Original ContributionPPARα ligands inhibit radiation-induced microglial inflammatory responses by negatively regulating NF-κB and AP-1 pathways
Introduction
Brain metastases represent a significant cause of morbidity and mortality, and are the most common intracranial tumors in adults, occurring in 10 to 30% of adult cancer patients [1], [2]. The annual incidence appears to be rising as a result of an aging population, improved treatment of systemic disease, and the use of advanced imaging techniques such as magnetic resonance imaging to detect smaller metastases in asymptomatic patients [1]. Radiation therapy, administered in the form of large-field partial or whole brain irradiation (WBI), is the primary mode of treatment for brain metastases; over 170,000 patients will receive WBI/year in the United States [3], [4]. However, late delayed effects of brain irradiation characterized by a progressive cognitive impairment occur in up to 50% of brain tumor patients who are long-term survivors [5] (> 6 months postirradiation). Currently there are neither long-term treatments nor any preventive strategies to alleviate this radiation-induced morbidity [1].
Although the exact pathogenic mechanisms of radiation-induced brain injury are not known, a growing body of data suggests that oxidative stress/proinflammatory responses might play a role [6]. An acute molecular response characterized by increased expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), intracellular adhesion molecule-1 (ICAM-1), cyclooxygenase-2 (Cox-2), and activation of transcription factors such as nuclear factor kappa B (NF-κB) is observed within hours of irradiating the rodent brain [7], [8], [9]. In addition, a chronic elevation of TNFα has been observed in the mouse brain up to 6 months postirradiation [10].
Microglia, the immune cells of the brain, are key mediators of neuroinflammation. They represent about 10% of the total glial population in the central nervous system [11]. In the ramified state, microglia actively survey the microenvironment and ensure normal central nervous system activity by secreting neurotrophic factors such as neuronal growth factor (NGF) [12]. However, they can become activated by a variety of stimuli and release a host of proinflammatory cytokines, chemokines and reactive oxygen/nitrogen oxide species (ROS/RNOS) [13]. Although microglial activation plays an important role in phagocytosis of dead cells in the central nervous system, prolonged activation leads to a sustained inflammatory status in the central nervous system [14]. Microglial activation has been implicated in several neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease, and Parkinson's disease [14].
In vitro studies suggest that irradiating microglia leads to a marked increase in expression of proinflammatory genes including TNFα, IL-1β, IL-6, and Cox-2 [15], [16], [17]. Radiation-induced expression of microglial TNFα and IL-1β has been shown to enhance ICAM-1 expression in nonirradiated astrocytes [16]. These studies are supported by in vivo experiments in rodents which indicate that brain irradiation leads to a marked increase in microglial activation associated with both a concomitant decrease in neurogenesis in the subgranular zone (SGZ) of the hippocampus and spatial memory retention deficits [18], [19]. Further, administration of the anti-inflammatory drug indomethacin decreased radiation-induced microglial activation and was associated with an improvement in hippocampal neurogenesis [20]. These data suggest that the efficacy of anti-inflammatory therapies to mitigate radiation-induced brain injury may involve inhibition of radiation-induced microglial activation.
Peroxisomal proliferator-activated receptor alpha (PPARα) is one of the three nuclear receptor subtypes belonging to the PPAR family [21]. Following activation, PPARs regulate gene transcription by binding to specific consensus sequences termed PPAR response elements (PPREs) in the promoter regions of genes as a heterodimer with the retinoid X receptor (RXR) [21]. PPARα is activated by both natural ligands such as certain long-chain fatty acids and eicosanoids and synthetic ligands such as hypolipidemic fibrates [22]. PPARα is predominantly expressed in tissues that catabolize high amounts of fatty acids such as the liver, kidney, and heart [23], and regulates many metabolic pathways, including activation of fatty acid β-oxidation and apolipoprotein expression [22], [24], [25]. More recently, PPARα has been shown to play a major role in regulating inflammatory processes. Administration of fibrates to patients with a moderate hyperlipidemia decreased plasma concentrations of proinflammatory mediators such as IL-6, TNF-α, Interferon-γ (IFN-γ), fibrinogen, and C-reactive protein [25]. PPARα ligands can negatively impact atherogenesis and vascular thrombus formation, in part, by repressing Tissue factor and TNF-α expression in T lymphocytes and macrophages [26], [27]. In addition, PPARα has been shown to mediate its anti-inflammatory activities, in part, via downregulation of activator protein-1 (AP-1) and NF-κB signaling pathways [28].
In the brain, PPARα is expressed in multiple cell types including the microglia [29]. PPARα agonists have been shown to inhibit the production of nitric oxide and secretion of proinflammatory cytokines including TNFα, IL-1β, and IL-6 in both cytokine and LPS-stimulated microglia [29], [30], [31]. The role of PPARα in radiation-induced brain injury is unknown. We hypothesized that activation of PPARα could modulate the inflammatory and/or oxidative stress responses of the microglia following radiation. In the current study, we report that pretreatment of microglial cells with PPARα agonists prevented the radiation-induced increases in TNFα and IL-1β gene expression and Cox-2 protein levels, in part, by modulating the activity of AP-1 and NF-κB transcription factors.
Section snippets
Cell culture and reagents
The immortalized BV-2 murine microglial cell line was cultured in high glucose DMEM (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine, 100 IU/mL penicillin, and 100 mg/mL streptomycin. These cells display phenotypic and functional properties of reactive microglial cells and resemble nonactivated primary microglial cells [32]. Cells were maintained at 37 °C with 10% CO2/90% air mixture and the culture medium was replaced with serum-free
BV-2 cells possess a functional PPARα
In order to confirm the suitability of the BV-2 cells for our studies, we cotransfected these cells with a PPRE-driven reporter plasmid construct along with a renilla vector and performed luciferase activity assays 24 h after treatment with the PPARα agonists GW7647 and Fenofibrate. Incubating BV-2 cells with GW7647 (1 and 10 μM) and Fenofibrate (100 μM) increased the luciferase activity 2-fold, suggesting that PPARα is functional in these cells (data not shown). Since GW7647 and Fenofibrate
Discussion
These studies indicate that irradiating BV-2 cells leads to an increase in intracellular ROS generation. Increases in ROS levels could amplify the proinflammatory responses of the microglia though effects on kinase signaling pathways and transcription factor activation [44]. Consistent with this, we also observed increased TNFα and IL-1β gene expression and Cox-2 protein levels following irradiation, extending previous reports [15], [16], [17]. Further, the current data not only confirm that
Acknowledgments
This work was supported by NIH Grant CA112593 (MER). We thank Dr. Linda van Eldik, Northwestern University, USA, for generously providing the BV-2 cells, originally developed by Dr. V. Bocchini, Univeristy of Perugia, Italy. We thank Denise Gibo [Brain Tumor Center of Excellence Wake Forest University School of Medicine (WFUSM)] and Dr. Carol Milligan (Department of Neurobiology and Anatomy, WFUSM) for providing antibodies against total c-jun and phosphorylated c-jun, respectively. We also
References (66)
- et al.
Radiation enhances tumor necrosis factor alpha production by murine brain cells
Brain Res.
(1991) - et al.
Induction of acute phase gene expression by brain irradiation
Int. J. Radiat. Oncol. Biol. Phys.
(1995) - et al.
Cyclooxygenase-2 modulates brain inflammation-related gene expression in central nervous system radiation injury
Brain Res. Mol. Brain Res.
(2002) - et al.
TNF alpha and IL-1beta mediate intercellular adhesion molecule-1 induction via microglia-astrocyte interaction in CNS radiation injury
J. Neuroimmunol.
(1999) - et al.
Ionizing radiation induces astrocyte gliosis through microglia activation
Neurobiol. Dis.
(2006) - et al.
Radiation-induced impairment of hippocampal neurogenesis is associated with cognitive deficits in young mice
Exp. Neurol.
(2004) - et al.
Peroxisome proliferator-activated receptor alpha negatively regulates the vascular inflammatory gene response by negative cross-talk with transcription factors NF-kappaB and AP-1
J. Biol. Chem.
(1999) - et al.
Peroxisome proliferator-activated receptor agonist regulation of glial activation: relevance to CNS inflammatory disorders
Neurochem. Int.
(2006) - et al.
Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus
J. Neuroimmunol.
(1990) - et al.
Inhibiting catalase activity sensitizes 36B10 rat glioma cells to oxidative stress
Free Radic. Biol. Med.
(2007)
8-hydroxydeoxyguanosine suppresses NO production and COX-2 activity via Rac1/STATs signaling in LPS-induced brain microglia
Free Radic. Biol. Med.
Activation of stress-responsive promoters by ionizing radiation for deployment in targeted gene therapy
DNA Repair (Amst.)
Dexamethasone inhibits IL-1 beta gene expression in LPS-stimulated RAW 264.7 cells by blocking NF-kappa B/Rel and AP-1 activation
Immunopharmacology
Interleukin (IL)-1 beta, IL-2, IL-4, IL-6 and transforming growth factor-alpha levels are elevated in ventricular cerebrospinal fluid in juvenile parkinsonism and Parkinson's disease
Neurosci. Lett.
Omega-3 fatty acid ethyl-eicosapentaenoate, but not soybean oil, attenuates memory impairment induced by central IL-1beta administration
J. Lipid Res.
Global suppression of IL-6-induced acute phase response gene expression after chronic in vivo treatment with the peroxisome proliferator-activated receptor-alpha activator fenofibrate
J. Biol. Chem.
Peroxisomal proliferation protects from beta-amyloid neurodegeneration
J. Biol. Chem.
NADPH oxidase mediates radiation-induced oxidative stress in rat brain microvascular endothelial cells
Free Radic. Biol. Med.
Peroxisome proliferator-activated receptor-gamma agonists inhibit the activation of microglia and astrocytes: implications for multiple sclerosis
J. Neuroimmunol.
Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice
Neurosci. Lett.
Whole-brain radiotherapy in the management of brain metastasis
J. Clin. Oncol.
Metastatic Brain Cancer
Radiosurgery for single and multiple brain metastasis
Brain Metastases
Neurobehavioral sequelae of cranial irradiation in adults: a review of radiation-induced encephalopathy
J. Clin. Oncol.
Oxidative damage pathways in relation to normal tissue injury
Br. J. Radiol.
NF kappa B activity and target gene expression in the rat brain after one and two exposures to ionizing radiation
Radiat. Oncol. Invest.
Delayed molecular responses to brain irradiation
Int. J. Radiat. Biol.
Microglia
Metab. Brain Dis.
Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function
J. Neurosci.
Microglia as a source and target of cytokines
Glia
Microglia in health and disease
J. Neurosci. Res.
Irradiation induces neural precursor-cell dysfunction
Nat. Med.
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