LPS stimulation activates IKK and different MAP kinase pathways, as well as the PI3K‐Akt‐mTOR‐p70 S6k pathway, a negative regulator of these MyD88‐dependent intracellular signals. Here, we show that ...Cot/tpl2, a MAP3K responsible for the activation of the MKK1‐Erk1/2, controls P‐Ser473 Akt and P‐Thr389 p70 S6k phosphorylation in LPS‐stimulated macrophages. Analysis of the intracellular signalling in Cot/tpl2 KO macrophages versus WT macrophages reveals lower IκBα recovery and higher phosphorylation of JNK and p38α after 1 h of LPS stimulation. Moreover, Cot/tpl2 deficiency increases LPS‐induced NO synthase 2 (NOS2) expression in macrophages. Inhibition of the PI3K pathway abolishes the differences in IκBα and NOS2 expression between Cot/tpl2 KO and WT macrophages following LPS administration. Furthermore, in zymosan‐ and polyI:C‐stimulated macrophages, Cot/tpl2 mediates P‐Ser473 Akt phosphorylation, increases IκBα levels and decreases NOS2 expression. In conclusion, these data reveal a novel role for the Cot/tpl2 pathway in mediating TLR activation of the Akt‐mTOR‐p70 S6k pathway, allowing Cot/tpl2 to fine‐control the activation state of other signalling pathways.
Cyclooxygenase-2 (COX-2) expression has been detected in human hepatoma cell lines and in human hepatocellular carcinoma (HCC); however, the contribution of COX-2 to the development of HCC remains ...controversial. COX-2 expression is higher in the non-tumoral tissue and inversely correlates with the differentiation grade of the tumor. COX-2 expression depends on the interplay between different cellular pathways involving both transcriptional and post-transcriptional regulation. The aim of this work was to assess whether COX-2 could be regulated by microRNAs in human hepatoma cell lines and in human HCC specimens since these molecules contribute to the regulation of genes implicated in cell growth and differentiation. Our results show that miR-16 silences COX-2 expression in hepatoma cells by two mechanisms: a) by binding directly to the microRNA response element (MRE) in the COX-2 3'-UTR promoting translational suppression of COX-2 mRNA; b) by decreasing the levels of the RNA-binding protein Human Antigen R (HuR). Furthermore, ectopic expression of miR-16 inhibits cell proliferation, promotes cell apoptosis and suppresses the ability of hepatoma cells to develop tumors in nude mice, partially through targeting COX-2. Moreover a reduced miR-16 expression tends to correlate to high levels of COX-2 protein in liver from patients affected by HCC. Our data show an important role for miR-16 as a post-transcriptional regulator of COX-2 in HCC and suggest the potential therapeutic application of miR-16 in those HCC with a high COX-2 expression.
Macrophages regulate essential aspects of innate immunity against pathogens. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for ...host defense. The liver X receptors (LXRα, LXRβ) are ligand‐dependent nuclear receptors that direct gene expression important for cholesterol metabolism and inflammation, but little is known about the individual roles of LXRα and LXRβ in antimicrobial responses. Here, the results demonstrate that induction of LXRα transcription by prolonged exposure to lipopolysaccharide (LPS) supports inflammatory gene expression in macrophages. LXRα transcription is induced by NF‐κB and type‐I interferon downstream of TLR4 activation. Moreover, LPS triggers a reprogramming of the LXRα cistrome that promotes cytokine and chemokine gene expression through direct LXRα binding to DNA consensus sequences within cis‐regulatory regions including enhancers. LXRα‐deficient macrophages present fewer binding of p65 NF‐κB and reduced histone H3K27 acetylation at enhancers of secondary inflammatory response genes. Mice lacking LXRα in the hematopoietic compartment show impaired responses to bacterial endotoxin in peritonitis models, exhibiting reduced neutrophil infiltration and decreased expansion and inflammatory activation of recruited F4/80lo‐MHC‐IIhi peritoneal macrophages. Together, these results uncover a previously unrecognized function for LXRα‐dependent transcriptional cis‐activation of secondary inflammatory gene expression in macrophages and the host response to microbial ligands.
The inflammatory response is a defense mechanism of the innate immune system against infections and other injuries. In response to microbial components, macrophages activate primary and secondary inflammatory gene programs crucial for host defense. De la Rosa et al. provide evidence that inflammation triggers a reprogramming of the transcription factor liver X receptor α (LXRα), that sustains cytokine and chemokine gene expression through direct binding to DNA consensus sequences and collaboration with NF‐κB at enhancers of secondary inflammatory response genes. This novel TLR4‐LXRα‐NF‐κB axis supports macrophage inflammatory gene expression and in vivo immune‐cell recruitment during inflammatory responses to microbial ligands, positioning LXRα as a key factor in the transcriptional regulation in the late stages of inflammation.
Macrophages are immune cells that play crucial roles in host defense against pathogens by triggering their exceptional phagocytic and inflammatory functions. Macrophages that reside in healthy ...tissues also accomplish important tasks to preserve organ homeostasis, including lipid uptake/efflux or apoptotic-cell clearance. Both homeostatic and inflammatory functions of macrophages require the precise stability of lipid-rich microdomains located at the cell membrane for the initiation of downstream signaling cascades. Caveolin-1 (Cav-1) is the main protein responsible for the biogenesis of caveolae and plays an important role in vascular inflammation and atherosclerosis. The Liver X receptors (LXRs) are key transcription factors for cholesterol efflux and inflammatory gene responses in macrophages. Although the role of Cav-1 in cellular cholesterol homeostasis and vascular inflammation has been reported, the connection between LXR transcriptional activity and Cav-1 expression and function in macrophages has not been investigated. Here, using gain and loss of function approaches, we demonstrate that LXR-dependent transcriptional pathways modulate Cav-1 expression and compartmentation within the membrane during macrophage activation. As a result, Cav-1 participates in LXR-dependent cholesterol efflux and the control of inflammatory responses. Together, our data show modulation of the LXR-Cav-1 axis could be exploited to control exacerbated inflammation and cholesterol overload in the macrophage during the pathogenesis of lipid and immune disorders, such as atherosclerosis.
The use of animals lacking genes or expressing genes under the control of cell-specific promoters has signifi cantly increased our knowledge of the genetic and molecular basis of ...physiopathology,allowing testing of functional hypotheses and validation of biochemical and pharmacologic approaches in order to understand cell function.However,with unexpected frequency,gene knockout animals and,more commonly,animal models of transgenesis give experimental support to even opposite conclusions on gene function.Her...
Abstract 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 E 2 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.
Atherothrombosis, the main cause of acute coronary syndromes (ACS), is characterized by the rupture of the atherosclerotic plaque followed by the formation of thrombi. Fatal plaque rupture sites show ...large necrotic cores combined with high levels of inflammation and thin layers of collagen. Plaque necrosis due to the death of macrophages and smooth muscle cells (SMCs) remains critical in the process. To determine the contribution of the innate immunity receptor NOD1 to the stability of atherosclerotic plaque,
and
atherosclerosis prone mice were placed on a high-fat diet for 16 weeks to assess post-mortem advanced atherosclerosis in the aortic sinus. The proliferation and apoptosis activity were analyzed, as well as the foam cell formation capacity in these lesions and in primary cultures of macrophages and vascular SMCs obtained from both groups of mice. Our results reinforce the preeminent role for NOD1 in human atherosclerosis. Advanced plaque analysis in the
atherosclerosis model suggests that NOD1 deficiency may decrease the risk of atherothrombosis by decreasing leukocyte infiltration and reducing macrophage apoptosis. Furthermore,
SMCs exhibit higher proliferation rates and decreased apoptotic activity, contributing to thicker fibrous caps with reduced content of pro-thrombotic collagen. These findings demonstrate a direct link between NOD1 and plaque vulnerability through effects on both macrophages and SMCs, suggesting promising insights for early detection of biomarkers for treating patients before ACS occurs.
The innate immune system is responsible for the initial response of an organism to potentially harmful stressors, pathogens or tissue injury, and accordingly plays an essential role in the ...pathogenesis of many inflammatory processes, including some cardiovascular diseases. Toll like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors (NLRs) are pattern recognition receptors that play an important role in the induction of innate immune and inflammatory responses. There is a line of evidence supporting that activation of TLRs contributes to the development and progression of cardiovascular diseases but less is known regarding the role of NLRs. Here we demonstrate the presence of the NLR member NOD1 (nucleotide-binding oligomerization domain containing 1) in the murine heart. Activation of NOD1 with the specific agonist C12-iEDAP, but not with the inactive analogue iE-Lys, induces a time- and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in whole hearts. At the cellular level, both native cardiomyocytes and cardiac fibroblasts expressed NOD1. The NLR activation in cardiomyocytes was associated with NF-κB activation and induction of apoptosis. NOD1 stimulation in fibroblasts was linked to NF-κB activation and to increased expression of pro-fibrotic mediators. The down-regulation of NOD1 by specific siRNAs blunted the effect of iEDAP on the pro-fibrotic TGF-β pathway and cell apoptosis. In conclusion, our report uncovers a new pro-inflammatory target that is expressed in the heart, NOD1. The specific activation of this NLR induces cardiac dysfunction and modulates cardiac fibrosis and cardiomyocyte apoptosis, pathological processes involved in several cardiac diseases such as heart failure.
Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2 (COX-2) is mainly overexpressed in CRC but less is known regarding ...post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans.
To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile.
Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge (Barcelona, Spain) and Germans Trias i Pujol University Hospital (Campus Can Ruti) (Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F (PNGase F). Prostaglandin E
(PGE
) levels were determined using a specific ELISA.
H high resolution magic angle spinning (HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer (nano LC-MS/MS) using a QExactive HF orbitrap MS.
Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients
the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE
production in tumors. These results were corroborated
performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE
levels. Moreover, HRMAS analysis indicated that tumor tissue has altered metabolic features
non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity, DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing.
In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile.
Cyclooxygenase (COX) catalyzes the first step in prostanoid biosynthesis and exists as two isoforms. COX-1 is a constitutive enzyme involved in physiological processes, whereas COX-2 is induced by a ...variety of stimuli. MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. Although it is known that COX-2 expression is regulated by miRNAs, there are no data regarding COX-2 involvement in miRNA regulation. Considering our previous results showing that COX-2 expression in hepatocytes protects against insulin resistance, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs implicated in insulin signaling in liver cells. Our results provide evidence of the molecular basis for a novel function of COX-2 in miRNA processing. COX-2 represses miRNA 23b (miR-23b), miR-146b, and miR-183 expression in liver cells by increasing the level of DEAD-box helicase p68 (DDX5) through phosphatidylinositol 3-kinase (PI3K)/p300 signaling and by modulating the enzymatic function of the Drosha (RNase type III) complex through its physical association with DDX5. The decrease of miR-183 expression promotes protection against insulin resistance by increasing insulin receptor substrate 1 (IRS1) levels. These results indicate that the modulation of miRNA processing by COX-2 is a key event in insulin signaling in liver and has potential clinical implications for the management of various hepatic dysfunctions.
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