Sustained release of prostaglandin E2 in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca2+-mobilization

The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2is involved in the regulation of P2Y signaling in these cells.This work was supported by Grants BFU2011-24760 and BFU2011-24743 from MINECO, S2010/BMD-2378 from Comunidad de Madrid, Red de Investigación Cardiovascular, RIC, RD12/0042/0019, and Fundación Marcelino Botín (to María Teresa Miras-Portugal). RIC and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Sustained release of prostaglandin E2 in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca2+-mobilization

The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2is involved in the regulation of P2Y signaling in these cells.This work was supported by Grants BFU2011-24760 and BFU2011-24743 from MINECO, S2010/BMD-2378 from Comunidad de Madrid, Red de Investigación Cardiovascular, RIC, RD12/0042/0019, and Fundación Marcelino Botín (to María Teresa Miras-Portugal). RIC and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Crosstalk between signal transduction and metabolism in macrophages: metabolic effects of MEK inhibition

Resumen del trabajo presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. Here, the metabolic profile associated with the MEK/ERK cascade in macrophages was studied and the effect of its inhibition on the specific metabolic pattern of lipopolysaccharide (LPS) stimulation was characterized. A [1,2-13C2]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production, but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate (Fru-2,6-P2) concentration. The oxidative and non-oxidative pentose phosphate pathways (PPP) and the ratio between them also decreased. However, tricarboxylic acid (TCA) cycle flux did not change significantly. LPS activation led to the opposite responses, although all these were suppressed by PD325901. However, LPS also induced a small decrease in PPP fluxes and an increase in glutamine consumption that were not affected by PD325901. We conclude that inhibition of the ERK1/2 cascade interferes with central metabolism, and this crosstalk between signal transduction and metabolism also occurs in the presence of LPS.Peer reviewe

Anti-inflammatory actions of acanthoic acid-related diterpenes involve activation of the PI3K p110γ/δ subunits and inhibition of NF-κB

et al.The effect of acanthoic acid analogs on the response to proinflammatory challenge was investigated. Some pimarane diterpenes are known activators of the LXRαβ nuclear receptors, but we show here that they also exert a rapid, potent, and selective activation of the p110γ and p110δ subunits of PI3K. Combination of these effects results in an important attenuation of the global transcriptional response to LPS in macrophages. PI3K/Akt activation leads to inhibition of the LPS-dependent stimulation of IKK/NF-κB and p38 and ERK MAPKs. Macrophages from LXRαβ-deficient mice exhibited an inhibition of these pathways similar to the corresponding wild-type cells. Silencing or inhibition of p110γ/δ suppressed the effect of these diterpenes (DTPs) on IKK/NF-κB and MAPKs signaling. Taken together, these data show a multitarget anti-inflammatory mechanism by these DTPs including a selective activation of PI3K isoenzymes.This work was supported by grants BFU2011/24760 and RD12/0042/0019 from MINECO and RIC (ISCIII), Spain, and S2010/BMD2378 from Comunidad de Madrid to L.B.; SAF2011-29244 and S2010/BMD2350 to A.C.; and NIH grant R44AI49014 awarded to M.A.P.Peer Reviewe

HIF-1α and PFKFB3 mediate a tight relationship between proinflammatory activation and anerobic metabolism in atherosclerotic macrophages

PMCID: PMC4441599.-- et al.[Objective]: Although it is accepted that macrophage glycolysis is upregulated under hypoxic conditions, it is not known whether this is linked to a similar increase in macrophage proinflammatory activation and whether specific energy demands regulate cell viability in the atheromatous plaque. [Approach and Results]: We studied the interplay between macrophage energy metabolism, polarization, and viability in the context of atherosclerosis. Cultured human and murine macrophages and an in vivo murine model of atherosclerosis were used to evaluate the mechanisms underlying metabolic and inflammatory activity of macrophages in the different atherosclerotic conditions analyzed. We observed that macrophage energetics and inflammatory activation are closely and linearly related, resulting in dynamic calibration of glycolysis to keep pace with inflammatory activity. In addition, we show that macrophage glycolysis and proinflammatory activation mainly depend on hypoxia-inducible factor and on its impact on glucose uptake, and on the expression of hexokinase II and ubiquitous 6-phosphofructo-2-kinase. As a consequence, hypoxia potentiates inflammation and glycolysis mainly via these pathways. Moreover, when macrophages' ability to increase glycolysis through 6-phosphofructo-2-kinase is experimentally attenuated, cell viability is reduced if subjected to proinflammatory or hypoxic conditions, but unaffected under control conditions. In addition to this, granulocyte-macrophage colony-stimulating factor enhances anerobic glycolysis while exerting a mild proinflammatory activation. [Conclusions]: These findings, in human and murine cells and in an animal model, show that hypoxia potentiates macrophage glycolytic flux in concert with a proportional upregulation of proinflammatory activity, in a manner that is dependent on both hypoxia-inducible factor -1α and 6-phosphofructo-2-kinase.Peer Reviewe

Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge

Inhibition of protein tyrosine phosphatase 1B (PTP1B) has been suggested as an attractive target to improve insulin sensitivity in different cell types. In the present work, we have investigated the effect of PTP1B deficiency on the response of human and murine macrophages. Using in vitro and in vivo approaches in mice and silencing PTP1B in human macrophages with specific siRNAs, we have demonstrated that PTP1B deficiency increases the effects of pro-inflammatory stimuli in both human and rodent macrophages at the time that decreases the response to alternative stimulation. Moreover, the absence of PTP1B induces a loss of viability in resting macrophages and mainly after activation through the classic pathway. Analysis of early gene expression in macrophages treated with pro-inflammatory stimuli confirmed this exacerbated inflammatory response in PTP1Bdeficient macrophages. Microarray analysis in samples from wild-type and PTP1B-deficient macrophages obtained after 24 h of pro-inflammatory stimulation showed an activation of the p53 pathway, including the excision base repair pathway and the insulin signaling pathway in the absence of PTP1B. In animal models of lipopolysaccharide (LPS) and D-galactosamine challenge as a way to reveal in vivo inflammatory responses, animals lacking PTP1B exhibited a higher rate of death. Moreover, these animals showed an enhanced response to irradiation, in agreement with the data obtained in the microarray analysis. In summary, these results indicate that, although inhibition of PTP1B has potential benefits for the treatment of diabetes, it accentuates pro-inflammatory responses compromising at least macrophage viability.This work was supported by grants BFU2011-24760 and SAF2012-33283 from MINECO, S2010/BMD-2378 and S2010/BMD-2423 from Comunidad de Madrid and FIS-RIC RD06/0014/0025 and EFSD and Amylin Paul Langerhans Grant. Ciberdem and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Relevance of the MEK/ERK signaling pathway in the metabolism of activated macrophages: A metabolomic approach

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.This work was supported by Grants SAF2008-00164, BFU2011-24760, and PIB2010BZ-00540 from Spanish Ministry of Science and Innovation, Red Temática de Investigación Cooperativa en Cáncer, the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation and European Regional Development Fund “Una manera de hacer Europa” ISCIII-RTICC (RD6/0020/0046), and FIS-RECAVA (RD06/0014/0006) and CIBERehd founded by Instituto de Salud Carlos III, the European Commission (FP7) Etherpath KBBE Grant Agreement 222639, and by Agència de Gestió d'Ajuts Universitaris i de Recerca-Generalitat de Catalunya (Grant 2009SGR1308, 2009 CTP 00026, and Icrea Academia award 2010 to M.C.).Peer Reviewe

Selective impairment of P2Y signaling by prostaglandin E2 in macrophages: Implications for Ca2+-dependent responses

Extracellular nucleotides have been recognized as important modulators of inflammation via their action on specific pyrimidine receptors (P2). This regulation coexists with the temporal framework of proinflammatory and proresolution mediators released by the cells involved in the inflammatory response, including macrophages. Under proinflammatory conditions, the expression of cyclooxygenase-2 leads to the release of large amounts of PGs, such as PGE2, that exert their effects through EP receptors and other intracellular targets. The effect of these PGs on P2 receptors expressed in murine and human macrophages was investigated. In thioglycollate-elicited and alternatively activated macrophages, PGE2 selectively impairs P2Y but not P2X7 Ca2+ mobilization. This effect is absent in LPS-activated cells and is specific for PGE2 because it cannot be reproduced by other PGs with cyclopentenone structure. The inhibition of P2Y responses by PGE2 involves the activation of nPKCs (PKCε) and PKD that can be abrogated by selective inhibitors or by expression of dominant-negative forms of PKD. The inhibition of P2Y signaling by PGE2 has an impact on the cell migration elicited by P2Y agonists in thioglycollate-elicited and alternatively activated macrophages, which provide new clues to understand the resolution phase of inflammation, when accumulation of PGE2, anti-inflammatory and proresolving mediators occurs.This work was supported by Grants BFU2011-24760 and BFU2011-24743 from Ministerio de Economía y Competitividad, Grant S2010/BMD-2378 from Comunidad de Madrid, and Fondo de Investigación Sanitaria-Red Cardiovascular (RECAVA) Grant RD06/0014/0025 and Fundación Marcelino Botín (to M.T.M.-P.).Peer Reviewe

Anti-inflammatory actions of acanthoic acid-related diterpenes involve activation of the PI3K p110γ/δ subunits and inhibition of NF-κB.

The effect of acanthoic acid analogs on the response to proinflammatory challenge was investigated. Some pimarane diterpenes are known activators of the LXRαβ nuclear receptors, but we show here that they also exert a rapid, potent, and selective activation of the p110γ and p110δ subunits of PI3K. Combination of these effects results in an important attenuation of the global transcriptional response to LPS in macrophages. PI3K/Akt activation leads to inhibition of the LPS-dependent stimulation of IKK/NF-κB and p38 and ERK MAPKs. Macrophages from LXRαβ-deficient mice exhibited an inhibition of these pathways similar to the corresponding wild-type cells. Silencing or inhibition of p110γ/δ suppressed the effect of these diterpenes (DTPs) on IKK/NF-κB and MAPKs signaling. Taken together, these data show a multitarget anti-inflammatory mechanism by these DTPs including a selective activation of PI3K isoenzymes