Takashi Ichiyama

α-Melanocyte-Stimulating Hormone Inhibits NF-κB Activation and IκBα Degradation in Human Glioma Cells and in Experimental Brain Inflammation

Abstract The neuropeptide α-melanocyte-stimulating hormone (α-MSH) modulates production of proinflammatory cytokines in brain tissue and in peripheral inflammatory cells. Transcription of the genes for these proinflammatory cytokines is regulated by the nuclear factor κB (NF-κB). NF-κB is also activated by proinflammatory cytokines. Degradation of the cytoplasmic inhibitor IκBα protein results in activation of NF-κB. Because of increasing evidence that NF-κB is involved in brain injury and inflammation and neurodegenerative disease, we examined whether α-MSH inhibits activation of NF-κB and limits degradation of IκBα protein induced by lipopolysaccharide (LPS) in human glioma cells (A-172) and in mouse brain. Electrophoretic mobility shift assays of nuclear extracts from A-172 cells and whole mouse brains stimulated with LPS revealed that α-MSH does suppress NF-κB activation. Western blot analysis demonstrated that α-MSH preserved expression of IκBα protein in vitro (glioma cells) and in vivo (brain tissue). Chloramphenicol acetyltransferase assay indicated that α-MSH suppresses NF-κB-dependent reporter gene expression induced by LPS in A-172 cells. The findings are consistent with the possibility that the anti-inflammatory action of α-MSH in CNS inflammation occurs via modulation of NF-κB activation by peptide-induced inhibition of degradation of IκBα protein.

Systemically administered α-melanocyte-stimulating peptides inhibit NF-κB activation in experimental brain inflammation

Abstract The neuropeptide α-melanocyte-stimulating hormone (α-MSH) and its C-terminal tripeptide α-MSH11-13 modulate production of proinflammatory cytokines and inhibit inflammation. We examined whether systemic α-MSH and α-MSH11-13 inhibit activation of the nuclear transcription factor, nuclear factor kappa B (NF-κB), a factor that is essential to expression of proinflammatory cytokines, in experimental murine brain inflammation induced by lipopolysaccharide. Electrophoretic mobility shift assays of nuclear extracts demonstrated that parenteral α-MSH inhibited NF-κB activation. Western blot analysis revealed that this inhibition was linked to α-MSH-induced preservation of expression of IκBα protein in the brain. The effects of α-MSH on NF-κB and IκBα were paralleled by pretreatment with α-MSH11-13. Similar effects of the two peptides were observed in mice with nonfunctional melanocortin 1 receptors (MC1R), ruling out the possibility that this receptor subtype is essential to the influence on NF-κB. These findings indicate that α-MSH peptides given systemically can inhibit NF-κB activation induced in acute brain inflammation even in the absence of MC1R.

Inhibition of peripheral NF-κB activation by central action of α-melanocyte-stimulating hormone

Abstract With the rise in the field of neuroimmunomodulation research, there is increased recognition of the influence of the nervous system and neuropeptides in peripheral disease. The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a neuroimmunomodulatory agent that modulates production of proinflammatory cytokines and inhibits peripheral inflammation via actions on CNS receptors. We examined whether central α-MSH operates by inhibiting activation of the nuclear factor kappa B (NF-κB) that is essential to the expression of proinflammatory cytokines and development of inflammation in the periphery. Electrophoretic mobility shift assays of nuclear extracts from the murine foot pad injected with TNF-α demonstrated that centrally administered α-MSH does inhibit NF-κB activation. Western blot analysis revealed that this inhibition was linked to central α-MSH-induced preservation of expression of IκBα protein in the peripheral tissue. The NF-κB and IκBα effects were inhibited in mice with spinal cord transection. Intraperitoneal (ip) injection of the nonspecific β-adrenergic receptor blocker propranolol, and of a specific β 2 -adrenergic receptor antagonist, likewise prevented these effects of central α-MSH; blockade of cholinergic, α-adrenergic, or β 1 -adrenergic receptors did not. Centrally administered α-MSH inhibited peripheral NF-κB activation and IκBα degradation even in mice with nonfunctional melanocortin 1 receptors (MC1R). These findings indicate that α-MSH can act centrally to inhibit NF-κB activation in peripheral acute inflammation via a descending neural pathway. The pathway involves β 2 -adrenergic receptors, but does not require activation of MC1R within the brain.

Mechanisms of Antiinflammatory Action of α‐MSH Peptides: In Vivo and in Vitro Evidence

ABSTRACT: α‐Melanocyte stimulating hormone (α‐MSH) modulates all forms of inflammation by acting on peripheral inflammatory cells, glial inflammatory cells, and on CNS receptors that activate descending antiinflammatory neural pathways. The multiple actions of this ancient peptide suggest that there is no singular biochemical mechanism through which it exerts its antiinflammatory activity. However, research on IL‐10 deficient and Agouti protein hypersecreting mice provide new insights into the actions of the peptide in living animals. Studies of cultured human astrocytes, whole murine brain, and human monocyte/macrophages indicate that a primary effect of the peptide is modulation of activation of the nuclear transcription factor κB. The latter influence may underlie the established reduction of gene expression and production of proinflammatory peptides and inducible nitric oxide by α‐MSH peptides.

Ketamine suppresses endotoxin-induced NF-κB expression

Purpose: Ketamine reduces endotoxin-induced production of proinflammatory cytokines, including tumour necrosis factor- α (TNF), in several types of inflammatory cells, including monocytes and macrophages. Transcription of the genes that encode production of these proinflammatory cytokines is regulated by nuclear factor-kappa B (NF-κB). Cytoplasmic B protein is activated by endotoxin (LPS) as well as by TNF, allowing B protein to migrate into the cell nucleus to activate gene transcription for these inflammatory mediators. Because NF-κB is likely involved in brain injury and inflammatory neurodegenerative disease, such as multiple sclerosis, we examined whether ketamine inhibits LPS-induced activation of NF-κB in human glioma cellsin vitro and intact mouse brain cellsin vivo.Methods: Endotoxin-induced NF-κB expression in both the human glioma cellsin vitro and the intact mouse brain cellsin vivo was determined by electrophoretic mobility shift assays (EMSA) of nuclear extracts and measurement of NF-κB expression by densitometry. Endotoxin was injected intracerebroventricularlyin vivo and intact brain was harvested. Klenow fragment labeling was used to identify NF-κB protein for both thein vivo andvitro experiments.Results: Endotoxin treatment increased NF-κB expression (P<0.05) bothin vivo andvitro compared with control (untretaed) cells. Ketamine suppressed endotoxin-induced neuronal NF-κB activation in a dose-dependent manner (P<0.05, except for the 10−5M concentrationin vitro) bothin vivo andvitro.Conclusion: Ketamine inhibits endotoxin-induced NF-κB expression in brain cellsin vivo andvitro and it is suggested that this may have implications in the neuroprotective effects of ketamine reported by other investigators.RésuméObjectif: La kétamine réduit la production de cytokines pro-inflammatoires induite par endotoxine, y compris le facteur nécrosant des tumeurs (TNF), dans certains types de cellules inflammatoires comprenant les monocytes et les macrophages. La transcription des gènes qui encodent la production de ces cytokines pro-inflammatoires est réglée par le facteur-kappa B nucléaire (NF-6B). La protéine cytoplasmique 6B est activée par l’endotoxine (LPS) et par le TNF et peut ainsi migrer dans le noyau cellulaire et activer la transcription génique pour ces médiateurs de l’inflammation. Comme le NF-6B participe probablement aux lésions cérébrales et aux maladies inflammatoires neurodégénératives, dont la sclérose en plaques, notre but était de savoir si la kétamine inhibe l’activation de NF-6B induit par LPS dans des cellules de gliome humainin vitro et dans des cellules cérébrales intactes de sourisin vivo.Méthode: L’expression du NF-6B induite par endotoxine dans les cellules humainesin vitro et dans les cellules de sourisin vivo a été déterminée par une étude de retardement de la mobilité électrophorétique (ERME) d’extraits nucléaires et la mesure de l’expression du NF-6B a été faite par densitométrie. L’endotoxine a été injectée dans les ventricules cérébrauxin vivo et du tissu cérébral intact a été prélevé. Le marquage de fragments de Klenow a été utilisé pour identifier la protéine du NF-6B des deux expériencesin vivo etvitro.Résultats: Le traitement avec l’endotoxine a augmenté l’expression du NF-6B (P<0,05) des cellulesin vivo etin vitro comparées aux cellules témoin (non traitées). La kétamine a supprimé l’activation neuronale de NF-6B induite par endotoxine d’une façon dose-dépendante (P<0,05, sauf pour une concentration de 10−5Min vitro) des cellulesin vivo etin vitro.Conclusion: La kétamine inhibe l’expression de NF-6B induite par endotoxine dans des cellules cérébralesin vivo etin vitro et on croit que cela pourrait contribuer aux effets neuroprotecteurs de la kétamine dont parlent d’autres chercheurs.

The Neuroimmunomodulatory Peptide α-MSH

Abstract: Alpha‐melanocyte‐stimulating hormone (α‐MSH), a neuroimmunomodulatory peptide of ancient origin, is known to be involved in the control of host responses. In inflammatory cells, in the periphery and within the central nervous system, α‐MSH modulates the production and action of proinflammatory cytokines. This broad influence occurs via endogenous α‐MSH (melanocortin) receptors. The key to this anti‐inflammatory influence is inhibition of NF‐κB. Indeed α‐MSH inhibits activation of this nuclear factor through preservation of IκBα, which binds to NF‐κB and prevents its migration to the nucleus. Cells transfected with α‐MSH plasmid vector are resistant to challenge with bacterial lipopolysaccharide. The peptide also act on central melanocortin receptors to modulate inflammation in the periphery. In brief, α‐MSH and certain of its fragments such as α‐MSH [11‐13] KPV modulate inflammation via three general actions: direct actions on peripheral host cells; actions on inflammatory cells within the brain to modulate local reactions; and descending neural anti‐inflammatory pathways that control inflammation in peripheral tissues.

NF-κB activation is inhibited in human pulmonary epithelial cells transfected with α-melanocyte-stimulating hormone vector

Abstract α-Melanocyte-stimulating hormone (α-MSH) modulates inflammation. We investigated the influence of α-MSH on NF-κB activation in human pulmonary epithelial cells (A549) using a plasmid vector encoding α-MSH (pCMV-ssMSH). Electrophoretic mobility shift assays demonstrated that NF-κB activation induced by lipopolysaccharide was inhibited in A549 cells transfected with pCMV-ssMSH. Western blot analysis revealed that this inhibition was linked to preservation of expression of IκBα protein. Chloramphenicol acetyltransferase assay indicated that NF-κB-dependent reporter gene expression was suppressed in A549 cells transfected with pCMV-ssMSH. The findings indicate that anti-inflammatory actions are exerted via modulation of NF-κB activation by preservation of IκBα protein in human pulmonary epithelial cells transfected with α-MSH vector. We showed a possibility of gene therapy for chronic inflammatory lung diseases.