing genetic resources.
Assuntos
Família Multigênica , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Tiamina/metabolismo , Leveduras/fisiologia , Candida glabrata/fisiologia , Meio Ambiente , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Duplicação Gênica , Regulação Fúngica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Hidrólise , Filogenia , Saccharomycetales/fisiologia , Especificidade por Substrato , Leveduras/classificaçãoRESUMO
The phosphorylated form of thiamine (Vitamin B1), thiamine pyrophosphate (TPP) is essential for the metabolism of amino acids and carbohydrates in all organisms. Plants and microorganisms, such as yeast, synthesize thiamine de novo whereas animals do not. The thiamine signal transduction (THI) pathway in Saccharomyces cerevisiae is well characterized. The ~10 genes required for thiamine biosynthesis and uptake are transcriptionally upregulated during thiamine starvation by THI2, THI3, and PDC2. Candida glabrata, a human commensal and opportunistic pathogen, is closely related to S. cerevisiae but is missing half of the biosynthetic pathway, which limits its ability to make thiamine. We investigated the changes to the THI pathway in C. glabrata, confirming orthologous functions. We found that C. glabrata is unable to synthesize the pyrimidine subunit of thiamine as well as the thiamine precursor vitamin B6. In addition, THI2 (the gene encoding a transcription factor) is not present in C. glabrata, indicating a difference in the transcriptional regulation of the pathway. Although the pathway is upregulated by thiamine starvation in both species, C. glabrata appears to upregulate genes involved in thiamine uptake to a greater extent than S. cerevisiae. However, the altered regulation of the THI pathway does not alter the concentration of thiamine and its vitamers in the two species as measured by HPLC. Finally, we demonstrate potential consequences to having a partial decay of the THI biosynthetic and regulatory pathway. When the two species are co-cultured, the presence of thiamine allows C. glabrata to rapidly outcompete S. cerevisiae, while absence of thiamine allows S. cerevisiae to outcompete C. glabrata. This simplification of the THI pathway in C. glabrata suggests its environment provides thiamine and/or its precursors to cells, whereas S. cerevisiae is not as reliant on environmental sources of thiamine.