Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation.

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcepsilon-receptor antibodies. Antigens, anti-IgE and anti-FcepsilonRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE(2)) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosis factor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.

PGE2-induced colon cancer growth is mediated by mTORC1.

The inflammatory prostaglandin E2 (PGE2) cytokine plays a key role in the development of colon cancer. Several studies have shown that PGE2 directly induces the growth of colon cancer cells and furthermore promotes tumor angiogenesis by increasing the production of the vascular endothelial growth factor (VEGF). The signaling intermediaries implicated in these processes have however not been fully characterized. In this report, we show that the mechanistic target of rapamycin complex 1 (mTORC1) plays an important role in PGE2-induced colon cancer cell responses. Indeed, stimulation of LS174T cells with PGE2 increased mTORC1 activity as observed by the augmentation of S6 ribosomal protein phosphorylation, a downstream effector of mTORC1. The PGE2 EP4 receptor was responsible for transducing the signal to mTORC1. Moreover, PGE2 increased colon cancer cell proliferation as well as the growth of colon cancer cell colonies grown in matrigel and blocking mTORC1 by rapamycin or ATP-competitive inhibitors of mTOR abrogated these effects. Similarly, the inhibition of mTORC1 by downregulation of its component raptor using RNA interference blocked PGE2-induced LS174T cell growth. Finally, stimulation of LS174T cells with PGE2 increased VEGF production which was also prevented by mTORC1 inhibition. Taken together, these results show that mTORC1 is an important signaling intermediary in PGE2 mediated colon cancer cell growth and VEGF production. They further support a role for mTORC1 in inflammation induced tumor growth.

Suppression of tumor angiogenesis through the inhibition of integrin function and signaling in endothelial cells: which side to target?

Tumor angiogenesis is an essential step in tumor progression and metastasis formation. Suppression of tumor angiogenesis results in the inhibition of tumor growth. Recent evidence indicates that vascular integrins, in particular alpha V beta 3, are important regulators of angiogenesis, including tumor angiogenesis. Integrin alpha V beta 3 antagonists, such as blocking antibodies or peptides, suppress tumor angiogenesis and tumor progression in many preclinical tumor models. The potential therapeutic efficacy of extracellular integrin antagonists in human cancer is currently being tested in clinical trials. Selective disruption of the tumor vasculature by high doses of tumor necrosis factor (TNF) and interferon gamma (IFN-gamma), and the antiangiogenic activity of nonsteroidal anti-inflammatory drugs are associated with the suppression of integrin alpha V beta 3 function and signaling in endothelial cells. Furthermore, expression of isolated integrin cytoplasmic domains disrupts integrin-dependent adhesion, resulting in endothelial cell detachment and apoptosis. These results confirm the critical role of vascular integrins in promoting endothelial cell survival and angiogenesis and suggest that intracellular targeting of integrin function and signaling may be an alternative strategy to extracellular integrin antagonists for the therapeutic inhibition of tumor angiogenesis.

15-hydroxyprostaglandin dehydrogenase is down-regulated in gastric cancer.

PURPOSE: We have investigated the expression and regulation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in gastric cancer. EXPERIMENTAL DESIGN: Clinical gastric adenocarcinoma samples were analyzed by immunohistochemistry and quantitative real-time PCR for protein and mRNA expression of 15-PGDH and for methylation status of 15-PGDH promoter. The effects of interleukin-1beta (IL-1beta) and epigenetic mechanisms on 15-PGDH regulation were assessed in gastric cancer cell lines. RESULTS: In a gastric cancer cell line with a very low 15-PGDH expression (TMK-1), the 15-PGDH promoter was methylated and treatment with a demethylating agent 5-aza-2'-deoxycytidine restored 15-PGDH expression. In a cell line with a relatively high basal level of 15-PGDH (MKN-28), IL-1beta repressed expression of 15-PGDH mRNA and protein. This effect of IL-1beta was at least in part attributed to inhibition of 15-PGDH promoter activity. SiRNA-mediated knockdown of 15-PGDH resulted in strong increase of prostaglandin E(2) production in MKN-28 cells and increased cell growth of these cells by 31% in anchorage-independent conditions. In clinical gastric adenocarcinoma specimens, 15-PGDH mRNA levels were 5-fold lower in gastric cancer samples when compared with paired nonneoplastic tissues (n = 26) and 15-PGDH protein was lost in 65% of gastric adenocarcinomas (n = 210). CONCLUSIONS: 15-PGDH is down-regulated in gastric cancer, which could potentially lead to accelerated tumor progression. Importantly, our data indicate that a proinflammatory cytokine linked to gastric carcinogenesis, IL-1beta, suppresses 15-PGDH expression at least partially by inhibiting promoter activity of the 15-PGDH gene.

G-protein coupled receptor-evoked glutamate exocytosis from astrocytes: role of prostaglandins.

Astrocytes are highly secretory cells, participating in rapid brain communication by releasing glutamate. Recent evidences have suggested that this process is largely mediated by Ca(2+)-dependent regulated exocytosis of VGLUT-positive vesicles. Here by taking advantage of VGLUT1-pHluorin and TIRF illumination, we characterized mechanisms of glutamate exocytosis evoked by endogenous transmitters (glutamate and ATP), which are known to stimulate Ca(2+) elevations in astrocytes. At first we characterized the VGLUT1-pHluorin expressing vesicles and found that VGLUT1-positive vesicles were a specific population of small synaptic-like microvesicles containing glutamate but which do not express VGLUT2. Endogenous mediators evoked a burst of exocytosis through activation of G-protein coupled receptors. Subsequent glutamate exocytosis was reduced by about 80% upon pharmacological blockade of the prostaglandin-forming enzyme, cyclooxygenase. On the other hand, receptor stimulation was accompanied by extracellular release of prostaglandin E2 (PGE2). Interestingly, administration of exogenous PGE2 produced per se rapid, store-dependent burst exocytosis of glutamatergic vesicles in astrocytes. Finally, when PGE2-neutralizing antibody was added to cell medium, transmitter-evoked exocytosis was again significantly reduced (by about 50%). Overall these data indicate that cyclooxygenase products are responsible for a major component of glutamate exocytosis in astrocytes and that large part of such component is sustained by autocrine/paracrine action of PGE2.

mTORC2 regulates PGE2-mediated endothelial cell survival and migration.

Prostaglandin E(2) (PGE(2)) promotes angiogenesis by in part inducing endothelial cell survival and migration. The present study examined the role of mTOR and its two complexes, mTORC1 and mTORC2, in PGE(2)-mediated endothelial cell responses. We used small interfering RNA (siRNA) to raptor or rictor to block mTORC1 or mTORC2, respectively. We observed that down-regulation of mTORC2 but not mTORC1 reduced baseline and PGE(2)-induced endothelial cell survival and migration. At the molecular level, we found that knockdown of mTORC2 inhibited PGE(2)-mediated Rac and Akt activation two important signaling intermediaries in endothelial cell migration and survival, respectively. In addition, inhibition of mTORC2 by prolonged exposure of endothelial cells to rapamycin also prevented PGE(2)-mediated endothelial cell survival and migration confirming the results obtained with the siRNA approach. Taken together these results show that mTORC2 but not mTORC1 is an important signaling intermediary in PGE(2)-mediated endothelial cell responses.

Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

PPARgamma but not PPARalpha ligands are potent repressors of major histocompatibility complex class II induction in atheroma-associated cells.

Peroxisome proliferator-activated receptors (PPARs) are essential in glucose and lipid metabolism and are implicated in metabolic disorders predisposing to atherosclerosis, such as diabetes and dyslipidemia. Conversely, antidiabetic glitazones and hypolipidemic fibrate drugs, known as PPARgamma and PPARalpha ligands, respectively, reduce the process of atherosclerotic lesion formation, which involves chronic immunoinflammatory processes. Major histocompatibility complex class II (MHC-II) molecules, expressed on the surface of specialized cells, are directly involved in the activation of T lymphocytes and in the control of the immune response. Interestingly, expression of MHC-II has recently been observed in atherosclerotic plaques, and it can be induced by the proinflammatory cytokine interferon-gamma (IFN-gamma) in vascular cells. To explore a possible role for PPAR ligands in the regulation of the immune response, we investigated whether PPAR activation affects MHC-II expression in atheroma-associated cells. In the present study, we demonstrate that PPARgamma but not PPARalpha ligands act as inhibitors of IFN-gamma-induced MHC-II expression and thus as repressors of MHC-II-mediated T-cell activation. All different types of PPARgamma ligands tested inhibit MHC-II. This effect of PPARgamma ligands is due to a specific inhibition of promoter IV of CIITA and does not concern constitutive expression of MHC-II. Thus, the beneficial effects of antidiabetic PPARgamma activators on atherosclerotic plaque development may be partly explained by their repression of MHC-II expression and subsequent inhibition of T-lymphocyte activation.

Non steroidal anti-inflammatory drugs and COX-2 inhibitors as anti-cancer therapeutics: hypes, hopes and reality.

Non-steroidal anti-inflammatory drugs (NSAIDs) and specific inhibitors of cyclooxygenase (COX)-2, are therapeutic groups widely used for the treatment of pain, inflammation and fever. There is growing experimental and clinical evidence indicating NSAIDs and COX-2 inhibitors also have anti-cancer activity. Epidemiological studies have shown that regular use of Aspirin and other NSAIDs reduces the risk of developing cancer, in particular of the colon. Molecular pathology studies have revealed that COX-2 is expressed by cancer cells and cells of the tumor stroma during tumor progression and in response to chemotherapy or radiotherapy. Experimental studies have demonstrated that COX-2 over expression promotes tumorigenesis, and that NSAIDs and COX-2 inhibitors suppress tumorigenesis and tumor progression. Clinical trials have shown that NSAIDs and COX-2 inhibitors suppress colon polyp formation and malignant progression in patients with familial adenomatous polyposis (FAP) syndrome. Recent advances in the understanding of the cellular and molecular mechanisms of the anti-cancer effects of NSAIDs and COX-2 inhibitors have demonstrated that these drugs target both tumor cells and the tumor vasculature. The therapeutic benefits of COX-2 inhibitors in the treatment of human cancer in combination with chemotherapy or radiotherapy are currently being tested in clinical trials. In this article we will review recent advances in the understanding of the anti-tumor mechanisms of these drugs and discuss their potential application in clinical oncology.

The renal hemodynamic effects of ibuprofen in the newborn rabbit.

In early childhood, nonsteroidal anti-inflammatory drugs are mainly used to either prevent or treat premature labor of the mother and patent ductus arteriosus of the newborn infant. The most frequently used prostaglandin-synthesis inhibitor is indomethacin. Fetuses exposed to indomethacin in utero have been born with renal developmental defects, and in both the unborn child and the term and premature newborn this drug may compromise renal glomerular function. The latter has in the past also been observed when i.v. indomethacin or i.v. acetylsalicylic acid (aspirin) were administered to newborn rabbits. The present experiments were designed to evaluate whether ibuprofen has less renal side effects than indomethacin, as claimed. Three groups of anesthetized, ventilated, normoxemic neonatal rabbits were infused with increasing doses of ibuprofen (0.02, 0.2, 2.0 mg/kg body weight) and the following renal parameters were measured: urine volume, urinary sodium excretion, GFR, and renal plasma flow. Renal blood flow, filtration fraction, and the renal vascular resistance were calculated according to standard formulae. Intravenous ibuprofen caused a dose-dependent, significant reduction in urine volume, GFR, and renal blood flow with a fall in filtration fraction in the animals receiving the highest dose of ibuprofen (2 mg/kg body weight). There was a very steep rise in renal vascular resistance. Urinary sodium excretion decreased. These experiments in neonatal rabbits clearly show that acute i.v. doses of ibuprofen also have significant renal hemodynamic and functional side effects, not less than seen previously with indomethacin.

Renal effects of selective cyclooxygenase-2 inhibition in normotensive salt-depleted subjects.

PURPOSE: To compare the renal hemodynamic and tubular effects of celecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2) to those of naproxen, a nonselective inhibitor of cyclooxygenases in salt-depleted subjects. METHODS AND SUBJECTS: Forty subjects were randomized into four parallel groups to receive 200 mg celecoxib twice a day, 400 mg celecoxib twice a day, 500 mg naproxen twice a day, or a placebo for 7 days according to a double-blind study design. Blood pressure, renal hemodynamics, and urinary water and electrolyte excretion were measured before and for 3 hours after drug intake on days 1 and 7. RESULTS: Celecoxib had no effect on systemic blood pressure, but short-term transient decreases in renal blood flow and glomerular filtration rate were found with the highest dose of 400 mg on day 1. On the first day, both celecoxib and naproxen decreased urine output (P < .05) and sodium, lithium, and potassium excretion (P < .01). On day 7, similar effects on water and sodium excretion were observed. During repeated administration, a significant sodium retention occurred during the first 3 days. CONCLUSION: In salt-depleted subjects, selective inhibition of COX-2 causes sodium and potassium retention. This suggests that an increased selectivity for COX-2 does not spare the kidney, at least during salt depletion.

Hypotensive effect of the active fragment derived from factor XII is mediated by an activation of the plasma kallikrein-kinin system.

Plasma protein fraction (PPF) contaminated by factor XII active fragment (XIIf) may cause hypotensive reactions when infused to patients. This study was planned to assess in conscious normotensive rats whether the blood pressure response to the factor XIIf is mediated by an activation of the plasma kallikrein-kinin system or by stimulation of prostaglandin synthesis. To test whether the factor XIIf-induced blood pressure fall is due partially to an enhanced generation of vasodilating prostaglandins, the blood pressure effect of XIIf (1 microgram i.v.) was investigated 15 min after treatment with indomethacin (5 mg i.v.), an inhibitor of cyclo-oxygenase. Factor XIIf reduced mean blood pressure similarly in indomethacin- and vehicle-treated rats (-23 +/- 4 mmHg, n = 5, and -23 +/- 5 mmHg, n = 4, respectively). Other rats received factor XIIf 15 min after depletion of circulating prekallikrein by the administration of dextran sulfate. Thirty minutes after a 0.25 mg i.v. dose of this agent, plasma prekallikrein activity averaged 0.12 +/- 0.015 mumol/min/ml (n = 6) as compared to 2.48 +/- 0.31 mumol/min/ml in control rats (n = 4, P less than .001). Factor XIIf decreased mean blood pressure by only 4 +/- 2 mm Hg in rats pretreated with dextran sulfate. Thus, it was possible to blunt the acute hypotensive effect of factor XIIf by depleting circulating prekallikrein, but not by inhibiting prostaglandin production. This strongly suggests that the blood pressure effects of factor XIIf is mediated by a stimulation of the plasma kallikrein-kinin system.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.