RESUMEN
Lipid mediators such as prostaglandin E2 (PGE2) play a central role during atherogenesis as a consequence of inflammation. PGE2 is produced from phospholipids by a cascade of enzymatic reactions involving phospholipase A2 (PLA2), cyclooxygenase (COX), and prostaglandin E synthase (PGES). It is released by several cell types, including vascular smooth muscle cells (VSMCs). Recent work has shown that the secretory PLA2-IIA (sPLA2-IIA), the most abundant isoform of secreted PLA2 in VSMCs, acts as a potent cytokine and activates VSMCs through a positive feedback loop. High mobility group protein 1 (HMGB1), also known as amphoterin, is a ubiquitous protein that plays various roles in the nucleus. HMGB1 is released by necrotic cells and by immune cells in response to various inflammatory mediators and acts as a potent proinflammatory cytokine. The present study investigates the role of HMGB1 in the activation of sPLA2-IIA expression and PGE2 production in VSMCs. Recombinant HMGB1 slightly activated the sPLA2-IIA, COX-2, and mPGES-1 genes but dramatically stimulated these genes in VSMCs that had been incubated with the proinflammatory cytokine IL-1beta for 24 h. This effect was accompanied by significantly increased PGE2 release. Induction of the three known receptors of HMGB1, namely RAGE, TLR-2, and TLR-4, by IL-1beta suggests that proinflammatory cytokines sensitize VSMCs to HMGB1. This provides new insights into the role of HMGB1 in VSMCs, suggesting it may be essential for the progression of atherosclerosis.
Asunto(s)
Dinoprostona/biosíntesis , Proteína HMGB1/farmacología , Interleucina-1/farmacología , Músculo Liso Vascular/metabolismo , Fosfolipasas A/metabolismo , Animales , Aterosclerosis/etiología , Activación Enzimática , Regulación de la Expresión Génica/efectos de los fármacos , Fosfolipasas A2 Grupo II , Humanos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Fosfolipasas A2 , Ratas , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Proteínas Recombinantes , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genéticaRESUMEN
OBJECTIVE: The inflammation that occurs during the development of atherosclerosis is characterized by a massive release of sPLA2-IIA (group IIA secretory phospholipase A2) from vascular smooth muscle cells (VSMCs). We have investigated the autocrine function of sPLA2-IIA in rat aortic and human VSMCs. METHODS AND RESULTS: We found that the transcription of the endogenous sPLA2-IIA gene increased by adding a cell supernatant containing human sPLA2-IIA proteins. We show that this effect was independent of the sPLA2 activity using sPLA2-IIA proteins lacking enzyme activity. Transient transfections with various sPLA2-IIA rat promoter-luciferase constructs demonstrated that the C/EBP, NK-kappaB, and Ets transcription factors are involved in the increase in sPLA2-IIA gene transcription. We also found the M-type sPLA2 receptor mRNA in VSMCs, and we showed that the sPLA2-luciferase reporter gene was induced by the specific agonist of the sPLA2 receptor, aminophenylmannopyranoside (APMP), and that this induction was mediated by the same transcription factor-binding sites. Finally, we used a sPLA2-IIA mutant unable to bind heparan-sulfate proteoglycans to show that the binding of wild-type sPLA2-IIA to proteoglycans is essential for the induction of an autocrine loop. CONCLUSIONS: We have thus identified new autocrine and paracrine pathways activating sPLA2-IIA gene expression in rat and human VSMCs.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Músculo Liso Vascular/enzimología , Fosfolipasas A/genética , Fosfolipasas A/metabolismo , Animales , Aorta Torácica/citología , Comunicación Autocrina/fisiología , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Fosfolipasas A2 Grupo II , Humanos , Masculino , Músculo Liso Vascular/citología , FN-kappa B/metabolismo , Comunicación Paracrina/fisiología , Fosfolipasas A2 , Unión Proteica , Proteoglicanos/metabolismo , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Fosfolipasa A2 , Activación Transcripcional/fisiología , Factores de Transcripción Winged-Helix/metabolismoRESUMEN
The inflammation that occurs during rheumatoid arthritis or atherosclerosis is characterized by the release of large amounts of sPLA(2) (group IIA secretory phospholipase A(2)). We have shown previously that the sPLA(2) promoter in SMC (smooth-muscle cells) is activated by interleukin-1beta and cAMP-signalling pathways, through the interplay of multiple transcription factors [Antonio, Brouillet, Janvier, Monne, Bereziat, Andreani, and Raymondjean (2002) Biochem. J. 368, 415-424]. In the present study, we have investigated the regulation of sPLA(2) gene expression in rat aortic SMCs by oxysterols. We found that oxysterol ligands that bind to the LXR (liver X receptor), including 25-HC (25-hydroxycholesterol) and 22( R )-HC, cause the accumulation of sPLA(2) mRNA and an increased enzyme activity. Transient transfection experiments demonstrated that the sPLA(2) promoter is synergistically activated by 22( R )-HC in combination with 9- cis -retinoic acid, a ligand for the LXR heterodimeric partner RXR (retinoid X receptor). Promoter activity was also increased in a sterol-responsive fashion when cells were co-transfected with LXRalpha/RXRalpha or LXRbeta/RXRalpha. Mutagenesis studies and gel mobility-shift assays revealed that LXR/RXR heterodimers regulate sPLA(2) transcription directly, by interacting with a degenerated LXRE (LXR response element) at position [-421/-406] of the sPLA(2) promoter. Chromatin immunoprecipitation revealed the in vivo occupancy of LXR on the sPLA(2) promoter. In addition, the orphan nuclear receptor LRH-1 (liver receptor homologue-1) potentiated the sterol-dependent regulation of the sPLA(2) promoter by binding to an identified promoter element (TCAAGGCTG). Finally, we have demonstrated that oxysterols act independent of interleukin-1beta and cAMP pathways to activate the sPLA(2) promoter. In the present study, we have identified a new pathway activating sPLA(2) gene expression in SMCs.
Asunto(s)
Hidroxicolesteroles/farmacología , Músculo Liso Vascular/enzimología , Fosfolipasas A/genética , Activación Transcripcional , Tretinoina/farmacología , Alitretinoína , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas de Unión al ADN , Inducción Enzimática , Fosfolipasas A2 Grupo II , Interleucina-1/farmacología , Receptores X del Hígado , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Receptores Nucleares Huérfanos , Fosfolipasas A/biosíntesis , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Regiones Promotoras Genéticas , ARN Mensajero/biosíntesis , Ratas , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Ácido Retinoico/metabolismo , Elementos de Respuesta , Receptores X Retinoide , Factores de Transcripción/metabolismoRESUMEN
The abundant secretion of type IIA secreted phospholipase A(2) (sPLA(2)) is a major feature of the inflammatory process of atherosclerosis. sPLA(2) is crucial for the development of inflammation, as it catalyses the production of lipid mediators and induces the proliferation of smooth muscle cells. We have analysed the activation of sPLA(2) transcription by cAMP and interleukin-1beta (IL-1beta), and shown that the 500 bp region upstream of the transcription start site of the rat sPLA(2) gene is implicated in activation by synergistically acting cAMP and IL-1beta. We transiently transfected and stimulated rat smooth muscle cells in primary culture and measured the promoter activities of serial and site-directed deletion mutants of sPLA(2)-luciferase constructs. A distal region, between -488 and -157 bp, bearing a CAAT/enhancer binding protein (C/EBP)-responsive element (-242 to -223) was sufficient for cAMP/protein kinase A-mediated sPLA(2) promoter activation. We find evidence for the first time that activation of the sPLA(2) promoter by IL-1beta requires activation of an Ets-responsive element in the -184 to -180 region of the distal promoter via the Ras pathway and a nuclear factor-kappaB site at positions -141 to -131 of the proximal promoter. We also used electrophoretic mobility shift assays to identify five binding sites for the Sp1 factor; a specific inhibitor of Sp1, mithramycin A, showed that this factor is crucial for the basal activity of the sPLA(2) promoter.