Amyloid beta peptide (25-35) activates protein kinase C leading to cyclooxygenase-2 induction and prostaglandin E2 release in primary midbrain astrocytes

Prostaglandins (PGs) are generated by the enzymatic activity of cyclooxygenase-1 and -2 (COX-1/2) and modulate several functions in the CNS such as the generation of fever, the sleep/wake cycle, and the perception of pain. Moreover, the induction of COX-2 and the generation of PGs has been linked to neuroinflammatory aspects of Alzheimer's disease (AD). Non-steroidal anti-inflammatory drugs (NSAIDs) that block COX enzymatic activity have been shown to reduce the incidence of AD in various epidemiological studies. While several reports investigated the expression of COX-2 in neurons and microglia, expression of COX-2 in astroglial cells has not been investigated in detail. Here we show that amyloid β peptide 25–35 (Aβ_25–35) induces COX-2 mRNA and protein synthesis and a subsequent release of prostaglandin E₂ (PGE₂) in primary midbrain astrocytes. We further demonstrate that protein kinase C (PKC) is involved in Aβ_25–35-induced COX-2/PGE₂ synthesis. PKC-inhibitors prevent Aβ_25–35-induced COX-2 and PGE₂ synthesis. Furthermore Aβ_25–35 rapidly induces the phosphorylation and enzymatic activation of PKC in primary rat midbrain glial cells and in primary human astrocytes from post mortem tissue. Our data suggest that the PKC isoforms and/or β are most probably involved in Aβ_25–35-induced expression of COX-2 in midbrain astrocytes. The potential role of astroglial cells in the phagocytosis of amyloid and the involvement of PGs in this process suggests that a modulation of PGs synthesis may be a putative target in the prevention of amyloid deposition.     

ERK1/2 and p38-MAPK signalling pathways, through MSK1, are involved in NF-kappaB transactivation during oxidative stress in skeletal myoblasts

Skeletal muscle is highly adapted to respond to oxidative imbalances, since it is continuously subjected to an increased production of reactive oxygen species (ROS) during exercise. Oxidative stress, however, has been associated with skeletal muscle atrophy and damage in many diseases. In this study, we examined whether MAPK and NF-κB pathways participate in the response of skeletal myoblasts to oxidative stress, and whether there is a cross talk between these pathways. H₂O₂ induced a strong activation of ERKs, JNKs and p38-MAPK in a time- and dose-dependent profile. ERK and JNK activation by H₂O₂, but not that of p38-MAPK, was mediated by Src kinase and, at least in part, by EGFR. H₂O₂ also stimulated a mild translocation of NF-κB to the nucleus, as well as a moderate phosphorylation of its endogenous cytoplasmic inhibitor IκB (at Ser32/36), without any significant decrease in IκB total levels. Moreover, oxidative stress induced a strong phosphorylation of NF-κB p65 subunit at Ser536 and Ser276. Inhibition of MAPK pathways by selective inhibitors did not appear to affect H₂O₂-induced nuclear translocation of NF-κB or the phosphorylation of IκB. In contrast, phosphorylation of p65 at Ser276 was found to be mediated by MSK1, a substrate of both ERKs and p38-MAPK. In conclusion, it seems that, during oxidative stress, NF-κB translocation to the nucleus is most likely not related with the MAPK activation, while p65 phosphorylations are in part mediated by MAPKs pathways, probably modifying signal specificity.     

PI 3-kinase and MAP kinase regulate bradykinin induced prostaglandin E(2) release in human pulmonary artery by modulating COX-2 activity

Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E₂ (PGE₂) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE₂ in a three step process involving phospholipase A₂ (PLA₂), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE₂ release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA₂ activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity.     

Antiinflammatory effects of the cyclopentenone isoprostane 15-A(2)-IsoP in human gestational tissues

Proinflammatory prostaglandins and cytokines are involved in the initiation of human labor and delivery. Although cyclopentenone prostaglandins regulate the formation of these prolabor mediators via nuclear factor-κB (NF-κB) and/or peroxisome proliferator-activated receptor-γ, recent evidence suggests that they do not exist in vivo. Cyclopentenone isoprostanes (IsoPs), which are highly reactive structural isomers of bioactive cyclopentenone prostaglandins, do exist physiologically and have been shown to inhibit the inflammatory response in macrophages. Therefore the aim of this study was to determine the effect of the synthetic cyclopentenone IosP 15-A₂-IsoP on the expression of prolabor mediators in human gestational tissues. Human placenta and gestational membranes (n = 5) were incubated in the absence or presence of 12.5, 25, and 50 μM 15-A₂-IsoP with 10 μg/ml lipopolysaccharide (LPS). Treatment of placenta and fetal membranes with 15-A₂-IsoP caused a dose-dependent decrease in LPS-stimulated release of the cytokines IL-1β, IL-6, IL-8, and TNF- and the prostaglandins PGE₂ and PGF₂. NF-κB p65 DNA binding activity was significantly inhibited by treatment with 50 μM 15-A₂-IsoP. Collectively, these data suggest that 15-A₂-IsoP exhibits antiinflammatory properties via antagonism of NF-κB activity. Cyclopentenone IsoPs may serve as negative feedback regulators of the inflammatory response in human gestational tissues.     

Angiotensin II induces matrix metalloproteinase-9 expression via a nuclear factor-kappaB-dependent pathway in vascular smooth muscle cells

Angiotensin II (AngII) is widely recognized as a critical regulator of the development of atherosclerosis. Matrix metalloproteinases (MMPs) are thought to participate in plaque destabilization through degradation of the extracellular matrix. In the present study, we investigated the potential mechanism of AngII-induced MMP-9 expression in vascular smooth muscle cells (VSMC). AngII upregulated the expression of MMP-9 significantly in VSMC obtained from rat aorta. RNAi-mediated knockdown of p65 and losartan, an inhibitor of AngII receptors subtype-1 (AT₁), could abolish AngII-induced MMP-9 expression. In addition, AngII induced the NF-κB binding activity via AT₁ and AT₂ receptors in VSMC, and AngII-induced activation of NF-κB is not associated with significant downregulation of IκB. In summary, this study demonstrates that AngII stimulates NF-κB nuclear translocation in VSMC via AT₁ and AT₂. AngII increases the expression of MMP-9 in VSMC, and AT₁ and NF-κB pathways have an important role in this response.     

Involvement of protein kinase C in the inhibition of lipopolysaccharide-induced nitric oxide production by thapsigargin in RAW 264.7 macrophages

This study explored the effects of inhibition of endoplasmic reticulum (ER) Ca²⁺-ATPase on lipopolysaccharide (LPS)-induced protein kinase C (PKC) activation, nuclear factor-κB (NF-κB) translocation, inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in RAW 264.7 macrophages. Thapsigargin (TG) irreversibly inhibits ER Ca²⁺-ATPase and LPS-induced NO production is reduced even after washout. TG also attenuated LPS-stimulated iNOS expression by using immunoblot analysis. However, another distinct fully reversible ER Ca²⁺-ATPase inhibitor, 2,5-di-tert-butylhydroquinone (DBHQ), ionophore A23187 and ionomycin could exert a similar effect to TG in increasing intracellular calcium concentration; however, these agents could not mimic TG in reducing iNOS expression and NO production. LPS increased PKC- and -β activation, and TG pretreatment attenuated LPS-stimulated PKC activation. Not did pretreatment with DBHQ, A23187 and ionomycin reduce LPS-stimulated PKC activation. Furthermore, NF-κB-specific DNA–protein-binding activity in the nuclear extracts was enhanced by treatment with LPS, and TG pretreatment attenuated LPS-stimulated NF-κB activation. None of DBHQ, A23187 and ionomycin pretreatment reduced LPS-stimulated NF-κB activation. These data suggest that persistent inhibition of ER Ca²⁺-ATPase by TG would influence calcium release from ER Ca²⁺ pools that was stimulated by the LPS activated signal processes, and might be the main mechanism for attenuating PKC and NF-κB activation that induces iNOS expression and NO production.     

Alteration of subcellular redox equilibrium and the consequent oxidative modification of nuclear factor kappaB are critical for anticancer cytotoxicity by emodin, a reactive oxygen species-producing agent

We previously found that emodin produced reactive oxygen species (ROS) intracellularly. In various tumor cells at low doses it enhances the cytotoxicity of As₂O₃, and at higher doses it renders cytotoxicity independently in vitro and in vivo. The effects involve redox-mediated inhibition of NF-κB activation. In this study, we focus on the mechanisms by which emodin inhibits NF-κB activation. Results in HeLa cells demonstrated that emodin at high doses or in combination with As₂O₃, via generation of ROS especially in the nucleus, altered subcellular redox equilibrium and thus oxidized the redox-sensitive site on NF-κB and prevented its binding to the target DNA. In vivo study showed that tumors exposed to the arsenic/emodin cotreatment had dramatically smaller sizes and weaker antioxidant capacity, compared with arsenic alone. NF-κB binding and transactivation were inhibited in these tumors. These data help in the understanding of the mechanisms by which manipulation of cellular redox and NF-κB activation may enhance chemotherapy.     

Molecular pharmacology of aromatase and its regulation by endogenous and exogenous agents

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin E₂ (PGE₂) increases intracellular cAMP levels and stimulates estrogen biosynthesis, and our recent studies have shown a strong linear association between CYP19 expression and the sum of COX-1 and COX-2 expression in breast cancer specimens. PGE₂ can bind to four receptor subtypes, EP₁–EP₄, which are coupled to different intracellular signaling pathways. In primary human breast stromal cell cultures, aromatase activity was significantly induced by PGE₂, dexamethasone, and agonists for the EP₁ and EP₂ receptor subtypes. An EP₁ antagonist, SC-19220, inhibited the induction of enzyme activity by PGE₂ or 17-phenyltrinor-PGE₂, an EP₁ agonist. Sulprostone, an EP₃ agonist, did not alter aromatase activity levels. Investigations are also underway on the regulation of aromatase by exogenous medicinal agents. Selective steroidal and nonsteroidal agents are effective in inhibiting breast tissue aromatase. The benzopyranone ring system is a molecular scaffold of considerable interest, and this scaffold is found in certain flavonoid natural products that have weak aromatase inhibitory activity. Our novel synthetic route for benzopyranones utilizes readily available salicylic acids and terminal alkynes as starting materials. The synthesis of flavones with diversity on the benzopyranone moiety and at the C-2 position occurs with good to excellent yields using these reaction conditions, resulting in an initial benzopyranone library of thirty compounds exhibiting enhanced and differential aromatase inhibition. Current medicinal chemistry efforts focus on diversifying the benzopyranone scaffold and utilizing combinatorial chemistry approaches to construct small benzopyranone libraries as potential aromatase inhibitors.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Preventive effect of 20(S)-ginsenoside Rg3 against lipopolysaccharide-induced hepatic and renal injury in rats

The preventive effect of 20(S)-ginsenoside Rg₃ (20(S)-Rg₃) on lipopolysaccharide (LPS)-induced oxidative tissue injury in rats was investigated in this study. The elevated serum nitrite/nitrate, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and creatinine levels in LPS-treated control rats were significantly decreased following 15 consecutive days of 20(S)-Rg₃ administration. In addition, thiobarbituric acid-reactive substance levels in the serum, liver and kidney were dose-dependently lower in 20(S)-Rg₃-treated groups than in the LPS-treated control group. The nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) protein expressions in the liver and kidney were significantly increased by LPS treatment. However, the 20(S)-Rg₃ administrations significantly decreased these protein expressions except for HO-1 in the liver. On the other hand, in the kidney, oral administration of 20(S)-Rg₃ showed a tendency to reduce NF-κB and iNOS protein expressions and also significantly reduced the elevated COX-2 and HO-1 protein expressions at a dose of 10 mg/kg body weight/day. All these results suggest the preventive effect of 20(S)-Rg₃ against LPS-induced acute oxidative damage in the liver and kidney and the preventive effect of 20(S)-Rg₃ administration against LPS toxicity was thought to be more predominant in the liver than kidney.     

Differential production of prostaglandins D2 and E2 and of unusual prostanoid by chick spinal cord and meninges in vitro

In order to specify the source of locally synthesized prostaglandin (PG) E₂ which is able to saturate the large class of low affinity PGE₂ receptors in chick spinal cord, bioconversion of [1-¹⁴C]arachidonic acid into prostanoids was studied in homogenates of chick spinal cord and meninges first without addition of exogenous glutathione (GSH). Homogenates of spinal cord produced ¹⁴C-labeled PGE₂, PGD₂ and PGF₂. Homogenates of meninges accumulated much larger amounts of [¹⁴C]PGE₂ than spinal cord and surprisingly a ¹⁴C-labeled arachidonate metabolite referred to as compound Y. Compound Y generation, which was inhibited by indomethacin and enhanced by esculetin, was therefore mediated through the cyclooxygenase pathway. The fact that no labeled compound Y was detected in homogenates incubated with [³H]PGD₂ or [³H]PGE₂ indicated that compound Y was not degradation product of PG_s. Secondly, after addition of exogenous GSH, ¹⁴C-labeled compound Y was totally converted into [¹⁴C]PGE₂. The compound Y which is converted into PGF_s after a strong reduction with NaBH₄ and into PGE₂ after a mild reduction with GSH-hemin system or SnCl₂ was therefore assumed to be a 15 hydroperoxy-PGE₂ (15 HP-PGE₂). These results suggest that PGE₂ can be synthesized in meninges either by the classical isomerization of PGH₂ or by isomerization of PGG₂ followed by a GSH-sensitive reaction.     

Angiotensin II induces NF-kappa B activation in HUVEC via the p38MAPK pathway

Angiotensin II (Ang II) is the main active peptide of the renin–angiotensin system (RAS), producing a number of inflammatory mediators that lead to endothelial dysfunction and the progression of atherosclerosis. Ang II-induced NF-κB nuclear translocation plays a pivotal role in this response. This study examines the NF-κB activation mechanism elicited by Ang II in human umbilical vein endothelial cells (HUVEC). Electrophoretic mobility shift assays and Western blotting revealed that Ang II, signaling via AT₁, produces a time-dependent increase in NF-κB DNA binding and IκB degradation. These results also demonstrate that Ang II leads to MAPK phosphorylation and p38MAPK pathway-induced NF-κB activation. Furthermore, AT₁ is required for p38MAPK phosphorylation induced by Ang II. This study provides evidence that Ang II elicits NF-κB activation via the p38MAPK pathway in HUVEC.     

Prostaglandin E1 specific binding in rhesus myometrium

Myometrial low speed supernatant prepared from non-pregnant rhesus uteri was incubated with ³H-Prostaglandin (PG) E₁ with or without addition of unlabelled prostaglandins. The uptake of ³H-PGE₁ was inhibited in a dose dependent fashion by PGE₂>PGE₁>PGA₁>PGF₂=PGA₁>PGB₁=PGB₂≥PGD₂. PGE₁ metabolites inhibited ³H-PGE₁ binding in the following order: 13,14-dihydro-PGE₁>13,14-dihydro-15-keto-PGE₁=15-keto-PGE₁. The specific binding of ³H-PGE₁ and ³H-PGF₂ was similarly affected by the temperature and time of incubation. Equilibrium binding constants determined using rhesus uteri obtained during the luteal phase of the menstrual cycle indicate the presence of high affinity PGE₁ binding sites with an average (n=3) apparent dissociation constant of 2.2 × 10⁻⁹M and a lower affinity PGE₁ binding site with a K_d 1 × 10⁻⁸M. No high affinity — low capacity ³H-PGF₂ sites could be demonstrated.

Relative uterine stimulating potencies of some natural prostaglandins and prostaglandin analogs tested after acute intravenous administration in mid-pregnant rhesus monkeys corresponded with the PGE₁ binding inhibition of the respective compound. The uterine stimulating potencies of the prostaglandin analogs tested were: (15S)-15-methyl-PGE₂=16,16-dimethyl-PGE₂>17-phenyl-18,19,20-trinor-P GE₂>16 phenoxy-17,18,19,20-tetranor-PGE₂=PGE₂=PGE₁=(15S)-15-methyl-PGE₂>PGF₂.