Gö6976 inhibits LPS-induced microglial TNFα release by suppressing p38 MAP kinase activation

doi:10.1016/S0306-4522(02)00356-1

Neuroscience

Volume 114, Issue 3, 11 October 2002, Pages 689-697

https://doi.org/10.1016/S0306-4522(02)00356-1 Get rights and content

Abstract

Microglial responses to endotoxin, including the synthesis of inflammatory factors, contribute to gliosis and neuron degeneration in cultured brain tissue. We have previously shown that Gö6976, a protein kinase C (PKC) inhibitor, suppressed the lipopolysaccharide (LPS)-induced production of inflammatory factors in microglia and afforded marked protection of neurons from glia-mediated cytotoxicity. The purpose of this study was to identify the signal transduction pathway underlying the neuroprotective effect of Gö6976. Gö6976 suppressed the LPS-induced release of tumor necrosis factor α (TNFα) in the microglial cell line, BV2. We show in this study the inhibitory effect of Gö6976 on TNFα release occurring through suppression of p38 mitogen-activated protein kinase (MAPK) phosphorylation and not through a PKC mechanism. While Gö6976 did not inhibit the activity of p38 MAPK directly, it did suppress its activation by phosphorylation, indicating the target of action of Gö6976 is a signaling event upstream of p38 MAPK. Although Gö6976 is considered a selective inhibitor of certain PKC isozymes, suppression of TNFα production was not mediated through inhibition of PKC activity. Gö6976 appears to play a novel role in neuroprotection by suppressing the release of pro-inflammatory factors by inhibiting the activation of p38 MAPK in microglia, rather than a PKC isoform.

Section snippets

Materials

LPS (Escherichia coli O111:B4) was from List Biological Laboratories (Campbell, CA, USA). Tissue culture plates (24-well) were Costar® from Corning (Corning, NY, USA). Fetal bovine serum, Dulbecco’s modified Eagle’s medium with Ham’s nutrient mixture F12, and genistein were obtained from Life Technologies (Gaithersburg, MD, USA). SB202190, SB202474, Gö6976, Ro31-8220 and H89 were purchased from Calbiochem-Novobiochem International (La Jolla, CA, USA). The mouse TNFα capture antibody, mouse TNFα

Gö6976 and SB202190 inhibited LPS-induced TNFα production by BV2 microglial cells

We examined the effects of protein kinase inhibitors on the increase in production of TNFα, a representative inflammatory factor, in murine microglial BV2 cell cultures stimulated with LPS. TNFα production increased to about 5 ng/ml 3 h after LPS exposure. Pretreatment with the indolocarbazole Gö6976, which protects neurons from endotoxin- or combined cytokine-induced cytotoxicity (Jeohn et al., 2000b), suppressed the LPS-induced increase in TNFα production significantly (Fig. 1A). The

Discussion

Microglial activation in response to such toxins as bacterial LPS, HIV coat protein gp120, and β-amyloid-related peptides leads to the synthesis of inflammation-related products, which have been associated with neuron degeneration in the brain (Dickson et al., 1993, Chao et al., 1995). We propose that Gö6976 may suppress the synthesis of inflammation-related factors in microglia by a PKC-independent mechanism. The results of this study suggest that Gö6976 suppressed LPS-induced TNFα production

Conclusion

Our results indicate that Gö6976 suppressed the phosphorylation of p38 MAPK and consequently, the p38 MAPK activity induced by LPS, and resulted in a decrease in LPS-induced TNFα secretion. The inhibitory function of Gö6976 on the phosphorylation of p38 MAPK was not mediated through the inhibition of any known PKC. Rather, one of the upstream signaling events prior to p38 MAPK, and other than known PKC isotypes, appears to be the functional target of Gö697. Further understanding of the

Acknowledgements

We thank Dr. R.C.C. Chang for valuable discussions and insightful comments on this work and Ms. Crystal Paras for editorial assistance.

References (39)

T. Akiyama et al.
Genistein, a specific inhibitor of tyrosine-specific protein kinases
J. Biol. Chem.
(1987)
E. Blasi et al.
Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus
J. Neuroimmunol.
(1990)
C.C. Chen et al.
Protein kinase C eta mediates lipopolysaccharide-induced nitric-oxide synthase expression in primary astrocytes
J. Biol. Chem.
(1998)
B.L. Fiebich et al.
Protein kinase C-mediated regulation of inducible nitric oxide synthase expression in cultured microglial cells
J. Neuroimmunol.
(1998)
E. Galea et al.
CD14 mediate endotoxin induction of nitric oxide synthase in cultured brain glial cells
J. Neuroimmunol.
(1996)
M. Gschwendt et al.
Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes
FEBS Lett.
(1996)
G.H. Jeohn et al.
Post-transcriptional inhibition of lipopolysaccharide-induced expression of inducible nitric oxide synthase by Go6976 in murine microglia
Brain Res. Mol. Brain Res.
(2000)
G.H. Jeohn et al.
The indolocarbazole Go6976 protects neurons from lipopolysaccharide/interferon-gamma-induced cytotoxicity in murine neuron/glia co-cultures
Brain Res. Mol. Brain Res.
(2000)
L.Y. Kong et al.
Protein tyrosine kinase inhibitors decrease lipopolysaccharide-induced proinflammatory cytokine production in mixed glia, microglia-enriched or astrocyte-enriched cultures
Neurochem. Int.
(1997)
J.M. Kyriakis et al.
Sounding the alarm: protein kinase cascades activated by stress and inflammation
J. Biol. Chem.
(1996)

Y.B. Lee et al.

p38 MAP kinase regulates TNF-alpha production in human astrocytes and microglia by multiple mechanisms

Cytokine

(2000)

G. Martiny-Baron et al.

Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976

J. Biol. Chem.

(1993)

T. Moriguchi et al.

A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3

J. Biol. Chem.

(1996)

L.A. O’Neill et al.

The IL-1 receptor/toll-like receptor superfamily: crucial receptors for inflammation and host defense

Immunol. Today

(2000)

H. Yang et al.

Cellular events mediated by lipopolysaccharide-stimulated toll-like receptor 4. MD-2 is required for activation of mitogen-activated protein kinases and Elk-1

J. Biol. Chem.

(2000)

C.D. Beaty et al.

Lipopolysaccharide-induced cytokine production in human monocytes: role of tyrosine phosphorylation in transmembrane signal transduction

Eur. J. Immunol.

(1994)

M.M. Behrens et al.

Go 6976 is a potent inhibitor of neurotrophin-receptor intrinsic tyrosine kinase

J. Neurochem.

(1999)

N.R. Bhat et al.

Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures

J. Neurosci.

(1998)

V. Bocchini et al.

An immortalized cell line expresses properties of activated microglial cells

J. Neurosci. Res.

(1992)

Cited by (45)

2-cyclohexylamino-5,8-dimethoxy-1,4-naphthoquinone inhibits LPS-induced BV2 microglial activation through MAPK/NF-kB signaling pathways
2016, Heliyon
Citation Excerpt :
All these phenomena suggest that ROS-dependent signaling pathways are most likely responsible for the inhibitory effects described above. MAPK family plays crucial roles in the LPS-induced pro-inflammatory products and neuroinflammation [31, 32, 33]. It was reported that DMNQ derivatives exhibit anti-tumor activity by inducing apoptosis via caspases and mitogen activated protein (MAP) kinase-dependent pathways [34, 35, 36].
To verify the effects of several 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) derivatives on LPS-induced NO production, cellular ROS levels and cytokine expression in BV-2 microglial cells.
An MTT assay and FACS flow cytometry were performed to assess the cellular viability and apoptosis and cellular ROS levels, respectively. To examine the expression of pro-inflammatory cytokines and cellular signaling pathways, semi-quantitative RT-PCR and Western blotting were also used in this study.
Among the six newly synthesized DMNQ derivatives, 2-cyclohexylamino-5,8-dimethoxy-1,4-naphthoquinone (R6) significantly inhibited the NO production, cellular ROS levels and the cytokines expression in BV-2 microglial cells, which stimulated by LPS. Signaling study showed that compound R6 treatment also significantly down-regulated the LPS-induced phosphorylation of MAPKs (ERK, JNK and p38) and decreased the degradation of IκB-α in BV2 microglial cells.
Our findings demonstrate that our newly synthesized compound derived from DMNQ, 2-cyclohexylamino-5,8-dimethoxy-1,4-naphthoquinone (R6), might be a therapeutic agent for the treatment of glia-mediated neuroinflammatory diseases.
ET-1 mediates the release of reactive oxygen species and TNF-α in lung tissue by protein kinase C α and β1
2016, Pharmacological Reports
Citation Excerpt :
These findings suggest that ET-1 increases TNF-α production in lung homogenates via PKC αβ activation. Jeohn et al. [38] showed that Gö6976 suppressed LPS- induced TNF-α production in glia cell. Recently Dvoriantchikova et al. [39] also observed a decrease in TNF-α of the medium from astrocyte cultures treated with Hsp70 in the presence of PKC inhibitor Gö6976.
The aim of this study was to determine the involvement of protein kinase C (PKC) in the ET-1 induced generation of reactive oxygen species and TNF-α in rat lungs.
Experiments were performed on 6 groups of rats: Group I: saline-treated control; Group II: saline followed by endothelin-1 (ET-1) (3 μg/kg); Group III: saline followed by selective PKC αβ1 inhibitor (Gö6976) (2 μg/kg); Group IV: Gö6976 (2 μg/kg) administered 30 min before ET-1 (3 μg/kg); Group V: saline followed by the PKC activator phorbol 12-myristate 13-acetate (PMA) (50 μg/kg); Group VI: Gö6976 (2 μg/kg) administered 30 min before PMA (50 μg/kg). After 5 h, the animals were euthanized and their lungs were isolated for measurements.
ET-1 resulted in increase in thiobarbituric acid reactive substances (TBARS) and hydrogen peroxide (H₂O₂) levels and lung edema, as well as a decrease in GSH/GSSG ratio compared to the controls. The level of TNF-α also was elevated in the presence of ET-1. Administration of Gö6976 30 min before ET-1 injection significantly decreased lung edema, as well as the concentrations of TBARS, H₂O₂ and TNF-α, but increased the GSH/GSSG redox ratio compared to ET-1. Conversely, PMA elevated lung edema and TBARS, H₂O₂ and TNF-α concentrations, but decreased the GSH/GSSG redox ratio compared to the control group. Treatment with Gö6976 significantly ameliorated the PMA-induced oxidative stress parameters, decreased tissue TNF-α level, and lung edema.
Endothelin-1 induces ROS generation, increases TNF-α level and lung edema via activation of PKC αβ1.
Electrical stimulation ameliorates light-induced photoreceptor degeneration in vitro via suppressing the proinflammatory effect of microglia and enhancing the neurotrophic potential of Müller cells
2012, Experimental Neurology
Citation Excerpt :
These findings imply that treatment based on blockage of microglial activation could be developed to support neuronal survival and tissue regeneration. A few such therapies have been reported including naloxone, tetrandrine, and protein kinase C (PKC) inhibitors (He et al., 2011; Jeohn et al., 2002; Ni et al., 2008). However, whether ES can prevent photoreceptor degeneration by targeting microglia activity remains to be determined.
Many types of electrical stimulation (ES) devices have been shown to promote the survival of degenerated neural cells, such as dopaminergic neurons in the medial forebrain bundle-transected rats, ischemic-injured cortical neurons and inner-and outer-nuclear-layer cells in degenerated retina. Using a rat photic injury model, our lab previously proved the neuroprotective effect of transcorneal electrical stimulation (TCES) on apoptotic photoreceptor cells. To delineate the mechanisms that might underlie this process, the effects of ES on light-damaged photoreceptor degeneration-induced microglia and Müller cell activation were investigated in the present in vitro study. Our data showed that ES (3 ms, 20 Hz, 300–1600 μA) increased survival among light-reared cone-derived cells (661W) cultured alongside microglia or Müller cells analyzed by LDH and TUNEL assays. The degree of rescue was found to depend on the different intensities of the ES current. The immunocytochemistry revealed that ES significantly decreased the numbers of activated microglia cells with ameboid shapes and increased the numbers of reactive gliotic Müller cells with larger soma when they were co-cultured with light-damaged 661W cells. Real-time RT-PCR and Western blotting indicated that ES which was applied to different co-cultures and 661W cell-conditioned media (661WCM)-treated glia cultures had a prominent inhibitive effect on the secretion of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in microglia and a positive regulative effect on the production of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) in Müller cells. The death rate of light-exposed 661W cells cultured with microglia was decreased significantly by the addition of neutralizing antibodies against IL-1β and TNF-α. On the other hand, the death rate of light-exposed 661W cells cultured with Müller cells was prominently increased when the co-culture was incubated in the presence of neutralizing antibody against BDNF while anti-CNTF neutralizing antibody did not induce additional exacerbation of the cell death among those 661W cells. These findings indicate the feasibility of using ES to create a nurturing environment for light-damaged photoreceptor cells. This environment is characterized by diminished microglial activation and fortified Müller cells reactive gliosis, which may have great potential in ameliorating photoreceptor damage. In this way, ES was here determined to be a novel, potent therapeutic option for delaying the progression of photoreceptor degeneration in patients suffering from retinitis pigmentosa (RP).
Anti-neuroinflammatory activity of kamebakaurin from Isodon japonicus via inhibition of c-Jun NH<inf>2</inf>-terminal kinase and p38 mitogen-activated protein kinase pathway in activated microglial cells
2011, Journal of Pharmacological Sciences
Compelling evidence supports the notion that the majority of neurodegenerative diseases are associated with microglia-mediated neuroinflammation. Therefore, quelling of microglial activation may lead to neuronal cell survival. The present study investigated the effects of Kamebakaurin (KMBK), a kaurane diterpene isolated from Isodon japonicus HARA (Labiatae), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated cytotoxicity in rat primary microglial cultures and the BV-2 cell line. KMBK significantly inhibited the LPS-induced production of nitric oxide (NO) in a concentration-dependent fashion in activated microglial cells. The mRNA and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxycenase-2 (COX-2) were also decreased dose-dependently. Furthermore KMBK inhibited the JNK and p38 mitogen-activated protein kinases (MAPKs) in LPS-stimulated BV-2 microglial cells. Considering the results obtained, the present study authenticated the potential benefits of KMBK as a therapeutic target in ameliorating microglia-mediated neuroinflammatory diseases.
Inflexin attenuates proinflammatory responses and nuclear factor-ΚB activation in LPS-treated microglia
2010, European Journal of Pharmacology
Activated microglia participate in neuroinflammation which contribute to neuronal damage. Suppression of microglial activation would have therapeutic benefits, which lead to alleviation of the progression of neurodegeneration. In this study, the inhibitory effects of inflexin, a putative antiinflammatory agent isolated from Isodon excisus (Max.) Kudo (Labiateae), on the production of proinflammatory mediators were investigated in the lipopolysaccharide (LPS)-stimulated microglia. Inflexin significantly inhibited the release of nitric oxide (NO). Consistently, both the mRNA and the protein levels for the inducible NO synthase were decreased by inflexin in a concentration-dependent manner. Inflexin also inhibited the expression of cyclooxygenase (COX)-2, but not the COX-1 and effectively reduced the LPS-induced expression of proinflammatory cytokines in a dose-dependent manner. Furthermore, inflexin inhibited the degradation of IκB-α and the activation of NF-κB, p65 and Akt, while the MAPKs signal pathway was not affected. Our data suggest that inflexin was able to suppress neuroinflammation via inhibition of NF-κB activation and Akt pathway indicating that inflexin may be developed as a potent therapeutic agent in treating neuroinflammatory diseases.
The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells
2008, Neurochemistry International
Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKCɛ of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca²⁺. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKCɛ, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKCɛ had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.

View all citing articles on Scopus

¹: On sabbatical leave from Division of Science, Truman State University, Kirksville, MO 63501, USA.

²: Present address: College of Pharmacy, Kangwon University, Chuncheon, South Korea.

View full text

Published by Elsevier Ltd.

Gö6976 inhibits LPS-induced microglial TNFα release by suppressing p38 MAP kinase activation

Abstract

Section snippets

Materials

Gö6976 and SB202190 inhibited LPS-induced TNFα production by BV2 microglial cells

Discussion

Conclusion

Acknowledgements

J. Biol. Chem.

J. Neuroimmunol.

J. Biol. Chem.

J. Neuroimmunol.

J. Neuroimmunol.

FEBS Lett.

Brain Res. Mol. Brain Res.

Brain Res. Mol. Brain Res.

Neurochem. Int.

J. Biol. Chem.

Cytokine

J. Biol. Chem.

J. Biol. Chem.

Immunol. Today

J. Biol. Chem.

Lipopolysaccharide-induced cytokine production in human monocytes: role of tyrosine phosphorylation in transmembrane signal transduction

Eur. J. Immunol.

Go 6976 is a potent inhibitor of neurotrophin-receptor intrinsic tyrosine kinase

J. Neurochem.

Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures

J. Neurosci.

An immortalized cell line expresses properties of activated microglial cells

J. Neurosci. Res.