RESUMO
The fundamental mechanisms that underlie platelet activation in atherothrombosis are still obscure. Oxidative stress is involved in central features of atherosclerosis. Platelet-derived microvesicles (PMVs) could be important mediators between oxidative stress and platelet activation. CD36 could be a receptor of PMVs, thus generating a PMV-CD36 complex. We aimed to investigate the detailed pathway by which oxidative damage contributes to platelet activation by the PMV-CD36 complex. We found that oxidized low-density lipoprotein stimulated the generation of PMVs. PMVs enhanced normal platelet activation, as assessed by the expression of integrin α(IIb)ß3, secretion of soluble P-selectin and platelet aggregation, but CD36-deficient platelets were not activated by PMVs. The function of the PMV-CD36 complex was mediated by the MKK4/JNK2 signaling axis. Meanwhile, PMVs increased the level of 8-iso-prostaglandin-F2α, a marker of oxidative stress, in a CD36- and phosphatidylserine-dependent manner. We concluded that PMVs are important mediators between oxidative stress and platelet activation. PMVs and CD36 may be effective targets for preventing platelet activation in cardiovascular diseases.
Assuntos
Plaquetas/metabolismo , Lipoproteínas LDL/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Plaquetas/citologia , Plaquetas/efeitos dos fármacos , Antígenos CD36/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Ativação Plaquetária/efeitos dos fármacosRESUMO
1. C/EBP homologueueueous protein (CHOP), an endoplasmic reticulum (ER) stress-inducible protein, has a critical role in regulation of the cell cycle and apoptosis by forming heterodimers with other C/EBP proteins. However, how CHOP function is regulated remains to be determined. The human homologue of Drosophila tribbles (TRIB3) is associated with CHOP and is upregulated by oxidized low-density lipoprotein (ox-LDL). The aim of the present study was to investigate the role of CHOP in ox-LDL-induced TRIB3 expression in macrophages. 2. Human monocyte-derived macrophages were treated with various concentrations of ox-LDL (0, 2.5, 5, 10, 25 and 50 microg/mL) or 2 microg/mL tunicamycin for 0, 4, 8, 16, 24 and 48 h or were transfected with CHOP or TRIB3 expression plasmid and TRIB3 targeting short interference RNA (siRNA). The expression of CHOP and activating transcription factor 4 (ATF4) mRNA in treated cells was detected by quantitative real-time polymerase chain reaction (PCR). 3. The expression of CHOP and ATF4 mRNA increased with increasing concentrations of ox-LDL and duration of time. The ox-LDL-induced expression of TRIB3 mRNA was upregulated later than the expression of CHOP and ATF4 mRNA. Overexpression of CHOP increased the mRNA expression of TRIB3, which was further increased in CHOP-overexpressing macrophages treated with ox-LDL. Overexpression of TRIB3 suppressed the expression of CHOP, whereas TRIB3 silencing increased CHOP expression following ox-LDL stimulation by a negative feedback mechanism. 4. In conclusion, the expression of ATF4 and CHOP is upregulated by ox-LDL in a dose- and time-dependent manner in naturally differentiated human macrophages. Oxidized LDL induces TRIB3 expression via an ATF4/CHOP-dependent ER stress pathway.
Assuntos
Fator 4 Ativador da Transcrição/fisiologia , Proteínas de Ciclo Celular/metabolismo , Lipoproteínas LDL/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/fisiologia , Regulação para Cima/efeitos dos fármacos , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Proteínas de Ciclo Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/farmacologia , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Tunicamicina/farmacologiaRESUMO
Myosin regulatory light chain (LC20) phosphorylation plays an important role in vascular smooth muscle contraction and cell migration. Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates LC20 (its only known substrate) exclusively at S19. Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in the regulation of LC20 phosphorylation via direct phosphorylation of LC20 at T18 and S19 and indirectly via phosphorylation of MYPT1 (the myosin targeting subunit of myosin light chain phosphatase, MLCP) and Par-4 (prostate-apoptosis response-4). Phosphorylation of MYPT1 at T696 and T853 inhibits MLCP activity whereas phosphorylation of Par-4 at T163 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition. To evaluate the roles of MLCK, ROCK and ZIPK in these phosphorylation events, we investigated the time courses of phosphorylation of LC20, MYPT1 and Par-4 in serum-stimulated human vascular smooth muscle cells (from coronary and umbilical arteries), and examined the effects of siRNA-mediated MLCK, ROCK and ZIPK knockdown and pharmacological inhibition on these phosphorylation events. Serum stimulation induced rapid phosphorylation of LC20 at T18 and S19, MYPT1 at T696 and T853, and Par-4 at T163, peaking within 30-120 s. MLCK knockdown or inhibition, or Ca2+ chelation with EGTA, had no effect on serum-induced LC20 phosphorylation. ROCK knockdown decreased the levels of phosphorylation of LC20 at T18 and S19, of MYPT1 at T696 and T853, and of Par-4 at T163, whereas ZIPK knockdown decreased LC20 diphosphorylation, but increased phosphorylation of MYPT1 at T696 and T853 and of Par-4 at T163. ROCK inhibition with GSK429286A reduced serum-induced phosphorylation of LC20 at T18 and S19, MYPT1 at T853 and Par-4 at T163, while ZIPK inhibition by HS38 reduced only LC20 diphosphorylation. We also demonstrated that serum stimulation induced phosphorylation (activation) of ZIPK, which was inhibited by ROCK and ZIPK down-regulation and inhibition. Finally, basal phosphorylation of LC20 in the absence of serum stimulation was unaffected by MLCK, ROCK or ZIPK knockdown or inhibition. We conclude that: (i) serum stimulation of cultured human arterial smooth muscle cells results in rapid phosphorylation of LC20, MYPT1, Par-4 and ZIPK, in contrast to the slower phosphorylation of kinases and other proteins involved in other signaling pathways (Akt, ERK1/2, p38 MAPK and HSP27), (ii) ROCK and ZIPK, but not MLCK, are involved in serum-induced phosphorylation of LC20, (iii) ROCK, but not ZIPK, directly phosphorylates MYPT1 at T853 and Par-4 at T163 in response to serum stimulation, (iv) ZIPK phosphorylation is enhanced by serum stimulation and involves phosphorylation by ROCK and autophosphorylation, and (v) basal phosphorylation of LC20 under serum-free conditions is not attributable to MLCK, ROCK or ZIPK.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Associadas com Morte Celular/metabolismo , Músculo Liso Vascular/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Miosinas/metabolismo , Quinases Associadas a rho/metabolismo , Proteínas Reguladoras de Apoptose/genética , Artérias/citologia , Artérias/metabolismo , Células Cultivadas , Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas Quinases Associadas com Morte Celular/genética , Humanos , Músculo Liso Vascular/citologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/genética , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosforilação , RNA Interferente Pequeno/genética , Soro/metabolismo , Transdução de Sinais , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/genéticaRESUMO
This study was designed to investigate the changes of urinary microvesicle-bound uromodulin and total urinary uromodulin levels in human urine and the correlations with the severity of diabetic kidney disease (DKD). 31 healthy subjects without diabetes and 100 patients with type 2 diabetes mellitus (T2DM) were included in this study. The patients with T2DM were divided into three groups based on the urinary albumin/creatinine ratio (UACR): normoalbuminuria group (DM, n = 46); microalbuminuria group (DN1, n = 32); and macroalbuminuria group (DN2, n = 22). We use a specific monoclonal antibody AD-1 to capture the urinary microvesicles. Urinary microvesicle-bound uromodulin and total urinary uromodulin levels were determined by enzyme-linked immunosorbent assay (ELISA). Our results showed that the levels of urinary microvesicle-bound uromodulin in DN1 and DN2 groups were significantly higher than those in control group and DM group (P < 0.01). Multiple stepwise linear regression analysis showed that UACR was independent determinant for urinary microvesicle-bound uromodulin (P < 0.05) but not for total urinary uromodulin. These findings suggest that the levels of urinary microvesicle-bound uromodulin are associated with the severity of DKD. The uromodulin in urinary microvesicles may be a specific marker of DKD and potentially may be used to predict the onset and/or monitor the progression of DKD.
Assuntos
Albuminúria/diagnóstico , Diabetes Mellitus Tipo 2/urina , Nefropatias Diabéticas/diagnóstico , Uromodulina/urina , Adulto , Idoso , Albuminúria/urina , Biomarcadores/urina , Nefropatias Diabéticas/urina , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Smooth muscle contraction is activated by phosphorylation at Ser-19 of LC20 (the 20 kDa light chains of myosin II) by Ca2+/calmodulin-dependent MLCK (myosin light-chain kinase). Diphosphorylation of LC20 at Ser-19 and Thr-18 is observed in smooth muscle tissues and cultured cells in response to various contractile stimuli, and in pathological circumstances associated with hypercontractility. MLCP (myosin light-chain phosphatase) inhibition can lead to LC20 diphosphorylation and Ca2+-independent contraction, which is not attributable to MLCK. Two kinases have emerged as candidates for Ca2+-independent LC20 diphosphorylation: ILK (integrin-linked kinase) and ZIPK (zipper-interacting protein kinase). Triton X-100-skinned rat caudal arterial smooth muscle was used to investigate the relative importance of ILK and ZIPK in Ca2+-independent, microcystin (phosphatase inhibitor)-induced LC20 diphosphorylation and contraction. Western blotting and in-gel kinase assays revealed that both kinases were retained in this preparation. Ca2+-independent contraction of calmodulin-depleted tissue in response to microcystin was resistant to MLCK inhibitors [AV25 (a 25-amino-acid peptide derived from the autoinhibitory domain of MLCK), ML-7, ML-9 and wortmannin], protein kinase C inhibitor (GF109203X) and Rho-associated kinase inhibitors (Y-27632 and H-1152), but blocked by the non-selective kinase inhibitor staurosporine. ZIPK was inhibited by AV25 (IC50 0.63+/-0.05 microM), whereas ILK was insensitive to AV25 (at concentrations as high as 100 microM). AV25 had no effect on Ca2+-independent, microcystin-induced LC20 mono- or di-phosphorylation, with a modest effect on force. We conclude that direct inhibition of MLCP in the absence of Ca2+ unmasks ILK activity, which phosphorylates LC20 at Ser-19 and Thr-18 to induce contraction. ILK is probably the kinase responsible for myosin diphosphorylation in vascular smooth muscle cells and tissues.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiologia , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cálcio/farmacologia , MAP Quinase Quinase Quinases , Microcistinas , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Peptídeos Cíclicos/farmacologia , Fosforilação/efeitos dos fármacos , RatosRESUMO
The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.
Assuntos
Vasos Coronários/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Oxazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Vasos Coronários/citologia , Humanos , Simulação de Acoplamento Molecular , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Oxazóis/química , Inibidores de Proteínas Quinases/química , Quinases Associadas a rho/química , Quinases Associadas a rho/metabolismoRESUMO
Rho-associated kinase (ROCK) and zipper-interacting protein kinase (ZIPK) have been implicated in diverse physiological functions. ROCK1 phosphorylates and activates ZIPK suggesting that at least some of these physiological functions may require both enzymes. To test the hypothesis that sequential activation of ROCK1 and ZIPK is commonly involved in regulatory pathways, we utilized siRNA to knock down ROCK1 and ZIPK in cultured human arterial smooth muscle cells (SMC). Microarray analysis using a whole-transcript expression chip identified changes in gene expression induced by ROCK1 and ZIPK knockdown. ROCK1 knockdown affected the expression of 553 genes, while ZIPK knockdown affected the expression of 390 genes. A high incidence of regulation of transcription regulator genes was observed in both knockdowns. Other affected groups included transporters, kinases, peptidases, transmembrane and G protein-coupled receptors, growth factors, phosphatases and ion channels. Only 76 differentially expressed genes were common to ROCK1 and ZIPK knockdown. Ingenuity Pathway Analysis identified five pathways shared between the two knockdowns. We focused on cytokine signaling pathways since ROCK1 knockdown up-regulated 5 and down-regulated 4 cytokine genes, in contrast to ZIPK knockdown, which affected the expression of only two cytokine genes (both down-regulated). IL-6 gene expression and secretion of IL-6 protein were up-regulated by ROCK1 knockdown, whereas ZIPK knockdown reduced IL-6 mRNA expression and IL-6 protein secretion and increased ROCK1 protein expression, suggesting that ROCK1 may inhibit IL-6 secretion. IL-1ß mRNA and protein levels were increased in response to ROCK1 knockdown. Differences in the effects of ROCK1 and ZIPK knockdown on cell cycle regulatory genes suggested that ROCK1 and ZIPK regulate the cell cycle by different mechanisms. ROCK1, but not ZIPK knockdown reduced the viability and inhibited proliferation of vascular SMC. We conclude that ROCK1 and ZIPK have diverse, but predominantly distinct regulatory functions in vascular SMC and that ROCK1-mediated activation of ZIPK is not involved in most of these functions.
Assuntos
Artérias , Proteínas Quinases Associadas com Morte Celular/genética , Regulação da Expressão Gênica , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Quinases Associadas a rho/genética , Proliferação de Células , Sobrevivência Celular/genética , Células Cultivadas , Análise por Conglomerados , Proteínas Quinases Associadas com Morte Celular/metabolismo , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Miosinas/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismoRESUMO
DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.
Assuntos
Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Proteínas de Fluorescência Verde/metabolismo , Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/enzimologia , Ligação Competitiva , Cálcio/metabolismo , Proteínas Quinases Associadas com Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/metabolismo , Proteínas de Fluorescência Verde/genética , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Íleo/citologia , Íleo/efeitos dos fármacos , Íleo/enzimologia , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/citologia , Músculo Liso/enzimologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/enzimologia , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Fosforilação , Cultura Primária de Células , Inibidores de Proteínas Quinases/química , Proteômica , Pirazóis/química , Pirimidinonas/química , Coelhos , Proteínas Recombinantes de Fusão/genéticaRESUMO
Insulin resistance triggers the developments of diabetes mellitus and atherosclerosis. Tribbles homolog 3 (TRIB3) is involved in insulin resistance. We aimed to investigate whether TRIB3 is implicated in diabetic atherosclerosis. Sixty 3-week-old apolipoprotein E (ApoE-/-)/LDR receptor (LDLR-/-) mice were randomly divided into chow and diabetes groups. Diabetes was induced by a high-fat and high-sugar diet combined with low-dose streptozotocin. Mice in both groups were randomly divided into vehicle and TRIB3-silencing groups. After transfection, all mice were killed to evaluate the effects of TRIB3 on atherosclerosis. Silence of TRIB3 markedly decreased insulin resistance (P=0.039) and glucose (P=0.019), regardless of diabetes. Ultrasonography-measured parameters were similar in both groups, with and without silence of TRIB3. However, silence of TRIB3 decreased the aortic atherosclerotic burden (P=1×10(-13)). Further study showed that in brachiocephalic lesions, fibrous cap thickness, cap-to-core ratio, collagen content, and the number of smooth muscle cells were significantly increased (P<0.01 for all) by silence of TRIB3, whereas lipid and macrophage contents remained unaltered, with the vulnerability index significantly reduced. Moreover, the numbers of apoptotic cells and macrophages in brachiocephalic lesions were both significantly decreased (P<0.01 for both). Macrophage migration was decreased (P=4×10(-4)) by knocking down TRIB3, whereas adhesion and phagocytosis were increased (P<0.05 for both). Silence of TRIB3 would diminish atherosclerotic burden and increase the plaque stability in diabetic mice.
Assuntos
Apolipoproteínas E/fisiologia , Aterosclerose/prevenção & controle , Proteínas de Ciclo Celular/fisiologia , Diabetes Mellitus Experimental/complicações , Placa Aterosclerótica/terapia , Receptores de LDL/fisiologia , Animais , Apoptose , Adesão Celular , Proteínas de Ciclo Celular/antagonistas & inibidores , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Inativação Gênica , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Knockout , Fosforilação , Placa Aterosclerótica/diagnóstico por imagem , Proteínas Proto-Oncogênicas c-akt/fisiologia , UltrassonografiaRESUMO
The present study was designed to identify the changes in microvesicle-dipeptidyl peptidase-IV (DPP IV) levels in human urine and serum, and to determine whether there were correlations with the severity of diabetic kidney disease (DKD). A total of 127 patients with type 2 diabetes mellitus (T2DM) were divided into three groups according to the urinary albumin/ creatinine ratio (UACR): microalbuminuria group (n = 50); macroalbuminuria group (n = 34) and normoalbuminuria group (n = 43), and 34 age- and sex-matched non-diabetic healthy subjects were selected as controls. Microvesicle-bound DPP IV and free urinary DPP IV were separated by a filtra-centrifugation method. The total microvesicles were captured by a specific monoclonal antibody, AD-1. DPP IV activity was determined by measuring the cleavage of chromogenic free 4-nitroaniline from Gly-Pro-p-nitroanilide at 405 nm with an ELISA plate reader. DPP IV protein levels were determined by ELISA and Western blot. Our results showed that the microvesicle-bound type was the major form of DPP IV in urine; the urinary microvesicle-DPP IV excretion of each T2DM group was significantly higher compared with controls. The urinary microvesicle-DPP IV level was positively correlated with UACR in patients with T2DM. These findings suggest that the urinary level of microvesicle-bound DPP IV is associated with the severity of DKD.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/enzimologia , Dipeptidil Peptidase 4/urina , Idoso , Albuminúria/diagnóstico , Albuminúria/enzimologia , Albuminúria/etiologia , Albuminúria/urina , Biomarcadores/sangue , Biomarcadores/urina , Western Blotting , Estudos de Casos e Controles , Centrifugação , Creatinina/urina , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/diagnóstico , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/urina , Dipeptidil Peptidase 4/sangue , Ensaio de Imunoadsorção Enzimática , Exossomos/enzimologia , Exossomos/ultraestrutura , Feminino , Humanos , Testes de Função Renal , Modelos Lineares , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Índice de Gravidade de Doença , Urinálise , Urina/química , Urina/citologiaRESUMO
BACKGROUND: Microvesicles are involved in different pathological processes such as inflammation, coagulation and tumor progression. We intended to establish an immunoaffinity capture method for detecting microvesicles and bioactive effectors carried on them using a specific homemade monoclonal antibody AD-1. By this method we investigated the association of inflammation with platelet activation in patients with nonvalvular atrial fibrillation (NVAF). METHODS: A case-control study of 90 Chinese subjects selected in 3 groups: control, paroxysmal AF, and persistent AF. After capturing the microvesicles of serum using a specific monoclonal antibody AD-1, the amounts of LAP, IL-1ß and P-selectin loaded on these microvesicles were quantified by either enzyme activity assay (LAP) or ELISA respectively. RESULTS: Compared with normal controls, the patients with persistent AF showed significantly increased serum levels of microvesicles (P<0.001), microvesicle-bound IL-1 ß (P=0.019) and microvesicle-bound P-selectin (P=0.001). The latter two were significantly correlated with each other (r(2)=0.371, r=0.616, P<0.001). The microvesicle-bound IL-1ß (ß=0.570, P<0.001) and body weight (ß=0.427, P=0.002) were as independent predictors of platelet activation. CONCLUSIONS: The method was easy and reproducible. Inflammation may be involved in the activation of platelets in NVAF.
Assuntos
Anticorpos Monoclonais , Fibrilação Atrial/sangue , Micropartículas Derivadas de Células/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Inflamação/diagnóstico , Ativação Plaquetária , Adulto , Idoso , Idoso de 80 Anos ou mais , Povo Asiático/genética , Estudos de Casos e Controles , Micropartículas Derivadas de Células/química , Feminino , Humanos , Inflamação/sangue , Interleucina-1beta/sangue , Masculino , Métodos , Pessoa de Meia-Idade , Selectina-P/sangueRESUMO
The objective of this study was to investigate the gene expression signature of monocyte/macrophages and the pleiotropic effects of atorvastatin on monocytes in atherosclerotic patients. Forty patients with coronary heart diseases were randomly assigned to double-blind therapy with either 20 or 80 mg per day of atorvastatin. Follow-up visits occurred at weeks 6 and 12, including complete chemistry and lipid analyses and quantification of 14 target genes in monocytes. After 12 weeks of therapy, both groups gained beneficial alterations in lipid profiles. Both groups experienced significant reductions in gene expression of lipoprotein-associated phospholipase A2, CD13, leptin receptor, matrix metalloproteases-1, legumain, and prolyl oligopeptidase after 12 weeks of therapy. Only tumor protein 53 was increased in the atorvastatin 80-mg group. Moreover, nonsignificant interactions between dosage and duration of therapy were found. The pleiotropic effects of statins in atherosclerotic patients include increased expression of genes involved in apoptosis of monocyte/macrophage, inhibition of inflammatory responses, antioxidant properties, prevention of foam cell formation, and stabilization of atherosclerotic plaques. This property fuels potential clinical significance.
Assuntos
Anticolesterolemiantes/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Ácidos Heptanoicos/uso terapêutico , Monócitos/metabolismo , Pirróis/uso terapêutico , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Administração Oral , Apoptose/efeitos dos fármacos , Aterosclerose/complicações , Atorvastatina , Antígenos CD13/metabolismo , Doença das Coronárias/tratamento farmacológico , Doença das Coronárias/etiologia , Doença das Coronárias/metabolismo , Cisteína Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Seguimentos , Expressão Gênica/efeitos dos fármacos , Ácidos Heptanoicos/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Pessoa de Meia-Idade , Monócitos/efeitos dos fármacos , Prolil Oligopeptidases , Receptores para Leptina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
Tribble3 (TRB3), a mammalian homolog of Drosophila tribbles, slows cell-cycle progression, and its expression is increased in response to various stresses. The aim of this study was to investigate the role of the TRB3 gene in macrophage apoptosis induced by oxidized low-density lipoprotein (ox-LDL). We found that, in human monocyte-derived macrophages, TRB3 is upregulated by ox-LDL in a dose- and time-dependent manner. The cell viability of TRB3-overexpressing macrophages was decreased, but apoptosis was increased and the level of activated caspase-3 increased. Factorial analyses revealed no significant interaction between TRB3 overexpression and ox-LDL stimulation with respect to macrophage apoptosis. Furthermore, TRB3-silenced macrophages showed decreased apoptosis, and TRB3-silenced cells treated with ox-LDL showed significantly increased apoptosis. Silencing of TRB3 and ox-LDL stimulation showed significant interaction for macrophage apoptosis, suggesting that TRB3 knockdown resisted the macrophage apoptosis induced by ox-LDL. Therefore, TRB3 in part mediates the macrophage apoptosis induced by ox-LDL, which suggests that TRB3 might be involved in vulnerable atherosclerotic plaque progression.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/fisiologia , Lipoproteínas LDL/fisiologia , Macrófagos/citologia , Monócitos/citologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Repressoras/fisiologia , Regulação para Cima/fisiologia , Sequência de Bases , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Humanos , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Proteínas Repressoras/genéticaRESUMO
AIM: To elucidate the effect and the mechanisms of curcumin on the expression of the human homeobox gene NKX3.1 in the prostate cancer cell LNCaP. METHODS: The expression change of NKX3.1 in cells incubated with varying concentrations of curcumin was observed by Western blotting and RT-PCR. A dual luciferase reporter assay was used to test the effect of curcumin on the activity of the NKX3.1 1040 bp promoter. Curcumin-treated cells disposed to a designated amount of androgen analog R1881 and the androgen receptor (AR) antagonist flutamide, then the expression of NKX3.1 or the activity of the NKX3.1 promoter were investigated by Western blotting or reporter gene assay, respectively. Finally, Western blotting and electrophoretic mobility shift assay were performed to demonstrate the effect of curcumin on the expression of AR and its binding activity to the androgen response element (ARE). RESULTS: Curcumin downregulated the expression of NKX3.1 and the activity of the NKX3.1 1040 bp promoter in LNCaP cells. R1881 increased the expression of NKX3.1, and the AR antagonist flutamide decreased the expression of NKX3.1 in LNCaP cells, while curcumin could inhibit androgen-AR mediated induction of NKX3.1 expression. Curcumin decreased the expression of AR and the binding activity to ARE directly. CONCLUSION: Curcumin could downregulate NKX3.1 expression in LNCaP cells. It could also inhibit the androgen-AR mediated induction of NKX3.1 expression by downregulating AR expression and blocking its DNA binding activity.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Curcumina/farmacologia , Proteínas de Homeodomínio/biossíntese , Neoplasias da Próstata/metabolismo , Fatores de Transcrição/biossíntese , Androgênios/farmacologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Humanos , Masculino , Metribolona/farmacologia , Receptores Androgênicos/biossíntese , Receptores Androgênicos/genética , Fatores de Transcrição/genética , TransfecçãoRESUMO
Integrin-linked kinase (ILK) has been implicated in Ca(2+)- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17 kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr-->Ala mutation at Thr(38) of CPI-17 and Thr(57) of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr(38) of CPI-17 or Thr(57) of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr(38) or Thr(57) respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca(2+) sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr(38)Ala and PHI-1-Thr(57)Ala treated with ILK in the presence of adenosine 5'-[gamma-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.
Assuntos
Proteínas Musculares/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Animais , Cálcio/metabolismo , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Proteínas Musculares/genética , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Octoxinol/farmacologia , Mapeamento de Peptídeos , Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/genética , Fosforilação , Mutação Puntual , Proteínas/genética , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: To investigate the difference of serum high molecular weight alkaline phosphatase (HMAP) levels between biliary atresia (BA) and neonatal hepatitis (NH), and to develop a new differential method and early diagnostic indicators for cholestatic jaundice in neonates. METHODS: Totally 31 patients with cholestatic jaundice seen between Aug. 2000 and Feb. 2002, including 15 cases with BA, 16 cases with NH, 30 healthy infants and 30 infants with non-cholestatic jaundice were enrolled in this study. Serum samples were obtained from each subject by using venipuncture. The samples were stored at -80 degrees C and analyzed within 6 months. A murine hybridoma producing monoclonal antibody to human high molecular weight alkaline phosphatase (MoAb HMAP-1) was prepared by using partially purified HMAP from human serum as the immunogen. The antibody did not cross-react with other alkaline phosphatase (ALP) isozymes. A monoclonal antibody immunocatalytic assay for HMAP in serum was developed by using MoAb HMAP-1 bound to nitrocellulose membrane discs. The serum total ALP (TALP) and gamma-GT were determined in the meantime, the hepatobiliary ultrasonography and scintigraphy were performed too. The data were analyzed with t test, chi-square test and percentage. Comparisons were made between BA and NH with their sensitivity and specificity in different methods. RESULTS: Serum HMAP was detected in 14 of 15 patients of BA, in 2 of 16 NH patients, while in none of the healthy control group. The positive ratios of serum HMAP in BA and NH were 93.3% and 12.5%, respectively (P < 0.005). The sensitivity and specificity of serum HMAP in BA and NH were 93.3% and 87.5%, respectively. The sensitivity and specificity of TALP, gamma-GT and hepatobiliary scintigraphy were 80.0%, 73.3%, 86.7% and 62.5%, 68.8%, 62.5%, respectively, which were clearly lower than those of serum HMAP. CONCLUSIONS: The determination of serum HMAP was more sensitive and specific than the other methods tested. Therefore the method can be used as a useful indicator for cholestatic jaundice in neonates, although it needs further study.
Assuntos
Fosfatase Alcalina/sangue , Icterícia Obstrutiva/diagnóstico , Diagnóstico Diferencial , Feminino , Humanos , Técnicas Imunoenzimáticas/métodos , Lactente , Recém-Nascido , Masculino , Sensibilidade e Especificidade , gama-Glutamiltransferase/sangueRESUMO
A variety of contractile agonists trigger activation of the small GTPase RhoA. An important target of activated RhoA in smooth muscle is Rho-associated kinase (ROK), one of the downstream targets that is the myosin binding subunit (MYPT1) of myosin light chain phosphatase (MLCP). Phosphorylation of MYPT1 at T695 by activated ROK results in a decrease in phosphatase activity of MLCP and an increase in myosin light chain (LC(20)) phosphorylation catalyzed by Ca(2)(+)/calmodulin-dependent myosin light chain kinase and/or a distinct Ca(2)(+)-independent kinase. LC(20) phosphorylation in turn triggers cross-bridge cycling and force development. ROK also phosphorylates the cytosolic protein CPI-17 (at T38), which thereby becomes a potent inhibitor of MLCP. The RhoA/ROK pathway has been implicated in the tonic phase of force maintenance in response to various agonists, with no evident role in the phasic response, suggesting this pathway as a potential target for antihypertensive therapy. Indeed, ROK inhibitors restore normal blood pressure in several rat hypertensive models.
Assuntos
Hipertensão/fisiopatologia , Contração Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteína rhoA de Ligação ao GTP/fisiologia , Animais , Cálcio/fisiologia , Humanos , Hipertensão/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Transdução de Sinais , Quinases Associadas a rhoRESUMO
Protein phosphatases play key roles in cellular regulation and are subjected to control by protein inhibitors whose activity is in turn regulated by phosphorylation. Here we investigated the possible regulation of phosphorylation-dependent type-1 protein phosphatase (PP1) inhibitors, CPI-17, PHI-1, and KEPI, by various kinases. Protein kinases A (PKA) and G (PKG) phosphorylated CPI-17 at the inhibitory site (T38), but not PHI-1 (T57). Phosphorylated CPI-17 inhibited the activity of both the PP1 catalytic subunit (PP1c) and the myosin phosphatase holoenzyme (MPH) with IC(50) values of 1-8 nM. PKA predominantly phosphorylated a site distinct from the inhibitory T73 in KEPI, whereas PKG was ineffective. Integrin-linked kinase phosphorylated KEPI (T73) and this dramatically increased inhibition of PP1c (IC(50)=0.1 nM) and MPH (IC(50)=8 nM). These results suggest that the regulatory phosphorylation of CPI-17 and KEPI may involve distinct kinases and signaling pathways.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Galinhas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Musculares/metabolismo , Mutação , Fosfatase de Miosina-de-Cadeia-Leve , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteína Fosfatase 1 , Proteínas/metabolismo , Fatores de Tempo , PerusRESUMO
A mechanism proposed for regulation of myosin phosphatase (MP) activity is phosphorylation of the myosin phosphatase target subunit (MYPT1). Integrin-linked kinase (ILK) is associated with the contractile machinery and can phosphorylate myosin at the myosin light-chain kinase sites. The possibility that ILK may also phosphorylate and regulate MP was investigated. ILK was associated with the MP holoenzyme, shown by Western blots and in-gel kinase assays. MYPT1 was phosphorylated by ILK and phosphorylation sites in the N- and C-terminal fragments of MYPT1 were detected. From sequence analyses, three sites were identified: a primary site at Thr(709), and two other sites at Thr(695) and Thr(495). One of the sites for cAMP-dependent protein kinase (PKA) was Ser(694). Assays with the catalytic subunit of type 1 phosphatase indicated that only the C-terminal fragment of MYPT1 phosphorylated by zipper-interacting protein kinase, and ILK inhibited activity. The phosphorylated N-terminal fragment activated phosphatase activity and phosphorylation by PKA was without effect. Using full-length MYPT1 constructs phosphorylated by various kinases it was shown that Rho kinase gave marked inhibition; ILK produced an intermediate level of inhibition, which was considerably reduced for the Thr(695)-->Ala mutant; and PKA had no effect. In summary, phosphorylation of the various sites indicated that Thr(695) was the major inhibitory site, Thr(709) had only a slight inhibitory effect and Ser(694) had no effect. The findings that ILK phosphorylated both MYPT1 and myosin and the association of ILK with MP suggest that ILK may influence cytoskeletal structure or function.