The Synthetic Cannabinoid R(+)WIN55,212-2 Augments Interferon-β Expression via Peroxisome Proliferator-activated Receptor-α*

Background: The synthetic cannabinoid R(+)WIN55,212-2 can regulate IFN-β expression. Results: R(+)WIN55,212-2 regulates IFN-β expression in a PPARα-dependent manner. Conclusion: PPARα mediates effects of R(+)WIN55,212-2 on IFN-β expression. Significance: PPARα contributes to protective effects of R(+)WIN55,212-2 in models of multiple sclerosis. We have demonstrated that R(+)WIN55,212-2, a synthetic cannabinoid that possesses cannabimimetic properties, acts as a novel regulator of Toll-like receptor 3 (TLR3) signaling to interferon (IFN) regulatory factor 3 (IRF3) activation and IFN-β expression, and this is critical for manifesting its protective effects in a murine multiple sclerosis model. Here we investigated the role of peroxisome proliferator-activated receptor-α (PPARα) in mediating the effects of R(+)WIN55,212-2 on this pathway. Data herein demonstrate that the TLR3 agonist poly(I:C) promotes IFN-β expression and R(+)WIN55,212-2 enhances TLR3-induced IFN-β expression in a stereoselective manner via PPARα. R(+)WIN55,212-2 promotes increased transactivation and expression of PPARα. Using the PPARα antagonist GW6471, we demonstrate that R(+)WIN55,212-2 acts via PPARα to activate JNK, activator protein-1, and positive regulatory domain IV to transcriptionally regulate the IFN-β promoter. Furthermore, GW6471 ameliorated the protective effects of R(+)WIN55,212-2 during the initial phase of experimental autoimmune encephalomyelitis. Overall, these findings define PPARα as an important mediator in manifesting the effects of R(+)WIN55,212-2 on the signaling cascade regulating IFN-β expression. The study adds to our molecular appreciation of potential therapeutic effects of R(+)WIN55,212-2 in multiple sclerosis.

brain barrier integrity, and repair of damage (1). The type I interferon (IFN), IFN-␤, is a front line therapy currently available to treat patients with MS (2), displaying beneficial effects on disability progression (3) and relapse rate (4). IFN-␤ exerts a diverse array of therapeutic mechanisms, with demonstrated effects on antigen presentation, co-stimulatory molecule expression, T cell proliferation, and leukocyte migration (5). Given its clinical efficacy, an increased understanding of novel mechanisms that regulate endogenous expression of IFN-␤ may provide important clues to new therapy development.
Transcriptional regulation of IFN-␤ requires the assembly of a transcription enhancer complex on four positive regulatory domains (PRDI to -IV) (6). PRDI-III domains are recognized by IFN regulatory factors (IRFs) (3 and 7), PRDII by nuclear factor-B (NF-B) and PRDIV by activator protein-1 (AP-1) (ATF-2/c-Jun), and these transcription factors act in co-operation to form an enhanceosome that drives efficient production of IFN-␤ (7). Intriguingly, we have recently demonstrated that the aminoalkylindole, R(ϩ)WIN55,212-2, regulates IFN-␤ expression by augmenting activation of IRF3 and this is critical for manifesting its protective effects in experimental autoimmune encephalomyelitis (EAE), an animal model of MS (8). R(ϩ)WIN55,212-2 is a potent synthetic cannabinoid receptor agonist and while capable of binding to both CB 1 and CB 2 cannabinoid receptors, it exhibits greater selectivity for CB 2 (9). However, we have demonstrated cannabinoid receptor-independent mechanisms of action for R(ϩ)WIN55,212-2, including those underlying its regulation of IFN-␤ expression (8). Such findings and others clearly indicate that R(ϩ)-WIN55,212-2 can work independently of the classical cannabinoid receptor system. Indeed, other candidate receptors exist for mediating cannabinoid effects, including the orphan receptor GPR55 (10) and transient receptor potential vanilloid type 1 (TRPV1) (11). However more recent interest has focused on the role of nuclear receptor superfamily of peroxisome proliferator-activated receptors (PPARs) as potential mediators of some cannabinoid activity (12). Interestingly cannabinoid-mediated anti-inflammatory propensity has been linked with PPAR activity (13)(14) and this is consistent with the anti-inflammatory effects of PPAR ligands (15)(16).
PPARs are ligand-activated nuclear receptors with effects on proliferation, metabolism and immunity (17). Three isoforms exist (PPAR␣, -␥, and -␦) which heterodimerize with the retinoid X receptor, activating transcription by binding a specific DNA element called the PPAR response element (PPRE) (17). The PPAR subtypes exhibit distinct tissue expression patterns (18), with expression characterized throughout the central nervous system (CNS) (19). PPAR agonists (thiazolidinediones) are used clinically in the treatment of diabetes and experimental evidence suggests potential clinical benefits for patients with neuroinflammatory disorders (20).
The anti-inflammatory properties of PPARs result, at least in part, from inhibition of transcription factors NF-B and AP-1, and subsequent regulation of chemokines, cytokines and adhesion molecules (16,(21)(22). Activation of transcription factors such as NF-B, in addition to members of the IRF family, is tightly controlled by TLRs, single transmembrane receptors involved in the recognition of conserved microbial motifs (23). Despite the integral role of TLRs in pathogen recognition, dysregulation of TLR signaling cascades is associated with inflammation (24). We have previously demonstrated that R(ϩ)WIN55,212-2 differentially regulates TLR signaling (TLR3 and TLR4), and in particular, this aminoalkylindole derivative acts as a novel regulator of TLR3 signaling to IRF3 and subsequent expression of IFN-␤ (8). Such effects of R(ϩ)WIN55,212-2, in particular its capacity to induce endogenous IFN-␤, offers an attractive additional option to the current use of exogenously administered IFN-␤ in MS. Indeed, a high percentage of patients fail to respond to current therapy, with treatment failure associated with production of neutralizing antibodies to IFN-␤ in some cases (25). However, cannabinoid receptor involvement was not associated with the effects of R(ϩ)WIN55,212-2 on endogenous IFN-␤ expression. Given previous reports that cannabinoid compounds may manifest at least some effects via PPAR␣ (13,26) we were particularly interested to explore the role of PPAR␣ in mediating the effects of R(ϩ)WIN55,212-2 on IFN-␤ expression. We show that R(ϩ)WIN55,212-2 promotes PPAR␣ transactivation and expression, and potentiates TLR3-induced IFN-␤ via a PPAR␣ mechanism. We further show that R(ϩ)WIN55,212-2 specifically targets the AP-1-binding enhancer element of the IFN-␤ promoter, and this effect of R(ϩ)WIN55,212-2 is reliant on the PPAR␣ isoform. This study thus identifies a novel role for PPAR␣ in regulating the signaling cascade leading to IFN-␤ expression. Cell Culture-Cell lines were maintained in DMEM supplemented with 10% FBS, 100 g/ml penicillin, and 100 g/ml streptomycin. Cells were maintained in a 37°C humidified atmosphere with 5% CO 2 . The neomycin analog G418 (500 g/ml) was used to select for the stably transfected TLR cell lines and maintenance of CD14 expression. Primary astrocytes were prepared as described previously (27) from the whole brain of 1-day-old C57/BL6 mice in accordance with the guidelines laid down by the local ethics committee (National University of Ireland Maynooth). Briefly, dissected brains were chopped, added to DMEM (Invitrogen), triturated, passed through a sterile mesh filter (40 m), and centrifuged (2,000 ϫ g for 3 min at 20°C). The pellet was resuspended in DMEM and plated onto T25 flasks. Medium was changed after 1, 5, and 8 days. Astrocytes were isolated from mixed glia at day 10 -14 by removing non-adherent cells with mechanical shaking and harvesting by trypsinization (0.25% trypsin, 0.02% EDTA). Cells were centrifuged (2,000 ϫ g for 5 min at 20°C), and the astrocyte-enriched pellet was resuspended in DMEM. Astrocytes were plated (2 ϫ 10 5 cells/ml) on 6-or 12-well plates and treated 24 h later.
Confocal Microscopic Analysis of PPAR␣-Primary astrocytes were seeded (1 ϫ 10 5 cells/ml) in 4-well chamber slides (Lab-Tek, Roskilde, Denmark) and grown for 24 h. Cells were treated with R(ϩ)WIN55,212-2 (20 M) or S(Ϫ)WIN55,212-2 (20 M) for times ranging from 2 to 8 h. Cells were fixed in 4% paraformaldehyde and blocked with 10% chicken serum (Vector Laboratories, Peterborough, UK) for 2 h. Cells were treated overnight at 4°C with goat polyclonal PPAR␣ antibody (1:100 in 5% chicken serum; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Cells were washed and incubated with chicken antigoat Alexa488 secondary antibody (1:500 in 5% chicken serum; Invitrogen) and DAPI (1.5 g/ml) in PBS, washed, and mounted (Vectashield, Vector Laboratories). All samples were viewed using an Olympus FluoView FV1000 confocal laserscanning microscope equipped with the appropriate filter sets. Acquired images were analyzed using the Olympus FV-10 ASW imaging software. Negative control experiments were performed by replacing the primary antibody with isotype control IgG (Millipore, Cork, Ireland) and using equal gain settings during acquisition and analysis.
Induction and Assessment of EAE and Treatment with Cannabinoid and PPAR␣ Antagonist-EAE was induced in mice as described (35). Female SJL/J mice (10 weeks old) were injected subcutaneously at two sites with two injections (100 l) of emulsified Freund's complete adjuvant containing 100 g of myelin proteolipid protein amino acids 139 -151 (PLP(139 -151)) and 200 g of Mycobacterium tuberculosis H37Ra followed 2 h later with 200 ng of pertussis toxin (Hooke Laboratories, Lawrence, MA) delivered intraperitoneally. R(ϩ)WIN55,212-2 was prepared in Cremophor El (Sigma) and PBS (20:80) and administered (20 mg/kg) intraperitoneally on days 0, 1, 2, 3, 4, and 5. The preparation and immunization of the synthetic cannabinoid R(ϩ)WIN55,212-2 (Sigma) was modified from previous studies (36). GW6471 was dissolved in DMSO and administered intraperitoneally (10 mg/kg) on days 0, 1, 2, 4, and 6 after PLP immunization. Control mice received Cremophor/PBS (20:80) as vehicle. Data are from 4 -8 mice/ group. To ensure objective clinical scoring, all mice had electronic data chips placed subcutaneously prior to the experiment and were subsequently tracked by a barcode reader (AVID, UK). An investigator blinded to the treatment of the mice scored all animals by barcode number, to determine the mean clinical score as follows: 0, normal; 1, limp tail or hind limb weakness; 2, limp tail and hind limb weakness, 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, moribund.
Statistical Analysis-Data are expressed as means with S.E., and the results represent three independent experiments. Statistical comparisons of different treatments were done by a oneway analysis of variance using a post hoc Student-Newman-Keuls test. Differences with a p value less than 0.05 were considered statistically significant.

R(ϩ)WIN55,212-2 Enhances TLR3-induced IFN-␤ Expression via PPAR␣-
We have previously demonstrated that R(ϩ)WIN55,212-2 negatively regulates the transactivation of NF-B and expression of proinflammatory cytokines (37) but can positively impact the TLR3-IRF3 signaling axis to enhance IFN-␤ expression in the presence of TLR3 stimulation (8). Given that such effects were shown to be mediated in a manner independent of cannabinoid receptors (8,37) and that this compound can exert some effects via PPAR␣ (13,38), we used the specific PPAR␣ antagonist GW6471 to assess the potential role of PPAR␣ in mediating the effects of R(ϩ)WIN55,212-2 on TLR3-induced activation of NF-B and IRF3 and expression of TLR-responsive genes. GW6471 failed to regulate the ability of R(ϩ)WIN55,212-2 to inhibit TLR3-induced activation of NF-B in HEK293-TLR3 cells (Fig. 1A) or the expression of the NF-B-responsive chemokine RANTES in astrocytes (Fig. 1B), indicating that these effects of R(ϩ)WIN55,212-2 are mediated in a manner independent of PPAR␣. Similarly, the potentiating effect of R(ϩ)WIN55,212-2 on TLR3-induced IRF3 activation in HEK293-TLR3 cells was not reliant on the PPAR␣ isoform, given that GW6471 failed to influence the effects of R(ϩ)WIN55,212-2 on IRF3 activation (Fig. 1C). The lack of a role for PPAR␣ in regulating IRF3 was further confirmed in HEK293 cells by demonstrating that GW6471 failed to regulate the previously described ability of R(ϩ)WIN55,212-2 to promote the increased nuclear localization of IRF3-GFP fusion protein (supplemental Fig. 1). However, pre-exposure to the PPAR␣ antagonist GW6471 attenuated the ability of R(ϩ)WIN55,212-2 to enhance poly(I:C)-induced activation of the IFN-␤ promoter (Fig. 1D) and to potentiate poly(I:C)-induced IFN-␤ mRNA expression in primary astrocytes (Fig. 1E) and HEK293-TLR3 cells (Fig. 1F), indicating that R(ϩ)WIN55,212-2 enhances TLR3-induced IFN-␤ expression via a PPAR␣ mechanism.
R(ϩ)WIN55,212-2 Promotes Increased Transactivation and Expression of PPAR␣-Because R(ϩ)WIN55,212-2 enhances IFN-␤ expression via a PPAR␣ mechanism, we next assessed the potential of R(ϩ)WIN55,212-2 to directly regulate the activation and expression of PPAR␣. R(ϩ)WIN55,212-2 was initially compared with the PPAR␣-specific agonist fenofibrate for its ability to transactivate PPAR␣ and increase the expression of a luciferase reporter gene that is regulated by a PPAR␣-responsive element. Fenofibrate and R(ϩ)WIN55,212-2 demonstrated a significant increase in PPAR␣-mediated transcription in HEK293 cells ( Fig. 2A) with both agents directly acting on PPAR␣ because their effects were abrogated by the PPAR␣ antagonist GW6471 (Fig. 2B). Furthermore, exposure of primary astrocytes to R(ϩ)WIN55,212-2 significantly enhanced the expression of PPAR␣ mRNA in a time-dependent manner, with mean maximal stimulatory effects observed at 8 -24 h post-aminoalkylindole exposure (Fig. 2C). The stimulatory effect of R(ϩ)WIN55,212-2 on PPAR expression was restricted to the ␣ isoform because it had no effect on PPAR␦ or PPAR␥ mRNA expression in astrocytes (data not shown). The enantiomeric form of R(ϩ)WIN55,212-2, S(Ϫ)WIN55,212-2 (39), failed to affect PPAR␣ mRNA expression (Fig. 2C), suggesting that a stereoselective mechanism underlies the stimulatory effects of R(ϩ)WIN55,212-2 on PPAR␣. The time-and stereoselective-dependent effects of R(ϩ)WIN55,212-2 on PPAR␣ expression were also confirmed at the protein level by immunoblotting of extracts from R(ϩ)WIN55,212-2-treated astrocytes (Fig. 2D). We also employed confocal microscopy to characterize the effects of R(ϩ)WIN55,212-2 on the cellular expression pattern and localization of PPAR␣. Exposure of astrocytes to R(ϩ)WIN55,212-2 promoted strong cellular expression of PPAR␣ from 8 h, and this was highly localized to the nucleus (Fig. 2E). These findings strongly indicate that R(ϩ)WIN55,212-2 positively regulates the activation and expression of PPAR␣, and this is consistent with the PPAR␣ dependence of R(ϩ)WIN55,212-2 with respect to its regulatory effects on IFN-␤.
PPAR␣ Targets the PRDIV Domain of the IFN-␤ Promoter-Given that R(ϩ)WIN55,212-2 can directly target PPAR␣ and augment TLR3-induced activation of the IFN-␤ promoter in a PPAR␣-dependent manner, we next examined if activation of PPAR␣ was sufficient to manifest such positive effects. We thus examined the ability of the PPAR␣-specific agonist fenofibrate to regulate activation of the IFN-␤ promoter. Fenofibrate aug-mented poly(I:C)-induced activation of the IFN-␤ promoter (Fig. 3A) and induction of IFN-␤ mRNA (Fig. 3B) in a dose-dependent manner in HEK293-TLR3 cells, indicating that activation of PPAR␣ is indeed sufficient to positively regulate IFN-␤. We next probed the mechanistic basis to the regulatory effects of PPAR␣ on the IFN-␤ promoter. Transcriptional activation of IFN-␤ requires assembly of a transcription enhancer complex on the four PRDs of its promoter. PRDI-III domains are recognized by IRF3/7, PRDII is recognized by NF-B, and PRDIV is recognized by AP-1 (7). We thus assessed the regulatory influence of PPAR␣ on each of these regulatory regions by measuring the effects of fenofibrate on poly(I:C) induction of a luciferase reporter gene regulated by individual PRDs. Fenofibrate, in a dose-dependent manner, inhibited poly(I:C)-induced activation of PRDI-III (Fig. 3C) and PRDII (Fig. 3D) but intriguingly augmented poly(I:C)-induced activation of PRDIV (Fig. 3E). Fenofibrate had no effect on cell viability (supplemental Fig. 2) at the concentrations tested. We next probed the direct effects of fenofibrate on the transcription factors that bind to each of the PRD regions. Fenofibrate, in a dose-dependent manner, inhibited poly(I:C)-induced IRF3-regulated luciferase (Fig. 3F), and this is consistent with the negative effects of fenofibrate on PRDI-III. Fenofibrate also showed strong inhibitory effects on poly(I:C)-induced activation of NF-B (Fig. 3G), and this provides a credible basis to the negative effects of fenofibrate on PRDII. Interestingly, fenofibrate induced activation of AP-1
HEK293 cells were thus transfected with a construct encoding a fusion protein of Jun and the DNA binding domain of the yeast protein Gal4 and a luciferase reporter construct regulated by a promoter containing a Gal4 binding motif. R(ϩ)WIN55,212-2 induced the transactivation of the Jun-Gal4 protein in a dosedependent and stereoselective manner (Fig. 4E) and by a mechanism that is dependent on PPAR␣ because the positive effects of R(ϩ)WIN55,212-2 were abrogated by GW6471 (Fig. 4F).

DISCUSSION
This study defines a non-cannabinoid-dependent mechanism of action for the aminoalkylindole R(ϩ)WIN55,212-2, in that this synthetic cannabinoid receptor agonist regulates IFN-␤ expression via the PPAR␣ nuclear receptor. In so doing, the study also highlights the highly novel role of PPAR␣ in regulating IFN-␤ expression. Previously, we have demonstrated that R(ϩ)WIN55,212-2 augments TLR3 signaling to IRF3 activation and IFN-␤ expression, and this contributes to the therapeutic effects of R(ϩ)WIN55,212-2 in animal models of MS (8). Here, we now demonstrate that R(ϩ)WIN55,212-2 also regulates IFN-␤ expression by targeting the AP-1 transcription factor and the PRDIV enhancer element of the IFN-␤ promoter, and importantly, these effects are manifested in a PPAR␣-dependent manner. In addition, the protective effects of R(ϩ)WIN55,212-2 during the acute phase of EAE were ameliorated by GW6471. Hence, these data identify PPAR␣ as a key receptor target for R(ϩ)WIN55,212-2 in mediating the positive effects of the latter on IFN-␤ expression and highlight PPAR␣ as a lead target to exploit in diseases, such as MS, that would benefit from augmentation of IFN-␤ expression.
IFN-␤ therapy is a current first line treatment of MS, decreasing relapse rate and modestly reducing disability accumulation (1). The mechanism(s) of action of IFN-␤ is complex with demonstrated effects on antigen presentation, co-stimulatory molecule expression, T cell proliferation, and leukocyte migration (5). The cell type-specific production of type I IFNs is controlled by the innate immune pattern recognition receptors, namely TLRs and retinoic acid-inducible gene I-like receptors (42). TLRs induce signaling via recruitment of the adaptor myeloid differentiation factor 88 (MyD88), with the exception of TLR3, which induces Myd88-independent signaling via Toll interleukin-1 receptor-domain-containing adaptor-inducing IFN-␤ (TRIF) protein (43). Such TRIF-mediated signaling promotes the phosphorylation and nuclear localization of tran-scription factors IRF3 and IRF7 and subsequent induction of type I IFNs (23,44). We have recently demonstrated that R(ϩ)WIN55,212-2 can impact this pathway, acting as a novel regulator of TLR3 and TLR4 signaling by inhibiting the proinflammatory signaling axis triggered by TLR3 and TLR4 while selectively augmenting TLR3-induced activation of IRF3 and expression of . This is consistent with data indicating that the plant-derived cannabinoids (45)(46), endocannabinoids (30), and synthetic cannabinoid receptor agonists (30,(47)(48) abrogate TLR4-induced proinflammatory mediator production in glia.
The concentrations of R(ϩ)WIN55,212-2 used are in line with those used in various anti-inflammatory paradigms in vitro (30 -32). Furthermore, the inability of the enantiomeric form of R(ϩ)WIN55,212-2 to mimic its effects argues for a stereoselective receptor-mediated process(es), and the present study provides strong evidence for a role for PPAR␣. Indeed, recent studies have pointed to a growing importance for PPARs in regulating TLR signaling. Thus, PPAR␥ ligands negatively impact TLR-induced inflammatory signaling in glia (62), monocytes (63), and macrophages (64), and recently PPAR␥ has been shown to negatively regulate induction of IFN-␤ in response to TLR3 and TLR4 stimulation (65). Furthermore, in vascular smooth muscle cells, fenofibrate blunts TLR4-induced activation of TRIF and IRF3 (66). Because R(ϩ)WIN55,212-2 (13,38) and other cannabinoid-based compounds (38,67) have been shown to bind and increase the transactivation capacity of PPAR␣, this receptor was selected as a potential lead target for mediating the effects of R(ϩ)WIN55,212-2 on IFN-␤ expression. Using the PPAR␣ antagonist GW6471, we confirm that R(ϩ)WIN55,212-2 regulates PPAR␣ transactivation to impart its regulatory role on IFN-␤ expression.
Fenofibrate exerts neuroprotective properties in rodent models of stroke (68), MS (69), and traumatic brain injury (70). Mechanistically, the activation of PPAR␣ has been shown to inhibit proinflammatory gene transcription by repressing the pivotal inflammatory transcription factor, NF-B (71), and this is supported by our findings indicating an inhibitory effect of fenofibrate on poly(I:C) induction of the NF-B reporter gene. It is noteworthy that the PPAR␥ or PPAR␦ agonists, ciglitazone and L-165,041, did not negatively regulate poly(I:C) induction of the NF-B reporter gene (supplemental Fig. 3), identifying activators of the ␣ subtype as potent inhibitors of this transcription factor. The present study also adds further complexity to the mechanism of action of PPAR␣ ligands, and the findings raise the possibility that the positive effects of PPAR␣ on TLR3induced expression of IFN-␤ may contribute to its in vivo effects. Indeed, data presented herein indicate that mice treated with R(ϩ)WIN55,212-2 showed reduced severity of EAE dur-ing both the first and second paralytic episodes. However, mice co-treated with R(ϩ)WIN55,212-2 and GW6471 showed reduced protection during the first paralytic episode, indicating that the effects of R(ϩ)WIN55,212-2 display some PPAR␣ dependence during this phase of the disease. An imbalance in the cytokine network has a role in the initiation of EAE, with CD4 ϩ T helper 1 (Th1) cells and Th 17 T cells suggested as having distinct and possibly complementary roles in disease onset (72). Both cannabinoid (73) and PPAR␣ receptors (74) are expressed on T cells, and given that R(ϩ)WIN55,212-2 may exert its anti-inflammatory properties in EAE by regulating T cell viability (56), whereas fenofibrate can regulate IL-17 and interferon-␥ expression in isolated T cells (75), it will be interesting to mechanistically delineate the role of PPARs in mediating the effects of cannabinoids in EAE.
The study highlights dual effects of R(ϩ)WIN55,212-2 on PPAR␣ in that it directly activates and also induces the expression of PPAR␣. Few data linking an alteration in the expression profile of PPARs with neuroinflammation are available. However, it is attractive to suggest that the anti-inflammatory effects of R(ϩ)WIN55,212-2 may not be restricted to directly activating PPAR␣ but may also be associated with its ability to regulate the expression profile of PPARs and so facilitate increased signaling by endogenous PPAR ligands. Indeed, the ability of R(ϩ)WIN55,212-2 to up-regulate the expression of PPAR␣ adds to previous studies describing a similar effect of this aminoalkylindole on PPAR␥ expression (58,59), suggesting that the impact of R(ϩ)WIN55,212-2 on proliferative and inflammatory pathways may be due to its effects on PPAR expression. It is also noteworthy that LPS can enhance PPAR␣ expression (76), and evidence from our group suggests that endogenous PPAR␣ expression is up-regulated in EAE spinal cord and in peripheral blood mononuclear cells isolated from MS patients (data not shown). Further experiments will determine if this represents an endogenous neuroprotective response, an attractive possibility given that an up-regulation in PPAR expression has been shown to inhibit proinflammatory signaling (77). R(ϩ)WIN55,212-2 has been linked to the activation of the mitogen-activated protein kinase (MAPK) family members, including ERK (78 -80), p38 (58,78,81), and JNK (58,(81)(82). In particular, the ability of R(ϩ)WIN55,212-2 to activate JNK in the CNS (82) is consistent with our findings in astrocytes. Using a specific JNK inhibitor, we demonstrate that this kinase is an upstream signaling intermediate targeted by R(ϩ)WIN55,212-2 in the cascade leading to IFN-␤ expression. Furthermore, given that MAPK cascades phosphorylate different residues on the PPAR isoforms to control receptor activity (83), it will be of interest to determine the complex role of MAPKs in determining the effect of R(ϩ)WIN55,212-2 on PPAR activation.
Our findings identify JNK phosphorylation and AP-1 activation as key mediators of the effects of R(ϩ)WIN55,212-2 and the PPAR␣ agonist fenofibrate. This is not without precedent because synthetic cannabinoids target AP-1 in the regulation of tyrosine hydroxylase gene transcription in neural cells (84), whereas administration of the endocannabinoid anandamide enhances AP-1 activity in vivo (85). Interestingly, the positive effects of R(ϩ)WIN55,212-2 on AP-1 are mediated by PPAR␣, and this is somewhat surprising given that evidence indicates that fenofibrate negatively regulates AP-1 activity in T cells (86) and vascular smooth muscle cells (87). Cell type specificity may account for this discrepancy, and further experiments will determine the mechanism by which R(ϩ)WIN55,212-2-induced PPAR␣ activation directly regulates JNK and AP-1 activation. It should be emphasized that not all of the effects of R(ϩ)WIN55,212-2 on the IFN-␤ promoter are mediated by PPAR␣. Indeed, the former can augment TLR3-induced activation of IRF3 in a PPAR-independent manner, whereas, in contrast, fenofibrate inhibits activation of IRF3 in response to poly(I:C). Thus, whereas R(ϩ)WIN55,212-2 employs PPAR␣ to promote activation of AP-1 and the PRDIV domain of the IFN-␤ promoter, it can also utilize a PPAR␣-independent mechanism that overrides any negative regulatory effects of PPAR␣ on IRF3 and the PRDI-III regions of the promoter.
In summary, our results show that R(ϩ)WIN55,212-2 acts via PPAR␣ to impact the JNK/AP-1 pathway, leading to activation of the PRDIV region of the IFN-␤ promoter (see Fig. 7). We also show that R(ϩ)WIN55,212-2, in a PPAR␣-independent manner, can augment activation of IRF3 and the PRDI-III regions of the IFN-␤ promoter. Despite being mechanistically distinct, such effects of R(ϩ)WIN55,212-2 on the PRDI-III and PRDIV regions of the IFN-␤ promoter will result in positive cooperativity and strong induction of IFN-␤. This adds significantly to our understanding of the mechanism(s) underlying the therapeutic effects of R(ϩ)WIN55,212-2 in autoimmune disorders, in particular MS. More importantly, it also enhances PPAR␣ as a lead therapeutic target to exploit in the treatment of diseases, like MS, that benefit from IFN-␤ augmentation.