Specific PKC isoforms regulate LPS-stimulated iNOS induction in murine microglial cells
© Wen et al; licensee BioMed Central Ltd. 2011
Received: 24 October 2010
Accepted: 21 April 2011
Published: 21 April 2011
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.
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.
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.
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.
Microglia are distributed throughout the central nervous system (CNS) as resting immunocompetent cells derived from a monocyte/macrophage lineage [1, 2]. When activated, microglia protect neurons by clearing toxic cell debris and pathogens, and acting as antigen presenting cells to induce innate immune responses . However, excessive activation of microglia can also release a variety of toxic factors including reactive oxygen species (ROS), reactive nitrogen species (RNS) and proinflammatory cytokines, which cause toxicity to the neighboring cells such as neurons and oligodendrocytes (OLs). A pathogenic role for nitric oxide has been implicated in many inflammatory and neurodegenerative diseases, including multiple sclerosis, stroke and traumatic brain injury [4–7]. Understanding the potential mechanisms that turn beneficial inflammatory responses into detrimental action is crucial for identifying therapeutic targets to intervene in self-sustained inflammatory cycles.
Nitric oxide (NO), generated from L-arginine by nitric oxide synthase (NOS), has been shown to be both a signaling and an effector molecule in diverse biological systems [8–10]. Among the three isoforms of NOS identified, neuronal NOS (nNOS) and endothelial NOS (eNOS) are Ca2+ dependent [8–13], and inducible NOS (iNOS) functions in a Ca2+-independent manner [10, 13]. Induction of iNOS occurs primarily in astrocytes and microglia in response to endotoxin or to proinflammatory cytokines, such as TNFα, IL-1β or IFNγ . Using pharmacological inhibitors and molecular approaches, studies have shown that NO can react with superoxide to form peroxynitrite in reactive microglia causing toxicity to neurons and OLs [15, 16]. Although it is known that activation of various transcription factors - such as STAT, NF-κB, AP-1, and C/ERP - can contribute to the production of NO [17–20], the signaling pathways regulating expression of iNOS and production of NO in the CNS are still not well understood.
Protein kinase C (PKC) is a family of serine/threonine kinases that regulate cellular responses elicited by hormones, neurotransmitters and growth factors . Based on differences in sequence homology between these isozymes and their requirements for cofactors, the PKC family is divided into conventional PKCs (cPKC: α, β and γ), novel PKCs (nPKC: δ, ε, η and θ) and atypical PKCs (aPKCs: ζ and λ/ι) [22, 23]. PKC isoforms are widely expressed in many cell types, including microglia/macrophages , and studies have shown that PKC activation is an important mediator of microglial activation [25, 26]. PKC inhibitors reduce NO synthesis from IFN-γ-treated microglia and PKC δ is able to regulate NF-κB activation and iNOS expression in mouse peritoneal macrophages . Because of the existence of various PKC isoforms and the ambiguity of action of PKC inhibitors, the role of specific PKC isoforms involved in the inflammatory response in microglia has not been elucidated. In this study we used murine microglial cell line BV-2 cells to examine the signaling pathways by which PKC activation leads to iNOS induction in LPS-activated microglia. Our results indicate that all PKC isoforms are expressed in BV-2 cells with a particularly high expression of nPKC. Although several PKC isoforms can mediate lipopolysaccharide- (LPS-) stimulated increases in iNOS expression, PKC δ and β are likely the major PKC isoforms responsible for PKC function in reactive microglia. Furthermore, we found that distinct mitogen activated protein kinases (MAPKs) are activated in response to specific PKC isoforms and result in iNOS induction. Elucidation of the signaling pathways mediated by the different PKC isoforms in iNOS expression in reactive microglia will facilitate the development of isoform-specific PKC inhibitors with the potential to avoid the side effects of pan-PKC inhibitors.
Fetal bovine serum (FBS) and Dulbecco's modified Eagle's medium (DMEM) were purchased from Invitrogen (Carlsbad, CA). The BV-2 cell line was a generous gift from Dr. Feng-Qiao Li, Cognosci Inc., NC. Bacterial LPS (Escherichia Coli O111:B4) was obtained from Sigma (St. Louis, MO). 2',7'-dichlorohydrofluorescein diacetate (DCF) was purchased from Molecular Probes, Inc. (Eugene, OR). Antibodies against phosphorylated and total p38, extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) were purchased from Cell Signaling Technology (Danvers, MA). Anti-iNOS antibody was purchased from BD biosciences (San Diego, CA). PKC siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Bisindolylmaleimide-1 (Bis-1), Rottlerin, GO6976, SB203580, SP600125 and U0126 were purchased from Calbiochem (Gibbstown, NJ). Transfection reagents were from Roche (Basel, Switzerland) and Luciferase assay kit was from Promega (Madison, WI).
Immortalized murine microglial cells (BV-2) were cultured in 100 mm dishes in DMEM containing 5% FBS, 1% penicillin/streptomycin at 37°C in an incubator with a humidified atmosphere of 95% air and 5% CO2.
Quantitative real-time PCR and reverse transcriptase PCR analysis
Primer sequences of mouse PKC isoforms.
5'-c c c a t t c c a g a a g g a g a t g a-3'
5'-t t c c t g t c a g c a a g c a t c a c-3'
5'-t c c c t g a t c c c a a a a g t g a g-3'
5'-a a c t t g a a c c a g c c a t c c a c-3'
5'-c a g a c c a a g g a c c a c c t g t t-3'
5'-g c a t a a a a c g t a g c c c g g t a-3'
5'-a c c a g g g c a t c a t c t a c a g g-3'
5'-c t t c c t c a t c t t c c c c a t c a-3'
5'-g a g g a c t g g a t t g a c c t g g a-3'
5'-a t c t c t g c a g t g g g a g c a g t-3'
5'-c a t c c c a c a c a a g t t c a a c g-3'
5'-a t a t t t c c g g g t t g g a g a c c-3'
5'-t a t g g c t t c a g c g t t g a c t g-3'
5'-c c t t t g g g t c c t t g t t g a g a-3'
5'-a t g g a c a a c c c c t t c t a c c c-3'
5'-g c g g a t g t c t c c t c t c a c t c-3'
5'-a a g t g g g t g g a c a g t g a a g g-3'
5'-c a g c t t c c t c c a t c t t c t g g-3'
PKC activity assay
The activity of PKC in BV-2 cells following LPS treatment was measured using a PKC activity assay kit from Assay Designs, Inc (Ann Arbor, MI). In brief, BV-2 cells cultured in 24-well plates were treated with 1 μg/ml LPS for 30 min and then washed with cold PBS twice and lysed with protein lysis buffer. Whole cell lysates were adjusted to equal protein concentrations with lysis buffer and the same volume of each sample was added to ELISA plates pre-coated with crebtide, a substrate that can be readily phosphorylated by PKC. ATP was added to each well to initiate reaction at 30°C for 90 min. After emptying the contents of each well, phosphospecific substrate antibody was added and incubated for 1 hr. The phosphorylated crebtide was quantitated following the manufacturer's instructions.
Western blot analysis
Whole cell lysates from cultured BV-2 cells were obtained by using ice-cold protein lysis buffer (containing 1 × TBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) with freshly added protease inhibitor cocktail and glycerophosphate and sodium orthovanadate. The lysates were subjected to centrifugation at 10,000 g for 10 min at 4°C. 5 μg of whole cell lysates were boiled for 5 min, and separated on Novex 4-12% Bis-Tris gel. Proteins were transferred to PVDF membrane using a Bio-Rad mini-trans-blot cell. Transferred blots were blocked by incubating the membranes with 5% BSA for 1 hr at room temperature to reduce non-specific binding. Blocked membranes were incubated with primary antibodies overnight. These antibodies include rabbit polyclonal anti-phosphorylated and total ERK1/2, JNK and p38 (all with dilution of 1:1000), mouse anti-iNOS (1:1000), mouse anti-PKC α, β, δ, ε and γ (BD Transduction Laboratories, 1:1000) and rabbit anti-PKC η, λ, θ and ζ polyclonal antibodies (Santa Cruz, all with 1:500 dilution). After washing with 1 × TBS-T (Tris-buffered saline containing 1% Tween 20), the membranes were incubated with goat anti-rabbit or goat anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (1:2000) for 1 hr at room temperature. Finally, the membranes were incubated in Chemiluminescence western blot detection reagents from Pierce (Rockford, IL) for 1 min and protein was visualized with Image Reader LAS-3000 software.
The level of accumulated nitrite in the medium was determined by the Greiss reaction. Briefly, 50 μl of Greiss reagent (3.9 mM N-(1-naphthyl)ethylenediamine/58 mM sulfanilamide/5% phosphoric acid) was added to 50 μl of culture supernatant in a 96-well plate. Absorbance was measured at wavelength 550 nm, and nitrite concentration was calculated from a standard curve of sodium nitrite.
In order to specify the role of each PKC isoform in iNOS induction by LPS-activated microglia, double-stranded siRNA oligonucleotides for each PKC isoform (purchased from Santa Cruz) were transfected into BV-2 cells with X-treme transfection reagent (60 nmol siRNA/well). The day before the transfection, BV-2 cells were split and plated into 24-well plates at a density of 2 × 105 cells/well to assure cells around 80% confluency at the time of transfection. The transfected cells were continuously incubated at 37°C for 48 hr before use for further experiments. siGLO RISC-free siRNA from Dharmacon was used as a negative control and its fluorescence was also used for evaluating transfection efficiency.
Plasmid transfection and luciferase assay
The reporter gene with NF-κB promoter was transfected into BV-2 cells. In brief, the cells were trypsinized and plated into 96-well plates at a density of 5 × 104 cells/well. The transfection was performed with FuGene HD transfection reagent. One microgram plasmid containing NF-κB promoter or GFP was mixed with 0.25 μl FuGene HD in a total volume of 5 μl of serum-free DMEM for each reaction. At 24 hr after transfection, cells were treated with LPS for 3 hr in the presence of various PKC and MAPK inhibitors. Assessment of luciferase activity in transfected cells was carried out with a luciferase reporter assay system from Promega following the manufacturer's instructions.
Data were analyzed for statistical significance using a two-tailed t test (comparison of two data sets) or with analysis of variance (ANOVA, comparison of multiple data sets). A significant difference was determined as p < 0.05. All experiments were performed in triplicate and have been repeated at least three times.
ALL PKC isoforms are present in microglia and activated by LPS
PKC inhibitors attenuate iNOS expression in reactive microglia
Activation of MAPK occurs downstream PKC, but upstream iNOS induction in reactive microglia
Activation of NF-κB contributes to PKC-mediated iNOS induction in reactive microglia
The differential role of PKC isoforms in LPS-induced iNOS production and MAPK activation in BV-2 cells
Overproduction of NO by enhanced iNOS induction has been tightly linked to neuroinflammatory and neurodegenerative diseases [38–40]. A better understanding of the signaling mechanisms involved in the regulation of microglial iNOS has potential therapeutic implications. Previous studies mostly used PKC activators and inhibitors to determine the role of PKC in the regulation of iNOS production in murine microglia [26–30]. However, the absence of selectivity and the potential off-target effects of these pharmacological agents limit the ability to further define isoform-specific functions of the various PKCs. In the present study, we have employed PKC isoform-specific siRNAs to delineate novel molecular signaling pathways linking PKC to iNOS induction in BV-2 cells when exposed to LPS.
Role of the PKC specific isoforms in LPS-induced iNOS production
The PKC family consists of at least 10 serine/threonine protein kinases originally characterized by their dependency on lipids for catalytic activity [41, 42]. The cPKCs require DAG and Ca2+, the nPKCs require DAG but not Ca2+, while the aPKCs require neither. The different modes of PKC regulation suggest that PKC isoforms may function differently in response to various stimuli. In BV-2 cells, pharmacological inhibition studies suggest that the nPKC and cPKC isoforms are integral to LPS-induced increases in iNOS expression and NO production (Figure 2), and isoform-specific siRNA knockdown confirms that PKC δ and PKC β are the major nPKC and cPKC isoforms involved in the regulation of LPS-induced iNOS production in murine microglia (Figure 7).
A number of studies have reported that particular PKC isoforms are involved in the production of NO in several different cell types [28, 43–45]. Here we demonstrate a principal role for PKC δ and PKC β in the response to LPS exposure in murine BV-2 cells. These results are not only consistent with previous studies showing that PKC δ activation is required for regulating the production of iNOS in mouse peritoneal macrophages , human leukemia cells  and BV-2 cells , but also for the first time suggest that PKC β might play an important role in LPS-induced iNOS production in BV-2 cells even with its low levels of expression. It might be concluded that the primary role of PKC δ results from its high expression relative to other PKC isoforms (Figures 1A and 1B). However, PKC β expression is relatively low (Figure 1A) suggesting that induction of iNOS is dependent not only on levels of expression, but also on the activation of distinct PKC isoforms. Interestingly, PKC α and ε have been shown to be the major PKC isoforms involved in the signaling pathways by which IFNγ induces iNOS expression in the same cell line . Collectively, these results suggest that distinct PKC isoforms are activated and implicated in the regulation of iNOS induction in a stimulus-specific manner.
Downstream components of PKC activation in LPS-induced iNOS expression
MAPKs. In the present study we also explored signaling pathways downstream of PKC that increase iNOS expression in response to LPS exposure. In general agreement with the observed effects of the three PKC inhibitors, rottlerin, GO6976, and Bis-1 (Figure 4D), knockdown of PKC δ, η, α and β expression reduces LPS-induced phosphorylation of ERK1/2 (Figures 8A and 8B), whereas downregulation of PKC β significantly inhibits LPS-induced phosphorylation of p38 (Figure 8B). No effect on phosphorylation of JNK is observed with individual cPKC or nPKC siRNA (Figures 8A and 8B). Taken together, these results provide strong evidence that ERK1/2 and p38 are the main signaling pathways through which distinct PKC isoforms regulate iNOS induction in response to LPS. Moreover, these results suggest that distinct MAPKs are activated by specific PKC isoforms.
It has been shown that both p38 and ERK1/2 can mediate iNOS expression in glial cells . However, the phosphorylation of ERK1/2 has been found to be involved in IFNγ-, but not in LPS-induced NO production, although NO production seems to be coupled to PKC δ activation under both stimulations . The discrepancy between this report and our current study is unclear, but may be attributable to differences in the stage of BV-2 cells used in these studies. The same group has recently found that paraquat toxicity to microglia is mediated by PKC δ- and ERK1/2- dependent ROS generation . The fact that neither nPKCs nor cPKCs affect JNK phosphorylation (Figure 6) suggests that JNK is not involved in the signaling pathway of iNOS induction coupling to PKC activation. Interestingly, PKC θ siRNA significantly blocks p38 phosphorylation (Figure 8A), although the commonly used nPKC inhibitor rottlerin has no inhibitory effect (Figure 4D). Similarly, GO6976 blocks JNK activation (Figure 4D) but the same phenomenon is not observed with the use of cPKC siRNAs (Figure 8B). These results further suggest that it might be misleading to draw conclusions on the role of specific PKC isoforms in the function of reactive microglia on the basis of pharmacological inhibition.
NF-κB. It is known that iNOS expression is transcriptionally regulated. Activation of p38 has been shown to regulate NF-κB, C/EBP, and ATF-2 to induce iNOS expression in rat astroglia . However, HIV-1 Tat-induced iNOS expression in human astrocytes is dependent on phosphorylation of ERK1/2 and transcriptional activation of C/EBP, but not NF-κB . These studies indicate that different transcription factors can be recruited via one or more kinase pathways with respect to different inducers of iNOS. In this study, we find that activation of NF-κB is required for iNOS induction through the application of CAY10470, an NF-κB-specific inhibitor (Figure 5). The observation that all of the PKC inhibitors - GO6976, rottlerin and Bis-1 - significantly block NF-κB activation strongly supports the conclusion that NF-κB activation is required for iNOS induction in LPS-treated BV-2 cells.
The authors wish to thank Dr. Thomas Flagg for his careful reading and critical comments and suggestions on the manuscript. This work was supported by grants from the Blast Lethality Injury and Research Program (R600-070-00000-00-106109), the National Multiple Sclerosis Society (RG3741) and start-up fund from the Uniformed Services University (R070 UX).
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