phosphorylation of extracellular signal-regulated kinases 1/2 predominantly enhanced in the...

ORIGINAL PAPER

Phosphorylation of Extracellular Signal-Regulated Kinases1/2 Predominantly Enhanced in the Microglia of the RatSpinal Cord Following Lipopolysaccharide Injection

Dan Zhou Æ Min Fei Æ Qin Shen Æ Chun Cheng Æ Youhua Wang ÆJian Zhao Æ Hai-Ou Liu Æ Linlin Sun Æ Yonghua Liu Æ Xiaowei Yu ÆAiguo Shen

Received: 29 September 2007 / Accepted: 2 February 2008 / Published online: 1 March 2008

� Springer Science+Business Media, LLC 2008

Abstract The present study was initiated to inves-

tigate the role of extracellular signal-regulated

kinases (ERK) 1/2 signaling pathway in the early

response of spinal cord to systemic inflammation by

using Western blotting and immunohistochemical

techniques in a rat model intraperitoneally injected

with 10 mg/kg of lipopolysaccharide (LPS). The

results showed that there was a considerable amount

of phosphorylated ERK 1/2 protein in the spinal cord

of inflamed animals killed under pentobarbital anes-

thesia. The result of Western blotting showed that the

phosphorylation level of ERK 1/2 in the spinal cord

was increased at one hour; then 12 and 24 h after LPS

injection the level decreased, while the total ERK 1/2

level seemed unchanged. The phosphorylated ERK

1/2 dominantly existed in the microglia cells of the

gray matter of spinal cord, as demonstrated with

double immunofluorescent staining 1 h after LPS

injection. Collectively, the present results suggest

that ERK signal pathway involve the cellular activa-

tion in the spinal cord following systemic inflam-

mation, with ERK mainly in microglia. The increase

of phosphorylation of ERK 1/2 in microglia of spinal

cord after LPS injection implicates that ERK signal-

ing pathway involves intracellular activity of

microglia responding to the inflammation.

Keywords ERK �Microglia � Lipopolysaccharide �Spinal cord � Rat

Introduction

Lipopolysaccharide (LPS), a component of the outer

membrane of Gram-negative bacteria, is a potent

inducer of systemic inflammation (Ulevitch and

Tobias 1995). In response to LPS and inflammatory

cytokines induced by LPS, the endothelium alters

vascular tone and permeability, absorbs infectious

insults, and directs leukocytes into the areas of

inflammation (Cines et al. 1998).

The extracellular signal-regulated kinases (ERK) 1

and 2 are a subfamily of mitogen-activated protein

Dan Zhou and Min Fei contributed equally to this work.

D. Zhou � C. Cheng � H.-O. Liu � L. Sun �Y. Liu � X. Yu � A. Shen (&)

The Jiangsu Province Key Lab of Neuroregeneration,

Nantong University, Nantong 226001, People’s Republic

of China

e-mail: [email protected]

D. Zhou � Q. Shen

Department of Biochemistry, Medical College of Nantong

University, Nantong 226001, People’s Republic of China

M. Fei

Institute of Medical Biotechnology, Soochow University,

Suzhou 215007, People’s Republic of China

Y. Wang � J. Zhao

Department of Orthopaedics, Affiliated Hospital

of Nantong University, Nantong 226001, People’s

Republic of China

123

Cell Mol Neurobiol (2008) 28:867–874

DOI 10.1007/s10571-008-9264-3

kinases (MAPK) that transduce extracellular stimuli

into intracellular post-translational and transcriptional

responses (Cano et al. 1995; Impey et al. 1999).

Members of the MAPK superfamily are doubly

phosphorylated on both the threonine and tyrosine

residues in the Thr-X-Tyr sequence within the

catalytic core of the enzyme by upstream MAPK

kinases and become active. Many factors, such as

mitogens, growth and neurotrophic factors, hor-

mones, neurotransmitters, inflammatory cytokines,

lipopolysaccharide, ultraviolet light, heat shock, and

osmotic change, activate various but specific mem-

bers of the MAPK superfamily, to produce short-term

functional (nontranscriptional) changes by phosphor-

ylating kinases, receptors and ion channels, or long-

term adaptive changes by activating transcriptional

factors such as the CREB (Cano et al. 1995), c-Jun

and c-Myc (Davis et al. 1994). Functions of ERKs

have been studied in animals in both physiological

and pathological states.

While there is growing evidence showing that LPS

can cause hyperalgesia (Watkins et al. 1994, 1995;

Reeve et al. 2000), little is known on the role of MAPK/

ERK pathways in this process. The aim of this study is to

gain the data for the activation of ERK 1/2 and its

expression pattern following the onset of LPS injection.

Material and Methods

Experimental Animals and Treatments

Male Sprague–Dawley (SD) rats weighing 220–275 g

(Department of Animal Center, Medical College of

Nantong University, Nantong, China) were housed in

plastic cages at 24 ± 1�C on a 12 h light–dark cycle

and given free access to laboratory chow and water.

All animal experiments were carried out in accordance

with the United States National Institutes of Health

Guide for the Care and Use of Laboratory Animals.

Lipopolysaccharide

Some of them were injected with 10 mg/kg LPS

(E. coli 055:B5, Sigma). LPS (2.2–2.75 mg) was

diluted in normal sodium (NS) at a volume of 0.5 ml

and was injected intraperitoneally at a dose of 10 mg/

kg without anesthesia. The controls were only

injected with NS (0.5 ml).

Western Blotting Analysis

Western blotting was prepared from normal spinal

cords or from inflamed spinal cords at 1, 3, 6, 12,

24 h after intraperitoneal injection of LPS (4 to 5 rats

each group). To obtain samples for Western blotting,

the normal or inflamed L3–L5 spinal cords segments

were excised and snap frozen at -70�C until use. To

prepare lysates, frozen spinal cord samples were

minced with eye scissors in ice. The samples were

then homogenized in lysis buffer (1% NP-40,

50 mM/l Tris, pH 7.5, 5 mM/l EDTA, 1% SDS, 1%

sodium deoxycholate, 1%Triton-X100, 1 mM/l

PMSF, 10 lg/ml aprotinin, and 1 lg/ml leupeptin)

and clarified by centrifuging for 20 min in a micro-

centrifuge at 4�C. After determination of its protein

concentration with the Bradford assay (Bio-Rad), the

resulting supernatant (50 lg of protein) was sub-

jected to 10% (w/v) SDS-polyacrylamide gel

electrophoresis (PAGE). The membrane was then

blocked with 5% nonfat milk and incubated with

primary antibody diluted in blocking buffer overnight

at 4�C. The primary antibodies used for Western

blotting were rabbit anti-phosphorylated ERK 1/2

(pERK) (1:500; Cell Signaling, Beverly, MA), rabbit

anti-total ERK 1/2 (tERK) (1:500, Cell Signaling),

after incubating with an anti-rabbit horseradish

peroxidase-conjugated secondary antibody (1:500,

Santa Cruz), protein was visualized using an

enhanced chemiluminescence system.The density of

specific pERK bands was measured with a computer-

assisted image analysis system (Adobe Systems, San

Jose, CA) and normalized against tERK level, and the

relative differences between control and treatment

groups were calculated and expressed as relative

increases by setting control as 1. Values are respon-

sible for at least three independent reactions.

Immunohistochemistry

After defined survival times, control and inflamed

rats (4 to 5 rats each group) were terminally

anesthetized and perfused through the ascending

aorta with saline, followed by 4% paraformaldehyde.

After perfusion, the normal and inflamed spinal cords

were removed and postfixed in the same fixative for

3 h and then replaced with 20% sucrose for 2–3 days,

following 30% sucrose for 2–3 days. Serial trans-

verse sections (14 lm) were cut through the L3–L5

868 Cell Mol Neurobiol (2008) 28:867–874

123

spinal region. All the sections were blocked with 10%

donkey serum with 0.3% Triton X-100 and 1% BSA

for 2 h at room temperature (RT) and incubated

overnight at 4�C with anti-pERK antibody (rabbit,

1:100; Cell Signaling), followed by incubation in

biotinylated secondary antibody (goat anti rabbit,

1:200; Vector Laboratories, Burlingame, CA). Stain-

ing was visualized with DAB (Vector Laboratories).

We used a blocking peptide (Cell Signaling) to

evaluate the specificity of pERK antibody reactivity

in immunohistochemistry protocols. Twice the vol-

ume of peptide as volume of antibody was used in

100-ll total volume. Incubate for a minimum of

30 min prior to adding the entire volume to the slide.

For double labeling, anti-pERK rabbit polyclonal

antibodies (1:100) were used in combination with

mouse mAbs recognizing NeuN (neuron marker,

1:600; Sigma, St. Louis, MO), GFAP (astrocytes

marker, 1:200; Sigma), CD11b (microglia marker,

1:50; Serotec, UK). Briefly, sections were incubated

with both primary antibodies overnight at 4�C,

followed by a mixture of FITC- and TRITC-conju-

gated secondary antibodies (FITC-Donkey anti

Rabbit, 1:100, Jackson Laboratory; TRITC-Donkey

anti Mouse, 1:100, Jackson Laboratory) for 2 h at

4�C. The stained sections were examined with a

Leica fluorescence microscope (Germany).

Quantification and Statistics

All data were analyzed with Stata 7.0 statistical

software. The OD of the immunoreactivity is repre-

sented as mean ± SEM. Student’s t-tests were used.

One-way ANOVA and Dunnett t tests were also used

for statistical analysis. P values less than 0.05 were

considered statistically significant.

Results

Western Blotting Analysis of ERK and pERK

Levels

Western blotting analysis showed that ERK existed in

the spinal cord at L3–L5 levels in normal rat

anesthetized with pentobarbital. The molecular

weight for ERK one and ERK two was 44 and

42 kDa, respectively. It appeared that blotting density

of tERK was almost unchanged 1–24 h after LPS

administration (Fig. 1a).

Western blotting also demonstrated that there were

considerable amounts of pERK immunoreactivities in

the L3–L5 spinal cord. The blotting density of pERK

was increased bilaterally 1 h after LPS injection

(P \ 0.01); however, 12 and 24 h later, the density of

pERK decreased (Fig. 1b).

Cellular Distribution of pERK-like

Immunoreactivity in the Lumbar Spinal Cord

One hour after LPS injection, immunohistochemistry

showed intensely stained pERK-like immunoreactiv-

ity (LI) glial cells in the lumbar segments (Fig. 2a).

pERK-LI cells were increased significantly in the

anterior horn (Fig. 2e), especially in the glia-like

cells of the dorsal funiculus (Fig. 2f) and the dorsal

horn (Fig. 2g) at a higher magnification. Then we

used a blocking peptide to evaluate the specificity of

pERK antibody reactivity (Fig. 2b). There was no

Fig. 1 Western blotting analysis showed the tERK and pERK

levels in the L3–L5 segments before (control) and 1, 3, 6, 12,

24 h after LPS administration (a). Note that phosphorylated

ERK1/2 levels (indicated by pERK1 and 2) increased at 1 h,

then decreased at 24 h. As for the density of tERK, there was

no detectable difference among these time points. The bar chart

below demonstrated the pERK which expressed as fold of the

tERK (b). Results are the mean ± SEM of three independent

sets of analyses. (*P \ 0.01, significantly different from the

control group and other time points)

Cell Mol Neurobiol (2008) 28:867–874 869

123

pERK-positive signal, demonstrating that pERK

antibody was specific. A few pERK-LI cells were

observed in the spinal cord of NS group (Fig. 2c) and

no positive cells located in negative control (Fig. 2d).

Immunofluorescence staining showed that there

were a few cells in the lumbar spinal cord expressing

pERK-LI in normal rat. In the anterior horn, pERK

staining was mainly localized to the cytoplasm of

neuronal soma (Fig. 3a), and a few pERK-LI glia

cells expressed in the dorsal horn (Fig. 3d). After 1 h

of LPS injection, there was a considerable number of

pERK-positive glia-like cells located in the anterior

horn (Fig. 3b) and dorsal horn (Fig. 3e), the number

of positive cells was reduced more than 12 h after

LPS administration especially in the anterior horn

(Fig. 3c) and the dorsal horn (Fig. 3f).

Double Immunofluorescent Staining for pERK

and CD11b, NeuN, and GFAP

To determine the cellular types which express pERK-

LI in rat spinal cord, the double immunofluorescent

staining was done in the group 1 h after LPS

administration. We had found that pERK-LI was

mostly located in microglia cell, especially in the

anterior horn (Fig. 4a–c), the central canal (Fig. 4d–

f), and the dorsal horn (Fig. 4g–i). We also found that

almost all pERK-positive cells were microglia cells

in the dorsal funiculus (Fig. 5). It was also shown that

no double staining of pERK and NeuN/GFAP was

observed (Figs. 6, 7), no matter in the anterior horn or

dorsal horn.

Discussion

The results of the present study demonstrated that

pERK protein level was induced in the spinal cord

within several time points after intraperitoneal

administration of LPS, as assessed by Western

blotting and immunohistochemistry. We first exam-

ined the pERK in the spinal cord at several time

points of LPS injection and found that it could be

detected and reached a peak in 1 h, and then the level

of pERK decreased at 12 h, and the result was

confirmed by immunohistochemistry, suggesting that

Fig. 2 Localizations of pERK-immunoreactive cells in the

spinal cord of LPS-injected rats at 1 h (a, e, f, g) and normal

sodium (NS)-treated rats (c). Intensely stained pERK-immu-

noreactive cells were detected in the anterior horn,

intermediate zone, dorsal horn, dorsal funiculus, and central

canal in the inflamed rats (a). Some moderately stained cells

were observed in the spinal cord of rats injected with NS (c).

There were no positive cells in the segments with blocking

peptide (b) or negative control (d). At a higher magnification of

the spinal cord (a) pERK-positive glia cells were located in the

anterior horn (e), especially in the dorsal funiculus (f) and

dorsal horn (g). AH, anterior horn; IN, intermediate zone; DH,

dorsal horn; DF, dorsal funiculus; C, central canal. Scale bars:

100 lm (a–c), 20 lm (d–g)


123

pERK may function in that context of events. From

these results, we know that pERK is expressed in rat

spinal cord in the early inflammation stage. As we

found that there was no change in the intensity of

tERK after LPS injection, implicating that the

up-regulation of the activation of ERK1/2 1 h fol-

lowing inflammation is probably due to the increased

phosphorylation of ERK1/2 other than an increase

of protein synthesis. Thus, it is possible that the

up-regulation of ERK1/2 activation occurs through

phosphorylation at the early stage.

Based on the results of double immunofluorescent

staining, the pERK-LI mostly locates in microglia

cells, so we conclude that pERK can have functions

Fig. 3 pERK was induced in a time-dependent manner in the

spinal cord. In the anterior horn, very little pERK-immunore-

active cells was detected in cells associated with the spinal cord

of rats as control (a), rapid increase of pERK-immunoreactive

cells were found at 1 h after LPS injection (b), the number of

positive cells was reduced at 12 h (c). As for the dorsal horn,

the tendency of pERK was the same as the anterior horn (d–f).The increased pERK-like immunoreactivity was mainly

located in the glia-like cells. Scale bars: 20 lm

Fig. 4 Double

immunofluorescent staining

for pERK (green) and

CD11b (red), a microglia

marker, in the rat L3–L5

spinal segments. Note that

in the inflamed rat, the

distribution of pERK-

immunoreactive cells in the

anterior horn (a–c), the

central canal (d–f), and

the dorsal horn (g–h) were

numerous and almost all the

positive cells were

microglia cells. i was the

overlapped high

magnification from part of gand h. The arrows pointed

to some pERK singly

stained microglia cells.

Scale bars: 20 lm (a–h),

5 lm (i)


123

Fig. 5 Photomicrographs showing the distributions of pERK

(green) and CD11b protein (red)-immunoreactive in the dorsal

funiculus of rat spinal cord at L3–L5 segments at 1 h after LPS

administration. Note that the distribution patterns of CD11b

and pERK were quite similar. Many of CD11b protein-positive

glial cells seem to be expressing pERK. Scale bar: 20 lm

Fig. 6 Double


for pERK and NeuN, a

neuronal marker, in the

anterior horn (a–c) and

dorsal horn (d–f) of the rat

L3–L5 spinal segments at

1 h after LPS

administration. The

overlappings of pERK

(a, d) and NeuN (b, e) were

shown in c and f,respectively. Note that there

was no co-localization

between them. Scale bar:

20 lm

Fig. 7 Double


for pERK and GFAP, an

astrocyte marker, in the

anterior horn (a–c), the

central canal (d–f), and

dorsal horn (g–i) of the rat

L3–L5 spinal segments at

1 h after LPS

administration. The

overlappings of pERK (a, d,

g) and GFAP (b, e, h) were

shown in c, f, and i,respectively. Note that there

was no co-localization

between them. Scale bar:

20 lm


123

through microglia in vivo during the early inflam-

mation stage. Through some research we know that

ERK signaling pathway involves intracellular activity

of microglia responding to the inflammation.

Microglia, together with astrocyte and oligoden-

drocyte, constitute the major population of glial cells

within the central nervous system (CNS). Microglial

cells are quite sensitive to even minor disturbances in

CNS homeostasis, and they become readily activated

during many neuropathological conditions (Davis

et al. 1994). During the process of activation,

microglia display conspicuous morphological and

functional plasticity, which involve changes in cell

morphology, number, cellular surface receptor

expression, and production of growth factors, cyto-

kines, tumor necrosis factor-a, as well as nitric oxide

(Streit 1996; Bhat and Zhang 1999). The increase of

phosphorylation of ERK 1/2 in microglia of spinal

cord after rhizotomy implicates that ERK signaling

pathway involves intracellular activity of microglia

responding to the experimental injury. Parenchymal

microglia are ubiquitously distributed in the CNS

where they comprise up to 20% of the total non-

neuronal cell population (Lawson et al. 1990). These

cells are thought to play a prominent role in

infectious, traumatic, inflammatory, ischemic, and

degenerative CNS disease processes. The role of

microglia as mediators of CNS inflammation is, in

part, promulgated through their ability to process and

present class II-restricted antigens to CD41 T cells

(Aloisi 1999; Hickey and Kimura 1988). There is

increasing evidence supporting a role for microglia in

the pathogenesis of pain. A microglial inhibitor,

minocycline, reduces neuropathic pain (Raghavendra

et al. 2003). But the role of ERK in microglia is still

poorly understood.

ERK is the best-studied member of the MAPK

family and plays a critical role in intracellular

signal transduction, neural plasticity, and inflam-

matory responses (Ji and Woolf 2001). The activa-

tion of ERK is known to lead to a variety of

functional changes in both neuronal and non-neuro-

nal cells.

ERK activation by inflammation is also involved

in regulating gene transcription and maintaining

persistent inflammatory pain (Ji et al. 2002). The

acute pain hypersensitivity established within min-

utes of intraplantar formalin can be reduced by

preventing ERK activation (Ji et al. 1999), an effect

that is too quick ([1 h) to be mediated by an

inhibition of transcription and is likely therefore to

represent some post-translational change downstream

of the activated ERK. At present, it is not clear what

the substrate for such post-translational change is, but

it may well be an ion channel or receptor, such as the

NMDA or AMPA receptor (Woolf and Salter 2000).

Such post-translational changes underlie the induc-

tion and maintenance of central sensitization, a

use-dependent plasticity that outlasts its initiating

stimulus by tens of minutes (Woolf 1983; Woolf and

Wall 1986). If inflammatory hypersensitivity were a

manifestation only of a central sensitization main-

tained by ongoing afferent input from the inflamed

tissue, then blocking the initiation of central sensiti-

zation, by inhibiting an ERK-mediated phosphor-

ylation, should reduce the hypersensitivity over a

period of tens of minutes as the key proteins would be

dephosphorylated. Since the ERK pathway activation

stimulates cell growth and proliferation (Boulton

et al. 1991; Segal and Greenberg 1996), and inflam-

mation induced microglial cell proliferation in the

gray matter of corresponding spinal cord segment, the

possibility is that MAPK signaling pathway-induced

microglia activation plays a key role in inflammatory

processes. The detailed role of ERK in these

processes needs to be explored in future.

Above all, we know that the ERK signaling

pathway exist in the rat spinal cord during early

inflammation stage, and the microglia cells may play

a role in this process. However, its physiological

function as well as the mechanism and significance

during the process remain to be further elucidated.

Acknowledgments This work was supported by the National

Natural Scientific Foundation of China Grant (No.30300099

and No. 30770488), Natural Scientific Foundation of Jiangsu

Province Grant (No. BK2003035 and No. BK2006547), College

and University Natural Scientific Research Programme of

Jiangsu Province (No. 03KJB180109 and No. 04KJB320114),

Technology Guidance Plan for Social Development of Jiangsu

Province Grant (BS2004526), Health Project of Jiangsu

Province (H200632), ‘‘Liu-Da-Ren-Cai-Gao-Feng’’ Financial

Assistance of Jiangsu Province Grant (No. 2).

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