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Life Sciences 73 (2003) 595–609

The regulation of inducible nitric oxide synthase gene expression

induced by lipopolysaccharide and tumor necrosis factor-a in

C6 cells: involvement of AP-1 and NFnB

Jin-Koo Lee, Seong-Soo Choi, Je-Seong Won, Hong-Won Suh*

Department of Pharmacology, College of Medicine and Institute of Natural Medicine, Hallym University, 1 Okchun-Dong,

Chunchon, Kangwon-Do, 200-702, South Korea

Received 25 September 2002; accepted 10 February 2003

Abstract

The roles of AP-1 and NFnB in the regulation of inducible nitric oxide synthase (iNOS) mRNA expression

induced by the combination of lipopolysaccharide and tumor necrosis factor-a (LT) in C6 cells were examined in

the present study. The iNOS mRNA level and NO release were increased by several cytokines alone or

combination treatments at 24 hr. LT-induced iNOS mRNA level was maximally increased at 6 hr and maintained at

higher level at least up to 24 hr. At 6 hr, iNOS protein level and NO release were also increased by LT. By western

blot analysis, AP-1, such as Fra-1, Jun B, and phospho-CREB protein levels were increased by LT and

translocation of NFnB p52 from the cytoplasm to the nucleus was increased. In addition, phosphorylations of

MAPKs (ERK 1/2, p38, JNK 1/2) were increased by LT. LT-induced iNOS mRNA level was inhibited by

PD98059 (MEK 1/2 inhibitor), SB203580 (p38 inhibitor), and cycloheximide (a protein synthesis blocker),

indicating that the phosphorylation of ERK 1/2 and p38, and on-going protein synthesis are necessary for LT-

induced iNOS expression. Electrophoretic mobility shift assay (EMSA) showed that AP-1 and NFnB DNA

binding activities were increased at 6 hr and these AP-1 and NFnB DNA bands increased by LTwere super-shifted

when Fra-1, Jun B, or NFnB p50 antibody was coincubated. These findings strongly suggest that, in C6 cells, Fra-

1, Jun B, NFnB p50, and NFnB p52 appear to be involved in the regulation of iNOS mRNA induced by LT.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Inducible nitric oxide; Cytokines; Gene regulation; AP-1; NFnB; C6 glioma cells

0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved.

doi:10.1016/S0024-3205(03)00317-5

* Corresponding author. Tel.: +82-33-240-1654; fax: +82-33-240-1652.

E-mail address: [email protected] (H.-W. Suh).

J.-K. Lee et al. / Life Sciences 73 (2003) 595–609596

Introduction

Nitric oxide (NO) is a short-lived molecule that mediates a wide range of biologic effects. Its

biological effects include vasorelaxation (Palmer et al., 1987), neuro-transmission (Garthwaite, 1991),

inhibition of platelet aggregation (Radomski et al., 1987), as well as microbial and tumor cell killing

(Nathan, 1992). The enzyme responsible for NO synthesis, nitric oxide synthase (NOS), converts L-

arginine to L-citrulline and NO (Marletta, 1993). In the brain, three genes encode NO synthase isoforms,

with significant differences in their regulation. Neuronal and endothelial NOS are constitutively

expressed in astrocytes and in subpopulation of neurons. Their activity is predominantly regulated

via intracellular calcium/calmodulin signals in response to hormone or neurotransmitter stimulation

(Bredt and Snyder, 1990; Busse and Mulsch, 1990). A third type of NOS [an inducible NOS (iNOS)],

which presents in microglia and astrocytes, is regulated at the transcriptional level in response to

cytokines and lipopolysaccharide (LPS), and does not require calcium for its activity (Jaffrey and

Snyder, 1995; Nathan, 1992). Although NO produced by iNOS accounts for the bacterial and

tumoricidal properties of microglia, it is a particular importance in the pathophysiologies of inflamma-

tory neurological diseases including demyelinating disorders (e.g. multiple sclerosis, experimental

allergic encephalopathy, and X-adrenoleukodystrophy) and in ischemia and traumatic injuries associated

with infiltrating microglia and the production of proinflammatory cytokines (Bo et al., 1994; Cross et al.,

1994; Hooper et al., 1997; Koprowski et al., 1993; Merrill et al., 1993; Mitrovic et al., 1994). It is now

increasingly clear that glial cells in the central nervous system also produce NO in response to the

induction of iNOS by bacterial LPS and series of cytokines including tumor necrosis factor-a (TNF-a),

interleukin-1h (IL-1h), and interferon-g (IFN-g). Astrocytes in the healthy brain do not express iNOS;

however, after the ischemic, traumatic, neurotoxic, or inflammatory damages, the activated astrocytes

express iNOS in rodent and human (Galea et al., 1992; Hu et al., 1995; Pahan et al., 1997a,b, 1998a).

Although NO derived from both astrocytes and microglia has been believed to be involved in

demyelinating disease and neuronal death during the ischemia and trauma (Bo et al., 1994; Merrill et

al., 1993; Mitrovic et al., 1994), little is known about the signal transduction pathway involved in iNOS

induction in astrocytes.

Previous studies have suggested that the inhibitors of tyrosine kinase suppress astroglial iNOS gene

expression and NO production induced by cytokines or bacterial LPS (Feinstein et al., 1994b; Simmons

and Murphy, 1994). Among tyrosine kinases, p38 mitogen activated protein kinase (MAPK) has been

suggested to mediate LPS- or cytokine-induced iNOS expression in primary rat and mouse astrocyte-

enriched cultures (Bhat et al., 1998; Da Silva et al., 1997). However, another MAPK, an extracellular

signal regulated protein kinase (ERK), is reported to be involved selectively in iNOS gene expression in

primary cultured rat astrocytes, but not in mouse (Bhat et al., 1998; Da Silva et al., 1997). This

differential dependency of iNOS gene expression on ERK pathway may be due to the difference of

signal transduction mechanisms between in rat and mouse astrocytes.

Computer-assisted analysis of rodent iNOS promoter and in vitro transfection assay has identified

potential cytokine-responsive transcriptional elements. These include activator protein-1 (AP-1), nuclear

factor kappa B (NFnB), cAMP response element (CRE), CAAT box/enhancer binding protein (C/EBP),

g-interferon (IFN) activating site (GAS), IFN stimulated response element (ISRE), and IFN regulatory

factors (IRFs) (Zhang et al., 1998). Among these elements, AP-1 and NFnB are ubiquitous transcrip-

tional factors and pleiotropic regulators of the inducible expression of many genes that encode proteins

involved in the modulation of inflammatory and host defense process (Angel and Karin, 1991; Schreck

J.-K. Lee et al. / Life Sciences 73 (2003) 595–609 597

et al., 1991; Thanos and Maniatis, 1995). AP-1 is encoded by a set of genes called ‘‘immediate early

genes’’ whose transcription is rapidly induced independently of de novo protein synthesis, following cell

stimulation. Indeed, in human lung carcinoma cell line, a transient transfection assay has suggested that

AP-1 activation is important for the induction of iNOS transcription.

NFnB family, which includes p50, p52, RelA (p65), RelB, c-Rel, v-Rel and dorsal and Dif

proteins (Baldwin, 1996), is sequestered in the cytoplasm through its binding with its inhibitors, p105

or InB-like proteins in resting state (Blackwell and Christman, 1997). Activation of NFnB by

cytokines or oxidative stress requires either the degradation of its cytoplasmic inhibitor InB-alpha or

proteolytic cleavage of p105 (Blackwell and Christman, 1997). Free NFnB dimers translocate to the

nucleus and activate the target gene, such as iNOS. Indeed, NFnB has been believed to be the most

important transcriptional factor in the regulation of iNOS transcription in various cell types including

astrocytes (Eberhardt et al., 1994; Lowenstein and Snyder, 1992; Pahan et al., 1998b; Sherman et al.,

1993).

Therefore, we now report that the inhibition of p38 and ERK1/2 attenuates combination of LPS plus

TNF-a (LT)-induced iNOS gene expression and NO production in C6 rat glioma cells. We examined the

possible involvement of AP-1 (Fra-1 and Jun B) or NFnB (p50 and p52) in the regulation of LT-induced

iNOS gene expression.

Methods

Drugs and Chemicals

LPS and CHX were purchased from sigma chemicals (MI, USA). PD98059 was purchased from New

England Biolabs Inc. (MA, USA). SB203580 was purchased from Calbiochem (CA, USA). TNF-a,

IFN-g, and IL-1h were purchased from R and D systems (Woongbee Meditech, Korea).

C6 glioma cell culture

C6 rat glioma cells obtained from Korean Cell Line Bank (Seoul) were maintained in DMEM/F-12

medium containing 10% fetal bovine serum and 2 mM gentamicin. Cells were plated on 25 cm2 culture

flask (Falcon, Franklin, NJ). The cultures were incubated at 37 jC in 5% CO2, and after 1 day, the

medium was completely changed to a fresh culture medium. At 70% confluency, the cells were

incubated with serum free DMEM/F-12 medium (DMEM-base medium for nitrite assay) for 24 hrs prior

to the incubation with LPS and other chemicals.

Nitrite assay

Synthesis of NO was determined by an assay of the culture supernatant for nitrite, a stable reaction

product of NO with molecular oxygen. Briefly, 100 Al of culture supernatant was allowed to react with

100 Al of Griess reagent (Sigma Chemicals) (Feinstein et al., 1994a) and incubated at the room

temperature for 15 min. The optical density of the assay samples was measured spectrophotometrically

at 570 nm. Fresh culture media served as the blank in all the experiments. Nitrite concentration was

calculated from a standard curve derived from the reaction of NaNO2 in the assay.

Isolation of total RNA and proteins

Total cellular RNAwas extracted from C6 rat glioma cells using a rapid guanidine thiocyanate-water

saturated phenol/chloroform extraction procedure and subsequent precipitation with acidic sodium

acetate (Chomczynski and Sacchi, 1987). Total cellular RNA in the aqueous phase was precipitated with

ice-cold isopropyl alcohol. Isolated RNA samples were subjected to spectrophotometric analysis at 260

nm and 280 nm. The separated organic layer was extracted twice with an equal volume of sterilized

(Millipore) water and proteins were precipitated by adding 2 volumes of absolute ethanol to the water-

extracted organic phase. The protein pellets were washed twice with cold absolute ethanol and dried. The

dried pellets were dissolved in a denaturing buffer (6 M guanidium chloride, 20 mM Tris-HCl [pH 8.0],

and 1 mM EDTA). The protein samples were dialyzed against a renaturing buffer (20 mM Tris-HCl [pH

8.0], 100 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, 5 mM MgCl2, 0.4 mM phenylmethylsulfonyl

fluoride, and 20% glycerol) at 4 jC. The concentration of protein was determined with the Coomassie

blue protein assay reagent (Pierce Chemical Co., Rockford, IL) using bovine serum albumin (BSA) as

the standard.

Preparation of DIG-labeled cRNA probes

The cRNA probes for iNOS (Galea et al., 1994) and cyclophilin (Danielson et al., 1988) were

synthesized in vitro from linearized expression vectors which contained SP6 or T7 viral promoter.

One Ag of linearized plasmid was mixed with RNA labelling mixture that containing ATP, CTP, GTP

and Dig-labeled-UTP, transcription buffer, and SP6 or T7 RNA polymerase. After incubation at 37

jC for 2 hrs, the mixture was co-incubated with DNase I (RNase free) at 37 jC for 15 min,

precipitated in ethanol containing lithium chloride at � 70 jC for 30 min, and washed with 70%

chilled ethanol.

Non-isotopic Northern blot analysis

Ten microgram of total RNA were denatured in 65 jC. The denatured RNA samples were

electrophoresed on 1% agarose-formaldehyde gels and transferred to nylon hybond-N hybridization

membrane sheets (Amersham, Buckinghamshire, England). After UV cross-linking, the membranes

were pre-hybridized at 68 jC for at least 1 hr in prehybridization buffer containing 50% formamide.

The DIG-labeled iNOS probe was added to prehybridization buffer. The membranes were incubated

overnight at 68 jC. Following hybridization, the membrane was washed in washing solution (0.1 �SSC and 0.1% SDS). The membranes were gently equilibrated in Buffer I (100 mM maleic acid [pH

7.5] and 150 mM NaCl) and agitated in Buffer II (1% blocking reagent in Buffer I) for 30–60 min. The

membranes were incubated with the diluted anti-DIG-alkaline phosphatase (1:10,000). After washing

the membranes in 0.3% Tween 20 (in Buffer I), the membranes were equilibrated in alkaline buffer.

Diluted CSPD (Boehringer Mannheim) (1:100 dilution) was spread over the surface of the membranes.

After incubation of the membranes at 37 jC for 15–20 min, the membranes were exposed to

Hyperfilm-ECL (Amersham) for detection of the chemiluminescent signal. For rehybridization, the

membranes were washed for overnight at 65 jC in 50 mM Tris-HCl (pH 8.0), 50% dimethylformamide

and 1% SDS to remove the hybridized probe and rehybridized to the DIG-labeled rat cyclophilin cRNA

probe, a gene encoding peptidyl-prolyl cis-trans isomerase which is constitutively expressed in most



mammalian tissues with the exception of skeletal muscle (Danielson et al., 1988; Takahashi et al.,

1989).

Western blot analysis

Cellular protein (50 Ag) was mixed with an equal volume of SDS loading buffer (20% glycerol, 100

mM tris-HCl [pH 6.8], 200 mM dithiothreitol, 4% SDS, 0.1% bromophenol blue), boiled for 3 min, and

separated by electrophoresis in 12% polyacrylamide gels as described (Laemmli, 1970). A pre-stained

rainbow protein mixture (Amersham Co., Arlington Heights, IL) was used as the molecular weight

standard. Proteins were transferred from acrylamide gel onto polyvinylidene difluoride filters (PDVF;

Amersham Pharmacia Biotech, England) accoriding to the previous published procedures (Towbin et al.,

1979). Electro-transferred to PDVF were first blocked with blocking buffer (3% skim milk, 1% BSA, 10

mM Trizma base [pH 8.0], and 150 mM NaCl) and then incubated with antisera against Fra-1 (1:1000)

(Santa Cruz Biotechnology, Santa Cruz, CA), Jun B (1:1000) (Santa Cruz Biotechnology), iNOS

(1:1000) (Santa Cruz Biotechnology), CREB (1:1000) (New England Biolabs, Beverly, MA), phospho-

CREB (1:1000) (New England Biolabs), p38MAPK/phospho-p38MAPK (1:1000) (New England

Biolabs), JNK/phospho-JNK (1:1000) (New England Biolabs), and ERK/phospho-ERK (1:1000)

(New England Biolabs) in blocking buffer for 4 h at room temperature. Filters were then washed with

Tris-buffered saline containing 0.3% Tween-20 (TBST) for 5 min and then incubated with the goat anti-

rabbit donkey IgG-horseradish peroxidase conjugate (1:5000) in blocking buffer at room temperature for

1 hr. After washing the filters with TBST, ECL-plus solution (Amersham Life Science Co., England)

was added. Then, the membranes were exposed to Hyperfilm-MP (Amersham) for detection of light

emission.

Preparation of nuclear extracts and non-isotopic electrophoretic mobility-shift assay

Nuclear extract from stimulated or unstimulated C6 cells (1�107 cells) were prepared using the

method of Dignam et al. (1983) with slight modification. The AP-1 (5V-CGC-TTG-ATG-ACT-CAG-CCG-GAA-3V) and NFnB (5V-AGT-TGA-GGG-GAC-TTT-CCC-AGG-C-3V) (Santa Cruz Biotechnol-

ogy) were annealed by incubation an equal molar concentration of each single-stranded oligonucleotide

in 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 200 mM NaCl at 95 jC for 10 min, and then the mixture

was allowed to cool to the room temperature. The DNA-binding assay was performed according to the

instructions in the manual provided with the DIG-Gel Shift Kit (Boehringer Mannheim). Binding

reactions were carried out at the room temperature for 20 min and reaction mixtures contained 30 Ag of

nuclear protein, 20 mM HEPES (pH 7.6), 30 mM KCl, 5 mMMgCl2, 1 mM EDTA, 1 mM dithiothreitol,

0.2% Tween 20, 50 mg/ml poly (dI-dC), and approximately 0.3 pmol of specified probe labeled with

DIG-ddUTP using terminal deoxynucleotidyl transferase (Boehringer Mannheim). Protein-DNA com-

plexes were separated from protein-free DNA by non-denaturing electrophoresis in 5% polyacrylamide

gels. Gels were run at a constant voltage (8 V/cm), and electro-blotted onto positively charged nylon

membranes. The membranes were baked at 80 jC for 15 min, washed with 0.3% Tween 20 in Buffer I,

and hybridized with the diluted anti-DIG-alkaline phosphatase (1:10,000) in Buffer II for 30 min. After

two washes for 15 min with 0.3% Tween 20 (in Buffer I), the membranes were equilibrated in alkaline

buffer for 2 min. The method for detection of chemiluminescence was identical to the method used for

the non-isotopic Northern blot analysis.

Statistical Analysis

The expression of iNOS mRNA induced by drugs was quantified with the Bio-profil Bio-1D

application (Vilber-Lourmat, France), and expressed as the percentage of the control iNOS mRNA

levels. A Student’s paired t-test was used to compare the relative abundance of mRNA in the

experimental and control groups.


Results

iNOS gene expression induced by LPS plus TNF-a (LT) in C6 cells

NO production and the regulation of iNOS mRNA level in rat C6 glioma cells were examined after the

treatment with LPS (500 ng/ml) plus TNF-a (20 ng/ml). As shown in Fig. 1A, a combination of LPS and

TNF-a stimulated NO production in C6 rat glioma cells. In Northern blot analysis, increased iNOS

mRNA level by combination treatment was in line with NO production in 24 hr (Fig. 1B). The

combination of LPS and TNF-a showed an additive effect for NO production in C6 glioma cells (Fig. 1A).

In C6 glioma cells, iNOS mRNA level began to increase at 1 hr, reached a maximum at 6 hr, and

maintained for up to 24 hr after the treatment with LPS (500 ng/ml) plus TNF-a (20 ng/ml) (Fig. 2A). In

Fig. 1. Effects of LPS and TNF-a on iNOS mRNA expression and NO production in C6 glioma cells. (A) Nitrite concentrations

were measured in culture media using the Griess reaction at 24 hr after combination treatment of LPS and TNF-a. (B) Northern

blot analysis was performed at 24 hr after the combination treatment of LPS and TNF-a. Final concentration was as follows:

LPS, 500 ng/ml; TNF-a, 20 ng/ml. The vertical bars in graphs indicate the S.E.M. (***P < 0.001; compared to the control

group, n = 3 independent experiments).

Fig. 2. Effect of LPS plus TNF-a on iNOS mRNA expression, iNOS protein level, and nitric oxide production in C6 glioma

cells. (A) Northern blot analysis was performed at specific time points (1, 6, 12, 24 hr) after treatment of LPS plus TNF-a. (B)

Western blot analysis was performed at the same time points after treatment with LPS (500 ng/ml) and TNF-a (20 ng/ml). Sixty

Ag of total cellular proteins was used for determination of iNOS protein level. A polyclonal antiserum against iNOS was used at

a 1:1000 dilution. The 130 kDa band indicates the iNOS protein. (C) Nitrite concentrations were measured in culture media

using the Griess reaction at specific time points (0, 10 min, 30 min, 1 hr, 3 hr, and 6 hr) after treatment of LPS plus TNF-a. The

vertical bars in graphs indicate the S.E.M. (*P < 0.05, **P < 0.01, and ***P < 0.001; compared to the control group, n = 3

independent experiments).


a short time-course study, western blot analysis using antibody against iNOS (130 kDa) protein was

carried out and showed that the iNOS protein level was increased in a time-dependent manner (Fig. 2B).

Furthermore, NO production was increased in a time-dependent manner in LPS plus TNF-a treated C6

glioma cells (Fig. 2C).

Effect of LPS plus TNF-a on the phosphorylation of MAPKs protein and several transcription factors

To examine the phosphorylation of MAPKs for the stimulation of LPS plus TNF-a, western blot

analysis using antibodies against ERK 1/2 (p44/p42 MAPK), p38 MAPK, and JNK 1/2 (p46/p54


MAPK) proteins and against their phosphorylated proteins were performed (Fig. 3). ERK 1/2, p38

MAPK, and JNK1/2 proteins were not affected by the stimulation of LPS plus TNF-a (data not shown).

However, phospho-ERK 1/2 began to increase at 10 min and was increased at a maximum at 1 hr and

returned to the basal level at 6 hr after the LPS plus TNF-a treatment. Phosphorylated p38 protein level

began to increase at 10 min and reached at a peak at 3 hr. At last, phosphorylated JNK 1/2 was increased

at 10 min.

Western blot analysis using antibodies against AP-1 proteins, such as, Fra-1 (35 kDa), Jun B (39 kDa)

and CREB/phospho-CREB (45 kDa) proteins was performed. Fra-1 and Jun B proto-oncoproteins were

remarkably increased 3–6 hrs after the stimulation with LPS plus TNF-a (Fig. 3). In contrast to the

increase of Fra-1 and Jun B proteins, Fra-2 and Jun D proteins were not affected by LPS plus TNF-a

treatment (data not shown). Total CREB protein level was not affected by LPS plus TNF-a treatment.

However, phospho-CREB protein level was increased at 1 hr and was returned to the control level at 3–

6 hr (Fig. 3).

Effects of PD98059, SB203580, and cycloheximide on LT-induced iNOS mRNA level

To examine the possible involvement of phospho-ERK 1/2 and phospho-p38 in the regulation of

iNOS expression, PD98059, a specific inhibitor of MEK1/2, which is upstream kinases of ERK1/2

Fig. 3. Effect of LPS plus TNF-a on the phosphorylation of MAPKs, AP-1, and phospho-CREB protein levels in C6 glioma

cells. Western blot analysis was performed at specific time points after treatment with LPS (500 ng/ml) plus TNF-a (20 ng/ml).

Fifty Ag of total cellular protein was used for determination of phospho-MAPKs (ERK, p38, and JNK), AP-1 (Fra-1 and Jun B),

and phospho-CREB. A polyclonal antiserum against phospho-ERK 1/2 (p44/p42), phospho-p38, phospho-JNK 1/2 (p46/p54),

Jun B, Fra-1, and phospho-CREB were used at a 1:1000 dilution.


(Payne et al., 1991) and SB203580, a specific inhibitor of p38 (Lee et al., 1994), were pretreated 30 min

prior to the combined LPS (500 ng/ml) and TNF-a (20 ng/ml) treatment. PD98059 (10 AM) partially

inhibited the increased iNOS mRNA level induced by LPS plus TNF-a stimulation at 3-6 hrs (Fig. 4A).

As shown in Fig. 4B, SB203580 (10 AM) caused a complete inhibition of increased iNOS mRNA level

induced by LPS plus TNF-a stimulation at 3–6 hrs (Fig. 4B). The dose of inhibitors was chosen on the

basis of preliminary studies and a previous publication (Won et al., 2001).

Fig. 4. Effect of PD98059 (A), SB203580 (B), and cycloheximide (C) on LPS plus TNF-a treated C6 glioma cells. Northern blot

analysis was performed at specific time points (3 and 6 hr) in the cells treated with control medium, LPS (500 ng/ml) plus TNF-a

(20 ng/ml), PD98059 (10 AM), SB203580 (10 AM), or cycloheximide (CHX; 15 AM), LPS plus TNF-a and PD98059, SB203580

or CHX together. PD98059, SB203580, and CHX were pre-treated for 30 min prior to the incubation of LPS plus TNF-a. The

vertical bars in graphs indicate the S.E.M. (*P < 0.05, **P < 0.01, and ***P < 0.001; compared to the control group, +P < 0.05,+ +P < 0.01 and + + +P < 0.001; compared to the LPS plus TNF-a-treated group, n = 3 independent experiments).

Fig. 5. Translocation of NFnB p52 from cytoplasm to nucleus in LPS plus TNF-a treated C6 glioma cells. twenty micrograms

of nuclear, cytosolic and total proteins, which were extracted at 30 min, 1 hr, and 3 hr after drug treatment were used for

determination of NFnB p52. A polyclonal antiserum against NFnB p52 was used at a 1:500 dilution.


To investigate whether on-going protein synthesis is required for LPS plus TNF-a-induced iNOS

mRNA expression, protein synthesis blocker, cycloheximide (CHX) was pretreated prior to 30 min the

LPS plus TNF-a treatment. iNOS mRNA level induced by LPS plus TNF-a treatment was inhibited by

CHX at 3–6 hr (Fig. 4C).

Fig. 6. AP-1 and NFnB DNA binding activities in LPS plus TNF-a treated C6 glioma cells. At 6 hr after LPS (500 ng/ml) plus

TNF-a (20 ng/ml) stimulation, the AP-1 (A) and NFnB (B) DNA binding activities were examined using gel shift analysis as

described in MATERIAL AND METHODS. For competition or supershift/attenuation assay, 50 X unlabelled AP-1 DNA

oligomers or two Ag of specific antiserum against c-Fos, c-Jun, Fra-1, or Jun B was added to each reaction. For NFnB DNA

binding activity, 50 X unlabelled NFnB DNA oligomers or two Ag of specific antiserum against p50, p52, c-Rel, or GR was

added to each reaction.

LPS plus TNF-a induces the translocation of NFjB p52

To elucidate the involvement of NFnB activation on iNOS mRNA expression induced by LPS plus

TNF-a treatment, we carried out western blot analysis from cytoplasm and nuclear proteins. As shown in

Fig. 5, NFnB p52 protein level was increased in nuclear fraction and decreased in cytosolic fraction at

0.5–3 hr. The protein level of total NFkB p52 was not changed by LPS plus TNF-a treatment.

Effect of LPS plus TNF-a on the AP-1 and NFjB DNA binding activities

To investigate the possible involvement of AP-1 or NFnB transcriptional factors in the regulation of

LPS plus TNF-a induced iNOS mRNA expression, the electrophoretic mobility shift assay was carried

out. In a quantitative study, DNA binding activities of AP-1 and NFnB were increased at all time points

(3 and 6 hr)(Data not shown). To examine the transcriptional factors involve in AP-1 and NFkB DNA

binding activities induced by LPS plus TNF-a treatment, we performed self-competition and supershift

assays using the various antibodies. In the self-competition study, AP-1 DNA binding activity induced

by LPS plus TNF-a treatment was decreased by unlabelled AP-1 DNA oligomer (cold AP-1), suggesting

that this DNA binding activity was AP-1 sequence specific (Fig. 6A). In a supershift study, antibodies

against Fra-1 and Jun B supershifted LT-induced AP-1 DNA binding activity (Fig. 6A). NFkB DNA

binding activity induced by LPS plus TNF-a treatment was also decreased by unlabelled NFkB DNA

oligomer (cold NFkB)(Fig. 6B). In a supershift study, antibody against p50 supershifted NFnB DNA

binding activity induced by LPS plus TNF-a treatment (Fig. 6B).


Discussion

The major findings in the present study are: (1) iNOS mRNA expression induced by LPS plus TNF-a

treatment might be mediated via the activation of ERK 1/2 and p38 and (2) new protein synthesis require

in iNOS mRNA expression induced by LPS plus TNF-a treatment. (3) iNOS mRNA expression induced

by LPS plus TNF-a treatment is also mediated by the activation AP-1 (Fra-1 and Jun B) and NFnB p50

transcription factors.

In the present study, we demonstrated signaling pathways for NO production and iNOS expression

induced by LT in C6 rat glioma cells. A combination of LPS and TNF-a increases NO production via iNOS

expression at 24 hr in C6 glioma cells. This finding is in line with the several previous studies using the

same cells (Feinstein et al., 1994a), as well as the primary cultured rat astrocytes (Simmons and Murphy,

1994). Furthermore, the necessity to stimulate cells with combined cytokines such as TNF-a plus IL-1h or

IFN-g or with cytokines combined with LPS to induce iNOS expression has been reported in several cell

types such as fibroblasts (Farivar and Brecher, 1996) and endothelial cells (Kanno et al., 1994), although a

single cytokine can induce iNOS expression in some other cell lines including hepatocytes (Geller et al.,

1993), islet cells (Heitmeier et al., 1997), and vascular smoothmuscle cells (Koide et al., 1993). Thus, in C6

rat glioma cells, co-stimulation with LPS and TNF-a was required to derive iNOS expression.

The exposure of C6 glioma cells to LPS plus TNF-a significantly increased the level of iNOS protein

level and NO production at 3–6 hr after treatment. These results suggest that iNOS expression induced

by LPS plus TNF-a treatment and its protein synthesis simultaneously generate. We have found for the

fist time that AP-1 transcription factors (such as Fra-1 and Jun B) and phosphorylated CREB were


increased by LPS plus TNF-a treatment in C6 glioma cells. Induction of these transcription factors may

cause an induction of iNOS mRNA through interacting with AP-1 or CREB and AP-1 or CRE DNA

binding domains located in iNOS promoter regions. As shown in Fig. 6A, AP-1 DNA binding activity

was increased by LPS plus TNF-a treatment. In a supershifted assay, supershifted bands observed in the

cells coincubated with antibody against Fra-1 or Jun B. These results are supported by several studies

that promoter region of iNOS gene contains AP-1, NFnB, CRE, C/EBP, GAS, ISRE, and IRFs (Zhang etal., 1998; Angel and Karin, 1991; Schreck et al., 1991; Thanos and Maniatis, 1995). This contention is

supported by the finding that AP-1 transcription factor is a complex composed of fos and jun proto-

oncogene families, which need to dimerize to promote the binding of the complex to the AP-1

recognition site and transcriptional activation of the target gene (Hunter and Karin, 1992).

We also found that translocation of p52 protein from the cytosol into nucleus was increased by LPS plus

TNF-a treatment. Furthermore, NFnB DNA binding activity also was increased by LPS plus TNF-a

treatment and the supershifted band observed in the cells cotreated with antibody against NFnB p50. These

results indicate that NFnB p50 and p52 may be important for the regulation of iNOS mRNA expression

induced by LPS plus TNF-a treatment. Recently, Nomura (2001) has reviewed that NFnB activation and

InB-a dynamism are involved in iNOS and chemokine induction in astroglial cells. However, the

involvement of NFnB p52 in iNOS mRNA expression observed in the present study is a new finding.

The roles of p38, JNK, and ERK must be considered in the regulation of iNOS expression, since those

kinases are shown to be activated by LPS plus TNF-a treatment with a slightly different kinetics as

revealed in the present study. The extent and kinetics of p38 as well as ERK and JNK kinase activation

did not provide the evidence for any additive or enhancing effects by LPS plus TNF-a treatment. This

result is in line with other study that p38 mediates iNOS mRNA expression in mouse astrocytes (Da

Silva et al., 1997). Furthermore, iNOS mRNA expression induced by LPS plus TNF-a treatment was

inhibited by PD98059 and SB203580 at 3–6 hrs (Fig. 4A and B). These results suggest that ERK1/2 and

p38 pathways may be important for the regulation of iNOS mRNA expression in C6 glioma cells.

Recently, we have reported that the LPS-induced NO production and iNOS mRNA expression are

inhibited dose-dependently by SB203580, but not PD98059 (Won et al., 2001). In the present study,

SB203580 completely blocked the iNOS mRNA expression induced by LPS plus TNF-a while

PD98059 showed a partial inhibition. It is suggested that p38 pathway may be more important for

the modulation of iNOS expression than ERK1/2 pathway.

To investigate whether on-going protein synthesis is required for LPS plus TNF-a-induced iNOS

mRNA expression, protein synthesis blocker, CHX was pretreated. LPS plus TNF-a-induced iNOS

mRNA expression was blocked by CHX pretreatment. This result suggests that iNOS mRNA expression

induced by LPS plus TNF-a treatment requires on-going protein synthesis.

In summary, the present study clearly shows that iNOS mRNA expression appears to be increased

via activating p38 and ERK1/2 MAPKs in LPS plus TNF-a-treated C6 rat glioma cells. Activations of

Fra-1, Jun B, phospho-CREB, and NFnB p50 and p52 proteins may serve as transcription factors for

the upregulation of iNOS mRNA expression when C6 rat glioma cells are stimulated with LPS and

TNF-a.

Acknowledgements

This research was supported by the Research Grant from Hallym University, South Korea.


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