RESUMEN
Epigenetic modulators, including histone methylases, demethylases, and deacetylases, have been implicated previously in the regulation of classical and alternative macrophage activation pathways. In this study, we show that the histone acetyl transferase (HAT) Kat6B (MYST4) is strongly suppressed (>80%) in macrophages by lipopolysaccharide (LPS) (M1 activation), while Kat6A, its partner in the MOZ/MORF complex, is reciprocally upregulated. This pattern of expression is not altered by LPS together with the adenosine receptor agonist NECA (M2d activation). This is despite the observation that miR-487b, a putative regulator of Kat6B expression, is mildly stimulated by LPS, but strongly suppressed by LPS/NECA. Other members of the MYST family of HATs (Kat5, Kat7, and Kat8) are unaffected by LPS treatment. Using the pLightswitch 3'UTR reporter plasmid, the miR-487b binding site in the Kat6b 3'UTR was found to play a role in the LPS-mediated suppression of Kat6B expression, but other as-yet unidentified factors are also involved. As Kat6B is a HAT that has the potential to modulate gene expression by its effects on chromatin accessibility, we are continuing our studies into the potential roles of this epigenetic modulator in macrophage activation pathways.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Western Blotting , Lisina Acetiltransferasa 5/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Transactivadores/metabolismoRESUMEN
We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cß-2 (PLCß-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCß-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCß-2 mRNA stability. The 3'UTR of the PLCß-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCß-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCß-2 by LPS was examined using macrophages from mice lacking TTP (TTP-/-). LPS suppressed PLCß-2 expression to the same extent in wild type (WT) and TTP-/- macrophages. Also, the rate of decay of PLCß-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP-/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCß-2 mRNA in macrophages.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato/fisiología , Macrófagos/metabolismo , Fosfolipasa C beta/genética , Estabilidad del ARN/efectos de los fármacos , Tristetraprolina/fisiología , Regiones no Traducidas 3'/genética , Animales , Células Cultivadas , Diabetes Mellitus Experimental , Lipopolisacáridos/farmacología , Ratones , Células RAW 264.7 , Proteínas de Unión al ARNRESUMEN
Murine macrophages are activated by interferon-γ (IFN-γ) and/or Toll-like receptor (TLR) agonists such as bacterial endotoxin (lipopolysaccharide [LPS]) to express an inflammatory (M1) phenotype characterized by the expression of nitric oxide synthase-2 (iNOS) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-12. In contrast, Th2 cytokines IL-4 and IL-13 activate macrophages by inducing the expression of arginase-1 and the anti-inflammatory cytokine IL-10 in an IL-4 receptor-α (IL-4Rα)-dependent manner. Macrophages activated in this way are designated as "alternatively activated" (M2a) macrophages. We have shown previously that adenosine A2A receptor (A(2A)R) agonists act synergistically with TLR2, TLR4, TLR7, and TLR9 agonists to switch macrophages into an "M2-like" phenotype that we have termed "M2d." Adenosine signaling suppresses the TLR-dependent expression of TNF-α, IL-12, IFN-γ, and several other inflammatory cytokines by macrophages and induces the expression of vascular endothelial growth factor (VEGF) and IL-10. We show here using mice lacking a functional IL-4Rα gene (IL-4Rα(-/-) mice) that this adenosine-mediated switch does not require IL-4Rα-dependent signaling. M2d macrophages express high levels of VEGF, IL-10, and iNOS, low levels of TNF-α and IL-12, and mildly elevated levels of arginase-1. In contrast, M2d macrophages do not express Ym1, Fizz1 (RELM-α), or CD206 at levels greater than those induced by LPS, and dectin-1 expression is suppressed. The use of these markers in vivo to identify "M2" macrophages thus provides an incomplete picture of macrophage functional status and should be viewed with caution.
Asunto(s)
Adenosina/metabolismo , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Neovascularización Fisiológica/inmunología , Agonistas del Receptor Purinérgico P1/farmacología , Receptor de Adenosina A2A/metabolismo , Adenosina/farmacología , Animales , Arginasa/biosíntesis , Diferenciación Celular , Células Cultivadas , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Subunidad alfa del Receptor de Interleucina-4/genética , Lectinas/biosíntesis , Lectinas Tipo C/biosíntesis , Macrófagos/efectos de los fármacos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Receptores de Superficie Celular/biosíntesis , Transducción de Señal , Factor de Necrosis Tumoral alfa/biosíntesis , Factor A de Crecimiento Endotelial Vascular/biosíntesis , beta-N-Acetilhexosaminidasas/biosíntesisRESUMEN
The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium-endothelium-epicardium). We previously described that Id1Id3 double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early lethality precluded the studies of the roles of Id in the postnatal heart. To elucidate postnatal roles of Id genes, we ablated the Id3 gene and conditionally ablated the Id1 gene in the endothelium to generate conditional KO (cKO) embryos. We observed cardiac phenotypes at birth and at 6 months of age. Half of the Id cKO mice died at birth. Postnatal demise was associated with cardiac enlargement and defects in the ventricular septum, trabeculation and vasculature. Surviving Id cKO mice exhibited fibrotic vasculature, cardiac enlargement and decreased cardiac function. An abnormal vascular response was also observed in the healing of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Thus, ablation of Id genes in the vasculature leads to distinct postnatal cardiac phenotypes. These findings provide important insights into the role/s of the endocardial network of the endothelial lineage in the development of cardiac disease, and highlight IGFbp3 as a potential link between Id and its vascular effectors.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Cardiopatías/metabolismo , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Proteínas Inhibidoras de la Diferenciación/metabolismo , Miocardio/metabolismo , Animales , Biomarcadores , Linaje de la Célula , Células Endoteliales/citología , Células Endoteliales/metabolismo , Perfilación de la Expresión Génica , Cardiopatías/patología , Proteína 1 Inhibidora de la Diferenciación/deficiencia , Proteínas Inhibidoras de la Diferenciación/deficiencia , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Ratones Noqueados , Miocardio/citología , Fenotipo , Cicatrización de HeridasRESUMEN
Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.
Asunto(s)
Blastocisto , Células Madre Embrionarias/trasplante , Terapia Genética/métodos , Distrofia Muscular Animal/terapia , Animales , Quimera , Distrofina/genética , Distrofina/fisiología , Proteínas Asociadas a la Distrofina/análisis , Transferencia de Embrión , Femenino , Operón Lac , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Microinyecciones , Músculo Esquelético/química , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Animal/embriología , Distrofia Muscular Animal/patología , Distrofia Muscular Animal/fisiopatología , Distrofia Muscular de Duchenne , RegeneraciónRESUMEN
Synergy between Toll-like receptor (TLR) and adenosine A2A receptor (A2AR) signaling switches macrophages from production of inflammatory cytokines such as tumor necrosis factor-alpha to production of the angiogenic growth factor vascular endothelial growth factor (VEGF). We show in this study that this switch critically requires signaling through MyD88, IRAK4, and TRAF6. Macrophages from mice lacking MyD88 (MyD88(-/-)) or IRAK4 (IRAK4(-/-)) lacked responsiveness to TLR agonists and did not respond to A2AR agonists by expressing VEGF. Suppression of TRAF6 expression with siRNA in RAW264.7 macrophages also blocked their response to TLR and A2AR agonists. Excisional skin wounds in MyD88(-/-) mice healed at a markedly slower rate than wounds in wild-type MyD88(+/+) mice, showing delayed contraction, decreased and delayed granulation tissue formation, and reduced new blood vessel density. Although macrophages accumulated to higher levels in MyD88(-/-) wounds than in controls, expression of VEGF and HIF1-alpha mRNAs was elevated in MyD88(+/+) wounds. CGS21680, an A2AR agonist, promoted repair in MyD88(+/+) wounds and stimulated angiogenesis but had no significant effect on healing of MyD88(-/-) wounds. These results suggest that the synergistic interaction between TLR and A(2A)R signaling observed in vitro that switches macrophages from an inflammatory to an angiogenic phenotype also plays a role in wound healing in vivo.
Asunto(s)
Macrófagos/inmunología , Factor 88 de Diferenciación Mieloide/fisiología , Neovascularización Fisiológica/inmunología , Receptor de Adenosina A2A/metabolismo , Cicatrización de Heridas/inmunología , Adenosina/análogos & derivados , Adenosina/farmacología , Agonistas del Receptor de Adenosina A2 , Animales , Línea Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Quinasas Asociadas a Receptores de Interleucina-1/genética , Macrófagos/efectos de los fármacos , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Neovascularización Fisiológica/genética , Fenetilaminas/farmacología , ARN Mensajero/análisis , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Factor 6 Asociado a Receptor de TNF/antagonistas & inhibidores , Factor 6 Asociado a Receptor de TNF/genética , Receptores Toll-Like/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Under normoxic conditions, macrophages from C57BL mice produce low levels of vascular endothelial growth factor (VEGF). Hypoxia stimulates VEGF expression by approximately 500%; interferon-gamma (IFN-gamma) with endotoxin [lipopolysaccharide (LPS)] also stimulates VEGF expression by approximately 50 to 150% in an inducible nitric oxide synthase (iNOS)-dependent manner. Treatment of normoxic macrophages with 5'-N-ethyl-carboxamido-adenosine (NECA), a nonselective adenosine A(2) receptor agonist, or with 2-[p-(2-carboxyethyl)-phenylethyl amino]-5'-N-ethyl-carboxamido-adenosine (CGS21680), a specific adenosine A(2A) receptor agonist, modestly increases VEGF expression, whereas 2-chloro-N(6)-cyclopentyl adenosine (CCPA), an adenosine A(1) agonist, does not. Treatment with LPS (0 to 1000 ng/ml), or with IFN-gamma (0 to 300 U/ml), does not affect VEGF expression. In the presence of LPS (EC(50) < 10 ng/ml), but not of IFN-gamma, both NECA and CGS21680 synergistically up-regulate VEGF expression by as much as 10-fold. This VEGF is biologically active in vivo in the rat corneal bioassay of angiogenesis. Inhibitors of iNOS do not affect this synergistic induction of VEGF, and macrophages from iNOS-/- mice produce similar levels of VEGF as wild-type mice, indicating that NO does not play a role in this induction. Under hypoxic conditions, VEGF expression is slightly increased by adenosine receptor agonists but adenosine A(2) or A(1) receptor antagonists 3,7-dimethyl-1-propargyl xanthine (DMPX), ZM241385, and 8-cyclopentyl-1,3-dipropylxanthine (DCPCX) do not modulate VEGF expression. VEGF expression is also not reduced in hypoxic macrophages from A(3)-/- and A(2A)-/- mice. Thus, VEGF expression by hypoxic macrophages does not seem to depend on endogenously released or exogenous adenosine. VEGF expression is strongly up-regulated by LPS/NECA in macrophages from A(3)-/- but not A(2A)-/- mice, confirming the role of adenosine A(2A) receptors in this pathway. LPS with NECA strongly up-regulates VEGF expression by macrophages from C(3)H/HeN mice (with intact Tlr4 receptors), but not by macrophages from C(3)H/HeJ mice (with mutated, functionally inactive Tlr4 receptors), implicating signaling through the Tlr4 pathway in this synergistic up-regulation. Finally, Western blot analysis of adenosine A(2A) receptor expression indicated that the synergistic interaction of LPS with A(2A) receptor agonists does not involve up-regulation of A(2A) receptors by LPS. These results indicate that in murine macrophages there is a novel pathway regulating VEGF production, that involves the synergistic interaction of adenosine A(2A) receptor agonists through A(2A) receptors with LPS through the Tlr4 pathway, resulting in the strong up-regulation of VEGF expression by macrophages in a hypoxia- and NO-independent manner.