Morphine mediates a proinflammatory phenotype via μ-opioid receptor–PKCɛ–Akt–ERK1/2 signaling pathway in activated microglial cells

Abstract

Anti-nociceptive tolerance to opioids severely limits their clinical efficacy for the treatment of chronic pain syndromes. Glia has a central role in the development of morphine tolerance. Here, we characterized the receptor-proximal signaling events that link μ-opioid receptors to activation of Akt and ERKs in lipopolysaccharide (LPS)-stimulated murine microglial cells with the aim to define the molecular mechanism contributing to the ability of morphine to increase inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in activated microglial cells. In particular, the role of PKCɛ isoform in μ-opioid-induced inflammatory response in microglia was investigated. The results indicate that morphine increases the LPS-induced expression and activation of PKCɛ and stimulates Akt pathway upstream of ERK1/2 and iNOS. Furthermore, we found that morphine enhanced the release of IL-1β, TNF-α, IL-6, and of NO via μ-opioid receptor–PKCɛ signaling pathway in activated microglial cells, mediating a proinflammatory phenotype in mouse microglial cells. Together, these data suggest that the modulation of μ-opioid receptor signaling on microglia through PKCɛ selective inhibition may provide a means to attenuate glial activation and, as a consequence, to treat opioid development of tolerance and dependence.

Introduction

Opioids are potent analgesics and are irreplaceable for the treatment of severe pain and in anesthesia. They mediate their effects via three receptors termed μ-, δ- and κ-opioid receptors. Among these, μ-opioid receptors play an outstanding role, because they mediate effects of morphine and most clinically used opioids [1], [2]. Recently, we have demonstrated that morphine and the μ-opioid receptor agonist DAMGO induce the activation of Akt and ERKs in microglial cells. In addition, μ-opioid receptors increase NO and pro-inflammatory cytokine secretion, e.g. IL-1β, TNF-α and IL-6, through Akt and ERK signaling [3]. However, the mechanisms that occur in the GPCR signaling by which μ-opioid receptors signal to ERKs have not been studied in detail.

Microglia, brain inflammatory cells, are activated in injured brain where they release inflammatory mediators such as nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and prostaglandins [4], [5], [6]. The inflammatory mediators could protect tissues from bacterial infection but also could potentiate the damage of neurons [7]. Activated microglial cells in the spinal cord may release proinflammatory cytokines and other substances thought to facilitate pain transmission [8], [9]. Therefore, pharmacological attenuation of glial activation represents a novel approach for controlling pain [10], [11]. Although the intracellular signaling molecules involved in microglial activation have not been fully understood, protein kinase C (PKC) has been considered as an important mediator of microglial activation [12], [13]. In particular, it has been reported that PKC inhibitors reduced NO release from lipopolysaccharide (LPS)-treated microglia [12], [13].

Among different effectors, PKC has attracted a great deal of attention because of its prominent role in synaptic plasticity and memory. PKC is an integral component of most GPCR signaling pathways to ERK/MAPK [14]. Accordingly, PKC was found to be an early signaling component in the opioid pathway to ERK [15], [16], [17]. Nevertheless, little is known about the actual isoforms involved in this pathway. PKC serine/threonine kinases require diacylclycerol (DAG) and phosphatidyl serine (PS) for their activation, while according to their dependency on Ca²⁺ are classified into conventional, Ca²⁺-dependent cPKCs α, β, and γ, and into novel, Ca²⁺-independent nPKCs δ, ɛ, η, θ; the newest and atypical PKCs, aPKCs ζ and λ/ι require only PS [18], [19]. Most PKC isoforms are widely expressed in the brain with higher concentrations in the cerebral structures and areas known to participate in memory processes. PKCɛ in particular, a prominent PKC in differentiating and differentiated neurons [20], [21], is considered critical in learning and memory, and in synaptic remodeling [22]. There is evidence that PKCɛ isoform is activated by opiates, raising the possibility that PKCɛ modulates behavioral responses to opiates. The interactions between the transition from acute to chronic pain and the development of opioid tolerance and dependence are mediated by PKCɛ [23]. Recently, it has been demonstrated that morphine uses the PKCɛ pathway to induce ERK phosphorylation and receptor desensitization [24]. Furthermore, PKC inhibitors can reduce morphine anti-nociceptive tolerance [25]. Mice that lack PKCɛ isozyme (PKCɛ^−/−) show enhanced responses to morphine [26] and increased analgesia and thermal behavioral tolerance to specific cannabinoid agonists [27]. Several aspects of the immune system in these animals were normal although macrophages were defective in the production of LPS-stimulated TNF-α, IL-1β, PGE₂, and NO. There were also deficits in LPS-stimulated MAPK and NF-κB activation [28].

Therefore, the present study was designed to identify in detail the receptor-proximal signaling events that link μ-opioid receptors to activation of Akt and ERKs in microglial cells and to define the molecular mechanism contributing to the ability of morphine to increase inflammatory mediators such as NO, TNF-α, IL-1β and IL-6 in activated microglial cells. In particular, the role of PKCɛ isoform in μ-opioid-induced inflammatory response in microglia has been here investigated.