Ohannes K. Melemedjian

Molecular mechanisms of glucocorticoid antiproliferative effects: antagonism of transcription factor activity by glucocorticoid receptor

Glucocorticoids (GCs) exert their anti‐inflammatory andimmunosuppressive effects by inhibiting the expression of cytokines andadhesion molecules. The molecular basis of GC action lies in theircapacity to diffuse through the cell membrane and bind their cytosolicGC receptor (GR), which subsequently undergoes nuclear translocationand modulates transcriptional activation through association withpromoter elements, GC response elements (GRE). GR also antagonized theactivity of transcription factors, including NF‐κB, NF‐AT, and AP‐1,through direct and indirect mechanisms. GCs induced the genetranscription and protein synthesis of the NF‐κB inhibitor, IκB. Activated GR antagonized transcription factor activity throughprotein:protein interaction. This involved complexing with andinhibition of transcription factor binding to DNA (simple model),association with factor bound at its DNA site (compositemodel), and/or through interaction of GRE‐bound GR withDNA‐bound transcription factor (transmodulation model). Finally, GRcompeted with transcription factors for nuclear coactivators(competition model), including CBP and p300. Remarkably, GR did notaffect the assembly of the preinitiation complex but acted proximallyin inhibiting transcription factor activity and thus transcriptionalinitiation.

IL-6- and NGF-Induced Rapid Control of Protein Synthesis and Nociceptive Plasticity via Convergent Signaling to the eIF4F Complex

Despite the emergence of translational control pathways as mediators of nociceptive sensitization, effector molecules and mechanisms responsible for modulating activity in these pathways in pain conditions are largely unknown. We demonstrate that two major algogens, the cytokine interleukin 6 (IL-6) and the neurotrophin nerve growth factor (NGF), which are intimately linked to nociceptive plasticity across preclinical models and human pain conditions, signal primarily through two distinct pathways to enhance translation in sensory neurons by converging onto the eukaryotic initiation factor (eIF) eIF4F complex. We directly demonstrate that the net result of IL-6 and NGF signaling is an enhancement of eIF4F complex formation and an induction of nascent protein synthesis in primary afferent neurons and their axons. Moreover, IL-6- and NGF-induced mechanical nociceptive plasticity is blocked by inhibitors of general and cap-dependent protein synthesis. These results establish IL-6- and NGF-mediated cap-dependent translation of local proteins as a new model for nociceptive plasticity.

Targeting adenosine monophosphate-activated protein kinase (AMPK) in preclinical models reveals a potential mechanism for the treatment of neuropathic pain

Neuropathic pain is a debilitating clinical condition with few efficacious treatments, warranting development of novel therapeutics. We hypothesized that dysregulated translation regulation pathways may underlie neuropathic pain. Peripheral nerve injury induced reorganization of translation machinery in the peripheral nervous system of rats and mice, including enhanced mTOR and ERK activity, increased phosphorylation of mTOR and ERK downstream targets, augmented eIF4F complex formation and enhanced nascent protein synthesis. The AMP activated protein kinase (AMPK) activators, metformin and A769662, inhibited translation regulation signaling pathways, eIF4F complex formation, nascent protein synthesis in injured nerves and sodium channel-dependent excitability of sensory neurons resulting in a resolution of neuropathic allodynia. Therefore, injury-induced dysregulation of translation control underlies pathology leading to neuropathic pain and reveals AMPK as a novel therapeutic target for the potential treatment of neuropathic pain.

An alternate mechanism of glucocorticoid anti‐proliferative effect: promotion of a Th2 cytokine‐secreting profile

Glucocorticoids (GCs) are used as immunosuppressive and anti‐inflammatory agents in organ transplantation and in treating autoimmune diseases and inflammatory disorders and they exert their effects by several mechanisms, the most significant of which is inhibition of cytokine production and action. Recent reports suggested that GCs inhibit cytokine expression indirectly through promotion of a T helper cell type 2 (Th2) cytokine‐secreting profile, thereby resulting in preferential blockade of pro‐inflammatory monokine and T helper cell type 1 (Th1) cytokine expression. The target of GCs appeared to be monocytes–macrophages, whereby altered regulation of interleukin (IL)‐1/IL‐1 receptor antagonist (IL‐1ra), coupled with profound blockade of IL‐12 synthesis and inhibition of interferon (IFN)‐Γ‐induced major histocompatibility complex (MHC) class II expression, lead to a preferential cognate stimulation of Th2 cells at the expense of Th1 cells. It is possible that this may have involved the expansion of a Th2‐cell pool or, in addition, frank stimulation of uncommitted naive CD4+ T cells toward the Th2 lineage. In addition, GCs may have blocked Th1 cytokine expression, thereby inhibiting ongoing Th1 cytokine secretion, and consequently provided for the unimpeded production of Th2 cytokines. Collectively, this indicates that, in exerting their anti‐proliferative effects, GCs act indirectly by altering Th1/Th2 cytokine balance, blocking the (pro‐inflammatory) Th1 program and favoring the (anti‐inflammatory) Th2 program.

Spinal protein kinase M £ underlies the maintenance mechanism of persistent nociceptive sensitization

Sensitization of the pain pathway is believed to promote clinical pain disorders. We hypothesized that the persistence of a sensitized state in the spinal dorsal horn might depend on the activity of protein kinase M ζ (PKMζ), an essential mechanism of late long-term potentiation (LTP). To test this hypothesis, we used intraplantar injections of interleukin-6 (IL-6) in mice to elicit a transient allodynic state that endured ∼3 d. After the resolution of IL-6-induced allodynia, a subsequent intraplantar injection of prostaglandin E2 (PGE2) or intrathecal injection of the metabotropic glutamate receptor 1/5 (mGluR1/5) agonist DHPG (dihydroxyphenylglycol) precipitated allodynia and/or nocifensive responses. Intraplantar injection of IL-6 followed immediately by intrathecal injection of a PKMζ inhibitor prevented the expression of subsequent PGE2-induced allodynia. Inhibitors of protein translation were effective in preventing PGE2-induced allodynia when given immediately after IL-6, but not after the initial allodynia had resolved. In contrast, spinal PKMζ inhibition completely abolished both prolonged allodynia to hindpaw PGE2 and enhanced nocifensive behaviors evoked by intrathecal mGluR1/5 agonist injection after the resolution of IL-6-induced allodynia. Moreover, spinal PKMζ inhibition prevented the enhanced response to subsequent stimuli following resolution of hypersensitivity induced by plantar incision. The present findings demonstrate that the spinal cord encodes an engram for persistent nociceptive sensitization that is analogous to molecular mechanisms of late LTP and suggest that spinally directed PKMζ inhibitors may offer therapeutic benefit for injury-induced pain states.

Resveratrol engages AMPK to attenuate ERK and mTOR signaling in sensory neurons and inhibits incision-induced acute and chronic pain

BackgroundDespite advances in our understanding of basic mechanisms driving post-surgical pain, treating incision-induced pain remains a major clinical challenge. Moreover, surgery has been implicated as a major cause of chronic pain conditions. Hence, more efficacious treatments are needed to inhibit incision-induced pain and prevent the transition to chronic pain following surgery. We reasoned that activators of AMP-activated protein kinase (AMPK) may represent a novel treatment avenue for the local treatment of incision-induced pain because AMPK activators inhibit ERK and mTOR signaling, two important pathways involved in the sensitization of peripheral nociceptors.ResultsTo test this hypothesis we used a potent and efficacious activator of AMPK, resveratrol. Our results demonstrate that resveratrol profoundly inhibits ERK and mTOR signaling in sensory neurons in a time- and concentration-dependent fashion and that these effects are mediated by AMPK activation and independent of sirtuin activity. Interleukin-6 (IL-6) is thought to play an important role in incision-induced pain and resveratrol potently inhibited IL-6-mediated signaling to ERK in sensory neurons and blocked IL-6-mediated allodynia in vivo through a local mechanism of action. Using a model of incision-induced allodynia in mice, we further demonstrate that local injection of resveratrol around the surgical wound strongly attenuates incision-induced allodynia. Intraplantar IL-6 injection and plantar incision induces persistent nociceptive sensitization to PGE2 injection into the affected paw after the resolution of allodynia to the initial stimulus. We further show that resveratrol treatment at the time of IL-6 injection or plantar incision completely blocks the development of persistent nociceptive sensitization consistent with the blockade of a transition to a chronic pain state by resveratrol treatment.ConclusionsThese results highlight the importance of signaling to translation control in peripheral sensitization of nociceptors and provide further evidence for activation of AMPK as a novel treatment avenue for acute and chronic pain states.

Morphine treatment accelerates sarcoma-induced bone pain, bone loss, and spontaneous fracture in a murine model of bone cancer

Abstract Metastatic bone cancer causes severe pain that is primarily treated with opioids. A model of bone cancer pain in which the progression of cancer pain and bone destruction is tightly controlled was used to evaluate the effects of sustained morphine treatment. In cancer‐treated mice, morphine enhanced, rather than diminished, spontaneous, and evoked pain; these effects were dose‐dependent and naloxone‐sensitive. SP and CGRP positive DRG cells did not differ between sarcoma or control mice, but were increased following morphine in both groups. Morphine increased ATF‐3 expression only in DRG cells of sarcoma mice. Morphine did not alter tumor growth in vitro or tumor burden in vivo but accelerated sarcoma‐induced bone destruction and doubled the incidence of spontaneous fracture in a dose‐ and naloxone‐sensitive manner. Morphine increased osteoclast activity and upregulated IL‐1β within the femurs of sarcoma‐treated mice suggesting enhancement of sarcoma‐induced osteolysis. These results indicate that sustained morphine increases pain, osteolysis, bone loss, and spontaneous fracture, as well as markers of neuronal damage in DRG cells and expression of pro‐inflammatory cytokines. Morphine treatment may result in “add‐on” mechanisms of pain beyond those engaged by sarcoma alone. While it is not known whether the present findings in this model of osteolytic sarcoma will generalize to other cancers or opioids, the data suggest a need for increased understanding of neurobiological consequences of prolonged opioid exposure which may allow improvements in the use of opiates in the effective management of cancer pain.

Sensitization of dural afferents underlies migraine-related behavior following meningeal application of interleukin-6 (IL-6).

BackgroundMigraine headache is one of the most common neurological disorders, but the pathophysiology contributing to migraine is poorly understood. Intracranial interleukin-6 (IL-6) levels have been shown to be elevated during migraine attacks, suggesting that this cytokine may facilitate pain signaling from the meninges and contribute to the development of headache.MethodsCutaneous allodynia was measured in rats following stimulation of the dura with IL-6 alone or in combination with the MEK inhibitor, U0126. The number of action potentials and latency to the first action potential peak in response to a ramp current stimulus as well as current threshold were measured in retrogradely-labeled dural afferents using patch-clamp electrophysiology. These recordings were performed in the presence of IL-6 alone or in combination with U0126. Association between ERK1 and Nav1.7 following IL-6 treatment was also measured by co-immunoprecipitation.ResultsHere we report that in awake animals, direct application of IL-6 to the dura produced dose-dependent facial and hindpaw allodynia. The MEK inhibitor U0126 blocked IL-6-induced allodynia indicating that IL-6 produced this behavioral effect through the MAP kinase pathway. In trigeminal neurons retrogradely labeled from the dura, IL-6 application decreased the current threshold for action potential firing. In response to a ramp current stimulus, cells treated with IL-6 showed an increase in the numbers of action potentials and a decrease in latency to the first spike, an effect consistent with phosphorylation of the sodium channel Nav1.7. Pretreatment with U0126 reversed hyperexcitability following IL-6 treatment. Moreover, co-immunoprecipitation experiments demonstrated an increased association between ERK1 and Nav1.7 following IL-6 treatment.ConclusionsOur results indicate that IL-6 enhances the excitability of dural afferents likely via ERK-mediated modulation of Nav1.7 and these responses contribute to migraine-related pain behavior in vivo. These data provide a cellular mechanism by which IL-6 in the meninges causes sensitization of dural afferents therefore contributing to the pathogenesis of migraine headache.

BDNF regulates atypical PKC at spinal synapses to initiate and maintain a centralized chronic pain state

BackgroundChronic pain is an important medical problem affecting hundreds of millions of people worldwide. Mechanisms underlying the maintenance of chronic pain states are poorly understood but the elucidation of such mechanisms have the potential to reveal novel therapeutics capable of reversing a chronic pain state. We have recently shown that the maintenance of a chronic pain state is dependent on an atypical PKC, PKMζ, but the mechanisms involved in controlling PKMζ in chronic pain are completely unknown. Here we have tested the hypothesis that brain derived neurotrophic factor (BDNF) regulates PKMζ, and possibly other aPKCs, to maintain a centralized chronic pain state.ResultsWe first demonstrate that although other kinases play a role in the initiation of persistent nociceptive sensitization, they are not involved in the maintenance of this chronic pain state indicating that a ZIP-reversible process is responsible for the maintenance of persistent sensitization. We further show that BDNF plays a critical role in initiating and maintaining persistent nociceptive sensitization and that this occurs via a ZIP-reversible process. Moreover, at spinal synapses, BDNF controls PKMζ and PKCλ nascent synthesis via mTORC1 and BDNF enhances PKMζ phosphorylaton. Finally, we show that BDNF signaling to PKMζ and PKCλ is conserved across CNS synapses demonstrating molecular links between pain and memory mechanisms.ConclusionsHence, BDNF is a key regulator of aPKC synthesis and phosphorylation and an essential mediator of the maintenance of a centralized chronic pain state. These findings point to BDNF regulation of aPKC as a potential therapeutic target for the permanent reversal of a chronic pain state.

Vascularization of the dorsal root ganglia and peripheral nerve of the mouse: Implications for chemical-induced peripheral sensory neuropathies

Although a variety of industrial chemicals, as well as several chemotherapeutic agents used to treat cancer or HIV, preferentially induce a peripheral sensory neuropathy what remains unclear is why these agents induce a sensory vs. a motor or mixed neuropathy. Previous studies have shown that the endothelial cells that vascularize the dorsal root ganglion (DRG), which houses the primary afferent sensory neurons, are unique in that they have large fenestrations and are permeable to a variety of low and high molecular weight agents. In the present report we used whole-mount preparations, immunohistochemistry, and confocal laser scanning microscopy to show that the cell body-rich area of the L4 mouse DRG has a 7 fold higher density of CD31+ capillaries than cell fiber rich area of the DRG or the distal or proximal aspect of the sciatic nerve. This dense vascularization, coupled with the high permeability of these capillaries, may synergistically contribute, and in part explain, why many potentially neurotoxic agents preferentially accumulate and injure cells within the DRG. Currently, cancer survivors and HIV patients constitute the largest and most rapidly expanding groups that have chemically induced peripheral sensory neuropathy. Understanding the unique aspects of the vascularization of the DRG and closing the endothelial fenestrations of the rich vascular bed of capillaries that vascularize the DRG before intravenous administration of anti-neoplastic or anti-HIV therapies, may offer a mechanism based approach to attenuate these chemically induced peripheral neuropathies in these patients.