Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells

Wen Jie, Ribeiro Rachel, Zhang, Zhang Yumin

Description

Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC) can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia. Methods Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS) was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF-κB in activated microglia was examined by determining NF-κB transcriptional response element- (TRE-) driven, promoter-mediated luciferase activity. Results Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC β attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF-κB activation that leads to the expression of iNOS in reactive microglia. Conclusions Our results identify PKC δ and β as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF-κB. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Wen et al . Journal of Neuroinflammation 2011, 8 :38 http://www.jneuroinflammation.com/content/8/1/38

JOURNAL OF NEUROINFLAMMATION

R E S E A R C H Open Access Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells 1 Jie Wen , Rachel Ribeiro 2 and Yumin Zhang 1,2*

Abstract Background: Excessive production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) in reactive microglia is a major contributor to initiation/exacerbation of inflammatory and degenerative neurological diseases. Previous studies have indicated that activation of protein kinase C (PKC) can lead to iNOS induction. Because of the existence of various PKC isoforms and the ambiguous specificity of PKC inhibitors, it is unclear whether all PKC isoforms or a specific subset are involved in the expression of iNOS by reactive microglia. In this study, we employed molecular approaches to characterize the role of each specific PKC isoform in the regulation of iNOS expression in murine microglia. Methods: Induction of iNOS in response to bacterial endotoxin lipopolysaccharide (LPS) was measured in BV-2 murine microglia treated with class-specific PKC inhibitors, or transfected with siRNA to silence specific PKC isoforms. iNOS expression and MAPK phosphorylation were evaluated by western blot. The role of NF- B in activated microglia was examined by determining NF- B transcriptional response element- (TRE-) driven, promoter- mediated luciferase activity. Results: Murine microglia expressed high levels of nPKCs, and expressed relatively low levels of cPKCs and aPKCs. All PKC inhibitors attenuated induction of iNOS in LPS-activated microglia. Knockdown of PKC δ and PKC b attenuated ERK1/2 and p38 phosphorylation, respectively, and blocked NF- B activation that leads to the expression of iNOS in reactive microglia. Conclusions: Our results identify PKC δ and b as the major PKC isoforms regulating iNOS expression in reactive microglia. The signaling pathways mediated by PKC involve phosphorylation of distinct MAPKs and activation of NF- B. These results may help in the design of novel and selective PKC inhibitors for the treatment of many inflammatory and neurological diseases in which production of NO plays a pathogenic role.

Background neighboring cells such as neurons and oligodendrocytes Microglia are distributed throughout the central nervous  (OLs). A pathogenic role for nitric oxide has been impli- system (CNS) as resting immunocompetent cells derived cated in many inflammatory and neurodegenerative dis- from a monocyte/macrophage lineage [1,2]. When acti- eases, including multiple sclerosis, stroke and traumatic vated, microglia protect neurons by clearing toxic cell brain injury [4-7]. Understanding the potential mechan- debris and pathogens, and acting as antigen presenting isms that turn beneficial inflammatory responses into cells to induce innate immune responses [3]. However, detrimental action is crucial for identifying therapeutic excessive activation of microglia can also release a vari-  targets to intervene in self-sustained inflammatory ety of toxic factors including reactive oxygen species cycles. (ROS), reactive nitrogen sp ecies (RNS) and proinflam- Nitric oxide (NO), generated from L-arginine by nitric matory cytokines, which cause toxicity to the oxide synthase (NOS), has been shown to be both a sig- naling and an effector molecule in diverse biological sys- * Correspondence: [e-mail]  s, Uniformed Services t d e e m n s tifi [ e 8 d - , 1 n 0 e ]. uro A n m al on N g OS th ( e nN th O r S e ) e an is d o e fo n r do m t s hel o i f al N N O O S S 1 Department of Anatomy, Physiology and Genetic i University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD (eNOS) are Ca 2+ dependent [8-13], and inducible NOS F 2 u 0 l 8 l 1 li 4 s , t U o S f A authorinformationisavailableattheendofthearticle (iNOS) functions in a Ca 2+ -independent manner [10,13]. © 2011 Wen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.