María José Díaz-Guerra

Inhibition of IκB Kinase and IκB Phosphorylation by 15-Deoxy-Δ12,14-Prostaglandin J2 in Activated Murine Macrophages

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Notch-1 Up-Regulation and Signaling following Macrophage Activation Modulates Gene Expression Patterns Known to Affect Antigen-Presenting Capacity and Cytotoxic Activity

Notch signaling has been extensively implicated in cell-fate determination along the development of the immune system. However, a role for Notch signaling in fully differentiated immune cells has not been clearly defined. We have analyzed the expression of Notch protein family members during macrophage activation. Resting macrophages express Notch-1, -2, and -4, as well as the Notch ligands Jagged-1 and -2. After treatment with LPS and/or IFN-γ, we observed a p38 MAPK-dependent increase in Notch-1 and Jagged-1 mRNA and protein levels. To study the role of Notch signaling in macrophage activation, we forced the transient expression of truncated, active intracellular Notch-1 (Notch-IC) proteins in Raw 264.7 cells and analyzed their effects on the activity of transcription factors involved in macrophage activation. Notch-IC increased STAT-1-dependent transcription. Furthermore, Raw 264.7 Notch-IC stable transfectants increased STAT1-dependent transcription in response to IFN-γ, leading to higher expression of IFN regulatory factor-1, suppressor of cytokine signaling-1, ICAM-1, and MHC class II proteins. This effect was independent from an increase of STAT1 Tyr or Ser phosphorylation. However, inducible NO synthase expression and NO production decreased under the same conditions. Our results show that Notch up-regulation and subsequent signaling following macrophage activation modulate gene expression patterns known to affect the function of mature macrophages.

Evidence for common mechanisms in the transcriptional control of type II nitric oxide synthase in isolated hepatocytes

Incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS), S-[2,3-bis(palmitoyloxy)-(2-R,S)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys4 (TPP), a synthetic lipopeptide present in bacterial cell wall lipoproteins, or with phorbol 12,13-dibutyrate (PDBu) induced an increase in nitric oxide synthesis through the expression of type II nitric oxide synthase (iNOS). Transfection of hepatocytes with a HindII fragment corresponding to the promoter region of the murine iNOS gene (from nucleotide −1588 to +165) resulted in the expression of the reporter gene when cells were stimulated with these factors. The transcription factors activated by these stimuli involved an increase in the nuclear content of proteins that bind to κB, AP-1, GAS, and SIE sequences. Inhibition of NF-κB activation with pyrrolidine dithiocarbamate eliminated the expression of iNOS in hepatocytes stimulated with LPS, TPP, or PDBu. In addition to this, transfection of hepatocytes with promoter mutants in which a sequential 2-base pair change within the κB sites was introduced (position −971 to −961 and −85 to −75, respectively), resulted in approximately 17 and 35%, respectively, of the activity of the naive promoter. Simultaneous mutation of both κB sites abolished the promoter activity. Analysis of the proteins involved in κB binding showed the presence of p50/p65 dimers in the nuclei of activated cells at the time that an important decrease of IκB-α was observed soon after cell stimulation with LPS, TPP, or PDBu. However, only LPS was able to decrease the amount of IκB-β. These results suggest that LPS, TPP, and PDBu, although activating different signal transduction pathways, use a common mechanism mediating iNOS expression in cultured hepatocytes.

Notch1 upregulates LPS-induced macrophage activation by increasing NF-κB activity

Macrophages present different Notch receptors and ligands on their surface. Following macrophage activation by LPS or other TLR ligands, Notch1 expression is upregulated. We report here that Notch signaling increases both basal and LPS‐induced NF‐κB activation, favoring the expression of genes implicated in the inflammatory response, such as the cytokines TNF‐α and IL‐6, or enzymes, such as iNOS. Delta4 seems to be the most effective ligand to induce Notch activation and increasing NF‐κB transcriptional activity in macrophages. We show that Notch1 signaling promotes NF‐κB translocation to the nucleus and DNA binding by increasing both phosphorylation of the IκB kinase α/β complex and the expression of some NF‐κB family members. Treatment of macrophages with the γ‐secretase inhibitor DAPT, which prevents the cleavage and activation of Notch receptors, inhibits all these processes, diminishing NF‐κB activity following LPS stimulation. Additionally, we show that the active intracellular Notch fragment can directly interact with TNF‐α and iNOS promoters. Our results suggest that Notch signaling results in an amplification of the macrophage‐dependent inflammatory response by enhancing NF‐κB signaling.

Up-regulation of protein kinase C-epsilon promotes the expression of cytokine-inducible nitric oxide synthase in RAW 264.7 cells.

Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -ε isotype but not with PKC-α, -β1, -δ, or constitutively active -α and -β1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the −1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-ε isotype. The mechanism by which PKC-ε induced iNOS expression involved the activation of nuclear factor binding to κB sites (NF-κB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-κB activation, and by the activation of κB sites in cells transfected with a vector containing a κB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-ε can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.

Rapid Up-regulation of IκBβ and Abrogation of NF-κB Activity in Peritoneal Macrophages Stimulated with Lipopolysaccharide

Lipopolysaccharide (LPS) administration to mice elicited the activation of nuclear factor κB (NF-κB) in several tissues including liver and macrophages. Maximal activation was observed 1 h after treatment but declined at 3 and 6 h. The levels of IκBα and IκBβ were analyzed during this period in an attempt to correlate NF-κB activity with IκB resynthesis. Degradation of IκBα was very rapid and was followed by recovery 1 h after LPS administration. IκBβ degradation, which has been associated with persistent NF-κB activation, was complete at 1 h. However, a rapid recovery of IκBβ in these tissues was observed at 3 h in parallel with the abrogation of NF-κB activity. Immunolocalization of newly synthesized IκBβ by confocal microscopy revealed its preferential accumulation in the cytosol. Analysis of IκBβ by Western blot using high resolution polyacrylamide gel electrophoresis showed the presence of two bands in cytosolic extracts of LPS-treated macrophages at 3 h, but only one band with the same mobility as the control was detected at 6 h. Moreover, treatment of extracts of resynthesized IκBβ with alkaline phosphatase resulted in the accumulation of the protein of slightly higher electrophoretic mobility, indicating the prevalence of a rapid phosphorylation of the newly synthesized IκBβ. At the mRNA level, up-regulation of IκBβ was observed in macrophages stimulated for 1 h with LPS. When the effect of pro-inflammatory cytokines was investigated, tumor necrosis factor α, but not interleukin-1 or interferon-γ, promoted an important degradation of IκBβ followed by an increase in the mRNA at 1 h. These results suggest the existence of LPS- and tumor necrosis factor α- specific pathways involved in a rapid IκBβ degradation and resynthesis and might explain the transient period of activation of NF-κB in these tissues upon stimulation with these factors. This rapid control of NF-κB function may contribute to the attenuation of the inflammatory response of these cells.

Mechanisms of the neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices

Acetylsalicylic acid (ASA, Aspirin) is an anti-inflammatory drug with a wide spectrum of pharmacological activities and multiple sites of action. Apart from its preventive actions against stroke due to its antithrombotic properties, recent data in the literature suggest that high concentrations of ASA also exert direct neuroprotective effects. We have used an in vitro model of brain ischaemia using rat forebrain slices deprived of oxygen and glucose to test ASA neuroprotective properties. We have found that ASA inhibits neuronal damage at concentrations lower than those previously reported (0.1-0.5 mM), and that these effects correlate with the inhibition of excitatory amino acid release, of NF-kappaB translocation to the nucleus and iNOS expression caused by ASA. All of these three mechanisms may mediate the neuroprotective effects of this drug. Our results also show that the effects of ASA are independent of COX inhibition. Taken together, our present findings show that ASA is neuroprotective in an in vitro model of brain ischaemia at doses close to those recommended for its antithrombotic effects.

Inhibition of Ikappa B Kinase and Ikappa B Phosphorylation by 15-Deoxy-Delta 12,14-Prostaglandin J2 in Activated Murine Macrophages

ABSTRACT Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Δ12,14-prostaglandin J2(15dPGJ2) inhibited the LPS- and IFN-γ-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J2, prostaglandin E2, or rosiglitazone, a peroxisomal proliferator-activated receptor γ ligand. Incubation of activated macrophages with 15dPGJ2 inhibited the degradation of IκBα and IκBβ and increased their levels in the nuclei. NF-κB activity, as well as the transcription of NF-κB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IκB phosphorylation showed an inhibition of IκB kinase by 15dPGJ2 in cells treated with LPS and IFN-γ, resulting in an impaired phosphorylation of IκBα, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IκB kinase with 2 μM 15dPGJ2 reduced by 83% the phosphorylation in serine 32 of IκBα, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IκB kinase complex. These results show rapid actions of 15dPGJ2, independent of peroxisomal proliferator receptor γ activation, in macrophages challenged with low doses of LPS and IFN-γ.

Inhibition of NOS‐2 expression in macrophages through the inactivation of NF‐κB by andalusol

Andalusol, ent‐6α,8α,18‐trihydroxy‐13(16),14‐labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844‐849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ). Incubation of J774 macrophages with andalusol (0.1–100 μM) inhibited the synthesis of nitrite caused by LPS (1 μg ml−1) in concentration and time‐dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h. Incubation of J774 cells with LPS resulted in the expression of NOS‐2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme decreased significantly in the presence of andalusol (IC50=10.5 μM), suggesting that this diterpene inhibited NOS‐2 expression. Andalusol inhibited nuclear factor κB activation, a transcription factor necessary for NOS‐2 expression in response to LPS and IFN‐γ. This compound also inhibited the degradation of IκBα favouring the retention of the inactive NF‐κB complexes in the cytosol. Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS‐2 expression in LPS‐activated macrophages. The present findings provide a mechanism by which the anti‐inflammatory properties of this diterpene could be mediated.