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

Abstract

BackgroundExcessive 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.MethodsInduction 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.ResultsMurine 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.ConclusionsOur 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.

Reversal of NO-induced nociceptive hypersensitivity by St. John’s wort and hypericin: NF-κB, CREB and STAT1 as molecular targets

“…In particular, we can suppose that PKC might act as an upstream regulator of NF-κB, STAT1 and CREB. The hypothesis of the activation of the PKC/NF-κB pathway following NO donors' administration, that contributes to the induction of allodynia/hyperalgesia, was confirmed by studies conducted on murine microglia showing that LPS-and peptidoglycan-induced iNOS production appears to be mediated by a signaling pathway involving the sequential of PKC and NF-κB activation (Bhatt et al 2010;Wen et al 2011). Similarly, we can suppose that PKC might be involved in the NO-induced supraspinal activation of STAT1.…”

Regulation of Postnatal Forebrain Amoeboid Microglial Cell Proliferation and Development by the Transcription Factor Runx1

“…8 A, lanes 1-3). To confirm these observations, primary microglia were treated with LPS, which causes robust microglia activation and iNOS upregulation (Sheng et al, 2011;Wen et al, 2011). LPS treatment led to a significant increase in iNOS expression in untransduced cells and considerably enhanced the upregulation of iNOS caused by adenovirus infection (Fig.…”

Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

“…LPS-activated microglia are known to express iNOS and release NO (Kumar et al, 2014;Wen et al, 2011). We therefore tested whether blocking iNOS with the specific inhibitor N-(3-(aminomethyl)benzyl)acetamidine dihydrochloride (1400 W) prevented uptake of live PC12 cells, and found that it partially inhibited this phagocytosis (Fig.…”