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1.
J Cell Sci ; 133(13)2020 07 09.
Artículo en Inglés | MEDLINE | ID: mdl-32482794

RESUMEN

Arterial remodeling in hypertension and intimal hyperplasia involves inflammation and disrupted flow, both of which contribute to smooth muscle cell dedifferentiation and proliferation. In this context, our previous results identified phosphoinositide 3-kinase γ (PI3Kγ) as an essential factor in inflammatory processes of the arterial wall. Here, we identify for the first time a kinase-independent role of nonhematopoietic PI3Kγ in the vascular wall during intimal hyperplasia using PI3Kγ-deleted mice and mice expressing a kinase-dead version of the enzyme. Moreover, we found that the absence of PI3Kγ in vascular smooth muscle cells (VSMCs) leads to modulation of cell proliferation, associated with an increase in intracellular cAMP levels. Real-time analysis of cAMP dynamics revealed that PI3Kγ modulates the degradation of cAMP in primary VSMCs independently of its kinase activity through regulation of the enzyme phosphodiesterase 4. Importantly, the use of an N-terminal competing peptide of PI3Kγ blocked primary VSMC proliferation. These data provide evidence for a kinase-independent role of PI3Kγ in arterial remodeling and reveal novel strategies targeting the docking function of PI3Kγ for the treatment of cardiovascular diseases.


Asunto(s)
Fosfatidilinositol 3-Quinasa , Fosfatidilinositol 3-Quinasas , Animales , Arterias , Proliferación Celular , Ratones , Miocitos del Músculo Liso , Fosfatidilinositol 3-Quinasas/genética
2.
Biochim Biophys Acta Mol Cell Res ; 1865(9): 1326-1340, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29940197

RESUMEN

Here, we cloned a new family of four adenylyl cyclase (AC) splice variants from interleukin-1ß (IL-1ß)-transdifferentiated vascular smooth muscle cells (VSMCs) encoding short forms of AC8 that we have named "AC8E-H". Using biosensor imaging and biochemical approaches, we showed that AC8E-H isoforms have no cyclase activity and act as dominant-negative regulators by forming heterodimers with other full-length ACs, impeding the traffic of functional units towards the plasma membrane. The existence of these dominant-negative isoforms may account for an unsuspected additional degree of cAMP signaling regulation. It also reconciles the induction of an AC in transdifferentiated VSMCs with the vasoprotective influence of cAMP. The generation of alternative splice variants of ACs may constitute a generalized strategy of adaptation to the cell's environment whose scope had so far been ignored in physiological and/or pathological contexts.


Asunto(s)
Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Empalme Alternativo , AMP Cíclico/metabolismo , Interleucina-1beta/farmacología , Músculo Liso Vascular/citología , Adenilil Ciclasas/química , Animales , Transdiferenciación Celular , Células Cultivadas , Clonación Molecular , Retículo Endoplásmico Rugoso/metabolismo , Células HEK293 , Humanos , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Multimerización de Proteína , Ratas
3.
FASEB J ; 32(9): 4972-4983, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29620941

RESUMEN

Cerebral amyloid angiopathy (CAA) is a major contributor to Alzheimer's disease (AD) pathogenesis. Like AD, CAA is often accompanied by marked inflammation, aggravating associated vasculopathies. No evidence-based prevention or treatment strategies are available. Here, we evaluate the possible beneficial effect of a diet enriched with docosahexaenoic acid (DHA), which is known to attenuate inflammation in CAA. Tg2576 mice, a transgenic model of AD/CAA, were fed a DHA-enriched diet starting at 2 mo of age and ending at 10, 14, or 18 mo of age. ß-Amyloid (Aß)-peptide deposition and bleeding were visualized by immunohistochemistry or histochemistry on coronal sections of the brain. DHA, arachidonic acid, and eicosanoid levels were measured by liquid chromatography/mass spectrometry or GC-MS. DHA-enriched diet throughout aging limits the accumulation of vascular Aß peptide deposits as well as the likelihood of microhemorrhages. There is a strong correlation between systemic 12-hydroxyeicosatetraenoic acid (HETE) levels and the size of the area affected by both vascular amyloid deposits and hemorrhages. The lowest levels of 12-HETE, a lipid-derived proinflammatory product of 12-lipoxygenase (LOX), were found in DHA-fed mice. In vitro experiments performed on amyloid vascular smooth muscle cells showed that a 12-LOX inhibitor almost completely blocked the Aß1-40 peptide-induced apoptosis of these cells. This study yet again highlights the important role of inflammation in CAA pathogenesis and identifies potential new targets for preventive care.-Hur, J., Mateo, V., Amalric, N., Babiak, M., Béréziat, G., Kanony-Truc, C., Clerc, T., Blaise, R., Limon, I. Cerebrovascular ß-amyloid deposition and associated microhemorrhages in a Tg2576 Alzheimer mouse model are reduced with a DHA-enriched diet.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Dieta/efectos adversos , Ácidos Docosahexaenoicos/farmacología , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Angiopatía Amiloide Cerebral/tratamiento farmacológico , Modelos Animales de Enfermedad , Ratones Transgénicos , Miocitos del Músculo Liso/metabolismo
4.
Biochim Biophys Acta ; 1853(12): 3235-47, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26403276

RESUMEN

Atherosclerosis development is associated with morphological changes to intimal cells, leading to a stellate cell phenotype. In this study, we aimed to determine whether and how key pro-atherogenic cytokines present in atherosclerotic plaques (IL-1ß, TNFα and IFNγ) could induce this phenotype, as these molecules are known to trigger the transdifferentiation of vascular smooth muscle cells (VSMCs). We found that, IL-1ß was the only major inflammatory mediator tested capable of inducing a stellate morphology in VSMCs. This finding was confirmed by staining for F-actin and vinculin at focal adhesions, as these two markers were disrupted only by IL-1ß. We then investigated the possible association of this IL-1ß-dependent change in morphology with an increase in intracellular cAMP concentration ([cAMP]), using the FRET-based biosensor for cAMP (T)Epac(VV). Experiments in the presence of IL-1ß or medium conditioned by IL-1ß-treated VSMCs and pharmacological tools demonstrated that the long-term increase in intracellular cAMP concentration was induced by the secretion of an autocrine/paracrine mediator, prostaglandin E2(PGE2), acting through the EP4 receptor. Finally, by knocking down the expression of the regulatory subunit PKAR1α, thereby reproducing the effects of IL-1ß and PGE2 on VSMCs, we demonstrated the contribution of PKA activity to the observed behavior of VSMCs.


Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Interleucina-1beta/metabolismo , Músculo Liso Vascular/citología , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Activación Enzimática , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Ratas
5.
Biochim Biophys Acta ; 1843(11): 2705-18, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25110346

RESUMEN

UNLABELLED: The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. IN CONCLUSION: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

6.
Biochim Biophys Acta Mol Cell Res ; 1871(2): 119645, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38016490

RESUMEN

Adenylyl Cyclase 8E (AC8E), which lacks part of M1 transmembrane domain, has been previously shown to dimerize with AC3 and down-regulate its activity, but the molecular mechanism of this inhibitory effect has remained elusive. Here, we first show that AC8E also inhibits AC2 and AC6, highlighting the functional importance of this novel regulatory mechanism in the cAMP signaling pathway across AC families. We then completed the partial structure of Bos taurus AC9 using combinations of comparative modeling and fold recognition methods, and used this as a template to build the first full 3D-models of AC8 and AC8E. These models evidenced that the lack of M1 transmembrane domain of AC8E shifts the N-terminal domain, which impacts the orientation of the helical domains, thus affecting the catalytic site. This was confirmed in living cells with cAMP imaging, where we showed that the N-terminal domain is required for reducing cAMP production. Our data also show that AC8E prevents the translocation of other ACs towards the plasma membrane, further reducing the cAMP responsiveness to extracellular signals. This newly discovered dual inhibitory mechanism provides an additional level of regulation of cAMP-dependent signals integration.


Asunto(s)
Adenilil Ciclasas , AMP Cíclico , Humanos , Animales , Bovinos , Adenilil Ciclasas/química , AMP Cíclico/metabolismo , Transducción de Señal , Dominio Catalítico , Membrana Celular/metabolismo
7.
J Biol Chem ; 287(30): 24978-89, 2012 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-22613711

RESUMEN

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1ß on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1ß-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1ß-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1ß, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.


Asunto(s)
Adenilil Ciclasas/biosíntesis , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Interleucina-1beta/farmacología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Adenilil Ciclasas/genética , Animales , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/patología , Transdiferenciación Celular , Modelos Animales de Enfermedad , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Masculino , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Ratas , Ratas Wistar , Receptores Notch/genética , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Transducción de Señal/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética
8.
Mech Dev ; 122(4): 573-85, 2005 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15804569

RESUMEN

Mice deficient for the homeobox gene Six1 display defects in limb muscles consistent with the Six1 expression in myogenic cells. In addition to its myogenic expression domain, Six1 has been described as being located in digit tendons and as being associated with connective tissue patterning in mouse limbs. With the aim of determining a possible involvement of Six1 in tendon development, we have carefully characterised the non-myogenic expression domain of the Six1 gene in mouse and chick limbs. In contrast to previous reports, we found that this non-myogenic domain is distinct from tendon primordia and from tendons defined by scleraxis expression. The non-myogenic domain of Six1 expression establishes normally in the absence of muscle, in Pax3-/- mutant limbs. Moreover, the expression of scleraxis is not affected in early Six1-/- mutant limbs. We conclude that the expression of the Six1 gene is not related to tendons and that Six1, at least on its own, is not involved in limb tendon formation in vertebrates. Finally, we found that the posterior domain of Six1 in connective tissue is adjacent to that of the secreted factor Sonic hedgehog and that Sonic hedgehog is necessary and sufficient for Six1 expression in posterior limb regions.


Asunto(s)
Tejido Conectivo/embriología , Tejido Conectivo/metabolismo , Extremidades/embriología , Proteínas de Homeodominio/metabolismo , Tendones/embriología , Transactivadores/metabolismo , Animales , Proteínas Aviares , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Tendones/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/genética
9.
J Vis Exp ; (116)2016 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-27805576

RESUMEN

The cerebral arterial circle (circulus arteriosus cerebri) or circle of Willis (CoW) is a circulatory anastomosis surrounding the optic chiasma and hypothalamus that supplies blood to the brain and surrounding structures. It has been implicated in several cerebrovascular disorders, including cerebral amyloid angiopathy (CAA)-associated vasculopathies, intracranial atherosclerosis and intracranial aneurysms. Studies of the molecular mechanisms underlying these diseases for the identification of novel drug targets for their prevention require animal models. Some of these models may be transgenic, whereas others will involve isolation of the cerebro-vasculature, including the CoW.The method described here is suitable for CoW isolation in any mouse lineage and has considerable potential for screening (expression of genes, protein production, posttranslational protein modifications, secretome analysis, etc.) studies on the large vessels of the mouse cerebro-vasculature. It can also be used for ex vivo studies, by adapting the organ bath system developed for isolated mouse olfactory arteries.


Asunto(s)
Círculo Arterial Cerebral , Aneurisma Intracraneal , Animales , Animales Modificados Genéticamente , Encéfalo , Circulación Cerebrovascular , Ratones , Técnicas de Cultivo de Tejidos
10.
Biol Aujourdhui ; 210(3): 153-166, 2016.
Artículo en Francés | MEDLINE | ID: mdl-27813476

RESUMEN

In response to various types of vascular stress, the smooth muscle cells of the vessel wall (VSMCs) change phenotype and acquire the capacity to react to abnormal signals. This phenomenon favors the involvement of these cells in the development of major vascular diseases, such as atherosclerosis, and some complications of angioplasty, such as restenosis. The cyclic adenosine monophosphate (cAMP) pathway plays a key role in the integration of stimuli from the immediate environment and in the development of cellular responses. The temporal and spatial subcellular compartmentalization of cAMP ensures that the signals transmitted are specific. This compartmentalization is dependent on the diversity of (1) proteins directly or indirectly regulating the synthesis, degradation or release of cAMP; (2) intracellular effectors of cAMP; (3) isoforms of all these proteins with unique biochemical properties and unique patterns of regulation and (4) the scaffolding proteins on which the macromolecular complexes are built. This review illustrates the ways in which changes in the profile of adenylyl cyclases (ACs) may play critical roles in signal integration, the response of muscle cells and pathological vascular remodeling. It also illustrates the relevance of the renewed consideration of ACs as potentially interesting treatment targets.


Asunto(s)
Adenilil Ciclasas/fisiología , Transdiferenciación Celular , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/fisiología , Remodelación Vascular/fisiología , Animales , AMP Cíclico/metabolismo , AMP Cíclico/fisiología , Humanos , Terapia Molecular Dirigida/tendencias , Transducción de Señal
11.
Aging Cell ; 12(3): 358-69, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23425004

RESUMEN

Several studies have shown that the accumulation of ß-amyloid peptides in the brain parenchyma or vessel wall generates an inflammatory environment. Some even suggest that there is a cause-and-effect relationship between inflammation and the development of Alzheimer's disease and/or cerebral amyloid angiopathy (CAA). Here, we studied the ability of wild-type Aß1-40 -peptide (the main amyloid peptide that accumulates in the vessel wall in sporadic forms of CAA) to modulate the phenotypic transition of vascular smooth muscle cells (VSMCs) toward an inflammatory/de-differentiated state. We found that Aß1-40 -peptide alone neither induces an inflammatory response, nor decreases the expression of contractile markers; however, the inflammatory response of VSMCs exposed to Aß1-40 -peptide prior to the addition of the pro-inflammatory cytokine IL-1ß is greatly intensified compared with IL-1ß-treated VSMCs previously un-exposed to Aß1-40 -peptide. Similar conclusions could be drawn when tracking the decline of contractile markers. Furthermore, we found that the mechanism of this potentiation highly depends on an Aß1-40 preactivation of the PI3 Kinase and possibly NFκB pathway; indeed, blocking the activation of these pathways during Aß1-40 -peptide treatment completely suppressed the observed potentiation. Finally, strengthening the possible in vivo relevance of our findings, we evidenced that endothelial cells exposed to Aß1-40 -peptide generate an inflammatory context and have similar effects than the ones described with IL-1ß. These results reinforce the idea that intraparietal amyloid deposits triggering adhesion molecules in endothelial cells, contribute to the transition of VSMCs to an inflammatory/de-differentiated phenotype. Therefore, we suggest that acute inflammatory episodes may increase vascular alterations and contribute to the ontogenesis of CAA.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/irrigación sanguínea , Desdiferenciación Celular , Inflamación/inmunología , Músculo Liso Vascular/citología , Músculo Liso Vascular/inmunología , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer , Animales , Células Cultivadas , Angiopatía Amiloide Cerebral/inmunología , Medios de Cultivo Condicionados , Activación Enzimática , Interleucina-1beta/farmacología , Ratones , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo
12.
Aging Cell ; 11(3): 384-93, 2012 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-22260497

RESUMEN

Cerebral amyloid angiopathy (CAA) is an important cause of intracerebral hemorrhages in the elderly, characterized by amyloid-ß (Aß) peptide accumulating in central nervous system blood vessels. Within the vessel walls, Aß-peptide deposits [composed mainly of wild-type (WT) Aß(1-40) peptide in sporadic forms] induce impaired adhesion of vascular smooth muscle cells (VSMCs) to the extracellular matrix (ECM) associated with their degeneration. This process often results in a loss of blood vessel wall integrity and ultimately translates into cerebral ischemia and microhemorrhages, both clinical features of CAA. In this study, we decipher the molecular mechanism of matrix metalloprotease (MMP)-2 activation in WT-Aß(1-40) -treated VSMC and provide evidence that MMP activity, although playing a critical role in cell detachment disrupting ECM components, is not involved in the WT-Aß(1-40) -induced degeneration of VSMCs. Indeed, whereas this peptide clearly induced VSMC apoptosis, neither preventing MMP-2 activity nor hampering the expression of membrane type1-MMP, or preventing tissue inhibitors of MMPs-2 (TIMP-2) recruitment (two proteins evidenced here as involved in MMP-2 activation), reduced the number of dead cells. Even the use of broad-range MMP inhibitors (GM6001 and Batimastat) did not affect WT-Aß(1-40) -induced cell apoptosis. Our results, in contrast to those obtained using the Aß(1-40) Dutch variant suggesting a link between MMP-2 activity, VSMC mortality and degradation of specific matrix components, indicate that the ontogenesis of the Dutch familial and sporadic forms of CAAs is different. ECM degradation and VSMC degeneration would be tightly connected in the Dutch familial form while being two independent processes in sporadic forms of CAA.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Angiopatía Amiloide Cerebral/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Músculo Liso Vascular/citología , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/fisiología , Muerte Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Angiopatía Amiloide Cerebral/enzimología , Angiopatía Amiloide Cerebral/patología , Humanos , Masculino , Datos de Secuencia Molecular , Músculo Liso Vascular/enzimología , Músculo Liso Vascular/metabolismo , Ratas , Ratas Wistar , Transfección
13.
Biochem Pharmacol ; 79(3): 407-12, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19728989

RESUMEN

The alpha(2B)-adrenoceptor (alpha(2B)-AR) mediates vasoconstriction and a common polymorphism (+901 Ins/Del), located in the coding region of the human alpha(2B)-AR gene (ADRA2B), has been demonstrated to affect receptor function in vitro. In this study, we have identified a novel polymorphism corresponding to the insertion of 12-nucleotides (GGGACGGCCCTG) at position -4825 relative to the start codon (-4825 del/ins) in the far upstream region of the ADRA2B promoter. The genotyping of 71 unrelated Finnish individuals showed that the -4825 ins polymorphism is common and in complete linkage with the Del polymorphism at position +901 and a G/C substitution at position -98. Transfection of various cell lines with luciferase constructs containing a 5.5 kb fragment of the ADRA2B promoter region indicated that the 12-nucleotide insertion at -4825 resulted in a large reduction of transcriptional activity. Electrophoretic mobility shift assays with oligonucleotide probes corresponding to the two ADRA2B alleles demonstrated that the region where the -4825 del/ins variation occurs binds nuclear proteins and that the 12-nucleotide insertion affects the pattern of bound transcription factors. Altogether, the present findings show that the previously identified +901 Del polymorphism is linked with a variation in the ADRA2B promoter that affects transcriptional activity in vitro. The molecular mechanisms underlying this effect are still unclear but a possible impact of the -4825 ins polymorphism on alpha(2B)-AR expression would merit to be examined in vivo as a diminution of promoter activity may limit the functional consequences of the +901 Del polymorphism.


Asunto(s)
Ligamiento Genético/genética , Polimorfismo Genético/genética , Regiones Promotoras Genéticas/genética , Receptores Adrenérgicos alfa 2/genética , Activación Transcripcional/genética , Animales , Secuencia de Bases , Línea Celular , Línea Celular Tumoral , Cricetinae , Eliminación de Gen , Células HeLa , Humanos , Masculino , Datos de Secuencia Molecular , Mutagénesis Insercional/métodos , Transcripción Genética/genética
14.
Arthritis Rheum ; 60(2): 428-39, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19180482

RESUMEN

OBJECTIVE: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.


Asunto(s)
Desdiferenciación Celular/genética , Condrocitos/fisiología , Metaloproteinasa 13 de la Matriz/genética , Receptor Notch1/genética , Animales , Biomarcadores/metabolismo , Cartílago Articular/citología , Células Cultivadas , Condrocitos/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Regulación Enzimológica de la Expresión Génica , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , ARN Mensajero/metabolismo , Receptor Notch1/biosíntesis , Transducción de Señal
15.
J Cell Sci ; 120(Pt 19): 3352-61, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17881497

RESUMEN

Atherogenesis begins with the transfer of monocytes from the lumen to the intimal layer of arteries. The paracrine activity acquired by these monocytes shifts vascular smooth muscle cells from a contractile-quiescent to a secretory-proliferative phenotype, allowing them to survive and migrate in the intima. Transformed and relocated, they also start to produce and/or secrete inflammatory enzymes, converting them into inflammatory cells. Activation of the Notch pathway, a crucial determinant of cell fate, regulates some of the new features acquired by these cells as it triggers vascular smooth muscle cells to grow and inhibits their death and migration. Here, we evaluate whether and how the Notch pathway regulates the cell transition towards an inflammatory or de-differentiated state. Activation of the Notch pathway by the notch ligand Delta1, as well as overexpression of the active form of Notch3, prevents this phenomenon [initiated by interleukin 1beta (IL-1beta)], whereas inhibiting the Notch pathway enhances the transition. IL-1beta decreases the expression of Notch3 and Notch target genes. As shown by using an IkappaBalpha-mutated form, the decrease of Notch3 signaling elements occurs subsequent to dissociation of the NF-kappaB complex. These results demonstrate that the Notch3 pathway is attenuated through NF-kappaB activation, allowing vascular smooth muscle cells to switch into an inflammatory state.


Asunto(s)
Inflamación/metabolismo , Interleucina-1beta/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/fisiología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Aorta/anatomía & histología , Aorta/metabolismo , Biomarcadores/metabolismo , Comunicación Celular/fisiología , Células Cultivadas , Dinoprostona/metabolismo , Regulación de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Interleucina-1beta/genética , Masculino , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/citología , Fosfolipasas A2/metabolismo , Ratas , Ratas Wistar , Receptor Notch3 , Receptores Notch/genética
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