Volume 2 Issue 1 - November 2, 2007
A Possible Therapeutic Strategy for CNS Repair: Reduction of iNOS Production in Activated Glia by Interference of PGE2/EP2 Action
Shun-Fen Tzeng

Department of Life Sciences, National Cheng Kung University, Taiwan
Glia. Jan 15;55(2):214-223 (2007)

Abstract--Glia are activated by inflamed microenviromental factors after injury to the central nervous system (CNS), which produce excessive amounts of NO by inducible nitric oxide synthase (iNOS) to induce neural cell death and hinder CNS repair. The production of iNOS is known to be regulated by a Ca2+-independent pathway. However, what is being described here is a novel mechanism involving both prostaglandin E2 (PGE2)-mediated cAMP/PKA/p38MAPK and PKA/IP3/Ca2+-dependent signaling pathways through the activation of its receptor, EP2, to upregulate astrocytic iNOS expression under the inflamed condition. The findings have provided important information to develop an effective strategy for CNS repair in the future.

Neural cells in the CNS (brain and spinal cord) are neurons and glia; the latter contains, astroglia, myelin-producing oligodendroglia and CNS-resident macrophages (microglia). Astroglia are the major glial cell type in the CNS. These cells regulate neuronal maturation, neuronal metabolic and neurotransmitter homeostasis, and synaptic plasticity in the developing and healthy adult CNS. Thus, it is thought that astroglia can protect neurons against CNS insults. However, astroglia can be activated in acute and chronic neurodegenerative diseases, which intensively produce proinflammatory mediators, such as cytokines, reactive oxygen species (ROS) and prostanoids (PGs), etc. Accordingly, astroglial activation is considered to involve neuroinflammation after CNS injury. These cells are also stimulated to become hypertrophy with upregulated expression of intermediate filaments (e.g., glial fibrillary acidic protein, vimentin, nestin). The process is so called as astrogliosis, which leads to the formation of glial scar as a physical barrier which makes axonal regeneration difficult (Fig. 1). Therefore, it is believed that inhibition of glial activation is the effective strategy for improving CNS repair.
Figure 1. A scheme showing the role of glial activation and glia scar in neurodegeneration (modified from Tzeng et al. 2005).

Similar to the behavior of microglia, activated astroglia produce high amounts of NO by iNOS in response to a series of cytokines including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) (Fig. 1). NO can be synthesized from L-arginine through NOS catalysis and has essential physiological functions such as vasodilation, respiration, and neurotransmission, etc. There are endothelial NOS (eNOS; NOS3), iNOS (NOS2) and neural NOS (nNOS; NOS1) being expressed in neurons and glia of the CNS. After injury to the CNS, NO is known to be remarkably produced by the action of iNOS in activated astroglia and microglia (Moncada and Bolanos 2006). It is known that the induction of iNOS is calcium-independent. NO from activated astroglia and microglia, reacts with superoxide anion (O2-) to produce peroxynitrite (ONOO-), which can cause the death of oligodendroglia and neurons in human CNS neurological disorders, such as stroke, traumatic brain injury (TBI), Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and spinal cord injury (SCI). It has been reported that S-nitrosylation of Parkin (an E3 ubiquitin ligase) by excessive NO to inhibit its ligase activity can trigger PD pathogenesis (Chung et al. 2004). In addition, peroxynitrite could damage neurons via the inhibition of mitochondrial complex I and cytochrome c oxidase to cause neuronal energy deficiency (Moncada and Bolanos 2006).

Arachidonic acid (AA) and its metabolites mediate the function of the normal CNS and inflammation in various neurodegenerative disorders (Tzeng et al. 2005). Prostanoids (PGs) are a large group of AA metabolites. Prostaglandin E2 (PGE2), the most abundant PG in the CNS, is involved in several pathological events, such as fever induction and pain hypersensitivity elicitation. Many studies have addressed that elevated PGE2 levels can be detected in the cerebrospinal fluid of patients with AD, stroke, ALS, SCI, and TBI. Activated glia are thought to serve as the producer for large amounts of PGs in the injured CNS. Evidence has indicated that exposure of astroglia to endotoxin lipopolysaccaride (LPS) resulted in an increase in cyclooxygenase 2 (COX-2) followed by a markedly increased levels of PGE2. ATP and phorbol 12-myristate 13-acetate have also been reported to markedly enhance PGE2 production in cytokine-stimulated astroglia.

PGE2 binds to multiple G protein-coupled receptors including high affinity E subtype receptors (EP1-4) and low affinity PG receptors, such as FP. Since all four EP receptors (EP1, EP2, EP3, and EP4) and FP are expressed in astroglia, PGE2 can affect astroglial function through the activation of its receptors. In collaboration with Professor Oi-Tong Mak in the Department of Life Sciences, we have found that PGE2 in synergy with TNF-α and IFN-γ increases astroglial iNOS expression through EP2 receptor-induced cellular signaling pathway, leading to improvement of astroglial activation (Hsiao et al. 2007). Our findings are described as follows.

1. PGE2 activates EP2 receptor in cytokine-primed glia to upregulate iNOS transcription.
Figure 2. The signaling cascades in PGE2/EP2-increased astroglial iNOS expression in the presence of TNF-α/IFN-γ (T/I). Together with proinflammatory cytokines (TNF-α and IFN-γ), PGE2 activates the cAMP/PKA cascade to induce p38MAPK activation and intracellular Ca2+ release via IP3 receptor; the latter in turn stimulate PKC activation. This stimulation subsequently activates transcription factor CREB, resulting in increased expression of iNOS in TNF-α/IFN-γ-treated astroglia (modified from Hsiao et al. 2007).

Treatment with TNF-α (10 ng/ml), IFN-γ (10 ng/ml), or PGE2 alone could not induce iNOS production in astroglia from cerebral cortices of neonatal day 1-2 Sprague-Dawley rats. However, astroglial iNOS expression could be induced by the addition of TNF-α together with IFN-γ (T/I). Moreover, iNOS and NO production were greatly enhanced in T/I-treated astroglia by the addition of PGE2 at the concentrations of 0.03-30 μM. Our further findings indicated that the addition of the EP2 agonist butaprost (100 nM) significantly elevated iNOS production in T/I-activated astroglia. This suggests that PGE2-induced increase of iNOS in T/I-treated astroglia was mediated by EP2-triggered cellular signaling. This is further confirmed by the finding that PGE2-induced increase of iNOS in T/I-treated astroglia was blocked by EP2 RNA interference using EP2-siRNA (Fig. 2). Similar to the observation in astroglia, iNOS expression in T/I-treated microglia or in T/I-treated mixed glia was enhanced by exposure to butaprost (Fig. 3). In addition, quantitative real-time polymerase chain reaction analysis (Q-PCR) has also indicated that PGE2/EP2 increased iNOS production in T/I-treated astroglia via upregulation of iNOS gene transcription. Moreover, electrophoretic mobility shift assay (EMSA) has indicated that the binding activity of the 21-bp oligomers derived from rat iNOS promoter in T/I-treated astrocytes was increased by PGE2 and butaprost. Since the 21-bp oligomer sequence used for EMSA contains a cAMP-responsive element (CRE) site (5’-TGACG-3’), we tested to see whether PGE2/EP2 could activate transcription factor CRE binding protein B (CREB). The results have shown that the addition of anti-CREB antibody inhibited iNOS promoter activity, verifying the fact that EP2 signaling increases T/I-induced production of astrocytic iNOS mainly through CREB activation.
Figure 3. EP2 agnoinst butaprost (EP2) increased iNOS expression in cytokine-treated mixed glia and microglia. Mixed glia and microglia isolated from cerebral cortices of neonatal day 1-2 Sprague-Dawley rats were stimulated for 24 h by TNF-α and IFN-γ (T/I) with or without the agonist for EP1 (17-phenyl-PGE2), EP2 (butaprost)or EP3 (sulprostone). The production of iNOS was analyzed by western blotting.

2. Blockage of EP2-induced PKA and calcium signaling reduces iNOS expression in cytokine-primed astroglia.
Our parallel experiments have also indicated that iNOS expression was upregulated by membrane permeable cAMP analogue (dbcAMP) in T/I-treated astroglia. EP2 is known to activate adenyl cyclase through the stimulatory G protein (Gs). It also increases intracellular cAMP which in turn triggers PKA-dependent signaling. In our study, we have found that EP2 action on iNOS upregulation in T/I-astroglia was suppressed by the addition of KT5720 (a selective inhibitor for PKA) and partially inhibited by SB203580 (an inhibitor for p38MAPK), indicating that iNOS production increased by EP2 activation in T/I-treated astroglia was mediated by cAMP/PKA/p38MAPK signaling (Fig. 2). Using the single cell calcium imaging system in NCKU Center of Bioscience and Biotechnology, we have also observed that butaprost or PCE2 can cause intracellular calcium rise in T/I-treated astroglia. This increase can be inhibited by the addition of BAPTA-AM (an intracellular calcium chelator), KT5720, staurosporine (a PKC inhibitor) or 2-APB (an IP3 blocker), but it can not be affected by EGTA (an extracellular calcium chelator). This finding indicates that EP2 also triggers the activation of IP3 and PKC though PKA-dependent signaling. Furthermore, iNOS production increased by EP2 activation in T/I-treated astroglia can be effectively inhibited by BAPTA-AM, staurosporine or 2-APB (Fig. 2). In summary, this is a novel mechanism which shows that calcium signaling is involved in the elevation of iNOS in T/I-activated astroglia by PGE2/EP2.

3. Concluding remarks
Our results indicate that EP2 activation by PGE2 can increase astrocytic iNOS production in the inflamed condition through cAMP/PKA signaling, which in turn triggers IP3/Ca2+ and p38MAPK signaling as well as CREB activation. PGE2/EP2 could participate in the process of the proinflammatory cytokine-induced astrocytic activation. Therefore, disruption of the PGE2/EP2 action could be an effective therapeutic strategy in the future for CNS repair in the future.

Chung KK, Thomas B, Li X, Pletnikova O, Troncoso JC, Marsh L, Dawson VL, Dawson TM. 2004. S-nitrosylation of parkin regulates ubiquitination and compromises parkin's protective function. Science 304(5675):1328-31.
Hsiao HY, Mak OT, Yang CS, Liu YP, Fang KM, Tzeng SF. 2007. TNF-alpha/IFN-gamma-induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2-evoked cAMP/PKA and intracellular calcium signaling. Glia 55(2):214-23.
Moncada S, Bolanos JP. 2006. Nitric oxide, cell bioenergetics and neurodegeneration. J Neurochem 97(6):1676-89.
Tzeng SF, Hsiao HY, Mak OT. 2005. Prostaglandins and cyclooxygenases in glial cells during brain inflammation. Curr Drug Targets Inflamm Allergy 4(3):335-40.
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