Inhibition of lipopolysaccharide-induced gene expression by liver X receptor ligands in macrophages involves interference with early growth response factor 1.

Liver X receptors (LXRs) are nuclear receptors that act as ligand-dependent transcription factors forming permissive heterodimers with retinoid X receptors (RXRs). In this study we aimed to assess the effect of LXR/RXR activation on the transcriptional induction of pro-inflammatory genes including cyclooxygenase-2 (COX-2) and microsomal prostaglandin E2 synthase-1 (mPGES-1) in activated macrophages. Our study shows that LXR ligands such as oxysterols, GW3965 or TO901317, as well as RXR ligands like 9cis retinoic acid or SR11237, decreased LPS-induced expression of COX-2 and mPGES-1. Consequently, LPS-dependent PGE2 production was substantially reduced in macrophages treated with LXR/RXR ligands. The inhibitory effects of LXR/RXR activation on LPS-induced expression of COX-2 and mPGES-1 in macrophages, occurred by a mechanism involving interference with transcriptional activation of these genes. LXR/RXR activation interfered with the activity of transcription factors essential in the up-regulation of the expression of pro-inflammatory genes in these cells, such as NFκB, but also Egr-1, which had not been previously associated with LXR-mediated gene repression. As this transcription factor is involved in the regulation of a variety of genes involved in inflammatory processes, LXR and RXR-mediated interference with Egr-1 signaling could represent an important event mediating the anti-inflammatory effects of these receptors in macrophages.

Cyclooxygenase-2 deficiency in macrophages leads to defective p110γ PI3K signaling and impairs cell adhesion and migration.

Cyclooxygenase (Cox)-2 dependent PGs modulate several functions in many pathophysiological processes, including migration of immune cells. In this study, we addressed the role of Cox-2 in macrophage migration by using in vivo and in vitro models. Upon thioglycolate challenge, CD11b(+) F4/80(+) macrophages showed a diminished ability to migrate to the peritoneal cavity in cox-2(-/-) mice. In vivo migration of cox-2(-/-) macrophages from the peritoneal cavity to lymph nodes, as well as cell adhesion to the mesothelium, was reduced in response to LPS. In vitro migration of cox-2(-/-) macrophages toward MCP-1, RANTES, MIP-1α, or MIP-1ß, as well as cell adhesion to ICAM-1 or fibronectin, was impaired. Defects in cell migration were not due to changes in chemokine receptor expression. Remarkably, cox-2(-/-) macrophages showed a deficiency in focal adhesion formation, with reduced phosphorylation of paxillin (Tyr(188)). Interestingly, expression of the p110γ catalytic subunit of PI3K was severely reduced in the absence of Cox-2, leading to defective Akt phosphorylation, as well as cdc42 and Rac-1 activation. Our results indicate that the paxillin/p110γ-PI3K/Cdc42/Rac1 axis is defective in cox-2(-/-) macrophages, which results in impaired cell adhesion and migration.

Inhibitory effects of sigma-2 receptor agonists on T lymphocyte activation.

Sigma (σ) receptor ligands are essentially known for their effects on the nervous system although recent studies have shown their potential effects modulating some other pathophysiological processes as cell proliferation, cancer, and the immune response. Here, we have analyzed the actions of σ-1 and σ-2 receptors ligands on T cell activation. Our results show that treatment of Jurkat T cells with σ-2 agonists decreased the induction of the expression of Interleukin (IL)-2, Tumor necrosis factor (TNF)-α, and Cyclooxygenase (COX)-2 by activated T cells in a dose-dependent manner. These effects take place at the transcriptional level since σ-2 agonists BD-737 and CB-184 diminished the activity of the promoters of those genes. Those immunosuppressive effects could be attributable to interference with transcription factor activation. Induced transcription mediated by Nuclear factor (NF)-κB or Nuclear Factor of Activated T cells (NFAT) was inhibited by σ-2 agonists. These effects seem to be specific for σ-2 agonists as no significant effects on T cell activation by σ-1 ligands PRE-084 and BD-1063 were found. Our results provide new insights into the immunomodulatory actions of σ ligands and describe a new property of σ-2 agonists, through inhibition of activation of transcription factors as NFAT by which these compounds are regulating gene expression. This may have important consequences on the possible therapeutic use of those compounds.

Involvement of PGE2 and the cAMP signalling pathway in the up-regulation of COX-2 and mPGES-1 expression in LPS-activated macrophages.

PG (prostaglandin) E2 plays an important role in the modulation of the immune response and the inflammatory process. In the present study, we describe a PGE2 positive feedback for COX (cyclo-oxygenase)-2 and mPGES-1 [microsomal PGES (PGE synthase)-1] expression in the macrophage cell line RAW 264.7. Our results show that PGE2 induces COX-2 and mPGES-1 expression, an effect mimicked by dbcAMP (dibutyryl-cAMP) or forskolin. Furthermore, the cAMP signalling pathway co-operates with LPS (lipopolysaccharide) in the induction of COX-2 and mPGES-1 transcriptional activation. Analysis of the involvement of PGE receptors [EPs (E-prostanoids)] showed that incubation with EP2 agonists up-regulated both COX2 and mPGES-1 mRNA levels. Moreover, EP2 receptor overexpression enhanced the transcriptional activation of COX2 and mPGES-1 promoters. This induction was repressed by the PKA (protein kinase A) inhibitor H89. Activation of the PGE2/EP2/PKA signalling pathway induced the phosphorylation of CREB [CRE (cAMP-response element)-binding protein] in macrophages and stimulated the specific binding of this transcription factor to COX2 and mPGES-1 promoters. Deletion or mutation of potential CRE sites in both promoters diminished their transcriptional activity. In summary, the results of the present study demonstrate that activation of PKA/CREB signalling through the EP2 receptor by PGE2 plays a key role in the expression of COX-2 and mPGES-1 in activated macrophages.

Cyclooxygenase-independent inhibitory effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole cyclooxygenase-2 inhibitors.

Anti-inflammatory efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) has been related to their properties as inhibitors of cyclooxygenase (COX)-mediated prostaglandin (PG) synthesis. However, recent studies have suggested that variations of the in vivo anti-inflammatory actions among different NSAIDs could not be solely explained by COX inhibition. Here, we have analyzed the effects on T cell activation of novel 4,5-dihydro-3 trifluoromethyl pyrazole anti-inflammatory drugs with different potencies as COX-2 inhibitors, namely E-6087, E-6232, E-6231, E-6036 and E-6259 as well as the chemically related COX-2 inhibitor Celecoxib. These drugs inhibited mitogen-mediated T cell proliferation as well as Interleukin (IL)-2, tumor necrosis factor (TNF)-α and Interferon (IFN)-γ synthesis by activated T cells, independently of their ability to inhibit COX-2 enzymatic activity. Immunosuppressive effects of these drugs seem to be due to their interference on transcription factor activation as induced transcription from Nuclear Factor (NF)-κB and Nuclear Factor of Activated T cells (NFAT)-dependent enhancers was inhibited in a dose-dependent manner, being the latter effect the most sensitive to the action of those compounds. Both NFAT dephosphorylation, required for its nuclear translocation, as well as transcriptional activity of a GAL4-NFAT chimera were diminished in the presence of these compounds. These findings provide new insights into the molecular mechanisms involved in the immunomodulatory and anti-inflammatory actions of NSAIDs, which may have important implications in anti-inflammatory therapy, through inhibition of NFAT.

Inhibition of hypoxia inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis.

PURPOSE: 2-(3,4-dihydroxyphenil)-ethanol (DPE), a polyphenol present in olive oil, has been found to attenuate the growth of colon cancer cells, an effect presumably related to its anti-inflammatory activity. EXPERIMENTAL DESIGN: To further explore the effects of DPE on angiogenesis and tumor growth we investigated the in vivo efficacy of DPE in a HT-29 xenograft model and in vitro activities in colon cancer cells exposed to interleukin-1beta (IL-1beta) and prostaglandin E-2 (PGE-2). RESULTS: DPE (10 mg/kg/day for 14 days) inhibited tumor growth, reducing vessel lumina and blood perfusion to tumor, and diminished expression of hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF), and microsomal prostaglandin-E synthase-1 (mPGEs-1). In vitro, DPE (100 mumol/L) neither affected cell proliferation nor induced apoptosis in HT-29 and WiDr cells. DPE prevented the IL-1beta-mediated increase of mPGEs-1 expression and PGE-2 generation, as it did the silencing of HIF-1alpha. Moreover, DPE blocked mPGEs-1-dependent expression of VEGF and inhibited endothelial sprouting induced by tumor cells in a coculture system. PGE-2 triggers a feed-forward loop involving HIF-1alpha, which impinges on mPGEs-1 and VEGF expression, events prevented by DPE via extracellular signal-related kinase 1/2. The reduction of PGE-2 and VEGF levels, caused by DPE, was invariably associated with a marked decrease in HIF-1alpha expression and activity, independent of proteasome activity, indicating that the DPE effects on tumor growth and angiogenesis are dependent on the inhibition of HIF-1alpha translation. CONCLUSIONS: We show that the in vivo DPE antitumor effect is associated with anti-inflammatory and antiangiogenic activities resulting from the downregulation of the HIF-1alpha/mPGEs-1/VEGF axis.

Coordinated up-regulation of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 transcription by nuclear factor kappa B and early growth response-1 in macrophages.

Prostaglandin (PG) E(2) is a potent lipid mediator that plays an essential role in inflammation, fever and pain. It is produced from arachidonic acid (AA) by a cascade of enzymatic reactions involving cyclooxygenases (COX-1 and -2) and prostaglandin E synthases (cPGES, mPGES-1 and -2). Functional coupling of the inducible enzymes COX-2 and mPGES-1 has been proposed for increased production of PGE(2) in different cell types. PGE(2) produced by macrophages plays an essential role in the pathogenesis of inflammatory diseases. Here, we have investigated the mechanisms involved in the regulation of COX-2 and mPGES-1 expressions in murine macrophages upon bacterial lipopolysaccharide (LPS) treatment. LPS stimulation induced the coordinated synthesis of COX-2 and mPGES-1 that resulted in an enhanced production of PGE(2) in RAW 264.7 macrophages. Furthermore, we show the involvement of NF-kappaB and Egr-1 transcription factors in the transcriptional induction of these enzymes. LPS treatment promoted specific binding of NF-kappaB to both COX-2 and mPGES-1 promoters. Site-directed mutagenesis, electrophoretic mobility shift assays and ChIP assays allowed the identification of a sequence acting as a NF-kappaB recognition site in the murine mPGES-1 promoter. Furthermore, LPS induced the expression of Egr-1 that cooperated with NF-kappaB in the up-regulation of COX-2 and mPGES-1. Inhibition of Egr-1 expression reduced substantially LPS-mediated induction of COX-2 and mPGES-1 expression, resulting in a decrease in PGE(2) production. Our findings point out to Egr-1 and NF-kappaB cooperation as determinant for PGE2 synthesis by macrophages in inflammatory processes through the coordinated regulation of COX-2 and mPGES-1.

Role of peroxisome proliferator-activated receptor alpha in the control of cyclooxygenase 2 and vascular endothelial growth factor: involvement in tumor growth.

A growing body of evidence indicates that PPAR (peroxisome proliferator-activated receptor) alpha agonists might have therapeutic usefulness in antitumoral therapy by decreasing abnormal cell growth, and reducing tumoral angiogenesis. Most of the anti-inflammatory and antineoplastic properties of PPAR ligands are due to their inhibitory effects on transcription of a variety of genes involved in inflammation, cell growth and angiogenesis. Cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) are crucial agents in inflammatory and angiogenic processes. They also have been significantly associated to cell proliferation, tumor growth, and metastasis, promoting tumor-associated angiogenesis. Aberrant expression of VEGF and COX-2 has been observed in a variety of tumors, pointing to these proteins as important therapeutic targets in the treatment of pathological angiogenesis and tumor growth. This review summarizes the current understanding of the role of PPARalpha and its ligands in the regulation of COX-2 and VEGF gene expression in the context of tumor progression.

Cot/Tpl2 and PKCzeta cooperate in the regulation of the transcriptional activity of NFATc2 through the phosphorylation of its amino-terminal domain.

Nuclear factor of activated T cells (NFAT) plays a prominent role in gene transcription during the immune response. Growing evidence demonstrates the implication of inducible phosphorylation of the transactivation domain (TAD) of NFAT in transcriptional activation of genes. We have analyzed the regulation of NFATc2 activation by Cot/Tpl2 and protein kinase C zeta (PKCzeta) in T cells. Our results show that PKCzeta and Cot/Tpl2 cooperate in regulating the transactivation activity mediated by the amino-terminal domain of NFATc2. Neither Cot/Tpl2 kinase nor PKCzeta-mediated induction of the transactivation activity of NFATc2 was affected by cyclosporin-A treatment, supporting a calcineurin independent route in the signaling pathways mediating NFATc2 activation. Co-precipitation experiments showed physical interaction among Cot/Tpl2, PKCzeta and NFATc2. Analysis of the transactivation activity of deletions in the N-terminal region of NFATc2, suggested the involvement of amino acids 52-64 of NFATc2 in the induction of its transactivating function by PKCzeta. This kinase in vitro phosphorylates NFATc2 and deletion and mutational studies identified Ser53 and Ser56 (of the SPPS motif) as substrates for PKCzeta. Thus, our results suggest that PKCzeta phosphorylation of Ser53 and Ser56 in the N-terminal TAD from NFATc2 potentiates its transactivating function in human T cells.

Peroxisome-proliferator-activated receptor alpha agonists inhibit cyclo-oxygenase 2 and vascular endothelial growth factor transcriptional activation in human colorectal carcinoma cells via inhibition of activator protein-1.

Recent evidence indicates that PPAR (peroxisome-proliferator-activated receptor) alpha ligands possess anti-inflammatory and antitumoural properties owing to their inhibitory effects on the expression of genes that are involved in the inflammatory response. However, the precise molecular mechanisms underlying these effects are poorly understood. In the present study, we show that tumour promoter PMA-mediated induction of genes that are significantly associated with inflammation, tumour growth and metastasis, such as COX-2 (cyclo-oxygenase 2) and VEGF (vascular endothelial growth factor), is inhibited by PPARalpha ligands in the human colorectal carcinoma cell line SW620. PPARalpha activators LY-171883 and WY-14,643 were able to diminish transcriptional induction of COX-2 and VEGF by inhibiting AP-1 (activator protein-1)-mediated transcriptional activation induced by PMA or by c-Jun overexpression. The actions of these ligands on AP-1 activation and COX-2 and VEGF transcriptional induction were found to be dependent on PPARalpha expression. Our studies demonstrate the existence of a negative cross-talk between the PPARalpha- and AP-1-dependent signalling pathways in these cells. PPARalpha interfered with at least two steps within the pathway leading to AP-1 activation. First, PPARalpha activation impaired AP-1 binding to a consensus DNA sequence. Secondly, PPARalpha ligands inhibited c-Jun transactivating activity. Taken together, these findings provide new insight into the anti-inflammatory and anti-tumoural properties of PPARalpha activation, through the inhibition of the induction of AP-1-dependent genes that are involved in inflammation and tumour progression.

Up-regulation of cyclooxygenase-2 by interleukin-1beta in colon carcinoma cells.

Growing evidence shows that Interleukin (IL)-1beta and Cyclooxygenase 2 (COX-2) play a crucial role in the pathogenesis of inflammatory diseases and tumor growth, particularly in the gastrointestinal tract. Here, we have analyzed the regulation of COX-2 by IL-1beta in the human colon carcinoma cell line Caco-2, showing that COX-2 induction by this cytokine is due to both nuclear factor (NF)-kappaB-dependent transcriptional and p38 mitogen-activated protein kinase (MAPK)-mediated post-transcriptional mechanisms. Treatment of these cells with IL-1beta increased the levels of COX-2 mRNA and protein and hence the production of PGE2. IL-1beta induced NF-kappaB activation in Caco-2 cells, promoting the binding of this transcription factor to DNA and increasing NF-kappaB-dependent transcription. Inhibition of NF-kappaB activation diminished IL-1beta-mediated transcriptional activation of COX-2. Furthermore, mutation or deletion of a putative NF-kappaB binding site in the human COX-2 promoter greatly diminished its induction by IL-1beta. In addition, this cytokine induced a rapid increase in p38 MAPK activation. Interestingly, inhibition of p38 MAPK by SB203580 severely decreased induction of COX-2 expression by IL-1beta. p38 MAPK signalling was required for IL-1beta-dependent stabilization of COX-2 transcript. Given the importance of COX-2 expression in intestinal inflammation and colon carcinogenesis, these findings contribute to determine the key signalling pathways involved in the regulation of COX-2 expression in colorectal cells by inflammatory stimuli, such as IL-1beta.

Expression and function of the nuclear factor of activated T cells in colon carcinoma cells: involvement in the regulation of cyclooxygenase-2.

Increasing evidence shows a crucial role of the Ca2+/ calcineurin-mediated activation of the nuclear factor of activated T cells (NFAT) in the regulation of a variety of processes in nonimmune cells. Here we provide evidence that NFATc1 and NFATc2 are expressed in human colon carcinoma cell lines. These proteins are translocated from the cytoplasm to the nucleus upon treatment with a combination of phorbol 12-myristate 13-acetate plus the calcium ionophore A23187. Subsequent to translocation to the nucleus, NFATc1 and NFATc2 were able to bind to a NFAT response element in the DNA, regulating transcriptional activation of genes containing a NFAT-responsive element such as cyclooxygenase-2 (COX-2). COX-2 expression and prostaglandin E2 (PGE2) production were induced upon pharmacological stimuli leading to NFAT activation and blunted by inhibition of calcineurin phosphatase with cyclosporin A or tacrolimus (FK506). Expression of NFAT wild type protein or the active catalytic subunit of calcineurin transactivates COX-2 promoter activity, whereas a dominant negative mutant of NFAT inhibited COX-2 induction in colon carcinoma cell lines. Furthermore, mutation or deletion of NFAT binding sites in the human COX-2 promoter greatly diminished its induction by phorbol 12-myristate 13-acetate/calcium ionophore A23187. These findings demonstrate the presence and activation of NFAT in human colon carcinoma cells, with important implications in the regulation of genes involved in the transformed phenotype as COX-2.

Inhibition of activator protein 1 activation, vascular endothelial growth factor, and cyclooxygenase-2 expression by 15-deoxy-Delta12,14-prostaglandin J2 in colon carcinoma cells: evidence for a redox-sensitive peroxisome proliferator-activated receptor-gamma-independent mechanism.

Cyclooxygenase (COX)-2 and vascular endothelial growth factor (VEGF) are significantly associated with tumor growth and metastasis. Here we show that phorbol ester-mediated induction of VEGF and COX-2 expression in colon carcinoma cells is inhibited by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)). This cyclopentenone was able to inhibit activator protein1 (AP-1)-dependent transcriptional induction of COX-2 and VEGF promoters induced by phorbol 12-myristate 13-acetate (PMA) or c-Jun overexpression. 15d-PGJ(2) interfered with at least two steps within the signaling pathway leading to AP-1 activation. First, 15d-PGJ(2) impaired AP-1 binding to a consensus DNA sequence. Second, 15d-PGJ(2) selectively inhibited c-Jun NH(2) terminal kinase (JNK) but not extracellular signal-regulated kinase or p38 mitogen-activated protein kinase activation induced by PMA. This led to a decreased ability of JNK to phosphorylate c-Jun and to activate its transactivating activity. Inhibition of AP-1 activation and COX-2 or VEGF transcriptional induction by this cyclopentenone was found to be independent of peroxisome proliferator-activated receptor-gamma (PPARgamma) because it was not affected by either expression of a dominant negative form of PPARgamma or the use of a PPARgamma antagonist. In contrast, we have found that the effects of 15d-PGJ(2) on AP-1 activation may occur through its ability to induce intracellular oxidative stress. The antioxidant N-acetylcysteine significantly reversed the inhibition by 15d-PGJ(2) of AP-1 activity and COX-2 or VEGF transcriptional induction. Together, these findings provide new insight into the antitumoral properties of 15d-PGJ(2) through the inhibition of the induction of AP-1-dependent genes involved in tumor progression, such as COX-2 and VEGF.

Cyclooxygenase-2: a therapeutic target in angiogenesis.

Angiogenesis has a role in the pathogenesis of several disorders, including cancer, chronic inflammatory diseases and retinopathies. Recent evidence demonstrates that the production of prostanoids by cyclooxygenase-2 (COX-2) promotes the expression of pro-angiogenic factors. Furthermore, inhibition of COX-2 by non-steroidal anti-inflammatory drugs leads to restricted angiogenesis and downregulated production of pro-angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor. These findings suggest that COX enzymes could be important therapeutic targets in the treatment of pathological angiogenesis.

The hepatitis B virus X protein promotes tumor cell invasion by inducing membrane-type matrix metalloproteinase-1 and cyclooxygenase-2 expression.

Hepatocellular carcinoma is strongly associated with chronic infection by the hepatitis B virus (HBV) and has poor prognosis due to intrahepatic metastasis. HBx is often the only HBV protein detected in hepatic tumor cells; however, its contribution to tumor invasion and metastasis has not been established so far. In this work, we show that HBx enhances tumor cell invasion, both in vivo and in vitro. The increased invasive capacity induced by HBx is mediated by an upregulation of membrane-type 1 matrix metalloproteinase (MT1-MMP) expression, which in turn activates matrix metalloproteinase-2. Induction of both MT1-MMP expression and cell invasion by HBx is dependent on cyclooxygenase-2 (COX-2) activity. In addition, HBx upregulates the expression of COX-2, which is mediated by the transcriptional activation of the COX-2 gene promoter in a nuclear factor of activated T cell-dependent (NF-AT-dependent) manner. These results demonstrate the ability of HBx to promote tumor cell invasion by a mechanism involving the upregulation of MT1-MMP and COX-2 and provide new insights into the mechanism of action of this viral protein and its involvement in tumor metastasis and recurrence of hepatocellular carcinoma.