The role of the prostaglandin E2 receptors in vulnerability of oligodendrocyte precursor cells to death
© Carlson et al.; licensee BioMed Central. 2015
Received: 29 December 2014
Accepted: 14 May 2015
Published: 23 May 2015
Activity of cyclooxygenase 2 (COX-2) in mouse oligodendrocyte precursor cells (OPCs) modulates vulnerability to excitotoxic challenge. The mechanism by which COX-2 renders OPCs more sensitive to excitotoxicity is not known. In the present study, we examined the hypothesis that OPC excitotoxic death is augmented by COX-2-generated prostaglandin E2 (PGE2) acting on specific prostanoid receptors which could contribute to OPC death.
Dispersed OPC cultures prepared from mice brains were examined for expression of PGE2 receptors and the ability to generate PGE2 following activation of glutamate receptors with kainic acid (KA). OPC death in cultures was induced by either KA, 3′-O-(Benzoyl) benzoyl ATP (BzATP) (which stimulates the purinergic receptor P2X7), or TNFα, and the effects of EP3 receptor agonists and antagonists on OPC viability were examined.
Stimulation of OPC cultures with KA resulted in nearly a twofold increase in PGE2. OPCs expressed all four PGE receptors (EP1–EP4) as indicated by immunofluorescence and Western blot analyses; however, EP3 was the most abundantly expressed. The EP3 receptor was identified as a candidate contributing to OPC excitotoxic death based on pharmacological evidence. Treatment of OPCs with an EP1/EP3 agonist 17 phenyl-trinor PGE2 reversed protection from a COX-2 inhibitor while inhibition of EP3 receptor protected OPCs from excitotoxicity. Inhibition with an EP1 antagonist had no effect on OPC excitotoxic death. Moreover, inhibition of EP3 was protective against toxic stimulation with KA, BzATP, or TNFα.
Therefore, inhibitors of the EP3 receptor appear to enhance survival of OPCs following toxic challenge and may help facilitate remyelination.
Cyclooxygenase (COX) catalyzes the rate-limiting step in the synthesis of prostanoids from arachidonic acid . Two isoforms of COX have been identified: a constitutive form designated COX-1 and an inducible form, COX-2 . In the central nervous system (CNS), COX-2 expression is increased in neurons in response glutamate receptor activation [3, 4]. COX inhibitors termed non-steroidal anti-inflammatory drugs (NSAIDs) that are specific to COX-2 promote neuronal survival in vitro [2, 3] and in vivo  following induction of glutamate-receptor-mediated excitotoxic death.
Genetic evidence also indicates a role for COX-2 in excitotoxicity. Transgenic mice that over-express neuronal COX-2 are more susceptible to excitotoxicity  and age-associated neuronal loss . In contrast, COX-2 null (knockout) mice exhibit less neuronal death following ischemia or challenge with NMDA . Therefore, pharmacological and genetic evidence reveals that COX-2 expression and activity contributes to neuronal excitotoxic cell death. Using this analogy as a framework for the role of COX-2 in death of oligodendrocytes (OLs), we showed that COX-2 is induced in OLs and OPCs following glutamate receptor (GluR) activation and renders these cells more susceptible to excitotoxic death . We also have shown that COX-2 is expressed in dying OLs at the onset of demyelination in Theiler’s Murine Encephalomyelitis Virus (TMEV) model of multiple sclerosis (MS)  and in dying OLs in MS lesions . Additional studies have shown that COX-2 also contributes to OL vulnerability in the cuprizone model of demyelination . These studies suggest that COX-2 may have an important role in demyelinating diseases like MS.
Studies with COX-2 inhibitors in animal models of MS also support a role for COX-2 as a contributor to disease pathology [11, 12]. Two groups have reported that administration of COX-2 inhibitors in experimental autoimmune encephalomyelitis (EAE) diminished the severity and incidence of disease and decreased demyelination and inflammation [11, 12]. In both cases, the therapeutic effects in EAE were only observed when the COX-2 inhibitors were initiated immediately after immunization and maintained throughout the course of the study. In these cases, COX-2 inhibition in the induction phase of EAE was due in part to immunomodulatory effects resulting from suppression of T-cell signaling through interleukin-12 (IL-12) . In addition, our group has shown that COX-2 inhibitors reduce demyelination in the TMEV model of MS . A recent study by Esaki et al. examined the role of PGE2 receptor signaling in EAE and identified a role for EP2 and EP4 in peripheral immune response and increase of blood–brain barrier permeability in the initiation and progression of monophasic EAE using global knockouts of PG receptors . However, their studies do not address the potential contribution of PG receptors towards modulation of OPC viability and remyelination.
In EAE, excitotoxicity and axonal damage appear to contribute to the pathology of the disease, since α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) antagonists of GluRs can ameliorate the neurological deficits associated with the progression of the disease . This affect may in part be due to injury of OLs and OPCs which express GluRs of the AMPA and kainate classes and are also susceptible to glutamate-mediated excitotoxicity . This may be particularly important for OPCs since the susceptibility of OPCs to injury within the MS lesion environment can be a major limitation to remyelination in MS .
In this study, we examined whether prostanoids (PGs) such as PGE2 and their receptors contribute to excitotoxic death of OPCs. We examined whether PGE2 was made by OPCs and whether activation of specific PGE2 receptors contributes to the vulnerability of OPCs.
Tissue culture media and reagents along with the kainic acid and 3′-O-(Benzoyl) benzoyl ATP (BzATP) were purchased from Sigma Chemical Company (Saint Louis, MO). Recombinant mouse TNFα was purchased from R&D systems (Minneapolis, MN). Fetal bovine serum and horse serum were purchased from Hyclone (Logan, UT). All the COX-2 inhibitors (CAY 10452, NS398, and CAY 10404) and the EP2 agonist butaprost were purchased from Cayman Chemical Company (Ann Arbor, MI). The EP3 antagonist ONO-AE5-599 was provided by Ono Pharmaceuticals.
Immunofluorescence confocal microscopy
Immunoreactivity was assessed with primary antibodies to mouse antigens that included anti-EP1, EP2, EP3, and EP4 (Cayman Chemicals, Ann Arbor, MI). These antibodies have been shown to have high specificity towards each EP receptor with little to no detectable cross-reactivity between different EP receptors . Antibodies to Olig 1 were from Abcam (Cambridge, England). Primary antibodies were used at dilutions recommended by the manufacturers. Secondary fluorochrome antibodies for mouse were donkey fluorescein isothiocyanate (FITC)-conjugated anti-rabbit and Cy5-conjugated anti-mouse/rat (Jackson ImmunoResearch laboratories, West Grove, PA) and donkey FITC anti-goat, Cy5 anti-mouse, and C3 anti-rabbit. Secondary antibodies were used at concentrations recommended by the manufacturers. The primary antibodies were incubated overnight in a humidified chamber at 4 °C. Secondary antibodies were added for 1 h at room temperature. Background staining was assessed with negative controls consisting of 20 μg/ml normal mouse/rat serum and 30 μg/ml normal rabbit serum. After staining with antibodies then propidium Iodide (PI), coverslips were mounted onto the samples using ProLong Gold anti-fade mounting media (Molecular Probes Inc., Eugene, OR). The Personal Confocal Microscopy PCM-2000 (NIKON, Melville, NY) with argon, green, and red HeNe lasers was used to acquire images from the three different fluorochromes. The Simple Personal Confocal Image program (PCI, Compix, Cranberry Township, PA) was used to acquire digital images for the three different image channels. The FITC label was detected with the argon laser at 488 nm, Cy5 with the red argon laser at 633 nm, and Cy3 was visualized with the green HeNe laser at 563 nm. Tissues were individually scanned with each respective laser filter. All images were acquired using the multi-focal program (z-focus) to create a stereopsis image. The three different images were merged together to acquire the final three-colored image, and the PI image was converted to blue color during merge.
Dispersed oligodendrocyte cultures and toxicity assay
All aspects of animal handling and care were conducted with local Institutional Animal Care and Use Committee (IACUC) approval in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-approved facility (The Veterans Affairs Salt Lake City Health Care System Veterinary Medical Unit). Dispersed oligodendrocyte cultures were prepared from P1 mouse pups as in our earlier study  which was originally performed as described in . Contaminating microglia were depleted from the OPCs by mixing the cultures with magnetic particles containing anti-CD64 antibodies (BD Biosciences, San Jose, CA) and placing the culture tube next to a magnet while the OPC culture suspension is removed. OPCs were plated in 96 well plates and photographed using phase contrast microscopy prior to treatment with kainic acid. Purity of the cell culture was verified by staining a sister well with the OPC marker Olig 1. The same fields were photographed 24, 48, or 144 h after treatment with agents that induce OPC death. Each treatment group typically contained 200–400 cells. For toxicity experiments, oligodendrocytes were scored as living if the cell did not stain with PI. The percent survival was calculated by dividing the number of live cells not stained with PI observed after kainic acid (KA) treatment divided by the number of cells present in the same field prior to KA treatment. Three or more fields were captured (at a magnification of ×20) for each treatment group. This assay has been used to assess OPC survival  and is similar to our previous published assays to determine neuronal survival following excitotoxicity [3, 19]. The percent survival was calculated as percent control relative to the survival observed with no KA treatment. Background death was typically less than 25 %.
Measurement of prostaglandin E2 from cultured OPCs
The media on the OPC cultures was replaced with fresh pre-warmed media containing either KA or vehicle. After a 4-h incubation at 37 °C, the media was removed, immediately spun at 2000×g (to remove any cells or debris), and frozen at −80 °C for subsequent analyses. The concentration of KA used normally would yield a high amount of toxicity (80 % cells killed) after 24 h of treatment, but no cell death was apparent at this time. Frozen media samples were analyzed using PGE2 enzyme-linked immunosorbent assay (ELISA) kit from Cayman Chemical Company (Ann Arbor, MI). This assay can detect concentrations of PGE2 to as low as 15 pg/ml.
Western blot analyses of EP receptors
OPCs from cultures were rinsed in PBS and subsequently lysed in SDS sample buffer containing protease inhibitors (cOmplete ULTRA®-protease inhibitor, Roche, Mannheim, Germany). The samples were fractionated on a 10 % acrylamide gel by electrophoresis, then electro blot transferred to nitrocellulose and probed with the same rabbit antibodies to EP1–EP4 (used at a dilution of 1:200) along with mouse anti-oligodendrocyte-specific protein (OSP) (used at a dilution of 1:400) to normalize for loading. The blot was washed and then probed with anti-rabbit and anti-mouse secondary antibodies (Licor Biotechnology, Lincoln, NE) (used at a dilution of 1:5000) and visualized using the Li-COR imaging system. Densitometry scanning of the Western blot image files was performed using image J to quantify the relative intensity of each EP receptor, and this was normalized to OSP for each lane. The relative amounts were quantified from triplicate gels and assessed for statistical differences using ANOVA, Tukey-Kramer multiple comparison test.
Real-time PCR of EP3 transcripts
Total cellular RNA was isolated from cultured OPCs using RNeasy protocols (Qiagen, Limburg, Netherlands). First-strand cDNA was reverse-transcribed from 1.0 μg of total RNA using a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA). The EP3 splice variants and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified using primer pairs listed below using an ABI 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA). cDNA was mixed with Power SYBR® Green PCR Master Mix (Life Technologies, Carlsbad, CA) and the appropriate primers for the gene of interest. We used the comparative cycle threshold (CT) method (2∆∆CT) to calculate relative gene expression under experimental and control conditions normalized to GAPDH. The results are expressed as a fold change over-expression relative to the beta isoform which was the least abundant isoform . The primers from Zhang et al.  for each splice variant are listed below along with the size of the PCR product.
EP3alpha: sense 5′-GGATCATGTGTGTGCTGTCC-3′ and
antisense 5′-GCAGAACTTCCGAAGAAGGA 3′, 218 bp;
EP3beta: sense 5′-TGAACAACCTGAAGTGGACTTTC-3′ and
antisense 5′-ATTCTCAGACCCAGGGAAACAGG-3′, 60 bp;
EP3gamma: sense 5′-TTCGCTGAACCAGATCTTGGATC-3′ and
antisense 5′-TAGACAATGAGATGGCCTGCCCT-3′, 136 bp,
The primers for GAPDH were from Nozaki et al.  and are listed below.
GAPDH sense 5′-TGGCAAAGTGGAGATTGTTGCC-3′ and
GAPDH antisense 5′AAGATGGTGATGGGCTTCCCG-3′, 156 bp.
Data were analyzed using InStat3, a statistical software package (graph pad Prism, San Diego, CA). Assessment of significance of cell death between groups and differences in relative EP3 splice variants was done using ANOVA Tukey-Kramer multiple comparisons test or pair wise using the Student t-test. All comparisons satisfied the Kolmogorov and Smirnov assumption test for Gaussian distributions, thus allowing parametric analyses.
GluR activation stimulates synthesis of PGE2
Expression of PGE2 receptors in OPCs
EP receptor contribution to OPC viability
Contribution of EP receptors to excitotoxic death
Antagonism of EP3 also protects against excitotoxicity following activation of the purinergic receptor P2X7 and treatment with TNFα
Expression of EP3 in OLs in MS lesions
Protection of OPCs by activation of the EP2 receptor
In neurons, PGE2 receptors such as EP2 can contribute to increased viability following excitotoxic challenge . We next examined whether activation of EP2 in OPCs could be protective against excitotoxicity as is seen in neurons . Treatment of OPCs with the EP2-specific agonist butaprost increased the survival of OPCs following excitotoxic challenge with either KA or BzATP (Fig. 9). These findings show that EP2 can contribute to the viability of OPCs.
In this study, we examined how COX-2 could contribute to the excitotoxic vulnerability of OPCs through stimulation of components of the downstream mediator PGE2. We identified PGE2 and its receptor EP3 as a major contributor to excitotoxic death of OPCs based on three major lines of evidence: (1) activation of GluRs in OPCs stimulates the synthesis of PGE2 in culture, (2) activation of EP3 reverses the protective effect of a COX-2 inhibitor, and (3) inhibition of EP3 is protective against both glutamatergic and purinergic excitotoxic stimuli as well as inflammatory mediators such as TNFα. We also identified that activation of the prostaglandin EP2 receptor renders OPCs less vulnerable to excitotoxicity.
These results suggest that the contributions of EP receptors to OPC viability largely mirror the same type of effects seen in neurons where EP3 can contribute to excitotoxic death  and EP2 can have protective effects . However, in contrast to neurons where EP1 appears to be a major contributor to excitotoxic death, we observed no contribution of EP1 to the excitotoxic death of OPCs. It is not clear why EP1 does not appear to play a similar role in OPCs, but it is notable that EP1 did not appear to be as abundantly expressed in OPCs as EP3 (see Figs. 2 and 3). Alternatively, OPCs may lack key effector proteins required for the coupling activation of EP1 to second messenger cascades that are known to activate phospholipase C and intracellular calcium increases linked to neuronal death .
EP3 is a G-protein-coupled receptor (GPCR) linked to different second messenger pathways that could impart their effects on modulation of OPC viability following a toxic challenge [30, 31]. The major pathway linked to EP3 activation is mediated through the G protein Gi that is coupled to a decrease in cAMP . However, each of the three different splice variants of EP3 (α, β, and γ) can be coupled to either this pathway or an increase of intracellular calcium linked to the IP3 pathway. The γ variant is unique in that it can also be coupled to an increase in cAMP. We identified that all three isoforms are present in cultured OPCs but that the alpha variant was the most abundant species. This result indicates that activation of EP3 in OPCs could be linked to either changes in cAMP or to an increase in intracellular calcium. Subsequent studies should help to identify which signaling pathways contribute to OPC death.
Our results also indicate that activation of EP2 on OPCs by butaprost is protective against an excitotoxic challenge. Butaprost can also interact with the IP receptor , but since we could not detect IP expression in our OPC cultures (data not shown), butaprost is likely acting specifically on the EP2 receptor in our experiments. In other recent studies, EP2 activation may not necessarily be protective against other insults to OLs. An antagonist with modest specificity towards EP2 prevented OL apoptosis in the cuprizone model of demyelination . The discrepancy with our result could be due to the following: (1) differences in OPC and OL death pathways between excitotoxicity and cuprizone-mediated death, (2) the drug that was used may have inhibited other PG receptors other than EP2, or (3) inhibition of EP2 on other cells, such as microglia, may have contributed to OPC protection. It is important to note that another receptor antagonist with high specificity towards EP2 was recently shown to have neuroprotective effects following status epilepticus by acting on microglia .
Our results have important ramifications with respect to demyelinating diseases such as MS. It has recently been shown that susceptibility of OPCs within the MS lesion environment can be a major limitation to remyelination in MS . As such, therapies that increase OPC viability could be valuable for increasing OPC number and promoting remyelination which in turn could preserve axons and help limit the progression of disease. We know from our previous work that inhibition of COX-2 has a net effect of increasing OPC viability. However, COX-2 inhibition may not be optimal for promoting OPC survival as suggested by our findings demonstrating that PGE2 was able to have both detrimental actions through EP3 and beneficial effects through EP2. In this case, with respect to OPC viability, targeting downstream receptors may be more beneficial by allowing inhibition of just the detrimental receptor (EP3), while allowing activation of the beneficial receptor (EP2). Furthermore, other prostanoids may also have beneficial effects in MS. For instance, prostacyclin (PGI2) has recently been reported to promote migration of OPCs to demyelinated areas to help promote remyelination . Therefore, targeting the detrimental downstream receptors could have added benefits beyond inhibition of the entire repertoire of PGs synthesized with COX-2 inhibitors.
We found that EP3 contributes to the vulnerability of OPCs to injury across three different toxic challenges. Therefore, EP3 may be a general contributor to OPC vulnerability across a spectrum of insults and provide a rationale for inhibiting EP3 receptors to help promote OPC survival which in turn could lead to increased remyelination. Inhibition of EP3 may also have other affects beyond OPC survival which could be of benefit in MS. For example, global EP3 knockout mice have been reported to have decreased neuronal loss, reduced blood–brain damage, and reduced microglial activation in stroke models , all of which could be beneficial in MS. Future in vivo studies in demyelinating disease models will help delineate the potential role of EP3 in disease pathogenesis.
We have begun to assess whether EP3 may play a role in MS pathogenesis. In ongoing preliminary studies with MS autopsy CNS tissue, we have found that EP3 was extensively associated with OLs on the edge of a MS lesion and was absent in control spinal cord white matter (data not shown). Immunoreactivity of EP3 in the MS plaque was in a region of the spinal cord without inflammation by conventional histology and contained evidence of ongoing demyelination signified by the presence of MBP fragments which are indicative of myelin injury within a 14-day time period. Since EP3 was present in the active edges of MS plaques, it is consistent that this receptor may also play an important role in demyelination and remyelination in MS. Therefore, EP3 may be a potential target for therapies to limit disease progression in demyelinating diseases including MS.
We have recently begun to assess whether other prostanoids may also play a role similar to PGE2. We examined whether KA can stimulate OPCs to synthesize any of the other four major prostanoids. Prostaglandin F2α (PGF2α) was the only other prostanoid that was synthesized following treatment of OPCs with KA (data not shown). In ongoing studies, we have found that an inhibitor of the PGF2α receptor, FP, was also protective against KA-induced death (Carlson et al. manuscript in preparation). Further studies are under way to determine the interaction between EP3 and FP in modulation of OPC viability.
This study demonstrates that the PGE2 receptor EP3 contributes to susceptibility of OPCs to death. These results suggest that inhibitors of EP3 could limit OPC death and may help promote remyelination. EP3 inhibitors may be considered as potential new targets for therapies for MS.
This work was supported by Veterans Affairs Merit grants (to NGC and JWR) and a pilot grant from the Western Institute for Biomedical Research. We are thankful for technical assistance from Ken Hill and Susan Clawson. Comments on the manuscript were greatly appreciated from James Burns.
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