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Lipopolysaccharide-mediated Reactive Oxygen Species andSignal Transduction in the Regulation of IL-1 Gene Expression

Running title: LPS-mediated ROS and signals in the regulation of proIL-1/IL-1

Hsien-Yeh Hsu1 and Meng-Hsuan Wen1

Faculty of Medical Technology, Institute of Biotechnology in Medicine,National Yang-Ming University1, 112, Taipei, TAIWAN

Address reprint requests and correspondence to:

Dr. Hsien-Yeh HsuFaculty of Medical TechnologyInstitute of Biotechnology in MedicineNational Yang-Ming University155 Li-Nong Street, Shih-PaiTaipei, TAIWAN

Tel. number: 011-886-2-2826-7252FAX number: 011-886-2-2826-4092Email: hyhsu@ym.edu.tw

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on April 8, 2002 as Manuscript M111883200 by guest on July 30, 2019

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1The abbreviations used are: LPS, lipopolysaccharide; ROS, reactive oxygen species; IL-1,

interleukin-1β; proIL-1, prointerleukin-1β; NAC, N-acetyl-cysteine; DPI, diphenyleneiodonium

chloride; MAPK, mitogen activated protein kinase; ERK, extracellular signal-regulated kinase;

JNK, c-JUN NH2-terminal protein kinase; p38, p38 mitogen activated protein kinase; PI 3-kinase,

phosphatidylinositol 3 kinase; PAK, p21 activated kinase; PTK, protein tyrosine kinase; PTP,

protein tyrosine phosphorylation; ICE, interleukin 1 converting enzyme

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ABSTRACT

Lipopolysaccharide (LPS) stimulates macrophages to release inflammatory cytokines,

interleukin-1β (IL-1) and TNF. LPS-induced TNF suppresses scavenger receptor functions in

macrophages (van Lenten, B.J. and Fogelman, A.M. (1992) J. Immunol. 148, 112-116), which is

regulated by TNF-mediated protein kinases (Hsu, H.Y. and Twu, Y.C. (2000) J. Biol. Chem. 275,

41035-41048). To examine the molecular mechanism for LPS induction of IL-1 in macrophages, we

demonstrate that LPS quickly stimulated reactive oxygen species (ROS), and 3 h later induced

prointerleukin-1β (proIL-1, precursor of IL-1) production and IL-1 secretion. LPS stimulated proIL-

1 message/protein between 3 h and 10 h; however, there was a 40% reduction of proIL-1 in pre-

incubation of the antioxidant, N-acetyl-cysteine (NAC). Moreover, NAC moderated LPS-induced

IL-1 secretion partially via ICE. The maximal activity of LPS-induced ERK, JNK and p38 was 12-

fold (30 min), 5-fold (30 min) and 16-fold (15 min), respectively. In contrast, NAC reduced ERK

activity to 60% and decreased p38 activity to the basal level, but JNK activity was induced 2-fold.

Furthermore, the pharmacological antagonists LY294002, SB203580, curcumin, calphostin C and

PD98059 revealed the diverse roles of LPS-mediated protein kinases in proIL-1. On the other hand,

NAC and diphenyleneiodonium chloride partially inhibited LPS-induced Rac activity and protein

tyrosine kinase (PTK), indicating that LPS-mediated ROS and NADPH oxidase correspond to Rac

activation and IL-1 expression. Our findings establish for the first time that LPS-mediated PTK/PI-3

kinase/Rac/p38 pathways play a more important role than pathways of PTK/PKC/MEK/ERK and of

PTK/PI-3 kinase/Rac/JNK in the regulation of proIL-1/IL-1. The findings also further elucidate the

critical role of LPS-mediated ROS in signal transduction pathways. Our results suggest that

understanding LPS-transduced signals in IL-1 induction upon the antibacterial action of macrophages

should provide a therapeutic strategy for aberrant inflammatory responses leading to severe cellular

injury or concurrent multi-organ septic damage.

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Lipopolysaccharide (LPS, or endotoxin) is a potent activator in the immune system. It

induces a variety of immune responses to severe infection (3-5), such as local inflammation, antibody

production, and septic shock . In addition, LPS is the major mediator of the high morbidity and

mortality rates characteristic of Gram-negative bacteria, i.e., endotoxic shock (3; 5). The

architecture of LPS consists of three separated “building blocks” : lipid A, an inner core region, and

the O-specific side chain. These separate building blocks have widely different compositions and

structures that are reflected in their biological activities (6). In early immune response for LPS,

macrophages, which are the major cellular targets for LPS action, play a central role in host defense

with physical and immune responses against bacterial infection (4). It is thought that LPS-induced

signal transmission requires binding to specific cellular receptors, including toll-like receptors (TLRs)

(5; 7-9). While bacterial products are binding to TLRs on macrophages, the cells are activated and

stimulate a wide spectrum of host defensive systems. This requires the activation of multiple

signaling molecules in transduction pathways, for example, protein tyrosine kinase (PTK), LPS

receptor-associated serine/threonine kinase, Ras, Raf-1, IκB kinase, MEK, mitogen-activated protein

kinases (MAPKs) (4; 10-16), etc. These molecules may converge or diverge, and often have “cross-

talk” properties, which make signaling networks complicated and with mutual influence.

Subsequently, the signals further transduce to downstream pathways and activate numerous

transcription factors, including AP-1, NF-κB (13), and ATF-2 (17). This in turn triggers a large

amount of genes encoded for inflammatory mediators and cytokines. These elicited cytokines are

believed to be responsible for cell proliferation, differentiation, immuno-regulation and cytotoxicity

for bacteria and bacterial products (6).

As it is known that LPS has multiple and different effects on macrophages, including the

induction of inflammatory cytokines such as IL-1, IL-6 and TNF (12; 15; 18; 19). Although IL-1

is a potent inflammatory cytokine with various biological activities regulating host defense and

immune responses (20), there has been less investigation of LPS induction of IL-1. In the process

of IL-1 maturation, a precursor molecule referred to as prointerleukin-1β (proIL-1) is produced in

the cytosol of macrophages. Originally, proIL-1 is a 31-34 kD non-active form of cytokine, until it

is cleavaged via an enzymatic procession into a 17 kD mature functional form by IL-1 converting

enzyme (ICE), or caspase 1 (21-24). After the ICE cleavage, the active IL-1 is released and

exhibits its diverse biological functions.

As growth factors or cytokines, LPS induces MAPKs, including extracellular signal-

regulated kinase (ERK), c-JUN NH2-terminal protein kinase (JNK), and p38 mitogen activated

protein kinase (p38). These play key roles in the LPS-mediated signal transduction between

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extracellular membrane stimulation and the cytoplasmic response and nuclear activity of the

activation of the gene (11; 17). Specifically, ERK activation involves cytokine induction and

regulation during responses to bacterial products (25-27). In addition to responding to numerous

physiological and stress stimuli (28-31), JNK is considered to play roles in regulating the

expression of various stress-induced proteins and inflammatory cytokines (12;31). p38 is

activated in response to stress signals such as LPS, osmotic stress and pro-inflammatory cytokines

(14; 27; 32; 33). Previous studies have shown that the p38 pathway is critical for LPS-stimulated

cytokines release (25; 33), including IL-1 and TNF induction in monocytes (17). Moreover,

previous findings indicated that p38 activity could be regulated via Rho GTPase (Rac1) and PAK

(34; 35).

Reactive oxygen species (ROS) regulate multiple cellular functions such as DNA synthesis

(36), transcription factor activation (37), gene expression (38) and proliferation (39). The

induction of H2O2 can affect various gene expressions in macrophages ((40; 41) and this

manuscript). There are several indications that H2O2 may act as a cellular secondary messenger

(37; 42). H2O2 is considered to activate NF-κB (37), which regulates the expression of multiple

immune and inflammatory molecules. Therefore, for the cell immune system, H2O2 displays a

critical role in the host defense mechanism. Alternatively, the activation of ROS including H2O2

acts as a significant and adverse participant in abnormal inflammatory diseases. One of the

important sources of ROS in phagocytes, including macrophage, is NADPH oxidase activation

during phagocytosis (43). NADPH oxidase, which could be inhibited by diphenyleneiodonium

chloride (DPI), is a membrane-associated complex, which consists of a minimum of four proteins,

i.e., membrane-bound cytochrome b558, p47phox, p67phox and Rac2 in humans, or Rac1 in rodents

(43). Among these components, Rac is the most critical for a functional NADPH oxidase whose

activity is regulated by small GTP-binding proteins. In response to phagocytic stimuli, i.e., lipids,

soluble peptides and opsonized particulates, NADPH oxidase moves electrons from NADPH to

reduce O2 to O2.-. These superoxide free radicals are rapidly converted to H2O2 and O2 by cytosolic

superoxide dismutase (44; 45) and then sequentially to other products. Nevertheless, both the

mechanism for LPS induction ROS and the role of induced ROS in cytokine expression of

macrophages are still unclear.

To understand how ligation of LPS induces inflammatory cytokine IL-1 gene expression

in macrophages, we studied the molecular mechanism for LPS-mediated signal transduction

pathways for various protein kinases in the regulation of IL-1 gene expression. Here we

demonstrate for the first time that LPS regulation of proIL-1/IL-1 expression in macrophages

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J774A.1 is partially mediated by NADPH oxidase-derived ROS, as well as by accompanying the

PTK/PI-3 kinase/Rac1/p38 pathway, but less by the PKC/MEK/ERK pathway and the PTK/PI-3

kinase/Rac1/JNK pathway.

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EXPERIMENTAL PROCEDURES

Cell cultures---Murine macrophage J774A.1 cell (J774A.1) was obtained from ATCC

(Rockville, MD), propagated in RPMI 1640 medium supplemented with 10% heated-inactivated

fetal bovine serum (Hyclone Co., Logan, UT) and 2 mM L-glutamine (Life Technologies, Inc., MD)

and cultured in 37°C, 5% CO2 incubator, unless otherwise indicated.

Materials---Lipopolysaccharide (LPS, from E. coli 0111:B4), sodium orthovanadate,

phenylmethylsulfonyl fluoride (PMSF), bovine serum albumin (fraction V), diphenyleneiodonium

chloride (DPI), N-acetyl-cysteine (NAC) and curcumin were purchased from Sigma Co. (St. Louis,

MO). Calcium Phosphate Transfection reagent was purchased from InVitrogen Co. (Carlsbad,

CA). Immobilon PVDF membrane was obtained from Millipore Inc. (Bedford, MA). DuPont

non-radioactive Western Blot Chemiluminescence Reagent, Renaissance was purchased from

DuPont NEN Research Products Co. (Boston, MA). REZOl C&T was from PROtech Technology

Co. (Taipei, Taiwan). GeneAmp RNA PCR kit for RT-PCR amplification was purchased from

Perkin Elmer Inc. (Branchburg, NJ). Growth factors and antibodies: Anti-IL-1β, 3ZD

monoclonal antibody (a gift from the NIH, Bethesda, MD, USA to Dr. H.-Y. Hsu), anti-rabbit IgG-

HRP, anti-mouse IgG-HRP and protein A/G plus-agarose were obtained from Santa Cruz

Biotechnology (Santa Cruz, CA); anti-phosphotyrosine, clone 4G10 (mouse monoclonal IgG2bκ)

was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The CaspACE Assay

System, Fluorometric, was purchased from Promega Co. (Madison, WI). Kinase assay kits: the

p44/42 MAP Kinase Assay Kit, SAPK/JNK Assay Kit and p38 MAP Kinase Assay Kit were

purchased from Cell Signaling Technology (Beverly, MA). The Rac Activation Assay Kit was

purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein kinase inhibitors:

PD98059 was from Cell Signaling Technology; whereas calphostin C, and herbimycin A were from

Calbiochem-Novabiochem Corp. (La Jolla, CA), LY294002 and SB203580 were from Sigma Co.

Oligonucleotides: primers for proIL-1/IL-1, glyceraldehyde phosphate dehydrogenase (GAPDH)

were synthesized from local MD Bio. Inc. (Taipei, Taiwan). The dominant negative JNK

construct (DN-JNK) was a gift from Dr. Michael Karin (UCSD, San Diego, CA, USA) (46). The

dominant negative Rac1 construct (DN-Rac1) was a gift from Dr. S. Bagrodia (Cornell University,

Ithaca, NY, USA) (47).

Measurement of LPS-induced intracellular ROS---Intracellular ROS stimulated by LPS

was measured by detecting the fluorescent intensity of either 2’ , 7’ -dichlorofluorescein diacetate

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(DCFH) or the improved analogue carboxyl-DCFH (CM-DCFH) (Molecular Probes, Inc., Eugene,

OR) oxidized product, DCF (or CM-DCF), as described (48). Briefly, 0.5-1.0 x 106 J774A.1

cells/ml grown in serum- and phenol red-free RPMI medium (starvation medium) for 24 h, were

pre-incubated with 2 µmol/L of CM-DCFH and NAC at 37°C for 30 min in the dark. To these

were added fresh starvation medium containing LPS for additional incubation at the indicated times.

The relative fluorescent intensity of fluorophore CM-DCF, which was formed by peroxide

oxidation of the non-fluorescent precursor, was detected at an excitation wavelength of 485 nm and

an emission wavelength of 530 nm with a fluorometer, Cytofluor 2300 (Millipore Inc., Bedford,

MA). In contrast, CM-DCFH with starvation medium was used as a blank control.

Measurement of LPS-induced NO---Cells were seeded in a 1x107/100 mm plate containing

6 ml media. After 24 h incubation, the media were replaced by FBS-free medium 1 h prior to LPS

treatment, and the conditioned media were collected at the indicated times. For assaying NO, 50

µl conditioned media of each sample was mixed with 50 µl Griess reagent I (GR I: 1%

sulfanilamide dissolved in 5% H3PO4) in a 96-well assay plate at room temperature for 10 min.

This was followed by addition of Griess reagent II (GR II: 0.1% (w/v) N-1-

naphthylethylenediamine) at room temperature in the dark for another 10 min. The absorbance of

reaction (wavelength at 550 nm) in each well was measured by an MRX microplate reader (Dynex

Tech. Inc., Chantilly, VA.). A standard calibration curve for NaNO2 was constructed, and

calculation of the relative absorbance unit for the NO concentration of each sample was as

described (19).

RNA Isolation, RT and PCR Amplification for detecting the expression of proIL-1/IL-1,

Analysis of ICE Activity, Western blot Analysis, Assay Activity of ERK, JNK, p38 and Enzyme-

Linked Immunosorbent Assay (ELISA) for measurement of IL-1---All methods and procedures

followed the previous descriptions (34).

Measurement of LPS-induced Rac activity---The measurement of LPS-induced Rac

activity was followed the manufacturer’ s instructions from Upstate Biotechnology, Inc. Briefly,

cells were starved for 18 h, and then pretreated with or without NAC for 30 min prior to LPS

stimulation. The whole cell lysates were collected at the indicated times and immediately

incubated with PAK-1 PBD agarose for immunoprecipitating active PAK-1/Rac-GTP complex.

Because the Rac-GTP is able to associate with PAK-1, the amount of activated form of Rac was

detected via mouse monoclonal IgG2b anti-Rac by Western blot analysis.

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RESULTS

LPS-induced ROS in murine macrophage J774A.1 cells---To investigate LPS-induced ROS

in J774A.1 cells, detection of fluorescent oxidative product of CM-DCFH was used to examine cell

oxygen bursts (48). As shown in Fig. 1A, within several minutes, LPS (1 µg/ml) stimulation of

cells rapidly induced significant ROS, including more H2O2 production, when compared to that of

LPS-untreated control samples. Although, there was less, but still significant, H2O2 generation by

low doses of LPS (0.1 µg/ml) (data not shown). In contrast, pretreatment of NAC (10 mM), a

potent antioxidant, quickly reduced LPS-induced ROS release (Fig. 1A) in a dose-dependent

fashion (data not shown). The difference of LPS-induced ROS production in cells treated with or

without NAC continued for the longer testing period of up to 240 min (Fig. 1B). NAC

approximately attenuated 20% of H2O2 in cells at the steady state (Figs. 1A and 1B). Although, we

could not totally rule out the involvement of other ROS including superoxide anion (O2.-) (data not

shown). Since LPS induces NO (19; 49; 50), a member of the ROS superfamily which plays

multiple biological roles in macrophages, we extended our examination to measure LPS-induced

NO in our system. The cells were grown at various times in media with or without LPS, and the

released NO in conditioned media was measured. In the early stage of LPS stimulation, there was

no detectable NO released from the cells (Fig. 1C). After incubation of LPS for 7 h, cells

gradually produced NO at about 2 µM, and at 24 h the concentration of NO reached 22 µM. In

contrast, in cells co-incubated with NAC, LPS-induced NO reduced to 0.8 µM and 6.4 µM at 7 h

and 24 h, respectively (Fig. 1C).

LPS induction and the role of LPS-induced ROS in regulation of IL-1 gene expression: IL-1

secretion, proIL-1 protein production and IL-1/proIL-1 message induction as well as activation of

ICE---To detect the effect of LPS on inflammatory cytokine, IL-1 gene expression by macrophages,

we first used ELISA to quantitate mature IL-1 secretion in the conditioned medium of J774A.1 cells.

As shown in Fig. 2A, compared with untreated control cells, IL-1 secretion increased with a longer

LPS incubation time. For example, at 3 h and 24 h, the concentration of LPS-induced IL-1 was

about 5 pg/ml and 22 pg/ml, respectively. In contrast, there was less IL-1 released from NAC-

pretreated cells, i.e., 1 pg/ml (3 h) and 12 pg/ml (24 h), indicating that LPS-stimulated ROS

involves IL-1 secretion.

Next, we investigated whether the increased mature IL-1 was reflected by induction of a

precursor of IL-1, proIL-1 protein (molecular weight 34 kD) and of IL-1/proIL-1 message. A time

course study of LPS-induced proIL-1 protein was performed by Western blot assay. For 3-h LPS

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stimulation, in the absence of NAC, a 21-fold increase of proIL-1 protein was detected as compared

with the un-stimulated cells, peaking at 8 h and about 24-fold relative to the control sample. After

12 h, proIL-1 protein decreased, gradually returning to the basal level at around 18 h and 24 h (Fig.

2B). To further examine the role of LPS-stimulated ROS in proIL-1 expression, proIL-1 protein

was determined via using cells pre-incubated with NAC followed by LPS treatment. As shown in

Fig. 2B, NAC blockage of LPS-stimulated ROS decreased LPS-induced proIL-1 protein about 35 %

of NAC-free cells between 3 h and 12 h. However, after 18 h, there was no difference in proIL-1

protein with or without NAC treatment.

Next, using RT-PCR, for 3-h LPS stimulation, proIL-1 mRNA increased more than 30-fold,

as compared with un-treated cells. Between 8-h and 12-h, LPS-induced proIL-1 mRNA gradually

reduced (Fig. 2C). After 18 h, the increased proIL-1 mRNA returned to the basal level. In

contrast, in cells pretreated with NAC, followed by LPS stimulation between 3 h and 8 h, there was

generally about 70% to 40% of proIL-1 mRNA, as compared with NAC-free, LPS-treated samples

(Fig. 2C). This indicated that NAC decreased LPS-induced proIL-1 mRNA. A similar

regulation of proIL-1 mRNA detected by Northern analyses was compatible to the message

detection by RT-PCR (data not shown).

Post-transcriptional regulation and processing of the proIL-1 protein into a mature IL-1

secretion via ICE has been well reported in various cells, including macrophages, as in our previous

reports (22; 23; 34; 51). Since incubation of macrophages with LPS induced proIL-1 protein

simultaneously increased IL-1 secretion in a time-dependent fashion (Figs. 2B versus 2A), we

initially examined whether LPS affects ICE activity during IL-1 secretion. As shown in Fig. 2D,

LPS increased ICE activity up to 2-fold between 3 h and 12 h, peaking at a 4-fold increase by 18 h,

and sequentially returning to the basal level at 24 h, as compared to the control cells. In contrast,

there was no difference in ICE activity in NAC-pretreated cells under LPS stimulation, as compared

to the control cells.

LPS induces MAPKs activity as well as the effect of LPS-induced ROS on MAPKs activity--

-To dissect LPS-mediated signal transduction pathways in the regulation of proIL-1 protein and IL-

1 secretion, we systematically dissected LPS-stimulated MAPKs including ERK, JNK and p38.

Furthermore, we examined the role of LPS-induced ROS in mediating the activity of MAPKs.

Incubation of cells with LPS led to phosphorylation of Elk-1, a transcriptional factor, indicating

LPS activation of ERK (Fig. 3A). The experiments for time course of LPS-induced ERK activity

were also conducted. As detected by Western blot analysis with an antibody that specifically

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recognized the activated, serine 383-phosphorylated form of Elk-1 by activated ERK (52), a 4-fold

increase in ERK activity was detected at 15 min, reaching a maximum level of 12-fold at 30 min

and reducing to 6-fold after 60 min (Fig. 3A). In comparison, there was almost no ERK activity in

untreated control cells. To further examine whether LPS-induced ROS involves LPS mediation of

ERK activation, cells were pre-treated with NAC for 30 min prior to LPS incubation. Upon LPS

stimulation, ERK activity in NAC-pretreated cells increased by 2-fold (15 min) and 7-fold (30 min),

respectively, as compared to samples without LPS. The relatively lower ERK activity in NAC-

treated cells compared to that of NAC-free cells indicates that ROS is involved in the LPS-induced

ERK activity.

The inflammatory stress response of J774A.1 cells to LPS prompted us to investigate the

LPS-mediated JNK signaling pathway. Therefore, we examined whether LPS activates JNK in the

cells. Stimulation of cells with LPS leads to activation of JNK in a time-dependent fashion, as

determined by Western blot analysis via anti-phospho-c-Jun, which specifically recognizes the

activated serine 63-phosphorylated form of c-Jun (28; 53; 54). Upon 15-min LPS incubation of

cells, JNK activity gradually increased, with a maximum level of JNK activation at 30 min, about a

5-fold increase over that of untreated cells. Then after 60 min, it returned to the basal level (Fig.

3B). Moreover, experiments using NAC to study the effect of ROS on LPS-mediated JNK activity

were conducted. Surprisingly, JNK activity of NAC-treated cells is super-induced up to 9-fold at

both 15 min and 30 min, as compared with the control cells (Fig. 3B). The reason for induction of

higher JNK activity in NAC-treated cells than that of NAC-free cells (i.e., 9-fold versus 5-fold)

upon LPS stimulation is unclear and requires further investigation.

To explore additional LPS-mediated signal transduction pathways, we further examined

whether LPS induces p38 activity. This is another important member of the MAPK superfamily

related to inflammatory responses (32; 33). Upon LPS stimulation, the p38 activity gradually

increased, as detected by Western blot analysis with anti-phospho-ATF-2, an antibody that

specifically recognizes the activated threonine 71-phosphorylated form of ATF-2 (55) (Fig. 3C).

The result of time course study of LPS-induced p38 activity indicated that at around 15 min, the

activity of p38 in LPS-treated cells was approximately 15 times greater than those of the control

cells, and gradually decreased to about 10 and 8 times approximately at 60 min and 240 min,

respectively (Fig. 3C). In contrast, deprivation of LPS-induced ROS by pretreatment of cells with

NAC significantly reduced LPS-stimulated p38 activity to the basal level after about 30 min to 240

min, as compared to NAC-free cells (Fig. 3C). A quantitative analysis of LPS-induced ERK, JNK

and p38 activity to compare the MAPKs activity with and without NAC is summarized in Fig. 3D.

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The role of LPS-induced protein kinases (PK) in the regulation of proIL-1 protein

expression---We demonstrated above that LPS stimulates a battery of MAPK activity, comparable

to other recent reports (15; 26; 27; 32; 33; 56). To further elucidate the role of various LPS-

induced PK-mediated signaling pathways in the regulation of proIL-1 protein expression, we

utilized certain specific pharmacological antagonists such as LY294002, SB203580, curcumin,

calphostin C, and PD98059 that inhibit the activation of PI-3 kinase, p38, JNK, PKC and MEK1,

respectively. The dose response for specific PK inhibitors was monitored by directly assaying

individual kinase activity, and the effective working concentrations of PK inhibitors were

determined (data not shown) as previously reported (34). Cells were pre-incubated with these PK

inhibitors for 1 h prior to stimulation of LPS, and proIL-1 protein expression in indicated samples

were analyzed by Western blotting with anti-IL-1 monoclonal antibody. ProIL-1 protein

expression was reduced to 15% by LY294002, to 20% by SB203580, to 50% by curcumin, and to

70% by either calphostin C or by PD98059, as compared with proIL-1 in LPS-stimulated cells (Fig.

4A).

Moreover, experiments for transient transfection of dominant negative JNK (DN-JNK),

dominant negative Rac1 (DN-Rac1) or dominant negative PI 3-kinase (DN-PI3K) into J774A.1

cells were conducted in order to further investigate the role of PI 3-kinase/Rac in LPS induction of

proIL-1. Results of Western blot analyses show that after LPS stimulation, there was about 30%

less proIL-1 protein in DN-JNK or DN-Rac1 transfected cells than that of control cells, and 40%

less proIL-1 protein in DN-PI3K transfected cells as compared with control cells (Fig. 4B, sample 2

versus samples 4, 6 and 8). In addition, pre-incubation of NAC in the dominant negative

transfectants synergistically reduced proIL-1 expression, except for DN-JNK transfectant.

Specifically, with NAC, there was 30% of the proIL-1 protein left in mock cells, and 20% of the

proIL-1 left in either DN-Rac1- transfected cells or DN-PI3K-transfected cells, respectively (Fig.

4B, sample 3 versus samples 7 and 9). However, there was 50% of the proIL-1 protein left in DN-

JNK transfected cells (Fig. 4B, sample 3 versus sample 5).

LPS-induced Rac activity and the role of ROS in Rac activation---It has been reported that

LPS induces Rac/NAPDH oxidase-dependent ROS formation (57-59). To further investigate the

potential upstream signaling molecules involving in LPS-mediated transducing networks, we

focused on the activity of a relevant molecule, Rac1. Upon LPS stimulation, Rac1 activity was

elevated 2.5- to 3-fold between 5 min and 60 min (Fig. 5, samples 2, 3 and 4 versus 1). In contrast,

blocking LPS-induced ROS via pre-incubation of NAC consequently reduced Rac1 activity by 40%,

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as compared with untreated samples (Fig. 5, samples 2, 3 and 4 versus samples 6, 7 and 8).

Moreover, DPI, an NADPH oxidase effective inhibitor (60) significantly reduced LPS-triggered

Rac1 activation by 50% as compared with the samples without DPI-preincubation (Fig. 5, samples

2, 3 and 4 versus samples 10, 11 and 12).

LPS induces protein tyrosine phosphorylation (PTP) as well as the effect of LPS-induced

ROS and NADPH oxidase on PTP in J774A.1 cells---To elucidate the mechanism by which LPS

induces IL-1, the LPS-initiated upstream signal transduction pathway was examined. For this we

examined the LPS stimulation of PTP via performance of SDS-PAGE and Western blot analysis

with monoclonal anti-phosphotyrosine IgG (61). As shown in Fig. 6, incubation of J774A.1 cells

with LPS induced the appearance of many phosphotyrosyl proteins as compared to cells incubated

with media alone. For example, upon 15-min LPS stimulation of cells, some apparent tyrosine-

phosphorylated proteins of molecular weight (M.W.) 38, 42, 44, 52-75 and 100-120 kD (Fig. 6,

samples 1 versus 2) were observed. These phosphotyrosyl proteins were identified and

specifically immuno-reacted with anti-p38 IgG, anti- ERK IgG, anti-JNK IgG, anti-Lyn IgG and

anti-Src IgG, respectively (data not shown). In contrast, NAC and DPI were utilized to investigate

the influence of LPS-induced ROS and NADPH oxidase in the tyrosine phosphorylation of proteins.

Pretreatment of the cells with either NAC or DPI for 30 min prior to LPS stimulation resulted in a

decrease of LPS-induced tyrosine phosphorylation of various proteins (Fig. 6, sample 2 versus

samples 4 and 6), indicating the involvement of ROS in LPS-induced PTP.

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DISCUSSION

LPS, a potent activator for macrophages, stimulates ROS such as H2O2 ((40) and this

manuscript), superoxides ((62), and data not shown) and NO ((49) and this manuscript) as well as

inducing a battery of signal transductions leading to gene expression (10-16; 26; 63). However,

the relationship among LPS-induced ROS and mediated various PK activation and regulation of the

inflammatory cytokine IL-1 is unclear. Here, we analyze and define the molecular mechanisms

for LPS-stimulated ROS production, signal transductions and PK activity in regulation of IL-1

expression.

Using the CM-DCFH fluorescent method, we demonstrate that LPS immediately induced

H2O2 in J774A.1 cells, but that pretreatment of cells with NAC effectively inhibited LPS-induced

H2O2 elevation. We also report here that LPS induced detectable NO production, which was

obviously much later than both proIL-1/IL-1 induction (7 h versus 3 h, Figs. 1 and 2) and MAPKs

activation (7 h versus 1 h, Figs. 1 and 3). Although, NO is an important reactive oxygen molecule

mediating response of LPS in macrophages (49). Our current findings suggest that LPS-induced

ROS, rather than NO, play a more significant role in the activation of MAPKs and regulation of

proIL-1/IL-1 in our system (see discussion below). However, we could not totally rule out effects

of NO or crosstalk among NO and other ROS in these reactions.

Initially, the data from studies of ICE activity, ELISA, Western blot analysis and RT-PCR

showed that LPS-triggered H2O2 played an important role in the induction of IL-1 gene. To

investigate further the molecular mechanism for ROS effects, we found evidence that ROS

participated in IL-1/proIL-1 expression involving multiple levels of regulation. Specifically, in

the presence or absence of antioxidant NAC, our results indicate LPS-induced ROS regulation of

IL-1 expression, probably at proIL-1 message (transcriptional), at proIL-1 protein production

(translational) and at the processing of proIL-1 to mature IL-1 releasing via modification of ICE

activity (post-translational). In addition to the antioxidation of NAC involving the elevation of

intracellular glutathione levels (38; 40; 64; 65), NAC is able to block the activation of JNK in

response to TNF (2) or IFN co-stimulation with TNF in macrophages (66). Nevertheless, the

mechanism for NAC reduction of LPS-induced ICE activity related to IL-1 secretion requires

further study.

LPS stimulation of MAPKs (ERK, JNK and p38) activity and activation of transcription

factors (AP-1, NF-κB and ATF-2) have previously been reported (25; 26; 67-69). In addition,

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activation of MEK/ERK and p38 critical for LPS-induced cytokine production by

monocytes/macrophages have been documented (25; 26; 33). However, there is little discussion

of the molecular mechanism for LPS-mediated MAPKs in the regulation and expression of proIL-

1/IL-1. Here, we demonstrate for the first time that there were differences in the activation time

and in extent of activity for LPS-stimulated ERK, JNK and p38. Moreover, the effect of LPS-

induced ROS on the activity of specific MAPK was also diverse (Fig. 3). For example, NAC

blockage of LPS-induced ROS resulted in a decrease in ERK and p38 activity, but an increase in

JNK activity as compared to untreated cells. We summarize the effect of LPS-induced ROS on

the relative activity of LPS-induced MAPKs (Fig. 3D), which indicates that the existence of ROS

was more effective and important to p38 activity than either ERK or JNK activity in LPS-mediated

regulation of proIL-1/IL-1 expression. Nevertheless, when all of the three MAPK pathways were

activated simultaneously, a dramatic induction of IL-1 gene expression was observed, suggesting a

cooperative effect on regulation of IL-1 among these kinases (15). We further used various

pharmacological antagonists to PKs. It dissected the specific role of individual PK in the

regulation of proIL-1 expression (Fig. 4A). Additional studies of the use of dominant PK negative

transfectants verified that both further NAC and specific dominant negative PK synergistically

reduced LPS-induced proIL-1 protein expression (Fig. 4B). Although stimulation of a single PK

produced only a modest effect on proIL-1 protein, activation of each PK pathway was important for

complete induction of IL-1 gene expression.

Various studies have shown the activation of JNK and p38 were mediated via PI-3

kinase/Rac1/PAK signaling pathway (2; 47; 70). Rac1 is important to LPS-induced proIL-1 in at

least two aspects: first the role of Rac1 in NADPH oxidase assemble/function (71), and second its

role in PI-3 kinase/Rac1/PAK signaling pathway. Rac is known as an important component of

functional NADPH oxidase, which is localized at the plasma membrane or specific granule vesicle

membrane of phagocytes, and which produces O2.- upon phagocytosis processing (71). Because of

the DPI reduction of LPS-triggered Rac1 activation (Fig. 5B), our data implies that LPS activates

phagocytosis-related NADPH oxidase, which entangles Rac1 leading signals as the initial of ROS

(Fig. 7). Moreover, data on transient transfection of DN-Rac1 indicates partially reduced LPS-

mediated proIL-1 expression. Although, pre-incubation of NAC led to additional decreasing

proIL-1 (Fig. 4B), indicating the importance of Rac1 and/or NADPH oxidase and ROS in LPS-

induced proIL-1. Our finding of NADPH oxidase and Rac1 involving ROS production is

consistent with the recent report of Rac1-dependent ROS in LPS-mediated TNF secretion (58).

Indeed, our current results clearly demonstrate that the originality of LPS-induced ROS including

H2O2 as a secondary messenger is probably derived from NADPH oxidase, and less from LPS-

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damaged mitochondria in the short term (data not shown), or via other molecule(s), but which, if

any, of these mediate signal transductions and regulate proIL-1 protein and IL-1 secretion.

Ligation of LPS quickly induces diverse phosphotyrosyl proteins, leading to activation of

various downstream molecules (27). Although, there is no evidence that the receptors including

TLRs have a functional tyrosine kinase domain. Since alteration of protein tyrosine

phosphorylation (PTP) is one of the most important signaling events leading to cellular responses

(6), and to further understand how LPS is coupled to the initiation of early signaling by inducing

various PTP resulting in upregulation of IL-1 gene expression, we initially examined and identified

the LPS-induced protein tyrosine phosphorylation, including MAPKs (ERK, JNK and p38) and the

Src family (Src and Lyn). Furthermore, we investigated LPS ligation, showing the relationships

among LPS-induced ROS and NADPH oxidase and observed alteration of PTP. The results of

antioxidant of ROS (NAC) or NADPH oxidase inhibitor (DPI) reducing LPS-induced tyrosine

kinase activation suggest that ROS indeed involves LPS-induced protein tyrosine phosphorylation.

In summary, we are the first to demonstrate that in macrophages the relationship among

NADPH oxidase-derived ROS, protein tyrosine phosphorylation and the role of ROS in the

regulation of IL-1 gene expression during LPS stimulation. Our results dissect the molecular

mechanism of LPS-induced ROS regulation of proIL-1 protein and IL-1 secretion. This regulation

is at multiple levels and involves LPS-mediated PTK, PK and MAPK activity. Although, LPS-

mediated cooperation of multiple MAPKs induces IL-1 gene expression, we show for the first time

that the LPS-induced PTK/Rac1/PI-3 kinase/p38 pathway plays a relatively more important role

than the pathways of PTK/PKC/MEK/ERK and of PTK/ Rac1/PI-3 kinase/JNK in the specific

cytokine regulation (Fig. 7). Thus, understanding how LPS-mediated signal transduction

pathways in IL-1 induction is responsible for macrophage antibacterial action, may provide

improved treatments for severe inflammatory responses leading to cellular injury, multi-organ

damage and concurrent sepsis.

Acknowledgments--- This work was supported by research grants of NSC 90-2321-B-010-005 (H.-

Y. Hsu) from the National Science Council, Taiwan, and NHRI-EX90-8937SL (H.-Y. Hsu) from

the National Health Research Institutes, Taiwan, and A-91-B-FA09-2-4 (H.-Y. Hsu), Program for

Promoting University Academic Excellence from the Ministry of Education, Taiwan. We

gratefully acknowledge the DN-JNK construct from Dr. M. Karin (UCSD, San Diego, CA, USA),

DN-Rac1 construct from Dr. S. Bagrodia (Cornell University, Ithaca, NY, USA), and the Anti-IL-

1β, 3ZD monoclonal antibody from the NIH (Bethesda, MD, USA).

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FIGURE LEGENDS

Fig. 1. Effect of NAC on LPS-induced release of ROS and NO production. To detect release

of ROS, J774A.1 cells were pre-incubated with CM-DCFH (2 µM) and NAC (10 mM) for 30 min,

followed by substitution with medium containing LPS (1 µg/ml) for additional incubation for the

indicated times. The relative fluorescent intensity of fluorophore CM-DCF was then detected.

The data expressed is one of four representative experiments. LPS-induced release of ROS A.

over a short period between 0 and 15 min; B. over a long period between 0 and 240 min. C. To

detect LPS-induced NO production, cells (1x107/6 ml medium/100 mm plate) were stimulated with

LPS (0.1 µg/ml) and supernatants were collected after LPS stimulation at the time indicated, and

assayed for the NO concentration by the Griess method. The data is representative of three similar

experiments and expressed as mean ± S.E.

Fig. 2. Effect of LPS-induced ROS on LPS stimulation of proIL-1/IL-1 expression in

macrophage J774A.1 cells. A. Time course of LPS-induced IL-1 and inhibitory effect of NAC

on IL-1 secretion. Cells (1x107/6 ml medium) were stimulated with LPS (0.1 µg/ml) and the

supernatants were collected after stimulation at the time indicated. To analyze the effect of NAC

on LPS-induced IL-1 secretion, cells were incubated with NAC (10 mM) for 30 min prior to LPS

stimulation. Samples were assayed for the IL-1 content in an IL-1-specific ELISA, as described in

the Experimental Procedures section. The experiments were conducted three times and all data is

expressed as mean ± S.E. B. Time course and the effect of NAC on LPS-induced proIL-1 protein.

After cells were stimulated as described above for the indicated time interval, cell lysates were

analyzed by Western blotting with anti-IL-1 monoclonal antibody at a position of 34 kD for proIL-1.

ProIL-1 and β-actin (internal control for protein loading) are indicated as arrows on the right hand

side, and one of three experiments is presented (n = 3). C. Time course and the effect of NAC on

LPS-mediated proIL-1/IL-1 mRNA expression. Total RNA was isolated from treated or untreated

J774A.1 cells, as indicated. Ethidium bromide-stained agarose gel with proIL-1 at 563 bps and

normalized by comparison to RT-PCR of mRNA of glyceraldehyde phosphate dehydrogenase

(GADPH), a constitutively expressed gene at 450 bps. Arrows indicate RT-PCR products of

proIL-1 and GAPDH, respectively. The results shown are representative of three independent

experiments. Quantification of protein and mRNA expression was carried out by

PhosphorImager of each sample using ImageQuaNT software from Molecular Dynamics, which

is expressed as fold increase relative to the level of cells without LPS treatment (t = 0, activity of

control cells defined as 1). D. Time-dependent activation of LPS-induced ICE activity in J774A.1

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cells. Followed by LPS and NAC treatments, cell extracts (90 µg of protein) were incubated in

the presence of the fluorescence ICE substrate Ac-YVAD-AMC (50 µM) for 1 h at 30°C. ICE

activity was measured fluormetrically after subtracting cleavage with excitation at 360 nm and

emission at 460 nm, as previously described (34). All values shown are means of triplicate values

± S.E.

Fig. 3. LPS-induced ROS regulation of multiple MAPKs activation. J774A.1 cells were

treated as described in Fig. 2. Then, activated ERK, JNK and p38 were immunoprecipitated from

cell lysates using specific antibodies. In vitro kinase assays were performed using the

immunoprecipitants as kinases, as previously described (34). Recombinant Elk-1 fusion protein,

c-Jun fusion protein and ATF-2 fusion protein were used as substrates for ERK, JNK and p38,

respectively. LPS-induced ERK (A), JNK (B) and p38 (C) activity were monitored by

phosphorylation of substrate. These were measured by quantitative immunoblotting with

phospho-Elk-1 (Ser383) antibody, phospho-c-Jun (Ser63) antibody and phospho-ATF-2 (Thr71)

antibody, respectively. Comparison and quantitative analysis of LPS-induced ERK, JNK and p38

activity are represented in histograms (D). The values shown are the mean ± S.E. of quadruplicate

determinations. All data of relative increased activity is expressed as comparison with untreated

J774A.1 cells, (i.e., t = 0, activity of control cells defined as 1). These figures are representative

of four such independent experiments.

Fig. 4. Effect of protein kinase inhibitors on LPS-induced proIL-1 expression and role of

ROS in LPS-induced proIL-1 protein of dominant-negative JNK, Rac1 or PI3 K-transfected

cells. A. Effect of inhibitor of PKC (calphostin C), of MEK1 (PD98059), of JNK (curcumin), of

p38 (SB203580), of PI-3 kinase (LY294002) and NAC on LPS-induced proIL-1 expression. This

was followed by cells pre-treated with calphostin C (12.64 nM) (sample 2), PD98059 (50 µM)

(sample 3), curcumin (10 µM) (sample 4), SB203580 (1 µM) (sample 5), LY294002 (50 µM)

(sample 6) for 1 h or NAC (10 mM) (sample 7) for 30 min. After this, the pre-treated cells were

then incubated with LPS (0.1 µg/ml) for an additional 8 h. After incubation, samples were

subjected to Western blot analysis of proIL-1, as previous described in Fig. 2. The indicated

arrows on the right hand side represent positions of proIL-1 and β-actin (an internal control). This

experiment is representative of three similar experiments. Histograms represent quantification of

proIL-1 of each sample. All data of relative quantity are expressed as comparisons with untreated

control cells (sample 1, and activity of control cells defined as 1). B. Role of ROS in LPS-

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induced proIL-1 protein of dominant-negative JNK, Rac1 or PI3 K-transfected cells. J774A.1 was

transiently transfected with various dominant-negative constructs for 24 h, as described previously

(2; 34), then incubated with NAC prior to LPS-stimulation. ProIL-1 protein expression in cells

was analyzed with Western blotting as above A. The values shown are the mean ± S.E. of

triplicate determinations.

Fig. 5. The roles of LPS-induced ROS and NADPH oxidase in differential regulation of

LPS-mediated Rac1 activity. Cells were in the presence or absence of antioxidant (NAC, 10mM)

or NADPH oxidase inhibitor (DPI, 25 µM), followed by LPS stimulation at the indicated times.

The Rac1-GTP complex was immunoprecipitated from whole cell lysate by PAK-1 PBD as well as

active Rac1. Then, the immunoprecipitant was quantitatively measured, using the anti-Rac

antibody as described in the Experimental Procedures section or in the manufacturer’ s instructions.

The data of relative Rac1 activity is expressed as compared with untreated control cells (sample 1,

Rac1 activity is defined as 1). The mean ± S.E. of results from three replicate determinations and

a representative experiment is shown.

Fig. 6. LPS stimulation of protein tyrosine phosphorylation in J774A. 1 cells. Cells were

pretreated with NAC (10 mM) or DPI (25 µM) for 30 min prior to stimulation with or without LPS

(1 µg/ml). Briefly, Sample 1, un-stimulated control; sample 2, LPS 15 min; sample 3, NAC 30

min; sample 4, NAC 30 min sequentially with LPS 15 min; sample 5, DPI 30 min; sample 6, DPI

30 min sequentially with LPS 15 min. Cells were lysed and analyzed by Western blot with anti-

phosphotyrosine monoclonal antibody. The detailed method was as previously described (61).

The indicated bars on the left side represent molecular weight (kD), and those on the right hand

side represent some significant molecules, as discussed in text. The experiment is representative

of three similar experiments.

Fig. 7. The proposed model for LPS-mediated ROS and signal transduction pathways in

the regulation of proIL-1/IL-1 expression.

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Fig. 1

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0

5

10

15

20

25

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)

IL-1

con

cent

rati

on (

pg/m

l) ctr

LPS

NAC

LPS+NAC

A

B

proIL-1 (34 kD)

-NAC (control) +NAC

LPS (h) 0 3 8 12 18 24 0 3 8 12 18 24

actin

Fold 1 21 24 14 2 1 1 7 10 7 2 1

C

LPS (h) 0 3 8 12 18 24 0 3 8 12 18 24

Fold 1 31 26 8 1 1 1 23 11 6 1 1

proIL-1 (563 bp)

-NAC (control) +NAC

GAPDH (450 bp)

Fig. 2

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0

1

2

3

4

0 2 4 6 8 10 12 14 16 18 20 22 24Time (h)

Rel

ativ

e IC

E A

ctiv

ity

ctrLPSNACLPS+NAC

D

Fig. 2

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Phospho-Elk-1

LPS (min) 0 15 30 60 0 15 30 60

+NACA

-NAC (control)

BLPS (min) 0 15 30 60 0 15 30 60

Phospho-c-Jun

+NAC-NAC (control)

C

Phospho-ATF-2

LPS (min) 0 15 30 60 240 0 15 30 60 240

+NAC-NAC (control)

02468

101214161820

0 15 30 60 0 15 30 60

Rel

ativ

e M

AP

Ks

acti

vity

ERK

JNK

p38

LPS without NAC LPS with NAC

Time (min)

D

Fig. 3

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LPS + + + + + + +NAC - - - - - - +LY294002 - - - - - + -SB203580 - - - - + - -curcumin - - - + - - -PD98059 - - + - - - -calphostin C - + - - - - -

proIL-1

actin

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7

Rel

ativ

e pr

oIL

-1 p

rote

inA Sample 1 2 3 4 5 6 7

Fig. 4

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0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6 7 8 9

Rel

ativ

e P

roIL

-1 P

rote

in

Sample 1 2 3 4 5 6 7 8 9

proIL-1

NAC - - + - + - + - +LPS - + + + + + + + +

Moc

kDN-JN

K

DN-R

ac1

DN-PI3

KB

Fig. 4

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0

0.5

1

1.5

2

2.5

3

3.5

1 2 3 4 5 6 7 8 9 10 11 12

Rel

ativ

e R

ac 1

Act

ivit

y

Rac 1

LPS (min) 0 5 15 60 0 5 15 60 0 5 15 60

control +NAC +DPI

Sample 1 2 3 4 5 6 7 8 9 10 11 12

Fig. 5

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LPS (min) 0 15 0 15 0 15

control NAC DPI

M.W. (kD)

205

126

89

50

33

28

52-75 kD

100-120 kD

38 kD

Sample 1 2 3 4 5 6

44 kD42 kD

Fig. 6

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Fig. 7

Ligand (LPS)

JNK

PI-3 Kinase

proIL-1

membrane

IL-1

p38

c-Jun ATF-2Elk1

proIL-1 mRNA

nucleus

Protein tyrosine kinase

MEKK 1,2,3,4

MEK 3

PLCγ1

PKC

ERK1/ERK2

MEK 1

calphostin C

PD98059SB203580

TLR

LY294002DN-PI3K

curcuminDN-JNK

Rac1

PAK

DN-Rac 1

NADPHoxidase

ROS NAC

DPI

proIL-1/IL-1 DNA

ICE

Rac1

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Hsien-Yeh Hsu and Meng-Hsuan Wenregulation of IL-1 gene expression

Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the

published online April 8, 2002J. Biol. Chem. 

  10.1074/jbc.M111883200Access the most updated version of this article at doi:

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Additions and Corrections

Vol. 277 (2002) 22131–22139

Lipopolysaccharide-mediated reactive oxygen speciesand signal transduction in the regulation of inter-leukin-1 gene expression.

Hsien-Yeh Hsu and Meng-Hsuan Wen

Page 22134, Fig. 2A: The concentrations of IL-1 shown on thefigure are incorrect. The correct figure is shown below.

Page 22133: Under “Results,” in the first paragraph under“LPS Induction and the Role of LPS-induced ROS in Regulationof IL-1 Gene Expression: IL-1 Secretion, proIL-1 Protein Pro-duction, and IL-1/proIL-1 Message Induction as Well as Acti-vation of ICE,” the last two sentences, which originally state“For example, at 3 h and 24 h, the concentration of LPS-induced IL-1 was about 5 pg/ml and 22 pg/ml, respectively.”and “In contrast, there was less IL-1 released from NAC-pretreated cells, i.e. 1 pg/ml (3 h) and 12 pg/ml (24 h), indicatingthat LPS-stimulated ROS involves IL-1 secretion.’’ should bechanged to ‘‘50 pg/ml and 220 pg/ml” and “10 pg/ml (3 h) and120 pg/ml (24 h).” However, these errors do not affect theconclusions of this study.

FIG. 2

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 36, Issue of September 6, p. 33530, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

We suggest that subscribers photocopy these corrections and insert the photocopies at the appropriateplaces where the article to be corrected originally appeared. Authors are urged to introduce thesecorrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice ofthese corrections as prominently as they carried the original abstracts.

33530