Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.     

TGF beta 1 and TNF alpha potentiate nitric oxide production in astrocyte cultures by recruiting distinct subpopulations of cells to express NOS-2

Nitric oxide (NO) synthase-2 (NOS-2), a key source of NO at sites of neuroinflammation, is induced in astrocyte cultures treated with lipopolysaccharide (LPS) plus interferon-gamma (IFN gamma). A recent study examining the regulation of astrocytic NOS-2 expression demonstrated that transforming growth factor-beta1 (TGF beta 1) potentiated LPS plus IFN gamma-induced NOS-2 expression via expansion of the pool of astrocytes that express NOS-2. Results in the current report indicate that this population-based mechanism of increasing NOS-2 expression is not restricted to TGF beta 1, since it also accounts for the potentiation of NO production in astrocyte cultures by tumor necrosis factor-alpha (TNFalpha). In contrast to TGF beta 1, which required 24h preincubation for optimal potentiation of NO production, TNF alpha was maximally effective when added concurrently with LPS plus IFN gamma. Nevertheless, under conditions that optimally potentiated NO production, both cytokines recruited similar numbers of astrocytes to express NOS-2 (% NOS-2-positive cells after LPS plus IFN gamma alone or with TNFalpha or TGF beta 1 was 9.5+/-1.2, 25.3+/-2.9, and 32.4+/-3.0, respectively). Interestingly, stimulation of astrocytes in the presence of both TGF beta 1 and TNFalpha additively increased the number of astrocytes that expressed NOS-2 protein (% NOS-2-positive cells was 61.0+/-4.2) relative to each cytokine alone. Potentiation of NO production by either TNF alpha or TGF beta 1 was not ablated by neutralizing antibodies to TGF beta 1 or TNFalpha, respectively. Thus, the two cytokines act independently to recruit separate pools of astrocytes to express NOS-2. These results are consistent with the notion that astrocytes possess an innate heterogeneity with respect to responsiveness to these cytokines.     

RGS10 physically and functionally interacts with STIM2 and requires store-operated calcium entry to regulate pro-inflammatory gene expression in microglia

Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the calcium-dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies pro-inflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of pro-inflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.     

The effect of dehydroepiandrosterone on inflammatory response of astroglial cells

An increased interest in neuroinflammation is conditioned by its involvement in various pathological processes in the brain. Astrocytes play an important role in neuroinflammation, participating in its regulation, throwing out a large number of signaling molecules. Steroid compounds, actively produced by astrocytes, are of interest with regards to the regulation of inflammatory processes in the central nervous system. In the present work the effect of dehydroepiandrosterone (DHEA) on astroglial cells (cultured primary rat astrocytes) in a model of inflammation was studied. The inflammatory response was stimulated with lipopolysaccharide (LPS). Expression levels of pro-inflammatory factor TNFα, antinflammatory interleukin IL-10, and both pro- and antiinflammatory protein COX-2 were measured. The expression of IL-10, COX-2, and TNFα mRNA was determined by real-time PCR, COX-2 protein level by immunoblotting method, TNFα and IL-10 release by enzyme immunoassay. The effect of short-term (30 min) and long-term (24 h) exposure to DHEA was evaluated. It was shown that DHEA potentiates LPS-stimulated (1) increase in the IL-10 mRNA level; (2) IL-10 release; (3) does not affect TNFα level, and (4) exerts a weak pulsating bidirectional effect on COX-2. Using trilostane, an inhibitor of 3β-hydroxysteroid dehydrogenase, a key enzyme of DHEA metabolism, it was shown that DHEA metabolites make the main contribution to its effect. Thus, DHEA is of interest as a stimulant of anti-inflammatory processes in the brain.     

Follistatin-like protein 1 suppressed pro-inflammatory cytokines expression during neuroinflammation induced by lipopolysaccharide

Follistain-like protein 1 (FSTL1), has been recently demonstrated to be involved in the embryo development of nervous system and glioblastoma. However, the role of FSTL1 in neuroinflammation remains unexplored. In this study, the expression of FSTL1 in astrocytes was verified and its role was studied in neuroinflammation induced by in vivo intracerebroventricular (ICV) injection of lipopolysaccharide (LPS) or LPS treatment to astrocytes in vitro. FSTL1 was significantly induced after ICV LPS injection or LPS treatment. FSTL1 suppressed upregulation of pro-inflammatory cytokines in astrocytes after LPS treatment. Moreover, FSTL1 downregulated expression of pro-inflammatory cytokines through suppressing MAPK/p-ERK1/2 pathway in astrocytes. Our results suggest that FSTL1 may play an anti-inflammatory role in neuroinflammation mediated by astrocytes.     

NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso-N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 microM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-D-penicillamine or the NO synthase (NOS) inhibitor 1 mM N(G)-nitro-L-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 microg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 microM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF(1alpha) release. LPS-stimulated COX-2 gene expression was decreased by approximately 50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF(1alpha) release. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Intracisternal administration of transforming growth factor-beta evokes fever through the induction of cyclooxygenase-2 in brain endothelial cells

Transforming growth factor-beta (TGF-beta), a pleiotropic cytokine, regulates cell proliferation, differentiation, and apoptosis, and plays a key role in development and tissue homeostasis. TGF-beta functions as an anti-inflammatory cytokine because it suppresses microglia and B-lymphocyte functions, as well as the production of proinflammatory cytokines. However, we previously demonstrated that the intracisternal administration of TGF-beta induces fever like that produced by proinflammatory cytokines. In this study, we investigated the mechanism of TGF-beta-induced fever. The intracisternal administration of TGF-beta increased body temperature in a dose-dependent manner. Pretreatment with cyclooxygenase-2 (COX-2)-selective inhibitor significantly suppressed TGF-beta-induced fever. COX-2 is known as one of the rate-limiting enzymes of the PGE(2) synthesis pathway, suggesting that fever induced by TGF-beta is COX-2 and PGE(2) dependent. TGF-beta increased PGE(2) levels in cerebrospinal fluid and increased the expression of COX-2 in the brain. Double immunostaining of COX-2 and von Willebrand factor (vWF, an endothelial cell marker) revealed that COX-2-expressing cells were mainly endothelial cells. Although not all COX-2-immunoreactive cells express TGF-beta receptor, some COX-2-immunoreactive cells express activin receptor-like kinase-1 (ALK-1, an endothelial cell-specific TGF-beta receptor), suggesting that TGF-beta directly or indirectly acts on endothelial cells to induce COX-2 expression. These findings suggest a novel function of TGF-beta as a proinflammatory cytokine in the central nervous system.     

Retinoic acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2

Bacterial lipopolysaccharides (LPS) induce expression of multiple genes in endothelial cells, which are critical cellular effectors in various pathologic syndromes. Using subtractive hybridization to identify genes that are differentially induced in human endothelial cells treated with LPS, we found that retinoic acid-inducible gene I (RIG-I) is induced in endothelial cells stimulated with LPS. RIG-I encodes a protein belonging to the DExH-box family which has diverse roles in regulation of gene expression and cellular functions. Cyclooxygenase-2 (COX-2) is also induced in endothelial cells by LPS. Overexpression of RIG-I selectively upregulated expression of COX-2 and also induced COX-2 promoter activity. RIG-I is an inducible gene in stimulated endothelial cells that may have important roles in vascular pathology by virtue of its ability to regulate expression of the COX-2 gene product.