Beneficial effects of hepatic cyclooxygenase-2 expression against cholestatic injury after common bile duct ligation in mice.

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but little is known about the significance of COX-2 in cholestatic injury. This study was designed to elucidate the role of COX-2 expression in hepatocytes during the pathogenesis of obstructive cholestasis. METHODS: We used genetically modified mice constitutively expressing human COX-2 in hepatocytes. Transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either subjected to a mid-abdominal laparotomy or common bile duct ligation (BDL) for 2 or 5 days. Then, we explored the mechanisms underlying the role of COX-2 and its derived prostaglandins in liver function, and the synthesis and excretion of bile acids (BA) in response to cholestatic liver injury. RESULTS: After BDL, hCOX-2-Tg mice showed lower grades of hepatic necrosis and inflammation than Wt mice, in part by a reduced hepatic neutrophil recruitment associated with lower mRNA levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice displayed a differential metabolic pattern of BA synthesis that led to an improved clearance after BDL-induced accumulation. In addition, an enhanced response to the BDL-induced oxidative stress and hepatic apoptosis was observed. In vitro experiments using hepatic cells that stably express hCOX-2 confirmed the cytoprotective role of prostaglandin E2 against BA toxicity. CONCLUSIONS: Taken together, our data indicate that constitutive expression of COX-2 in hepatocytes ameliorates cholestatic liver injury in mice by reducing inflammation and cell damage and by modulating BA metabolism, pointing to a role for COX-2 as a defensive response against cholestasis-derived BA accumulation and injury.

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

BACKGROUND AND AIMS: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models. APPROACH AND RESULTS: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G1 /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations. CONCLUSIONS: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.

High-fat diet activates splenic NOD1 and enhances neutrophil recruitment and neutrophil extracellular traps release in the spleen of ApoE-deficient mice.

In the course of atherogenesis, the spleen plays an important role in the regulation of extramedullary hematopoiesis, and in the control of circulating immune cells, which contributes to plaque progression. Here, we have investigated the role of splenic nucleotide-binding oligomerization domain 1 (NOD1) in the recruitment of circulating immune cells, as well as the involvement of this immune organ in extramedullary hematopoiesis in mice fed on a high-fat high-cholesterol diet (HFD). Under HFD conditions, the absence of NOD1 enhances the mobilization of immune cells, mainly neutrophils, from the bone marrow to the blood. To determine the effect of NOD1-dependent mobilization of immune cells under pro-atherogenic conditions, Apoe-/- and Apoe-/-Nod1-/- mice fed on HFD for 4 weeks were used. Splenic NOD1 from Apoe-/- mice was activated after feeding HFD as inferred by the phosphorylation of the NOD1 downstream targets RIPK2 and TAK1. Moreover, this activation was accompanied by the release of neutrophil extracellular traps (NETs), as determined by the increase in the expression of peptidyl arginine deiminase 4, and the identification of citrullinated histone H3 in this organ. This formation of NETs was significantly reduced in Apoe-/-Nod1-/- mice. Indeed, the presence of Ly6G+ cells and the lipidic content in the spleen of mice deficient in Apoe and Nod1 was reduced when compared to the Apoe-/- counterparts, which suggests that the mobilization and activation of circulating immune cells are altered in the absence of NOD1. Furthermore, confirming previous studies, Apoe-/-Nod1-/- mice showed a reduced atherogenic disease, and diminished recruitment of neutrophils in the spleen, compared to Apoe-/- mice. However, splenic artery ligation reduced the atherogenic burden in Apoe-/- mice an effect that, unexpectedly was lost in Apoe-/-Nod1-/- mice. Together, these results suggest that neutrophil accumulation and activity in the spleen are driven in part by NOD1 activation in mice fed on HFD, contributing in this way to regulating atherogenic progression.

Unraveling the interplay between iron homeostasis, ferroptosis and extramedullary hematopoiesis.

Iron participates in myriad processes necessary to sustain life. During the past decades, great efforts have been made to understand iron regulation and function in health and disease. Indeed, iron is associated with both physiological (e.g., immune cell biology and function and hematopoiesis) and pathological (e.g., inflammatory and infectious diseases, ferroptosis and ferritinophagy) processes, yet few studies have addressed the potential functional link between iron, the aforementioned processes and extramedullary hematopoiesis, despite the obvious benefits that this could bring to clinical practice. Further investigation in this direction will shape the future development of individualized treatments for iron-linked diseases and chronic inflammatory disorders, including extramedullary hematopoiesis, metabolic syndrome, cardiovascular diseases and cancer.

Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.

The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury.

Functional Crosstalk between PCSK9 Internalization and Pro-Inflammatory Activation in Human Macrophages: Role of Reactive Oxygen Species Release.

Atherosclerosis is a cardiovascular disease caused mainly by dyslipidemia and is characterized by the formation of an atheroma plaque and chronic inflammation. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that induces the degradation of the LDL receptor (LDLR), which contributes to increased levels of LDL cholesterol and the progress of atherosclerosis. Given that macrophages are relevant components of the lipidic and inflammatory environment of atherosclerosis, we studied the effects of PCSK9 treatment on human macrophages. Our data show that human macrophages do not express PCSK9 but rapidly incorporate the circulating protein through the LDLR and also activate the pro-inflammatory TLR4 pathway. Both LDLR and TLR4 are internalized after incubation of macrophages with exogenous PCSK9. PCSK9 uptake increases the production of reactive oxygen species and reduces the expression of genes involved in lipid metabolism and cholesterol efflux, while enhancing the production of pro-inflammatory cytokines through a TLR4-dependent mechanism. Under these conditions, the viability of macrophages is compromised, leading to increased cell death. These results provide novel insights into the role of PCSK9 in the crosstalk of lipids and cholesterol metabolism through the LDLR and on the pro-inflammatory activation of macrophages through TLR4 signaling. These pathways are relevant in the outcome of atherosclerosis and highlight the relevance of PCSK9 as a therapeutic target for the treatment of cardiovascular diseases.

NOD1 in the interplay between microbiota and gastrointestinal immune adaptations.

Nucleotide-binding oligomerization domain 1 (NOD1), a pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals, has been linked to inflammatory pathologies. NOD1, which is expressed by immune and non-immune cells, is activated after recognizing microbe-associated molecular patterns (MAMPs). This recognition triggers host defense responses and both immune memory and tolerance can also be achieved during these processes. Since the gut microbiota is currently considered a master regulator of human physiology central in health and disease and the intestine metabolizes a wide range of nutrients, drugs and hormones, it is a fact that dysbiosis can alter tissues and organs homeostasis. These systemic alterations occur in response to gastrointestinal immune adaptations that are not yet fully understood. Even if previous evidence confirms the connection between the microbiota, the immune system and metabolic disorders, much remains to be discovered about the contribution of NOD1 to low-grade inflammatory pathologies such as obesity, diabetes and cardiovascular diseases. This review compiles the most recent findings in this area, while providing a dynamic and practical framework with future approaches for research and clinical applications on targeting NOD1. This knowledge can help to rate the consequences of the disease and to stratify the patients for therapeutic interventions.

Protective Role of Hepatocyte Cyclooxygenase-2 Expression Against Liver Ischemia-Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but the significance of COX-2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX-2 induction in hepatocytes against liver IRI. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were subjected to IRI. hCOX-2-Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX-2-Tg mice showed a significant attenuation of the IRI-induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX-2-Tg mice against IRI due to a preconditioning-derived increase in endogenous COX-2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2 ) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX-2 induction and the consequent rise of derived prostaglandins against IRI.

Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1.

Atherosclerosis is a chronic disease characterized by vascular lipid retention and inflammation, and pattern recognition receptors (PRRs) are important contributors in early stages of the disease. Given the implication of the intracellular PRR nucleotide-binding oligomerization domain 1 (NOD1) in cardiovascular diseases, we investigated its contribution to early atherosclerosis. We evidenced NOD1 induction in atherosclerotic human and mouse tissues, predominantly in vascular endothelial cells. Accordingly, NOD1 genetic inactivation in Apoe-/- mice reduced not only atherosclerosis burden, but also monocyte and neutrophil accumulation in atheromata. Of note, in the presence of either peptidoglycan or oxidized LDLs, endothelial NOD1 triggered VCAM-1 up-regulation through the RIP2-NF-κB axis in an autocrine manner, enhancing firm adhesion of both sets of myeloid cells to the inflamed micro- and macrovasculature in vivo. Our data define a major proatherogenic role for endothelial NOD1 in early leukocyte recruitment to the athero-prone vasculature, thus introducing NOD1 as an innovative therapeutic target and potential prognostic molecule.-González-Ramos, S., Paz-García, M., Rius, C., del Monte-Monge, A., Rodríguez, C., Fernández-García, V., Andrés, V., Martínez-González, J., Lasunción, M. A., Martín-Sanz, P., Soehnlein, O., Boscá, L. Endothelial NOD1 directs myeloid cell recruitment in atherosclerosis through VCAM-1.

NOD1 activation in cardiac fibroblasts induces myocardial fibrosis in a murine model of type 2 diabetes.

Cardiac fibrosis and chronic inflammation are common complications in type 2 diabetes mellitus (T2D). Since nucleotide oligomerization-binding domain 1 (NOD1), an innate immune receptor, is involved in the pathogenesis of insulin resistance and diabetes outcomes, we sought to investigate its involvement in cardiac fibrosis. Here, we show that selective staining of cardiac fibroblasts from T2D (db/db;db) mice exhibits up-regulation and activation of the NOD1 pathway, resulting in enhanced NF-κB and TGF-ß signalling. Activation of the TGF-ß pathway in cardiac fibroblasts from db mice was prevented after inhibition of NF-κB with BAY-11-7082 (BAY). Moreover, fibrosis progression in db mice was also prevented by BAY treatment. Enhanced TGF-ß signalling and cardiac fibrosis of db mice was dependent, at least in part, on the sequential activation of NOD1 and NF-κB since treatment of db mice with a selective NOD1 agonist induced activation of the TGF-ß pathway, but co-administration of a NOD1 agonist plus BAY, or a NOD1 inhibitor prevented the NOD1-induced fibrosis. Therefore, NOD1 is involved in cardiac fibrosis associated with diabetes, and establishes a new mechanism for the development of heart fibrosis linked to T2D.

Cyclooxygenase-2 expression in hepatocytes attenuates non-alcoholic steatohepatitis and liver fibrosis in mice.

Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl4) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl4 had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression.

Benznidazole, the trypanocidal drug used for Chagas disease, induces hepatic NRF2 activation and attenuates the inflammatory response in a murine model of sepsis.

Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.

HIF-1α and PFKFB3 Mediate a Tight Relationship Between Proinflammatory Activation and Anerobic Metabolism in Atherosclerotic Macrophages.

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.

Metabolic signatures linked to macrophage polarization: from glucose metabolism to oxidative phosphorylation.

Macrophages are present in a large variety of locations, playing distinct functions that are determined by its developmental origin and by the nature of the activators of the microenvironment. Macrophage activation can be classified as pro-inflammatory (M1 polarization) or anti-inflammatory-pro-resolution-deactivation (M2), these profiles coexisting in the course of the immune response and playing a relevant functional role in the onset of inflammation (Figure 1). Several groups have analysed the metabolic aspects associated with macrophage activation to answer the question about what changes in the regulation of energy metabolism and biosynthesis of anabolic precursors accompany the different types of polarization and to what extent they are necessary for the expression of the activation phenotypes. The interest of these studies is to regulate macrophage function by altering their metabolic activity in a 'therapeutic way'.

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 Ca(2+) 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.

NOD1 receptor is up-regulated in diabetic human and murine myocardium.

Type 2 diabetes has a complex pathology that involves a chronic inflammatory state. Emerging evidence suggests a link between the innate immune system receptor NOD1 (nucleotide-binding and oligomerization domain 1) and the pathogenesis of diabetes, in monocytes and hepatic and adipose tissues. The aim of the present study was to assess the role of NOD1 in the progression of diabetic cardiomyopathy. We have measured NOD1 protein in cardiac tissue from Type 2 diabetic (db) mice. Heart and isolated cardiomyocytes from db mice revealed a significant increase in NOD1, together with an up-regulation of nuclear factor κB (NF-κB) and increased apoptosis. Heart tissue also exhibited an enhanced expression of pro-inflammatory cytokines. Selective NOD1 activation with C12-γ-D-glutamyl-m-diaminopimelic acid (iEDAP) resulted in an increased NF-κB activation and apoptosis, demonstrating the involvement of NOD1 both in wild-type and db mice. Moreover, HL-1 cardiomyocytes exposed to elevated concentrations of glucose plus palmitate displayed an enhanced NF-κB activity and apoptotic profile, which was prevented by silencing of NOD1 expression. To address this issue in human pathology, NOD1 expression was evaluated in myocardium obtained from patients with Type 2 diabetes (T2DMH) and from normoglycaemic individuals without cardiovascular histories (NH). We have found that NOD1 was expressed in both NH and T2DMH; however, NOD1 expression was significantly pronounced in T2DMH. Furthermore, both the pro-inflammatory cytokine tumour necrosis factor α (TNF-α) and the apoptosis mediator caspase-3 were up-regulated in T2DMH samples. Taken together, our results define an active role for NOD1 in the heightened inflammatory environment associated with both experimental and human diabetic cardiac disease.

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.

Sustained release of prostaglandin E2 in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca²âº-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 PGE2 in 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, Ca(2+)-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2 when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2 was added exogenously to the culture medium. This Ca(2+)-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 PGE2 involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2 reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2 is involved in the regulation of P2Y signaling in these cells.

Cyclooxygenase-2 over-expression inhibits liver apoptosis induced by hyperglycemia.

Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.