RESUMEN

Although thioredoxin-interacting protein (TXNIP) is involved in a variety of biologic functions, the contribution of endothelial TXNIP has not been well defined. To investigate the endothelial function of TXNIP, we generated a TXNIP knockout mouse on the Cdh5-cre background (TXNIPfl/fl cdh5cre). Control (TXNIPfl/fl) and TXNIPfl/fl cdh5cre mice were fed a high protein-low carbohydrate (HP-LC) diet for 3 mo to induce metabolic stress. We found that TXNIPfl/fl and TXNIPfl/fl cdh5cre mice on an HP-LC diet displayed impaired glucose tolerance and dyslipidemia concretizing the metabolic stress induced. We evaluated the impact of this metabolic stress on mice with reduced endothelial TXNIP expression with regard to arterial structure and function. TXNIPfl/fl cdh5cre mice on an HP-LC diet exhibited less endothelial dysfunction than littermate mice on an HP-LC diet. These mice were protected from decreased aortic medial cell content, impaired aortic distensibility, and increased plasminogen activator inhibitor 1 secretion. This protective effect came with lower oxidative stress and lower inflammation, with a reduced NLRP3 inflammasome expression, leading to a decrease in cleaved IL-1ß. We also show the major role of TXNIP in inflammation with a knockdown model, using a TXNIP-specific, small interfering RNA included in a lipoplex. These findings demonstrate a key role for endothelial TXNIP in arterial impairments induced by metabolic stress, making endothelial TXNIP a potential therapeutic target.-Bedarida, T., Domingues, A., Baron, S., Ferreira, C., Vibert, F., Cottart, C.-H., Paul, J.-L., Escriou, V., Bigey, P., Gaussem, P., Leguillier, T., Nivet-Antoine, V. Reduced endothelial thioredoxin-interacting protein protects arteries from damage induced by metabolic stress in vivo.

Asunto(s)

Aorta/metabolismo , Proteínas Portadoras/metabolismo , Dislipidemias/metabolismo , Intolerancia a la Glucosa/metabolismo , Estrés Fisiológico , Tiorredoxinas/metabolismo , Animales , Aorta/patología , Proteínas Portadoras/genética , Dieta Baja en Carbohidratos/efectos adversos , Proteínas en la Dieta/efectos adversos , Proteínas en la Dieta/farmacología , Dislipidemias/inducido químicamente , Dislipidemias/genética , Dislipidemias/patología , Intolerancia a la Glucosa/inducido químicamente , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/patología , Inflamasomas/genética , Inflamasomas/metabolismo , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/biosíntesis , Serpina E2/biosíntesis , Tiorredoxinas/genética

RESUMEN

BACKGROUND AND PURPOSE: The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. METHODS: We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. RESULTS: Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. CONCLUSIONS: Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy.

Asunto(s)

Anemia de Células Falciformes/complicaciones , Fibrinolíticos/uso terapéutico , Hipoxia-Isquemia Encefálica/complicaciones , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Accidente Cerebrovascular/complicaciones , Accidente Cerebrovascular/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Anemia de Células Falciformes/diagnóstico por imagen , Animales , Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/fisiopatología , Infarto Cerebral/diagnóstico por imagen , Infarto Cerebral/genética , Circulación Cerebrovascular , Masculino , Ratones , Selectina-P/biosíntesis , Selectina-P/genética , Adhesividad Plaquetaria , Serpina E2/biosíntesis , Serpina E2/genética , Accidente Cerebrovascular/diagnóstico por imagen , Ultrasonografía Doppler

RESUMEN

The post translational modification of lysine acetylation is a key mechanism that regulates chromatin structure. Epigenetic readers, such as the BET domains, are responsible for reading histone lysine acetylation which is a hallmark of open chromatin structure, further providing a scaffold that can be accessed by RNA polymerases as well as transcription factors. Recently, several reports have assessed and highlighted the roles of epigenetic readers in various cellular contexts. However, little is known about their role in the regulation of inflammatory genes, which is critical in exquisitely tuning inflammatory responses to a variety of immune stimuli. In this study, we investigated the role of epigenetic readers BRD2 and BRD4 in the lipopolysaccharide (LPS)-induced immune responses in mouse primary astrocytes. Inflammatory stimulation by LPS showed that the levels of Brd2 mRNA and protein were increased, while Brd4 mRNA levels did not change. Knocking down of Brd2 mRNA using specific small interfering RNA (siRNA) in cultured mouse primary astrocytes inhibited LPS-induced mRNA expression and secretion of plasminogen activator inhibitor-1 (PAI-1). However, no other pro-inflammatory cytokines, such as Il-6, Il-1ß and Tnf-α, were affected. Indeed, treatment with bromodomain-containing protein inhibitor, JQ1, blocked Pai-1 mRNA expression through the inhibition of direct BRD2 protein-binding and active histone modification on Pai-1 promoter. Taken together, our data suggest that BRD2 is involved in the modulation of neuroinflammatory responses through PAI-1 and via the regulation of epigenetic reader BET protein, further providing a potential novel therapeutic strategy in neuroinflammatory diseases.

Asunto(s)

Astrocitos/metabolismo , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética/genética , Lipopolisacáridos/farmacología , Serpina E2/biosíntesis , Serpina E2/genética , Animales , Astrocitos/efectos de los fármacos , Azepinas/farmacología , Citocinas/biosíntesis , Citocinas/genética , Técnicas de Silenciamiento del Gen , Histonas/metabolismo , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Ratones , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Cultivo Primario de Células , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño , Serpina E2/antagonistas & inhibidores , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transfección , Triazoles/farmacología

RESUMEN

BACKGROUND: GJA1 and PTX3 were proposed as gene markers for oocyte and embryo developmental competence, while SERPINE2 was reported to be associated with pregnancy outcome. PRSS35, which is exclusively expressed in the ovary, may be correlated with oocyte competence. This study was conducted to evaluate the correlation of cumulus GJA1, PRSS35, PTX3, and SERPINE2 gene expression levels with oocyte maturation, fertilization, and early embryo development. METHODS: In total, 308 cumulus cell samples separated from individual cumulus-oocyte complex were obtained from 40 patients undergoing the intracytoplasmic sperm injection treatment procedure. Gene expression levels (mRNA levels) in cumulus cells were assessed using quantitative real-time polymerase chain reaction. RESULTS: Gene expression levels of GJA1 and SERPINE2 in cumulus cells surrounding mature oocytes were significantly lower than those in cumulus cells enclosing immature oocytes. PRSS35 mRNA levels in cumulus cells of fertilized oocytes were significantly higher than those in cumulus cells of unfertilized oocytes. GJA1 and SERPINE2 seemed to express higher mRNA levels, while PRSS35 showed lower expression in cumulus cells of oocytes that developed into embryos with good morphology; however, the expression levels of all three genes and PTX3 showed no significant differences between embryos with good or poor morphology. CONCLUSIONS: GJA1 and SERPINE2 represent potential gene markers associated with oocyte maturation. PRSS35 may be correlated with oocyte fertilization potential. However, GJA1, PRSS35, PTX3, and SERPINE2 may not be considered as marker genes for predicting embryo morphology.

Asunto(s)

Proteína C-Reactiva/biosíntesis , Conexina 43/biosíntesis , Células del Cúmulo/metabolismo , Fertilización/fisiología , Oogénesis/fisiología , Serina Proteasas/biosíntesis , Serpina E2/biosíntesis , Componente Amiloide P Sérico/biosíntesis , Biomarcadores/metabolismo , Proteína C-Reactiva/genética , Conexina 43/genética , Células del Cúmulo/citología , Desarrollo Embrionario/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Embarazo , Serina Proteasas/genética , Serpina E2/genética , Componente Amiloide P Sérico/genética , Inyecciones de Esperma Intracitoplasmáticas/métodos

RESUMEN

OBJECTIVES: Osteoarthritis (OA) is a chronic joint disease, characterized by a progressive loss of articular cartilage. During OA, proinflammatory cytokines, such as interleukin IL-1, induce the expression of matrix metalloproteinases (MMPs) in chondrocytes, contributing thus to the extracellular matrix (ECM) degradation. Members of Serpine family, including plasminogen activator inhibitors have been reported to participate in ECM regulation. The aim of this study was to assess the expression of serpin peptidase inhibitor clade E member 2 (SERPINE2), under basal conditions and in response to increasing doses of IL-1α, in human cultured chondrocytes. We also examined the effects of SERPINE2 on IL-1α-induced MMP-13 expression. For completeness, the signaling pathway involved in this process was also explored. METHODS: SERPINE2 mRNA and protein expression were evaluated by RT-qPCR and western blot analysis in human T/C-28a2 cell line and human primary chondrocytes. These cells were treated with human recombinant SERPINE2, alone or in combination with IL-1α. ERK 1/2, NFκB and AP-1 activation were assessed by western blot analysis. RESULTS: Human cultured chondrocytes express SERPINE2 in basal condition. This expression increased in response to IL-1α stimulation. In addition, recombinant SERPINE2 induced a clear inhibition of MMP-13 expression in IL-1α-stimulated chondrocytes. This inhibitory effect is likely regulated through a pathway involving ERK 1/2, NF-κB and AP-1. CONCLUSIONS: Taken together, these data demonstrate that SERPINE2 might prevent cartilage catabolism by inhibiting the expression of MMP-13, one of the most relevant collagenases, involved in cartilage breakdown in OA.

Asunto(s)

Interleucina-1alfa/biosíntesis , Metaloproteinasa 13 de la Matriz/biosíntesis , Osteoartritis/genética , Serpina E2/biosíntesis , Factor de Transcripción AP-1/biosíntesis , Cartílago Articular/crecimiento & desarrollo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Matriz Extracelular/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Interleucina-1alfa/genética , Sistema de Señalización de MAP Quinasas/genética , FN-kappa B/genética , Osteoartritis/fisiopatología , Cultivo Primario de Células , Serpina E2/genética , Factor de Transcripción AP-1/genética

RESUMEN

AIMS: bFGF is a potent mitogen of cells associated with fibrosis. Although ERK5 has been reported to play roles in the development of fibrosis, its roles in regulating bFGF-induced fibrotic responses are not understood, especially in lung fibroblasts. The authors investigated the role of ERK5 in bFGF induction of cell proliferation and in induction of PAI-1, a critical regulator of the pathological features of fibrosis, in lung fibroblasts. MAIN METHODS: The role played by ERK5 in bFGF-induced PAI-1 expression was elucidated by perturbing the ERK5 signaling pathway using a specific chemical inhibitor and siRNA of ERK5. The effects of ERK5 signal perturbation on PAI-1 expression were measured at multiple levels by Q-PCR, immunoblotting, ELISA, and reporter gene analysis. The role of MEF2 in bFGF-induced activation of PAI-1 promoter activity via ERK5 was measured using a biotin-labeled DNA pull-down assay, and the effects of ERK5 on the mitogenic effects of bFGF were assessed using a MTT assay. KEY FINDINGS: In both primary human lung fibroblast and lung fibroblast cell lines, inhibition of ERK5 blocked bFGF-induced PAI-1 expression at both mRNA and protein levels and inhibited bFGF-induced PAI-1 promoter activity induction by bFGF. Upon stimulation with bFGF, MEF2 directly bound to the consensus sequence of the MEF2 binding site in the PAI-1 promoter. In addition, bFGF-induced PAI-1 up-regulation was inhibited by MEF2 siRNA, and bFGF-induced fibroblast proliferation was blocked by inhibiting ERK5. SIGNIFICANCE: This study reveals a novel role for the ERK5-MEF2 cascade, linking bFGF-induced PAI-1 expression and subsequent mitogenic processes in lung fibroblasts.

Asunto(s)

Proliferación Celular/efectos de los fármacos , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Pulmón/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Serpina E2/biosíntesis , Animales , Línea Celular , Proliferación Celular/genética , Factor 2 de Crecimiento de Fibroblastos/genética , Fibroblastos/patología , Regulación de la Expresión Génica/genética , Humanos , Pulmón/patología , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Ratones , Proteína Quinasa 7 Activada por Mitógenos/genética , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Serpina E2/genética

RESUMEN

Pressure ulcers result from tissue hypoxia caused by external forces. Thrombosis due to external forces is considered important, and hypoxia inducible factor-1 (HIF-1) is a master regulator of pressure ulcer development. To date, however, their causal relationship has not been determined. This study therefore investigated the mutual relationship between thrombosis and HIF-1 activation in compressed mouse skin, based on a hypothesis that HIF-1 regulation by plasminogen activator inhibitor-1 (PAI-1) enhances thrombosis. Compression of mouse skin significantly increased the numbers of thrombi and HIF-1α-positive cells compared with control skin. A thrombosis inhibitor significantly reduced the numbers of HIF-1α-positive cells and an HIF-1 inhibitor significantly inhibited thrombosis in compressed skin tissue, suggesting a mutual relationship between thrombosis and HIF-1 activation. Compression of mouse skin also enhanced the level of Pai-1 messenger RNA expression, but this increase was significantly reduced by treatment with an HIF-1 inhibitor, whereas a thrombosis inhibitor had no effect. These results suggested the involvement of PAI-1 in HIF-1-enhanced thrombosis and that an additional factor participates in regulating Pai-1 expression in compressed skin. These findings may suggest new strategies in pressure ulcer management.

Asunto(s)

Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ARN Mensajero/genética , Serpina E2/genética , Piel/metabolismo , Trombosis/genética , Heridas y Lesiones/genética , Animales , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Inmunohistoquímica , Masculino , Ratones , Presión , Reacción en Cadena en Tiempo Real de la Polimerasa , Serpina E2/biosíntesis , Piel/lesiones , Piel/patología , Estrés Mecánico , Trombosis/etiología , Trombosis/metabolismo , Cicatrización de Heridas , Heridas y Lesiones/complicaciones , Heridas y Lesiones/metabolismo

RESUMEN

Nephrolithiasis is a common kidney disease and one of the major causes of chronic renal insufficiency. We develop and utilize a glyoxylate induced mouse model of kidney calcium oxalate crystal deposition for studying the pharmacological effects of fasudil, a Rho associated protein kinase (ROCK) specific inhibitor, on the kidney injury and fibrosis caused by calcium oxalate crystallization and deposition. Glyoxylate was administrated intraperitoneally to C57BL/6J mice for five consecutive days to establish a mouse model of kidney calcium oxalate crystal formation and deposition. The results showed that the protein expression levels of E-cad and Pan-ck were lower, and the protein expression levels of α-SMA and Vim were higher, in the kidney tissue of the glyoxylate induced model mice compared with the control mice. The changes in protein expression were weakened when the animals were pretreated with fasudil before glyoxylate administration. Expression of ROCK, PAI-1, and p-Smad proteins in the kidney tissue increased in response to glyoxylate treatment, and the increase was eased when the animals were pretreated with fasudil. Expression of Smad2 and Smad3 in the kidney tissue remained unchanged after glyoxylate administration. Cell apoptosis and proliferation in the kidney cortex and medulla were enhanced in response to the glyoxylate induced calcium oxalate crystal formation and deposition, and fasudil pre-treatment was able to attenuate the enhancement. The results suggest that Fasudil reduces the glyoxylate induced kidney calcium crystal formation and deposition and slows down the kidney fibrogenesis caused by calcium crystal deposition. The possible mechanism may be related the regulatory effects on Rho/ROCK signal transduction and epithelial-mesenchymal transition (EMT).

Asunto(s)

1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Fibrosis/tratamiento farmacológico , Nefrolitiasis/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Actinas/biosíntesis , Animales , Apoptosis/efectos de los fármacos , Cadherinas/biosíntesis , Oxalato de Calcio/metabolismo , Proliferación Celular/efectos de los fármacos , Cristalización , Modelos Animales de Enfermedad , Fibrosis/prevención & control , Glioxilatos , Riñón/patología , Ratones , Ratones Endogámicos C57BL , Nefrolitiasis/prevención & control , Serpina E2/biosíntesis , Proteína Smad2/biosíntesis , Proteína smad3/biosíntesis , Quinasas Asociadas a rho/biosíntesis

RESUMEN

Protease nexin 1 (PN1) is an endogenous serine protease inhibitor (SERPIN), expressed at high levels in the prostate, and capable of inhibiting the proliferation of prostate cancer cells. We previously showed that PN1-uPA complexes inhibited Sonic Hedgehog (SHH) signalling through engagement of the LRP receptor. Here, we describe an alternative anti-proliferative mechanism through which PN1 expression leads to apoptosis. In prostate cancer cells, increased expression of PN1 led to substantial reduction of XIAP levels and apoptosis mediated through the uPAR, but not the LRP receptor. The alterations in XIAP were effected in two ways 1) via alteration in the NF-κB pathway, a pathway known to signal XIAP transcription and 2) by promoting XIAP instability. The AKT pathway is known to phosphorylate XIAP at serine 87 leading to protein stability and PN1 expression is shown to interfere with this process. As a result of both mechanisms, programmed cell death is substantially increased. Consistent with these observations, reduced PN1 protein correlated with elevated p65/XIAP expression and with higher Gleason scores in human prostate tissue arrays. Thus, PN1 expression appears to differentially down-regulate distinct oncogenic pathways depending upon the cell surface receptor engaged by its complexes and demonstrates a novel molecular mechanism by which the protein can promote tumor cell apoptosis.

Asunto(s)

Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Serpina E2/biosíntesis , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Células HL-60 , Humanos , Células Jurkat , Masculino , Ratones , Ratones Noqueados , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/genética , Serpina E2/metabolismo , Serpina E2/farmacología , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/genética , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-ß signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fibroblastos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/metabolismo , Fosfoproteínas/metabolismo , Fibrosis Pulmonar/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas de Ciclo Celular , Femenino , Fibroblastos/patología , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Pulmón/patología , Masculino , Mecanotransducción Celular/genética , Ratones , Ratones Endogámicos NOD , Mutación , Células 3T3 NIH , Fosfoproteínas/genética , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patología , Serpina E2/biosíntesis , Serpina E2/genética , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Señalizadoras YAP

RESUMEN

Previous reports suggest that plasminogen activator inhibitor-1 (PAI-1) promotes airway remodeling and that human and mouse mast cells (MCs) are an important source of PAI-1. In the present study we investigated MC-epithelial cell (EC) interactions in the production of PAI-1. We stimulated the human MC line LAD2 with IgE-receptor cross-linking and collected the supernatants. We incubated the human bronchial EC line BEAS-2B with the LAD2 supernatants and measured the level of PAI-1. When the supernatants from IgE-stimulated LAD2 were added to BEAS-2B, there was a significant enhancement of PAI-1 production by BEAS-2B. When we treated the MC supernatants with a transforming growth factor (TGF)-ß1 neutralizing antibody, the MC-derived induction of PAI-1 from BEAS-2B was completely abrogated. Although TGF-ß1 mRNA was constitutively expressed in resting LAD2, it was not highly induced by IgE-mediated stimulation. Nonetheless, active TGF-ß1 protein was significantly increased in LAD2 after IgE-mediated stimulation. Active TGF-ß1 produced by primary cultured human MCs was significantly reduced in the presence of a chymase inhibitor, suggesting a role of MC chymase as an activator of latent TGF-ß1. This study indicates that stimulation of human MCs by IgE receptor cross-linking triggers activation of TGF-ß1, at least in part via chymase, which in turn induces the production of PAI-1 by bronchial ECs. Our data suggest that human MCs may play an important role in airway remodeling in asthma as a direct source of PAI-1 and by activating bronchial ECs to produce further PAI-1 via a TGF-ß1-mediated activation pathway.

Asunto(s)

Asma/metabolismo , Bronquios/metabolismo , Comunicación Celular , Células Epiteliales/metabolismo , Mastocitos/metabolismo , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Serpina E2/biosíntesis , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Asma/patología , Bronquios/patología , Línea Celular , Quimasas/metabolismo , Células Epiteliales/patología , Humanos , Inmunoglobulina E/metabolismo , Mastocitos/patología , Ratones , ARN Mensajero/biosíntesis

RESUMEN

High temperature requirement protein A1 (HtrA1) is a primarily secreted serine protease involved in a variety of cellular processes including transforming growth factor ß (TGF-ß) signaling. Loss of its activity causes cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), an inherited form of cerebral small vessel disease leading to early-onset stroke and premature dementia. Dysregulated TGF-ß signaling is considered to promote CARASIL pathogenesis, but the underlying molecular mechanisms are incompletely understood. Here we present evidence from mouse brain tissue and embryonic fibroblasts as well as patient skin fibroblasts for a facilitating role of HtrA1 in TGF-ß pathway activation. We identify latent TGF-ß binding protein 1 (LTBP-1), an extracellular matrix protein and key regulator of TGF-ß bioavailability, as a novel HtrA1 target. Cleavage occurs at physiological protease concentrations, is prevented under HtrA1-deficient conditions as well as by CARASIL mutations and disrupts both LTBP-1 binding to fibronectin and its incorporation into the extracellular matrix. Hence, our data suggest an attenuation of TGF-ß signaling caused by a lack of HtrA1-mediated LTBP-1 processing as mechanism underlying CARASIL pathogenesis.

Asunto(s)

Alopecia/genética , Infarto Cerebral/genética , Proteínas de Unión a TGF-beta Latente/fisiología , Leucoencefalopatías/genética , Serina Endopeptidasas/fisiología , Enfermedades de la Columna Vertebral/genética , Factor de Crecimiento Transformador beta1/fisiología , Alopecia/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Infarto Cerebral/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/biosíntesis , Factor de Crecimiento del Tejido Conjuntivo/genética , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Proteínas de Unión a TGF-beta Latente/genética , Leucoencefalopatías/metabolismo , Ratones , Ratones Noqueados , Mutación Missense , Mutación Puntual , Unión Proteica , Mapeo de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Serpina E2/biosíntesis , Serpina E2/genética , Transducción de Señal , Piel , Enfermedades de la Columna Vertebral/metabolismo , Transfección

RESUMEN

BACKGROUND: Adiponectin (APN) and plasminogen activator inhibitor-1 (Pai-1) are adipocytokines, and low levels of serum APN and high levels of PAI-1 are observed in obese patients. Moreover, both APN and Pai-1 are known to be involved in colorectal carcinogenesis. Recently, we demonstrated that serum Pai-1 levels are elevated in APN-deficient mice. We hypothesized that Pai-1 expression levels could be depressed by APN. Thus, we aimed to clarify the bi-directional regulatory mechanisms between APN and Pai-1. MATERIALS AND METHODS: We investigated the expression levels of APN and Pai-1 during 3T3-L1 pre-adipocyte differentiation, and examined the role of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)-γ on APN and Pai-1 expression at early and late differentiation stages. RESULTS: In the early phase of differentiation, Pai-1 expression increased and APN slightly decreased. Reduction of Pai-1 or activation of PPARγ resulted in elevation of APN, and supplementation of APN with activation of AMPK resulted in reduction of Pai-1. In the late phase of differentiation, APN increased its expression and Pai-1 decreased. Supplementation of Pai-1 resulted in a slight reduction of APN. CONCLUSION: It is suggested that APN and Pai-1 expressions are inversely-regulated. Understanding of the regulatory system between APN and Pai-1 may lead to finding novel methods for colorectal cancer prevention.

Asunto(s)

Adipocitos/metabolismo , Adiponectina/biosíntesis , Diferenciación Celular/genética , Serpina E2/biosíntesis , Células 3T3-L1 , Adiponectina/genética , Animales , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/prevención & control , Regulación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Serpina E2/genética

RESUMEN

Longevity assurance homolog 2 of yeast LAG1 (Lass2) gene is capable of suppressing the proliferation and metastasis of several types of tumours including liver cancer. In the present study, hepatocyte-specific Lass2-knockout (Lass2 KO) and wild-type (WT) mice were exposed to the carcinogen, diethylnitrosamine (DEN), to induced liver tumours. At week 23 following DEN injection, tumours were produced in 100% of the Lass2 KO mice and 21.4% of the WT mice. At week 40, 100% of the Lass2 KO mice and 78.6% of the WT mice developed tumours, with no distinct significant difference in tumour occurrences between the two genotypes; yet, tumours in the Lass2 KO mouse livers were more numerous and larger in size. Hepatocellular carcinoma (HCC) was confirmed by α-fetoprotein (AFP). PCNA and EdU assays indicated more active proliferation whereas TUNEL assay revealed decreased apoptosis in Lass2 KO livers, when compared with the WT control. The expression of plasminogen activator inhibitor type-1 (PAI-1), a tumour-promoting gene, in the liver tissues of the 2 genotypes was detected using qPCR and western blotting, showing that PAI-1 levels were significantly elevated in Lass2 KO livers at week 40 following DEN introduction. Moreover, the expression of PAI-1-related TGF-ß1, Smad-4 and -7 was detected, displaying an elevation in TGF-ß1 and Smad-4 (not including Smad-7) in the Lass2 KO livers. Our data demonstrates that i) Lass2 is a protective gene against DEN-induced liver tumourigenesis; and ii) upregulation of the TGF-ß1-Smad4-PAI-1 axis may contribute to the vulnerability of Lass2-knockout mice to DEN.

Asunto(s)

Carcinoma Hepatocelular/genética , Transformación Celular Neoplásica/genética , Dietilnitrosamina/farmacología , Neoplasias Hepáticas Experimentales/genética , Esfingosina N-Aciltransferasa/genética , Animales , Apoptosis/genética , Carcinoma Hepatocelular/inducido químicamente , Proliferación Celular , Transformación Celular Neoplásica/efectos de los fármacos , Hepatocitos/patología , Hígado/patología , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Serpina E2/biosíntesis , Proteína Smad4/biosíntesis , Factor de Crecimiento Transformador beta1/biosíntesis

RESUMEN

Links between shift work and increases in metabolic risk factors for cardiovascular diseases have been documented in detail, although the underlying causes remain obscure. Plasminogen activator inhibitor-1 (PAI-1) is a key regulator of fibrinolysis that is also associated with an increased risk of cardiovascular diseases. We examined the effect of experimental chronic circadian clock disruption on PAI-1 expression in mice. Mice were exposed to chronic phase shifts and fed with a high-fat/high-sucrose diet. Chronic phase shifts resulted in increased plasma PAI-1 level through inducing PAI-1 mRNA expression and decreasing tissue-type plasminogen activator (tPA) mRNA expression in the liver. Chronic circadian clock disruption might induce hypofibrinolysis and increase the risk of cardiovascular events by inducing the PAI-1 gene expression in obese individuals.

Asunto(s)

Enfermedades Cardiovasculares/etiología , Trastornos Cronobiológicos/sangre , Regulación de la Expresión Génica , Serpina E2/biosíntesis , Animales , Enfermedad Crónica , Trastornos Cronobiológicos/complicaciones , Trastornos Cronobiológicos/genética , Corticosterona/sangre , Dieta Alta en Grasa/efectos adversos , Sacarosa en la Dieta/toxicidad , Fibrinólisis , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Obesidad/complicaciones , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , Serpina E2/sangre , Serpina E2/genética , Trombofilia/etiología , Trombofilia/fisiopatología , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/genética

RESUMEN

Prostate adenocarcinoma (CaP) patients are classified into low-, intermediate-, and high-risk groups that reflect relative survival categories. While there are accepted treatment regimens for low- and high-risk patients, intermediate-risk patients pose a clinical dilemma, as treatment outcomes are highly variable for these individuals. A better understanding of the factors that regulate the progression of CaP is required to delineate risk. For example, aberrant activation of the Hedgehog (Hh) pathway is implicated in CaP progression. Here, we identify the serine protease inhibitor protease nexin 1 (PN1) as a negative regulator of Hh signaling in prostate. Using human CaP cell lines and a mouse xenograft model of CaP, we demonstrate that PN1 regulates Hh signaling by decreasing protein levels of the Hh ligand Sonic (SHH) and its downstream effectors. Furthermore, we show that SHH expression enhanced tumor growth while overexpression of PN1 inhibited tumor growth and angiogenesis in mice. Finally, using comparative genome hybridization, we found that genetic alterations in Hh pathway genes correlated with worse clinical outcomes in intermediate-risk CaP patients, indicating the importance of this pathway in CaP.

Asunto(s)

Adenocarcinoma/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas Hedgehog/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de la Próstata/metabolismo , Serpina E2/biosíntesis , Transducción de Señal , Adenocarcinoma/genética , Adenocarcinoma/patología , Animales , Línea Celular Tumoral , Proteínas Hedgehog/genética , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Serpina E2/genética , Trasplante Heterólogo

RESUMEN

Macrophages are activated by recognizing bacterial DNA and CpG-oligodeoxynucleotides (CpG-ODNs) through Toll-like receptor-9 (TLR-9). Plasminogen activator inhibitor-1 (PAI-1) has been shown to be an important factor in inflammation-induced macrophage migration which is essential for defense functions. The aim of this study was to demonstrate the molecular mechanism associated with the regulation of PAI-1 expression and its biological significance in CpG-ODN-stimulated mouse macrophages. Our results clearly show that PAI-1 expression in macrophages was highly up-regulated by CpG-ODN-stimulation in vitro and in vivo. The TLR-9-mediated stimulation of PAI-1 expression was independent of the NF-κB pathway and involved the synergistic activation of Sp1 and Elk-1 by the MEK1/2-ERK and JNK signaling pathways. The elevated PAI-1 expression resulted in significantly enhanced transmigration of RAW264.7 cells through vitronectin but not through fibronectin. We suggest that CpG-ODN plays a role in regulating macrophage migration by stimulating the expression of PAI-1, and the migration is modulated depending on the microenvironmental extracellular matrix components.

Asunto(s)

Macrófagos/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Serpina E2/biosíntesis , Animales , Línea Celular , Movimiento Celular/genética , Microambiente Celular , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibronectinas/metabolismo , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , FN-kappa B/metabolismo , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Serpina E2/genética , Serpina E2/metabolismo , Receptor Toll-Like 9/metabolismo , Regulación hacia Arriba , Vitronectina/metabolismo , Proteína Elk-1 con Dominio ets/metabolismo

RESUMEN

Multipotent mesenchymal stromal cells (MSCs) increase tissue plasminogen activator (tPA) activity in astrocytes of the ischemic boundary zone, leading to increased neurite outgrowth in the brain. To probe the mechanisms that underlie MSC-mediated activation of tPA, we investigated the morphogenetic gene, sonic hedgehog (Shh) pathway. In vitro oxygen and glucose deprivation and coculture of astrocytes and MSCs were used to mimic an in vivo ischemic condition. Both real-time-PCR and western blot showed that MSC coculture significantly increased the Shh level and concomitantly increased tPA and decreased plasminogen activator inhibitor 1 (PAI-1) levels in astrocytes. Inhibiting the Shh signaling pathway with cyclopamine blocked the increase of tPA and the decrease of PAI-1 expression in astrocytes subjected to MSC coculture or recombinant mouse Shh (rm-Shh) treatment. Both MSCs and rm-Shh decreased the transforming growth factor-ß1 level in astrocytes, and the Shh pathway inhibitor cyclopamine reversed these decreases. Both Shh-small-interfering RNA (siRNA) and Glil-siRNA downregulated Shh and Gli1 (a key mediator of the Shh transduction pathway) expression in cultured astrocytes and concomitantly decreased tPA expression and increased PAI-1 expression in these astrocytes after MSC or rm-Shh treatment. Our data indicate that MSCs increase astrocytic Shh, which subsequently increases tPA expression and decreases PAI-1 expression after ischemia.

Asunto(s)

Astrocitos/metabolismo , Proteínas Hedgehog/metabolismo , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Serpina E2/biosíntesis , Accidente Cerebrovascular , Activador de Tejido Plasminógeno/biosíntesis , Animales , Astrocitos/citología , Astrocitos/patología , Técnicas de Cultivo de Célula , Hipoxia de la Célula , Técnicas de Cocultivo , Medios de Cultivo , Regulación hacia Abajo , Glucosa/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Oxígeno/metabolismo , Receptor Cross-Talk , Transducción de Señal , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patología , Regulación hacia Arriba

RESUMEN

BACKGROUND: SERPINE2, one of the potent serpins belonging to the plasminogen activator (PA) system, is involved in the tissue remodeling. We previously demonstrated the expression patterns of Serpine2 in the mouse placenta and uterus, indicating that Serpine2 is a major PA inhibitor in the placenta and uterus during the estrous cycle, pregnancy, and lactation. In this study, we further investigated the expression pattern of SERPINE2 in the human placenta and explored possible functional roles of SERPINE2 in regulating trophoblast activity. METHODS: Placental tissues from various trimesters were collected for real-time reverse-transcription polymerase chain reaction quantification. Immunohistochemical staining was performed in placental tissues to assure localization of SERPINE2. SERPINE2 small interfering (si) RNA was applied to suppress its expression in villous explants and extravillous trophoblast-like 3A cells. Subsequent experiments to evaluate SERPINE2 levels, villous outgrowth, trophoblast invasion, and tube formation were performed. RESULTS: SERPINE2 messenger RNA was detected in the human placenta during pregnancy with the highest levels in the third trimester. The SERPINE2 protein was present in villous syncytiotrophoblasts and trophoblasts of chorionic villi for anti-SERPINE2 immunostaining. Extravillous trophoblasts in the chorionic plate and basal plate confronting the invasive face of anchoring villi were also positive. In most decidual cells, SERPINE2 was observed in the cytoplasm. In addition, fibrinoid deposit was weakly immunoreactive. Introduction of SERPINE2 siRNA into villous explants and trophoblast cells led to significantly reduced villous outgrowth, and trophoblastic migration and invasion. Moreover, capillary-like network formation of 3A cells in Matrigel was greatly attenuated by SERPINE2 siRNA and SERPINE2 antiserum. CONCLUSIONS: These data identify the temporal and spatial SERPINE2 distribution in the human placenta and suggest its possible role in modulating tissue remodeling of extravillous trophoblasts in the placenta during pregnancy.

Asunto(s)

Placenta/metabolismo , Serpina E2/biosíntesis , Serpina E2/fisiología , Movimiento Celular/fisiología , Femenino , Humanos , Embarazo , Primer Trimestre del Embarazo , Segundo Trimestre del Embarazo , Tercer Trimestre del Embarazo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Serpina E2/inmunología , Trofoblastos/fisiología

RESUMEN

BACKGROUND: SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs). PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle. METHODS: Seven patients who underwent a hysterectomy and samples of 120 archived patients' endometrial curettage or parts of the uterus that were formalin-fixed and embedded in paraffin. Western blotting was performed to evaluate the specificity and sensitivity of the antibody. Immunohistochemistry was conducted to localize the SERPINE2 expression site. Quantitative analysis was conducted to evaluate expression levels of SERPINE2 in various sub-phases of the menstrual cycle. RESULTS: The SERPINE2 protein was primarily detected in the uterine fluid during the mid- and late-secretory phases of the menstrual cycle. It was predominantly expressed in the luminal and glandular epithelium, less in the myometrium, and only dispersedly in certain stromal cells throughout the menstrual cycle. A quantitative analysis of expression levels of SERPINE2 in the glandular epithelium revealed that it was highly expressed in the endometrium during the secretory phase compared to the proliferative phase. CONCLUSIONS: The SERPINE2 protein is highly expressed in the endometrium during the secretory phase, indicating that it may participate in tissue remodeling involved in implantation.

Asunto(s)

Fase Luteínica/metabolismo , Serpina E2/biosíntesis , Líquidos Corporales/química , Endometrio/metabolismo , Femenino , Humanos , Útero/química